Uterine histomorphological and immunohistochemical investigation during follicular phase of estrous cycle in Saidi sheep

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Khormi, Mohammed A. Alfattah, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4790328/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Jan, 2025 Read the published version in BMC Veterinary Research → Version 1 posted 11 You are reading this latest preprint version Abstract Background Saidi sheep are one of the most important farm animals in Upper Egypt, particularly in the Assiut governorate. Since they can provide meat, milk, fiber, and skins from low-quality roughages, sheep are among the most economically valuable animals bred for food in Egypt. Regarding breeding, relatively little is known about the Saidi breed. The uterus is an important organ for reproduction in mammals. Therefore, the purpose of this work was to provide further details on the histological, histochemical, and immunohistochemical analyses of the uterus during the follicular phase of the estrous cycle. In order to examine the histological changes in the uterus, 11 healthy Saidi ewes (38.5 ± 2.03 kg weight) ranging in age from 2 to 5 years were used. Results In Saidi sheep, the uterine histological and immunological picture during follicular phase of estrous cycle was characterized by epithelial and stromal proliferation and apoptosis. Leucocytic recruitment (lymphocytes, plasma cells and mast cells) was also observed. The most prominent features of the follicular phase were uterine gland adenogenesis, vascular angiogenesis, and oxidative marker expression, epithelial, stromal and muscular expression of PRA. Conclusion This study provides new evidences of the uterine morphological and immunohistochemical picture of the Saidi sheep during the follicular phase of the estrus cycle. These findings have growing significance to understand the key mechanisms that is characteristic of successful reproduction in Saidi sheep in order to enhance fertility and reproductive health of this livestock species. Saidi sheep Uterus Endometrium Estrous cycle SOD2 GR PRA Mast cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Background Sheep are extensively raised as livestock worldwide because they yield a lot of meat, fat, milk, wool, and other useful products. Many studies on breeding have been focused on improving sheep reproductive performance and litter size [ 1 ]. The Saidi breed of Egyptian sheep was once thought to be the oldest breed of Egyptian sheep, and its breeding grounds are in Upper Egypt, south of Assiut. It has a long, thick tail and its fleece is typically dark brown in color [ 2 ]. Due to its high reproductive performance the demand for this breed increases [ 3 – 5 ]. Saidi ewes had almost no seasonal variation in their reproductive cycles, despite a decrease in estrous activity in the spring [ 6 ]. The uterus is an important organ for reproduction in mammals. The uterus provides a microenvironment required for receptivity, implantation and for growth and development of the conceptus. The uterus is essential for the following processes: (1) transportation, storage, and maturation of spermatozoa; (2) synthesis of prostaglandin F2α, the luteolysin required for ovarian cyclicity in domestic animals; (3) offering of an embryotrophic environment for conceptus (embryo/fetus and associated extraembryonic membranes) growth and development; and (4) pickup of the conceptus at parturition [ 7 – 9 ]. Sheep have a bicornuate uterus with a small common corpus and single cervix. Histologically, the uterus in ewe was formed of endometrium, myometrium and perimetrium. The main cyclic changes were occurred in endometrium and to lesser extent in myometrium [ 10 – 12 ]. The key mechanisms that is characteristic of successful reproduction in Saidi sheep and gaps in knowledge that must be the subject of research in order to enhance fertility and reproductive health of this livestock species. Relatively little is known about the uterine picture of the Saidi sheep during the follicular phase of the estrous cycle. So, the aim of the current study is to give more details on the histological, histochemical, and immunohistochemical analyses of the uterus during the follicular phase of the estrous cycle in order to enhance fertility and reproductive health of this breed of sheep. Materials and Methods Animals and samples: Uteri were collected from eleven apparently healthy slaughtered (according to Islamic religion) Saidi sheep aged 2 to 5 years and weighted (38.5 ±2.03 Kg) at Assiut abattoir, Assiut governorate, Egypt. Histological examination: Uteri (n=11) were fixed in 10 % neutral buffered formalin. The formalin-fixed samples were dehydrated in ascending grades of ethanol, cleared in methyl benzoate, and embedded in paraplast. Paraffin sections at 5 μm in thickness were cut and stained with the following histological stains: Haematoxylin and Eosin for general histological examination of the uterus [13] Periodic acid Schiff (PAS) technique for demonstration of glycoprotein [13]. Masson’s trichrome technique for staining collagen fibers [14]. Picro-Sirius red technique to differentiate between mature and immature collagen fibers [15, 16] Orcien stain for detection of the distribution of elastic fibers in the uterus [17] Oxidative stress detection: Immunohistochemistry of glutathione reductase and superoxide dismutase 2. Paraffin-embedded tissue sections were deparaffinized, rehydrated, and rinsed in phosphate buffered saline (PBS). Then slides were placed in 10 mM sodium citrate buffer (pH 6.0) for antigen retrieval at (95–98 °C) in a water bath for 20 min. Endogenous peroxidase was blocked by incubating the slides in 3% hydrogen peroxide for 10 min at room temperature. This followed by washing the slides in PBS (3 times for 5 min each). The sections were incubated overnight in a humid chamber with rabbit polyclonal antibodies. For immunohistochemical detection of glutathione reductase (GR) and superoxide dismutase 2 (SOD2) in the ovary, we used polyclonal anti-glutathione reductase and anti-superoxide dismutase 2 antibodies, respectively (Chongqing Biospes Co., Ltd, China) and Power-Stain™ 1.0 Poly horseradish peroxidase (HRP) DAB Kit (Genemed Biotechnologies, Inc, 458 Carlton Ct. South San Francisco, CA 94080, USA) [18]. Negative image analysis was performed to assess the complex color micrographs that were obtained and to give more details[19, 20]. Apoptosis detection: TUNEL assay Detection of apoptosis was done using In Situ Cell Death Detection Kit, Fluorescein (Sigma-Aldrich, USA). Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay was designed to detect apoptotic cells that undergo extensive DNA fragmentation during the late stages of apoptosis. This method was depending up on the ability of TdT to label blunt ends of double-stranded DNA breaks independent of a template. The protocol we used as the previous published protocol [21]. Slides were rinsed with PBS and directly analyzed under a fluorescence microscope. Immunoexpression of progesterone receptor alpha (PRA): The protocol used was according to the company instructions and as our previous study [22]. The fixed ovaries were dehydrated in ethanol, cleared in methyl benzoate and then embedded in paraplast. Sections (5 μm) of paraplast-embedded tissue were dewaxed by xylene. Subsequently, rehydration by 100%, 95%, 80% and 70% ethanol, and slides were rinsed in PBS (pH 7.4). Endogenous peroxidase were prevented by adding 3% hydrogen peroxide followed by washing in PBS. For antigen detections, the slides were placed in 10 mM sodium citrate buffer (pH 6.0) at (95–98 °C) in a water bath for 20 min followed by cooling at room temperature. Sections were then rinsed in PBS. Immunoexpression of progesterone receptor alpha was done by using; progesterone receptor rabbit pAb (Catalog No.: A0321), ABclonal, USA. Sections were then incubated with the primary antibodies for 30-60 min at room temperature. The slides were washed with PBS then follow the company instructions of poly Q stain 2 step detection system goat anti-mouse/rabbit HRP, peroxidase quench, DAB kit, quartett, Germany. The sections were counterstained in Harris hematoxylin for 30 s. Then sections were dehydrated by ethanol 95%, and ethanol 100%, cleared of in xylene, and mounted with DPX. Immunohistochemical detection of mast cells: For detection of mast cell, we used Mast Cell Tryptase (3G3) Monoclonal Antibody (Bioss antibodies) and Poly Q stain 2-step detection system, goat anti-mouse/rabbit HRP, peroxidase quench, and DAB kit from Quartett, Germany. The protocol used was according to the company instructions and as our previous study [22]. All staining slides were examined by an Olympus BX51 microscope and the photographs were taken by an Olympus DP72 camera adapted into the microscope. Results General uterine histomorphological characters during follicular phase of estrous cycle in Saidi sheep: Microscopical analysis of the uterus of Saidi sheep during the follicular phase of the estrous cycle revealed that the uterine wall was composed of three layers: the inner layer; endometrium (mucosa), the middle layer; myometrium (muscular layer), and the outer layer; perimetrium (serosa). The endometrium had mucosal folds, caruncles and narrow uterine lumen. The caruncles were non glandular, highly cellular and highly vascular endometrial elevation or projections. The endometrium formed of lamina epithelialis of pseudostratified columnar epithelium with intraepithelial lymphocytes and connective tissue lamina propria contained uterine glands, fibroblasts, collagen fibers, blood vessels and leucocytic infiltrations especially lymphocytes and plasma cells (Fig. 1A-D). The current study showed that the follicular phase of estrous cycle was characterized by epithelial proliferation and many epithelial invaginations which form the uterine glands (uterine gland adenogenesis). The uterine glands in Saidi sheep during the follicular phase of estrous cycle were highly branched and highly coiled and formed of columnar epithelium which surrounded by myoepithelial cells and had intraepithelial lymphocytes (Fig. 1E-H).. The myometrium was formed of a thick, inner circular layer and a thin, outer longitudinal layer of smooth muscle fibers. A well vascularized stratum vascularis with many branches of the uterine artery was observed in the outer most layer of inner circular myometrium. The outer perimetrium was composed of a mesothelial layer and sub mesothelial connective tissue (Fig. 1I). Glycoprotein localization in the Saidi sheep uterus during follicular phase of estrous cycle : Glycoprotein localization in the sheep uterus during follicular phase of estrous cycle revealed that the endometrium had PAS positive epithelial basement membrane of lamina epithelialis and uterine glands. No PAS positive secretion in the uterine glands could be observed. PAS positive internal and external elastic lamina in the stratum vascularis could be detected (Fig. 2A-C). Elastic and collagen fibers distribution in the Saidi sheep uterus during follicular phase of estrous cycle : Elastic fibers distribution in the sheep uterus during follicular phase of estrous cycle revealed that few elastic fibers were observed in the sub epithelial and between the uterine glands in the endometrium. Furthermore few elastic fibers between the smooth muscle fibers in the myometrium were observed. However many elastic membranes were observed in the external elastic lamina of endometrial blood vessels and in the internal and external elastic lamina of the blood vessels of the tunica vascularis (Fig. 2D-F). While the collagen fibers distribution in the sheep uterus during follicular phase of estrous cycle revealed that the endometrium had moderate amount of collagen fibers which found between the uterine glands and few amounts in the caruncles. Whereas the myometrium had few amount of collagen fibers in-between the smooth muscle fibers of the inner circular and outer longitudinal layers and moderate amount of collagen fibers around blood vessels in the stratum vascularis (Fig. 2G-I and Fig. 3A & B). GR and SOD2 immunostaining in the Saidi sheep uterus during follicular phase of estrous cycle : GR immunostaining in the sheep uterus during the follicular phase of estrous cycle showed slight GR immunostaining in the lamina epithelialis and in the stroma cells of lamina propria and negative GR immunostaining in the uterine glands. Furthermore, slight GR immunostaining in the smooth muscle fibers of the blood vessels of the stratum vascularis and negative GR immunostaining in the smooth muscle fibers of the inner circular layer of the myometrium could be demonstrated (Fig. 4A-C). Whereas, slight SOD2 immunoexpression were observed in the lamina epithelialis, stroma cells and the smooth muscle fibers of the blood vessels of the lamina propria in addition the uterine glands expressed no SOD2 immunostaining (Fig. 4D-H). Slight SOD2 immunostaining in the smooth muscle fibers of blood vessels of stratum vascularis and negative SOD2 immunostaining in the smooth muscle fibers of the inner circular layer of the myometrium were also noticed (Fig. 4I). Fig. 5 was a negative image of Fig. 4 to give more detailed of GR and SOD2 immunoexpression. Apoptosis in the Saidi sheep uterus during follicular phase of estrous cycle : TUNEL assay immunofluorescence in the sheep uterus during the follicular phase of estrous cycle explored some apoptotic endometrial glandular epithelial cells (Fig. 6A) and some apoptotic endometrial stromal cells (Fig. 6B). While the inner circular layer of myometrium showed few or no apoptotic cells (Fig. 6B). Apoptotic endometrial stromal cells in the caruncles were also observed in addition to apoptotic smooth muscle fibers of the blood vessels of the stratum vascularis (Fig. 6D). PRA immunolocalization in the Saidi sheep uterus during follicular phase of estrous cycle : PRA immunolocalization in the sheep uterus during the follicular phase of estrous cycle revealed strong PRA immunolocalization in the lamina epithelialis and strong to moderate PRA immunolocalization in the sub-epithelial stroma cells (Fig. 7A). Also moderate PRA immunostaining was observed in the stromal cells and endothelial cells of blood vessels in the caruncles (Fig. 7B). The columnar epithelial cells of the uterine glands expressed strong to moderate PRA (Fig. 7C) while the smooth muscle fibers of the inner circular layer of the myometrium expressed moderate PRA (Fig. 7D). Moreover, the endothelium and the smooth muscle fibers of the blood vessels of the stratum vascularis showed moderate PRA immunolocalization (Fig. 7E). On the other hand, mild PRA immunoexpression was noted in the smooth muscle fibers and blood vessel endothelial cells in the myometrium's outer longitudinal layer. Whereas there was mild PRA immunolocalization in the perimetrial mesothelial cells and in the perimetrial blood vessel endothelial cells (Fig. 7F). Mast cells detection in the Saidi sheep uterus during follicular phase of estrous cycle : Interestingly, tryptase-positive immunostaining mast cells were recruited in the deep lamina propria of the caruncles during the follicular phase of the estrous cycle (Fig. 8A). Although there were no mast cells seen in the superficial lamina propria underneath the lamina epithelialis (Fig. 8B). Mast cells were rounded or oval cells with rounded central or eccentric nucleus and filled with tryptase positive immunostaining granules (Fig. 8C). Some degranulated mast cells were seen close to the macrophage in the endometrial lamina propria (Fig. 8D). Mast cells were also observed in-between uterine glands (Fig. 9A) and in the inner circular smooth muscle layer of the myometrium (Fig. 9B). Moreover they were noticed around the blood vessels of the stratum vascularis (Fig. 9C) and in the outer longitudinal smooth muscle layer of the myometrium (Fig. 9D). Discussion Microscopical analysis of the uterus of Saidi sheep during the follicular phase of the estrous cycle revealed that the uterine wall was composed of three layers: the inner endometrium, middle myometrium and the outer perimetrium. The endometrium had mucosal folds, caruncles and narrow uterine lumen. The endometrium formed of lamina epithelialis of pseudostratified columnar epithelium with intraepithelial lymphocytes and connective tissue lamina propria contained uterine glands, fibroblasts, collagen fibers, blood vessels and leucocytic infiltrations especially lymphocytes and plasma cells. The uterus (horns and body) in ruminants was lined with the simple columnar to pseudostratified columnar epithelium. The mean height of the epithelium was less in follicular phase [ 23 – 25 ]. In buffalo the endometrium was lined with three types of columnar cells, i.e. ciliated, non-ciliated cells and basal cells [ 25 ]. The endometrial stroma (propria submucosa) consisted of fibro-reticular connective tissue, stromal cells and blood vessels. Its cellular components comprised of stromal cells, fibroblasts, mesenchymal cells, neutrophils and lymphocytes. The stromal cells' nuclei were elliptical, oval, or circular in shape. In the follicular phase, the stroma was very crowded and swollen [ 24 , 25 ]. The endometrium showed a period of growth preceded by vascularization during the follicular phase of the estrous cycle in sheep[ 26 ]. Leukocytes invaded the functional layer on Day 7 of estrous cycle in cow [ 27 ]. The bovine luminal epithelium changes during the estrous cycle through a remodeling process [ 28 ]. The endometrium in adult ruminants (sheep, goat, buffalo and cattle) consists of a glandular caruncles and glandular intercaruncular areas [ 10 ]. The caruncles were non glandular, highly cellular and highly vascular endometrial elevation or projections. The locations of superficial implantation and placentation are occurred in caruncular regions. Interdigitation and branching morphogenetic growth of placental cotyledons with endometrial caruncles creates placentomes in synepitheliochorial placentation observed in ruminants. Placentomes are primarily involved in fetal-maternal gas exchange and the placenta's absorption of micronutrients for hemotrophic nutrition of the fetus [ 10 ]. The current study showed that the follicular phase of estrous cycle in Saidi sheep was characterized by epithelial proliferation and many epithelial invaginations which form the uterine glands (uterine gland adenogenesis). Herein, the uterine glands during the follicular phase of estrous cycle were highly branched and highly coiled and formed of columnar epithelium which surrounded by myoepithelial cells and had intraepithelial lymphocytes. Similar results were obtained by [ 23 , 24 ] in goat during follicular phase of estrous cycle. While uterine glands in sheep and pigs, are tightly coiled, heavily branched tubular glands, uterine glands in mice are comparatively simple tubes with little branching [ 29 ]. These glands have occasionally penetrated and reached the stratum vascularis. Proliferation of the endometrial glands were observed in the follicular phase [ 23 ] as a result of glandular epithelium mitoses [ 27 ]. Uterine gland development, or adenogenesis, is uniquely a postnatal event in sheep and pigs and involves differentiation and budding of glandular epithelium from luminal epithelium, followed by invagination and extensive tubular coiling and branching morphogenesis throughout the uterine stroma to the myometrium. Uterine adenogenesis is regulated by both intrinsic transcription factors and extrinsic factors from the pituitary, ovary, and mammary gland (lactocrine) [ 30 , 31 ]. To support the effects of certain hormones and growth factors, this mechanism necessitates site-specific changes in cell proliferation and extracellular matrix (ECM) remodeling in addition to paracrine cell-cell and cell-ECM interactions. According to studies on uterine development in newborn ungulates, prolactin, estradiol-17b, and their receptors are implicated in mechanisms controlling endometrial adenogenesis. When the functionalis is rebuilt from the basalis endometrium during menstruation, these hormones also seem to control endometrial gland development in menstrual primates and humans [ 30 ]. The endometrium of all mammalian uteri contains glands that produce, transport, and release chemicals necessary for the conceptus's survival and development (the embryo/fetus and related extraembryonic tissues). Adult ruminants' endometrium is composed of several a glandular caruncular regions and intercaruncular areas, each of which contained hundreds of glands per uterine wall cross-section [ 10 , 30 , 32 ]. The establishment of uterine receptivity, blastocyst implantation, and stromal cell decidualization all depend on uterine glands and their secretions. Similar to this, in humans, uterine glands and the secretory products they produce are probably important regulators of the uterine receptivity, blastocyst implantation and growth and development of the conceptus throughout the first trimester [ 32 , 33 ]. The survival and development of the periimplantation conceptus depend on the endometrial glands and their secretions [ 34 , 35 ]. The myometrium was formed of a thick, inner circular layer and a thin, outer longitudinal layer of smooth muscle fibers. A well vascularized stratum vascularis with many branches of the uterine artery was observed in the outer most layer of inner circular myometrium [ 10 , 24 ]. The main blood supply to the uterus is provided by the uterine arteries, which are found inside the myometrium. During the proliferative phase, the subepithelial capillary plexus has the highest vascular length density. Endothelial proliferation is the main mechanism of endometrial angiogenesis during the proliferative phase influenced by estrogen. Estradiol stimulates vascular permeability, angiogenesis, and endothelial cell proliferation in response to VEGF[ 36 ]. The estrous cycle is regulated in large part by the uterine blood supply. The two main hormones influencing blood flow in the arteries feeding the uterus are estrogens and progesterone. Vasodilation and vasoconstriction are regulated, complemented, and supported by the following factors: PGE2, LH, oxytocin, cytokines, neurotransmitters, and other local blood flow regulators [ 37 ]. The process of endometrial angiogenesis is strictly regulated. The endometrium and the macrophages that reside there can produce most of the key cytokines and factors that are currently known to be involved in the regulation of angiogenesis [ 38 ]. Some of these factors which expressed throughout the menstrual cycle include: vascular endothelial growth factor (VEGF) [ 39 , 40 ], fibroblast growth factor (FGF) [ 41 ] transforming growth factor-α (TGF-α) [ 42 ], interleukin (IL)-1 and IL-6 [ 43 ], epidermal growth factor (EGF) [ 44 ] and IL-8 [ 45 ]. Uterine vascular remodeling is important to the cycling and early pregnant endometrium. These vascular changes are strongly mediated by maternal regulatory factors, including ovarian hormones, VEGF, angiopoietins, Notch, and uterine natural killer cells [ 36 ]. We found that the endometrium had PAS positive epithelial basement membrane of the surface lamina epithelialis and uterine glands. This agree with the finding of [ 23 ] in Bakerwali Goat. No PAS positive secretion in the uterine glands could be observed during the follicular phase of estrous cycle. The intense PAS positivity was seen at supranuclear zone of the secretory glandular epithelium during the luteal phase [ 23 ]. The current study showed that few elastic fibers were observed in the endometrium (in the sub epithelial connective tissue and between the uterine glands) and between the smooth muscle fibers in the myometrium. However many elastic membranes were observed in the internal and external elastic lamina of the blood vessels of the tunica vascularis. Similar finding were observed in human [ 46 ] and mice [ 47 ] uterus. Elastic fibers in the uterus are mainly located in the myometrium and perimetrium while the endometrium contains few elastic fibers[ 47 ]. The uterine elasticity is likely maintained without excess stress being placed on the developing fetus by these thin sheets of elastic membranes and elastic fibrils [ 48 ]. Elastic fibers are resistant to tensile stresses and have persistently variable functions based on the needs of the microenvironment in which they are found [ 49 ]. Our findings revealed that the collagen fibers were more thickly distributed in the lamina propria of the uterine endometrium close to the endometrial glands and were located between the muscles [ 49 ]. While the intercellular matrix of the endometrial stroma contained a moderate amount of collagen fibers [ 25 ]. It had been suggested that the variability of the connective tissue thread distribution in the uterus may have a role in the fertilization process [ 49 ]. Because collagen fibers are found in the stroma and between the muscles, they enable the uterus to contract and stretch [ 49 ]. Collagen fiber visualization could make it easier to assess the thickness of the connective tissue that envelops endometrial glands. Elevated density may lead to degeneration and loss of function by impairing the flow of nutrients and endocrine signaling molecules from blood arteries to the glandular epithelium [ 50 ]. Our results showed slight GR immunostaining in the lamina epithelialis and in the stroma cells of lamina propria. Furthermore, slight GR immunostaining in the smooth muscle fibers of the blood vessels of the stratum vascularis. Whereas, slight SOD2 immunoexpression were observed in the lamina epithelialis, stroma cells and the smooth muscle fibers of the blood vessels of the lamina propria. Slight SOD2 immunostaining in the smooth muscle fibers of blood vessels of stratum vascularis were also noticed. Estradiol and progesterone control uterine glutathione reductase, which may be crucial in preserving the uterus's lowered glutathione levels. This molecule may be necessary in detoxification reactions involving H2O2 and electrophylic chemicals as well as for the regulation of the redox state of thiol groups. Glutathione reductase is stimulated by estrogens, which contributes to their antioxidant properties[ 51 ]. The uterus and fallopian tube contain antioxidants that aid in removing excess reactive oxygen species (ROS), creating the ideal environment for embryonic growth. To get rid of the ROS that cytokines and inflammation produce in mitochondria, SOD2 content is raised [ 52 ]. In addition there was a close relation between increased ROS and apoptosis. SOD2 and GR expression help to control apoptosis in the uterus during estrous cycle [ 53 ]. Our finding by using TUNEL assay immunofluorescence in the sheep uterus during the follicular phase of estrous cycle explored some apoptotic endometrial glandular epithelial cells and some apoptotic endometrial stromal cells. While the inner circular layer of myometrium showed few apoptotic cells. Apoptotic endometrial stromal cells in the caruncles were also observed in addition to apoptotic smooth muscle fibers of the blood vessels of the stratum vascularis. Apoptotic cell death had been demonstrated in hamster and rat uterine epithelium during the estrous cycle. There was an inverse correlation between cell death and cell proliferation in rat uterine and vaginal epithelial cells during the estrous cycle. Uterine epithelial cell proliferation, differentiation, and death are regulated by estrogen and progesterone [ 54 , 55 ]. In human uterus apoptotic uterine cells were scattered in the functional layer of the early proliferative endometrium [ 55 ]. In contrast, in the dog uterus a high apoptotic index was not detected in the surface epithelium and there was no significant correlation between the apoptotic index in any cell group and progesterone concentrations [ 56 ]. It was postulated that epithelial cell apoptosis is regulated by estrogen while stromal cell apoptosis is under the control of progesterone [ 57 ]. Dynamic changes in the porcine endometrium during the estrous cycle are a type of homeostasis through control of cell proliferation and exclusion. Homeostasis of the uterus is closely related to apoptosis and involving many hormones and cyctokines [ 58 ]. Our results indicate that apoptosis might have crucial role in the regulation of the estrous cycle in Saidi sheep. The current study revealed PRA immunolocalization in the lamina epithelialis, stroma cells, endothelial cells, columnar epithelial cells of the uterine glands and in the smooth muscle fibers of the myometrium. Moreover, the endothelium and the smooth muscle fibers of the blood vessels of the stratum vascularis showed also PRA immunolocalization. On the other hand, there was PRA immunolocalization in the perimetrial mesothelial cells and in the perimetrial endothelial cells. Similar findings were obtained in rabbit uterus during psedopregnancy [ 59 ]. Progesterone, a critical steroid hormone in the reproductive system, plays vital roles during the follicular phase of the estrous cycle [ 60 ]. Although traditionally associated with the luteal phase, emerging research highlights its significant functions in the follicular phase [ 61 ]. Progesterone receptors (PRs), which exist in two main isoforms, PR-A and PR-B, are differentially expressed in ovarian follicles and uterus [ 62 ]. Their expression is dynamically regulated by fluctuating hormone levels throughout the cycle. During the follicular phase, PR-A predominantly mediates progesterone’s inhibitory effects on follicular atresia, promoting the survival of developing follicles. Conversely, PR-B is implicated in the regulation of ovulatory processes. Studies have shown that PR knockout models exhibit impaired follicular development and ovulation, underscoring the importance of these receptors. The PR-A is also essential for uterine decidualization and implantation [ 62 ]. The interaction between PRs and other intracellular signaling pathways, such as the PI3K/AKT pathway, further illustrates the complexity of progesterone's role in folliculogenesis [ 63 ]. Progesterone, via acting through PR-A control the development and function of the endometrium and modifies cells essential for implantation and the establishment and maintenance of pregnancy. During pregnancy, progesterone via the PRs stimulats myometrial relaxation and cervical closure [ 64 , 65 ]. Progesterone modulates follicular development and ovulation through its interactions with progesterone receptors (PRs) [ 66 ]. During the follicular phase, low levels of progesterone and its receptors are necessary to prime the ovarian follicles for growth and maturation. Progesterone (P4) works synergistically with estrogen to regulate the expression of genes involved in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) receptors, crucial for follicular responsiveness to gonadotropins [ 67 ]. It was discovered that the plane of nutrition, the estrous cycle phase, and/or FSH all had an impact on the percentage of PR-positive uterine cells and/or staining intensity [ 68 ]. Changes in endometrial functions in superovulated models may arise from direct or indirect FSH action pathways. FSH exerts its indirect effects through binding to ovarian receptors and stimulating the synthesis of the estrogen and P4 [ 68 , 69 ] which in turn control endometrial activities [ 70 – 72 ]. Remarkably, during the menstrual cycle, the endometrial lining has high expression of the functional FSH receptor (FSHR). Increased expression of FSHR in the human endometrium can shed light on the potential direct effects of FSH on endometrial regeneration, which is primarily sustained by tissue-resident endometrial stem cells [ 73 – 76 ]. Through a complicated paracrine signaling network, the progesterone receptor controls glandular growth, decidualization, implantation, and the maintenance of a healthy uterus [ 70 – 72 ]. The role of progesterone and its receptors during the follicular phase is crucial for maintaining the delicate hormonal balance required for successful ovulation. By modulating the expression of enzymes like matrix metalloproteinases (MMPs), which are involved in follicular rupture, progesterone ensures that the dominant follicle reaches full maturation and is capable of releasing a viable oocyte [ 77 ]. We suggested that proper ovarian functions during follicular phase are necessary for proper uterine functions during both follicular phase and implantation. Additionally, other findings suggest that progesterone's actions extend beyond the ovaries and uterus, influencing the hypothalamic-pituitary-gonadal axis to fine-tune gonadotropin release, further demonstrating its integral role in reproductive physiology [ 78 ]. Interestingly, tryptase-positive immunostaining mast cells were recruited in the deep lamina propria of the caruncles during the follicular phase of the estrous cycle. Some degranulated mast cells were seen close to the macrophage in the endometrial lamina propria. Mast cells were also observed in-between uterine glands and smooth muscle fibers of the myometrium. Moreover they were noticed around the blood vessels of the stratum vascularis. MCs during the follicular phase of the estrous cycle play a crucial role in secreting substances that promote tissue remodeling [ 79 ]. Histamine is one of these important substances released from uterine mast cells, influencing ovulation, embryo implantation, and myometrium contractility leading to successful implantation and ultimately to parturition [ 80 – 82 ]. However, spatiotemporally expression of MCs in the female reproductive tract has been reported in other studies [ 82 – 92 ]. In the uterus, they change in number and structure depending on the hormonal level variations during the menstrual or estrous cycle [ 84 , 93 – 95 ]. An investigation under a light microscope has revealed alterations in the characteristics and location of MCs in mice, rats, hamsters, cows, and guinea pigs uterine tissues during pregnancy and estrous cycle [ 96 – 99 ]. In human endometrium, MCs were decreased in the stromal tissue [ 100 , 101 ]. Notably, the number and activity of MCs are correlated with estrogen concentrations in the uterine tissue of sows and rats [ 96 , 102 ], while they are correlated with progesterone concentrations in the canine uterus [ 82 ]. Nevertheless, to preserve regular reproductive processes and create the best environment for potential implantation, all uterine cell types interact with one another through junctacrine, paracrine, or endocrine pathways [ 68 ]. Conclusion This study provides new insight of the uterine histomorphological and immunohistochemical picture of the Saidi sheep during the follicular phase of the estrus cycle. These findings have growing significance to understand the key mechanisms that is characteristic of successful reproduction in Saidi sheep in order to enhance fertility and reproductive health of this livestock species and help to use advanced reproductive techinques. Declarations Ethics approval and consent to participate The experimental protocol was approved by the Local Ethical Committee and by the Institutional Review Board of Molecular Biology Research and studies Institute, Assiut University (22-2023-0028) and was carried out in accordance with relevant guidelines and regulations. This research was done in compliance with the ARRIVE guidelines and regulations (https:// arriv eguid elines. org). All national and institutional guidelines for animal care and use have been followed throughout the study procedures. Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. Funding Not applicable Authors' contributions M.A.: Conceptualization, Methodology, Investigation, Data curation, Writing—original draft, Writing—review & editing, and Formal analysis. M.A.K.: Writing—review & editing, Validation. M.A.A.: Writing—review & editing, Validation. M.S.H.: Conceptualization, Methodology, Writing—review& editing, Validation. All authors reviewed the manuscript and approved the final version for publication. Acknowledgements Not applicable References Wang H, Feng X, Muhatai G, Wang L. Expression profile analysis of sheep ovary after superovulation and estrus synchronisation treatment. Vet Med Sci. 2022;8:1276–87. Germot A, Khodary MG, Othman OE-M, Petit D. Shedding Light on the Origin of Egyptian Sheep Breeds by Evolutionary Comparison of Mitochondrial D-Loop. Animals. 2022;12:2738. El-Homosi FF, Abd El-Hafiz GA. REPRODUCTIVE PERPORMANCE OF OSSIMI AND SAIDI SHEEP UNDER TWO PRE PUBERTAL PLANES OF NUTRITION. Assiut Vet Med J. 1982;10.1:59–66. 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Cite Share Download PDF Status: Published Journal Publication published 13 Jan, 2025 Read the published version in BMC Veterinary Research → Version 1 posted Editorial decision: Revision requested 27 Sep, 2024 Reviews received at journal 09 Sep, 2024 Reviewers agreed at journal 03 Sep, 2024 Reviewers agreed at journal 29 Aug, 2024 Reviews received at journal 20 Aug, 2024 Reviewers agreed at journal 07 Aug, 2024 Reviewers invited by journal 07 Aug, 2024 Editor invited by journal 25 Jul, 2024 Editor assigned by journal 24 Jul, 2024 Submission checks completed at journal 24 Jul, 2024 First submitted to journal 23 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4790328","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":342656198,"identity":"531df29f-e933-4f1e-b4d3-4e7ecdc6d7f0","order_by":0,"name":"Mahmoud Abd-Elkareem","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYFACHhDBDGfY8SPJGRClJVmygYGxgSQtjBsOENCi23726IaPbdZwBrPxjeTjDxhqDucxsDdvk2CoqEXXYnYmL+3mzLZ0OIPP7EZaYgPDscPFDDzHyiQYzhzH0HIgx+w2b9thOIPZ7MwZwwYGtsOJDRI5ZhKMbccwtJx/A9UCZTBu7jn/sYHhH1CL/Bugln+YWm7AbIEyGDew9zA2MLaBbOEBammowdTyxuzmjHPpPDBGssTxNsMZiX3piW08acUWCccOYDosx+zGhzJrORjDjr+Z+cGHD9+sE/vZD2+88aGmDltAgwAPKjcBiNkgjMO4tOAGOG0ZBaNgFIyCEQMA+Qt3nq9vDJAAAAAASUVORK5CYII=","orcid":"","institution":"Assiut University","correspondingAuthor":true,"prefix":"","firstName":"Mahmoud","middleName":"","lastName":"Abd-Elkareem","suffix":""},{"id":342656199,"identity":"00ec97c1-6529-4141-90cb-9b28e403ffed","order_by":1,"name":"Mohsen A. 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A \u0026amp; B: Showing the endometrium with mucosal folds and caruncles and narrow uterine lumen. C: Showing the endometrium formed of lamina epithelialis (EP) of pseudostratified columnar epithelium with intraepithelial lymphocytes (arrowheads) and connective tissue lamina propria contained fibroblasts, collagen fibers and lymphocytes (arrow). D: lamina propria contained many blood vessels (BV) and leucocytic infiltrations (L). E: Showing many epithelial (EP) invaginations (arrowheads) which form the uterine glands (arrowhead). F \u0026amp; G: Showing lamina propria with blood vessels (BV) and highly branched and highly coiled uterine glands which formed of columnar epithelium (Co) and surrounded by myoepithelial cells (arrow). H: Showing intraepithelial lymphocytes (arrowheads) between the columnar cells (Co) of the uterine glands (UG), plasma cells and leucocytic infiltration in lamina propria. I: \u0026nbsp;Showing well vascularized tunica vascularis with many branches of the uterine artery (A) in the outer most layer of inner circular myometrium (IC). Note the outer longitudinal (OL) muscle fibers of myometrium and perimetrium. Original magnification; A: X12.5, scale bar = 1mm, B \u0026amp; I: X40, scale bar = 500 μm, C, D, G \u0026amp; H: X400, scale bar = 50 μm, E \u0026amp; F: X 100, scale bar = 200 μm, haematoxylin and eosin stain\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4790328/v1/4287e72c5b0e6af9aa5a405f.png"},{"id":62949842,"identity":"32c1c5b7-4814-4749-bb75-d5d6e0507f8b","added_by":"auto","created_at":"2024-08-21 11:03:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2515542,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrograph of the sheep uterus during the follicular phase of estrous cycle. A \u0026amp; B: Showing the endometrium with PAS positive epithelial basement membrane (arrows) of lamina epithelialis (Ep) and uterine glands (UG). Note no PAS positive secretion in the uterine glands. C: Showing well-vascularized tunica vascularis with PAS positive internal (arrow) and external (arrowhead) elastic lamina in the outer most layer of inner circular myometrium (IC). Note the outer longitudinal (OL) muscle fibers of the myometrium and the perimetrium (Pr). D \u0026amp; E: Showing the endometrium in the sub epithelial and between the uterine glands (UG) with few elastic fibers (arrow) except for blood vessels; external elastic lamina (arrowhead) of endometrial blood vessels (BV). F: Showing the myometrium with few elastic fibers between the smooth muscle fibers (forked tail arrow) except for blood vessels of stratum vascularis; in the internal (arrow) and external (arrowhead) elastic lamina. G \u0026amp; H: Showing the endometrium with moderate amount of collagen fibers (red color, CF) between the uterine glands (UG) and few amounts in the caruncle. I: Showing the myometrium with few amount of collagen fibers (red color) in between the smooth muscle fibers of the inner circular (IC) and outer longitudinal (OL) layers. Note the moderate amount of collagen fibers (CF) around blood vessels in the stratum vascularis (SV). Original magnification; A: X200, scale bar = 100 µm, B: X100, scale bar = 200 µm, C: X40, scale bar = 500 µm, PAS \u0026amp; Hx. D: X200, scale bar = 100 µm, E: X100, scale bar = 200 µm, F: X200, scale bar = 100 µm, Orcien stain. G: X40, scale bar = 500 µm, H: X100, scale bar = 200 µm, I: X40, scale bar = 500 µm, Picro-Sirius red technique.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4790328/v1/3114f66c94404369725cf951.png"},{"id":62949220,"identity":"9521c683-9233-4811-9fc6-5a489dc0d23f","added_by":"auto","created_at":"2024-08-21 10:55:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1027034,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrograph illustrating the collagen fibers distribution in the sheep uterus during the follicular phase of estrous cycle. A \u0026amp; B: Showing the lamina propria of the endometrium with moderate amount of collagen fibers (blue color with Masson's trichrome technique and red color with Picro-Sirius red technique, CF) between the uterine glands (UG) and few amounts in the caruncle. Original magnification; A \u0026amp; B: X40, scale bar = 500 µm, A: Masson's trichrome technique, B: Picro-Sirius red technique.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4790328/v1/baea4b08384002b18b794855.png"},{"id":62949228,"identity":"13cfc1c4-83d8-4978-a44a-5fdb61361763","added_by":"auto","created_at":"2024-08-21 10:55:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3966307,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrograph of GR immunostaining in the sheep uterus during the follicular phase of estrous cycle. A \u0026amp; B: Showing slight GR immunostaining in the lamina epithelialis (Ep) and in the stroma cells of lamina propria (LP) and negative GR immunostaining in the uterine glands (UG). D: Showing slight GR immunostaining in the smooth muscle fibers of the blood vessels of the stratum vascularis (SV) and negative GR immunostaining in the smooth muscle fibers of the inner circular layer (IC) of the myometrium. D-H: Showing slight SOD2 immunostaining in the lamina epithelialis (Ep) and in the stroma cells and the smooth muscle fibers of blood vessels (BV) of the lamina propria (LP) and negative SOD2 immunostaining in the uterine glands (UG). I: Showing slight SOD2 immunostaining in the smooth muscle fibers of blood vessels of stratum vascularis (SV) and negative SOD2 immunostaining in the smooth muscle fibers of the inner circular layer (IC) of the myometrium. Original magnification; A, B, C, E, H \u0026amp; I: X400, scale bar = 50 µm, D, F \u0026amp; G: X200, scale bar = 100 µm.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4790328/v1/1293d6a51d65b4a711df7ed2.png"},{"id":62949219,"identity":"2a0beb57-6f67-4928-be2d-d3dd2e5549e0","added_by":"auto","created_at":"2024-08-21 10:55:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2528493,"visible":true,"origin":"","legend":"\u003cp\u003eNegative image of Fig. 4.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4790328/v1/c246566cb090599d8ca6c0fa.png"},{"id":62950463,"identity":"bea4ffaa-6360-4355-9c82-cc4d3409ceb9","added_by":"auto","created_at":"2024-08-21 11:11:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1263360,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrograph of TUNEL assay immunofluorescence in the sheep uterus during the follicular phase of estrous cycle. A: Showing apoptotic endometrial glandular epithelial cells (arrowheads). B: Showing apoptotic endometrial stromal cells (arrowheads). Note inner circular layer (IC) of myometrium with few or no apoptotic cells. C: Showing apoptotic endometrial stromal cells (arrowheads) in the caruncle. D: Showing apoptotic smooth muscle fibers (arrowheads) of the blood vessels of the stratum vascularis (SV). Original magnification; A-D: X400, scale bar = 50 µm.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4790328/v1/10cd85bfc3b893bd0eae14c3.png"},{"id":62949852,"identity":"c097a0d7-a8a1-4cee-a56c-9293478c1d47","added_by":"auto","created_at":"2024-08-21 11:03:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2439052,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrograph of PRA immunolocalization in the sheep uterus during the follicular phase of estrous cycle. A: Showing strong PRA immunolocalization in the lamina epithelialis (Ep) and strong to moderate PRA immunolocalization in the sub epithelial stroma cells (S). B: Showing moderate PRA immunolocalization in the stromal cells and endothelial cells (arrowhead) of blood vessels in the caruncles. C: Showing strong to moderate PRA immunolocalization in the columnar epithelial cells (C) of the uterine glands (UG). D: Showing moderate PRA immunolocalization in the smooth muscle fibers (SMF) of the inner circular layer of the myometrium. E: Showing moderate PRA immunolocalization in the endothelium (E) and smooth muscle fibers (SMF) of the blood vessels of the stratum vascularis (SV). F: Showing moderate PRA immunolocalization in the smooth muscle fibers and endothelial cells (E) of blood vessels (BV) in the outer longitudinal layer (OL) of the myometrium and moderate PRA immunolocalization in the mesothelial cells (M) and in the endothelial cells (E) of blood vessels (BV) of the perimetrium. Original magnification; A-F: X400, scale bar = 50 µm.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4790328/v1/3395e1d51258315bf8173c08.png"},{"id":62949226,"identity":"c52d0fb6-ca8f-45b3-843a-05d4524cf046","added_by":"auto","created_at":"2024-08-21 10:55:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1340139,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrograph of mast cells immunolocalization in the sheep uterus during the follicular phase of estrous cycle. \u003cstrong\u003eA:\u003c/strong\u003e Showing tryptase positive immunostaining mast cells (arrowheads) in the deep lamina propria of the caruncles. \u003cstrong\u003eB:\u003c/strong\u003e Showing the superficial lamina propria (LP) under the lamina epithelialis (Ep) with stroma cells (S) and no mast cells. \u003cstrong\u003eC:\u003c/strong\u003eShowing mast cells (MC) filled with tryptase positive immunostaining granules. \u003cstrong\u003eD:\u003c/strong\u003eShowing degranulated mast cells (DMC) near the macrophage (Mc) in the endometrial lamina propria. Original magnification; A-D: X1000, scale bar = 20 µm.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4790328/v1/36e406deeefa0b5b151a171b.png"},{"id":62949843,"identity":"9b844f9a-e8ab-408b-a5d2-c0353bcb2c22","added_by":"auto","created_at":"2024-08-21 11:03:30","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1133765,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrograph of mast cells immunolocalization in the sheep uterus during the follicular phase of estrous cycle. \u003cstrong\u003eA:\u003c/strong\u003e Showing tryptase positive immunostaining mast cells (MC) in-between uterine glands (UG). \u003cstrong\u003eB:\u003c/strong\u003e Showing tryptase positive immunostaining mast cells (MC) in the inner circular smooth muscle layer of the myometrium. \u003cstrong\u003eC:\u003c/strong\u003e Showing tryptase positive immunostaining mast cells (arrowheads) around the blood vessels of the stratum vascularis (SV). \u003cstrong\u003eD:\u003c/strong\u003eShowing tryptase positive immunostaining mast cells (MC) in the outer longitudinal smooth muscle layer of the myometrium. Original magnification; A: X400, scale bar = 50 µm, B \u0026amp; D: X1000, Scale bar = 20 µm, C: X100, scale bar = 200 µm.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4790328/v1/9e2955f02e0d2d479e8bc723.png"},{"id":62949225,"identity":"2db3eb92-fc70-4d21-b1a1-f924ed72bd1f","added_by":"auto","created_at":"2024-08-21 10:55:30","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2350116,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the Results section.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-4790328/v1/44bd73edc39bfee0b80ca78f.png"},{"id":74284764,"identity":"6986e993-da91-4ba1-882b-6e6a20d4cb38","added_by":"auto","created_at":"2025-01-20 16:12:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":31528926,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4790328/v1/2e9f6153-b986-45a0-806d-fc439745de87.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Uterine histomorphological and immunohistochemical investigation during follicular phase of estrous cycle in Saidi sheep","fulltext":[{"header":"Background","content":"\u003cp\u003eSheep are extensively raised as livestock worldwide because they yield a lot of meat, fat, milk, wool, and other useful products. Many studies on breeding have been focused on improving sheep reproductive performance and litter size [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The Saidi breed of Egyptian sheep was once thought to be the oldest breed of Egyptian sheep, and its breeding grounds are in Upper Egypt, south of Assiut. It has a long, thick tail and its fleece is typically dark brown in color [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Due to its high reproductive performance the demand for this breed increases [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Saidi ewes had almost no seasonal variation in their reproductive cycles, despite a decrease in estrous activity in the spring [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe uterus is an important organ for reproduction in mammals. The uterus provides a microenvironment required for receptivity, implantation and for growth and development of the conceptus. The uterus is essential for the following processes: (1) transportation, storage, and maturation of spermatozoa; (2) synthesis of prostaglandin F2α, the luteolysin required for ovarian cyclicity in domestic animals; (3) offering of an embryotrophic environment for conceptus (embryo/fetus and associated extraembryonic membranes) growth and development; and (4) pickup of the conceptus at parturition [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Sheep have a bicornuate uterus with a small common corpus and single cervix. Histologically, the uterus in ewe was formed of endometrium, myometrium and perimetrium. The main cyclic changes were occurred in endometrium and to lesser extent in myometrium [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe key mechanisms that is characteristic of successful reproduction in Saidi sheep and gaps in knowledge that must be the subject of research in order to enhance fertility and reproductive health of this livestock species. Relatively little is known about the uterine picture of the Saidi sheep during the follicular phase of the estrous cycle. So, the aim of the current study is to give more details on the histological, histochemical, and immunohistochemical analyses of the uterus during the follicular phase of the estrous cycle in order to enhance fertility and reproductive health of this breed of sheep.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals and samples:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUteri were collected from eleven apparently healthy slaughtered (according to Islamic religion) Saidi sheep aged 2 to 5 years and weighted (38.5 \u0026plusmn;2.03 Kg) at Assiut abattoir, Assiut governorate, Egypt.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological examination:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUteri (n=11) were fixed in 10 % neutral buffered formalin. The formalin-fixed samples were dehydrated in ascending grades of ethanol, cleared in methyl benzoate, and embedded in paraplast. Paraffin sections at 5 \u0026mu;m in thickness were cut and stained with the following histological stains:\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eHaematoxylin and Eosin for general histological examination of the uterus [13]\u003c/li\u003e\n \u003cli\u003ePeriodic acid Schiff (PAS) technique for demonstration of glycoprotein [13].\u003c/li\u003e\n \u003cli\u003eMasson\u0026rsquo;s trichrome technique for staining collagen fibers [14].\u003c/li\u003e\n \u003cli\u003ePicro-Sirius red technique to differentiate between mature and immature\u003cem\u003e\u0026nbsp;\u003c/em\u003ecollagen fibers\u003cem\u003e\u0026nbsp;\u003c/em\u003e[15, 16]\u003c/li\u003e\n \u003cli\u003eOrcien stain for detection of the distribution of elastic fibers in the uterus [17]\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eOxidative stress detection:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry of glutathione reductase and superoxide\u003cem\u003e\u0026nbsp;\u003c/em\u003edismutase 2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParaffin-embedded tissue sections were deparaffinized, rehydrated, and rinsed in phosphate buffered saline (PBS). Then slides were placed in 10 mM sodium citrate buffer (pH 6.0) for antigen retrieval at (95\u0026ndash;98 \u0026deg;C) in a water bath for 20 min. Endogenous peroxidase was blocked by incubating the slides in 3% hydrogen peroxide for 10 min at room temperature. This followed by washing the slides in PBS (3 times for 5 min each). The sections were incubated overnight in a humid chamber with rabbit polyclonal antibodies. For immunohistochemical detection of glutathione reductase (GR) and superoxide dismutase 2 (SOD2) in the ovary, we used polyclonal anti-glutathione reductase and anti-superoxide dismutase 2 antibodies, respectively (Chongqing Biospes Co., Ltd, China) and Power-Stain\u0026trade; 1.0 Poly horseradish peroxidase (HRP) DAB Kit (Genemed Biotechnologies, Inc, 458 Carlton Ct. South San Francisco, CA 94080, USA) [18].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNegative image analysis\u0026nbsp;\u003c/strong\u003ewas performed to assess the complex color micrographs that were obtained and to give more details[19, 20].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptosis detection:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTUNEL assay\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDetection of apoptosis was done using \u003cem\u003eIn Situ\u0026nbsp;\u003c/em\u003eCell Death Detection Kit, Fluorescein (Sigma-Aldrich, USA). Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay was designed to detect apoptotic cells that undergo extensive DNA fragmentation during the late stages of apoptosis. This method was depending up on the ability of TdT to label blunt ends of double-stranded DNA breaks independent of a template. The protocol we used as the previous published protocol [21]. Slides were rinsed with PBS and directly analyzed under a fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoexpression of progesterone receptor alpha (PRA):\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protocol used was according to the company instructions and as our previous study [22]. The fixed ovaries were dehydrated in ethanol, cleared in methyl benzoate and then embedded in paraplast. Sections (5 \u0026mu;m) of paraplast-embedded tissue were dewaxed by xylene. Subsequently, rehydration by 100%, 95%, 80% and 70% ethanol, and slides were rinsed in PBS (pH 7.4). Endogenous peroxidase were prevented by adding 3% hydrogen peroxide followed by washing in PBS. For antigen detections, the slides were placed in 10 mM sodium citrate buffer (pH 6.0) at (95\u0026ndash;98 \u0026deg;C) in a water bath for 20 min followed by cooling at room temperature. Sections were then rinsed in PBS. Immunoexpression of progesterone receptor alpha was done by using; progesterone receptor rabbit pAb (Catalog No.: A0321), ABclonal, USA. Sections were then incubated with the primary antibodies for 30-60 min at room temperature. The slides were washed with PBS then follow the company instructions of poly Q stain 2 step detection system goat anti-mouse/rabbit HRP, peroxidase quench, DAB kit, quartett, Germany. The sections were counterstained in Harris hematoxylin for 30 s. Then sections were dehydrated by ethanol 95%, and ethanol 100%, cleared of in xylene, and mounted with DPX.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical detection of mast cells:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor detection of mast cell, we used Mast Cell Tryptase (3G3) Monoclonal Antibody (Bioss antibodies) and Poly Q stain 2-step detection system, goat anti-mouse/rabbit HRP, peroxidase quench, and DAB kit from Quartett, Germany. The protocol used was according to the company instructions and as our previous study [22].\u003c/p\u003e\n\u003cp\u003eAll staining slides were examined by an Olympus BX51 microscope and the photographs were taken by an Olympus DP72 camera adapted into the microscope.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eGeneral uterine histomorphological characters during follicular phase of estrous cycle in Saidi sheep:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicroscopical analysis of the uterus of Saidi sheep during the follicular phase of the estrous cycle revealed that the uterine wall was composed of three layers: the inner layer; endometrium (mucosa), the middle layer; myometrium (muscular layer), and the outer layer; perimetrium (serosa). The endometrium had mucosal folds, caruncles and narrow uterine lumen. The caruncles were non glandular, highly cellular and highly vascular endometrial elevation or projections. The endometrium formed of lamina epithelialis of pseudostratified columnar epithelium with intraepithelial lymphocytes and connective tissue lamina propria contained uterine glands, fibroblasts, collagen fibers, blood vessels and leucocytic infiltrations especially lymphocytes and plasma cells (Fig. 1A-D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe current study showed that the follicular phase of estrous cycle was characterized by epithelial proliferation and many epithelial invaginations which form the uterine glands (uterine gland adenogenesis). The uterine glands in Saidi sheep during the follicular phase of estrous cycle were highly branched and highly coiled and formed of columnar epithelium which surrounded by myoepithelial cells and had intraepithelial lymphocytes (Fig. 1E-H)..\u003c/p\u003e\n\u003cp\u003eThe myometrium was formed of a thick, inner circular layer and a thin, outer longitudinal layer of smooth muscle fibers. A well vascularized stratum vascularis with many branches of the uterine artery was observed in the outer most layer of inner circular myometrium. The outer perimetrium was composed of a mesothelial layer and sub mesothelial connective tissue (Fig. 1I).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlycoprotein localization in the Saidi sheep uterus during follicular phase of estrous cycle\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGlycoprotein localization in the sheep uterus during follicular phase of estrous cycle revealed that the endometrium had\u0026nbsp;PAS positive epithelial basement membrane\u0026nbsp;of lamina epithelialis and uterine glands. No PAS positive secretion in the uterine glands could be observed.\u0026nbsp;PAS positive internal and external elastic lamina\u0026nbsp;in the stratum vascularis could be detected (Fig. 2A-C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElastic and collagen fibers distribution in the Saidi sheep uterus during follicular phase of estrous cycle\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElastic fibers distribution in the sheep uterus during follicular phase of estrous cycle revealed that few elastic fibers were observed in the sub epithelial and between the uterine glands in the endometrium. Furthermore few elastic fibers between the smooth muscle fibers in the myometrium were observed. However many elastic membranes were observed in the\u0026nbsp;external elastic lamina\u0026nbsp;of endometrial blood vessels and in the\u0026nbsp;internal and external elastic lamina\u0026nbsp;of the blood vessels of the tunica vascularis (Fig. 2D-F).\u003c/p\u003e\n\u003cp\u003eWhile the collagen fibers distribution in the sheep uterus during follicular phase of estrous cycle revealed that the endometrium had moderate amount of collagen fibers which found between the uterine glands and few amounts in the caruncles. Whereas the myometrium had few amount of collagen fibers in-between the smooth muscle fibers of the inner circular and outer longitudinal layers and moderate amount of collagen fibers around blood vessels in the stratum vascularis (Fig. 2G-I and Fig. 3A \u0026amp; B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGR and SOD2 immunostaining\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ein the Saidi sheep uterus during follicular phase of estrous cycle\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGR immunostaining in the sheep uterus during the follicular phase of estrous cycle showed slight GR immunostaining in the lamina epithelialis and in the stroma cells of lamina propria and negative GR immunostaining in the uterine glands. Furthermore, slight GR immunostaining in the smooth muscle fibers of the blood vessels of the stratum vascularis and negative GR immunostaining in the smooth muscle fibers of the inner circular layer of the myometrium could be demonstrated (Fig. 4A-C).\u003c/p\u003e\n\u003cp\u003eWhereas, slight SOD2 immunoexpression were observed in the lamina epithelialis, stroma cells and the smooth muscle fibers of the blood vessels of the lamina propria in addition the uterine glands expressed no SOD2 immunostaining (Fig. 4D-H). Slight SOD2 immunostaining in the smooth muscle fibers of blood vessels of stratum vascularis and negative SOD2 immunostaining in the smooth muscle fibers of the inner circular layer of the myometrium were also noticed (Fig. 4I). Fig. 5 was a negative image of Fig. 4 to give more detailed of GR and SOD2 immunoexpression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptosis in the Saidi sheep uterus during follicular phase of estrous cycle\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTUNEL assay immunofluorescence in the sheep uterus during the follicular phase of estrous cycle explored some apoptotic endometrial glandular epithelial cells (Fig. 6A) and some apoptotic endometrial stromal cells (Fig. 6B). \u0026nbsp;While the inner circular layer of myometrium showed few or no apoptotic cells (Fig. 6B). Apoptotic endometrial stromal cells in the caruncles were also observed in addition to apoptotic smooth muscle fibers of the blood vessels of the stratum vascularis (Fig. 6D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePRA immunolocalization\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ein the Saidi sheep uterus during follicular phase of estrous cycle\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePRA immunolocalization in the sheep uterus during the follicular phase of estrous cycle revealed strong PRA immunolocalization in the lamina epithelialis and strong to moderate PRA immunolocalization in the sub-epithelial stroma cells (Fig. 7A). Also moderate PRA immunostaining was observed in the stromal cells and endothelial cells of blood vessels in the caruncles (Fig. 7B). The columnar epithelial cells of the uterine glands expressed strong to moderate PRA (Fig. 7C) while the smooth muscle fibers of the inner circular layer of the myometrium expressed moderate PRA (Fig. 7D). Moreover, the endothelium and the smooth muscle fibers of the blood vessels of the stratum vascularis showed moderate PRA immunolocalization (Fig. 7E). On the other hand, mild PRA immunoexpression was noted in the smooth muscle fibers and blood vessel endothelial cells in the myometrium\u0026apos;s outer longitudinal layer. Whereas there was mild PRA immunolocalization in the perimetrial mesothelial cells and in the perimetrial blood vessel endothelial cells (Fig. 7F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMast cells detection in the Saidi sheep uterus during follicular phase of estrous cycle\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInterestingly, tryptase-positive immunostaining mast cells were recruited in the deep lamina propria of the caruncles during the follicular phase of the estrous cycle (Fig. 8A). Although there were no mast cells seen in the superficial lamina propria underneath the lamina epithelialis (Fig. 8B).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMast cells were rounded or oval cells with rounded central or eccentric nucleus and filled with tryptase positive immunostaining granules (Fig. 8C). Some degranulated mast cells were seen close to the macrophage in the endometrial lamina propria (Fig. 8D).\u003c/p\u003e\n\u003cp\u003eMast cells were also observed in-between uterine glands (Fig. 9A) and in the inner circular smooth muscle layer of the myometrium (Fig. 9B). Moreover they were noticed\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003earound the blood vessels of the stratum vascularis (Fig. 9C) and in the outer longitudinal smooth muscle layer of the myometrium (Fig. 9D).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMicroscopical analysis of the uterus of Saidi sheep during the follicular phase of the estrous cycle revealed that the uterine wall was composed of three layers: the inner endometrium, middle myometrium and the outer perimetrium. The endometrium had mucosal folds, caruncles and narrow uterine lumen. The endometrium formed of lamina epithelialis of pseudostratified columnar epithelium with intraepithelial lymphocytes and connective tissue lamina propria contained uterine glands, fibroblasts, collagen fibers, blood vessels and leucocytic infiltrations especially lymphocytes and plasma cells. The uterus (horns and body) in ruminants was lined with the simple columnar to pseudostratified columnar epithelium. The mean height of the epithelium was less in follicular phase [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In buffalo the endometrium was lined with three types of columnar cells, i.e. ciliated, non-ciliated cells and basal cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The endometrial stroma (propria submucosa) consisted of fibro-reticular connective tissue, stromal cells and blood vessels. Its cellular components comprised of stromal cells, fibroblasts, mesenchymal cells, neutrophils and lymphocytes. The stromal cells' nuclei were elliptical, oval, or circular in shape. In the follicular phase, the stroma was very crowded and swollen [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The endometrium showed a period of growth preceded by vascularization during the follicular phase of the estrous cycle in sheep[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Leukocytes invaded the functional layer on Day 7 of estrous cycle in cow [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The bovine luminal epithelium changes during the estrous cycle through a remodeling process [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe endometrium in adult ruminants (sheep, goat, buffalo and cattle) consists of a glandular caruncles and glandular intercaruncular areas [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The caruncles were non glandular, highly cellular and highly vascular endometrial elevation or projections. The locations of superficial implantation and placentation are occurred in caruncular regions. Interdigitation and branching morphogenetic growth of placental cotyledons with endometrial caruncles creates placentomes in synepitheliochorial placentation observed in ruminants. Placentomes are primarily involved in fetal-maternal gas exchange and the placenta's absorption of micronutrients for hemotrophic nutrition of the fetus [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe current study showed that the follicular phase of estrous cycle in Saidi sheep was characterized by epithelial proliferation and many epithelial invaginations which form the uterine glands (uterine gland adenogenesis). Herein, the uterine glands during the follicular phase of estrous cycle were highly branched and highly coiled and formed of columnar epithelium which surrounded by myoepithelial cells and had intraepithelial lymphocytes. Similar results were obtained by [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] in goat during follicular phase of estrous cycle. While uterine glands in sheep and pigs, are tightly coiled, heavily branched tubular glands, uterine glands in mice are comparatively simple tubes with little branching [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These glands have occasionally penetrated and reached the stratum vascularis. Proliferation of the endometrial glands were observed in the follicular phase [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] as a result of glandular epithelium mitoses [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUterine gland development, or adenogenesis, is uniquely a postnatal event in sheep and pigs and involves differentiation and budding of glandular epithelium from luminal epithelium, followed by invagination and extensive tubular coiling and branching morphogenesis throughout the uterine stroma to the myometrium. Uterine adenogenesis is regulated by both intrinsic transcription factors and extrinsic factors from the pituitary, ovary, and mammary gland (lactocrine) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. To support the effects of certain hormones and growth factors, this mechanism necessitates site-specific changes in cell proliferation and extracellular matrix (ECM) remodeling in addition to paracrine cell-cell and cell-ECM interactions. According to studies on uterine development in newborn ungulates, prolactin, estradiol-17b, and their receptors are implicated in mechanisms controlling endometrial adenogenesis. When the functionalis is rebuilt from the basalis endometrium during menstruation, these hormones also seem to control endometrial gland development in menstrual primates and humans [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe endometrium of all mammalian uteri contains glands that produce, transport, and release chemicals necessary for the conceptus's survival and development (the embryo/fetus and related extraembryonic tissues). Adult ruminants' endometrium is composed of several a glandular caruncular regions and intercaruncular areas, each of which contained hundreds of glands per uterine wall cross-section [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The establishment of uterine receptivity, blastocyst implantation, and stromal cell decidualization all depend on uterine glands and their secretions. Similar to this, in humans, uterine glands and the secretory products they produce are probably important regulators of the uterine receptivity, blastocyst implantation and growth and development of the conceptus throughout the first trimester [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The survival and development of the periimplantation conceptus depend on the endometrial glands and their secretions [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe myometrium was formed of a thick, inner circular layer and a thin, outer longitudinal layer of smooth muscle fibers. A well vascularized stratum vascularis with many branches of the uterine artery was observed in the outer most layer of inner circular myometrium [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe main blood supply to the uterus is provided by the uterine arteries, which are found inside the myometrium. During the proliferative phase, the subepithelial capillary plexus has the highest vascular length density. Endothelial proliferation is the main mechanism of endometrial angiogenesis during the proliferative phase influenced by estrogen. Estradiol stimulates vascular permeability, angiogenesis, and endothelial cell proliferation in response to VEGF[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The estrous cycle is regulated in large part by the uterine blood supply. The two main hormones influencing blood flow in the arteries feeding the uterus are estrogens and progesterone. Vasodilation and vasoconstriction are regulated, complemented, and supported by the following factors: PGE2, LH, oxytocin, cytokines, neurotransmitters, and other local blood flow regulators [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe process of endometrial angiogenesis is strictly regulated. The endometrium and the macrophages that reside there can produce most of the key cytokines and factors that are currently known to be involved in the regulation of angiogenesis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Some of these factors which expressed throughout the menstrual cycle include: vascular endothelial growth factor (VEGF) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], fibroblast growth factor (FGF) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] transforming growth factor-α (TGF-α) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], interleukin (IL)-1 and IL-6 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], epidermal growth factor (EGF) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and IL-8 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Uterine vascular remodeling is important to the cycling and early pregnant endometrium. These vascular changes are strongly mediated by maternal regulatory factors, including ovarian hormones, VEGF, angiopoietins, Notch, and uterine natural killer cells [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe found that the endometrium had PAS positive epithelial basement membrane of the surface lamina epithelialis and uterine glands. This agree with the finding of [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] in Bakerwali Goat. No PAS positive secretion in the uterine glands could be observed during the follicular phase of estrous cycle. The intense PAS positivity was seen at supranuclear zone of the secretory glandular epithelium during the luteal phase [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe current study showed that few elastic fibers were observed in the endometrium (in the sub epithelial connective tissue and between the uterine glands) and between the smooth muscle fibers in the myometrium. However many elastic membranes were observed in the internal and external elastic lamina of the blood vessels of the tunica vascularis. Similar finding were observed in human [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and mice [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] uterus. Elastic fibers in the uterus are mainly located in the myometrium and perimetrium while the endometrium contains few elastic fibers[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The uterine elasticity is likely maintained without excess stress being placed on the developing fetus by these thin sheets of elastic membranes and elastic fibrils [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Elastic fibers are resistant to tensile stresses and have persistently variable functions based on the needs of the microenvironment in which they are found [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur findings revealed that the collagen fibers were more thickly distributed in the lamina propria of the uterine endometrium close to the endometrial glands and were located between the muscles [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. While the intercellular matrix of the endometrial stroma contained a moderate amount of collagen fibers [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. It had been suggested that the variability of the connective tissue thread distribution in the uterus may have a role in the fertilization process [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Because collagen fibers are found in the stroma and between the muscles, they enable the uterus to contract and stretch [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Collagen fiber visualization could make it easier to assess the thickness of the connective tissue that envelops endometrial glands. Elevated density may lead to degeneration and loss of function by impairing the flow of nutrients and endocrine signaling molecules from blood arteries to the glandular epithelium [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur results showed slight GR immunostaining in the lamina epithelialis and in the stroma cells of lamina propria. Furthermore, slight GR immunostaining in the smooth muscle fibers of the blood vessels of the stratum vascularis. Whereas, slight SOD2 immunoexpression were observed in the lamina epithelialis, stroma cells and the smooth muscle fibers of the blood vessels of the lamina propria. Slight SOD2 immunostaining in the smooth muscle fibers of blood vessels of stratum vascularis were also noticed. Estradiol and progesterone control uterine glutathione reductase, which may be crucial in preserving the uterus's lowered glutathione levels. This molecule may be necessary in detoxification reactions involving H2O2 and electrophylic chemicals as well as for the regulation of the redox state of thiol groups. Glutathione reductase is stimulated by estrogens, which contributes to their antioxidant properties[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The uterus and fallopian tube contain antioxidants that aid in removing excess reactive oxygen species (ROS), creating the ideal environment for embryonic growth. To get rid of the ROS that cytokines and inflammation produce in mitochondria, SOD2 content is raised [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In addition there was a close relation between increased ROS and apoptosis. SOD2 and GR expression help to control apoptosis in the uterus during estrous cycle [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur finding by using TUNEL assay immunofluorescence in the sheep uterus during the follicular phase of estrous cycle explored some apoptotic endometrial glandular epithelial cells and some apoptotic endometrial stromal cells. While the inner circular layer of myometrium showed few apoptotic cells. Apoptotic endometrial stromal cells in the caruncles were also observed in addition to apoptotic smooth muscle fibers of the blood vessels of the stratum vascularis. Apoptotic cell death had been demonstrated in hamster and rat uterine epithelium during the estrous cycle. There was an inverse correlation between cell death and cell proliferation in rat uterine and vaginal epithelial cells during the estrous cycle. Uterine epithelial cell proliferation, differentiation, and death are regulated by estrogen and progesterone [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In human uterus apoptotic uterine cells were scattered in the functional layer of the early proliferative endometrium [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In contrast, in the dog uterus a high apoptotic index was not detected in the surface epithelium and there was no significant correlation between the apoptotic index in any cell group and progesterone concentrations [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. It was postulated that epithelial cell apoptosis is regulated by estrogen while stromal cell apoptosis is under the control of progesterone [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Dynamic changes in the porcine endometrium during the estrous cycle are a type of homeostasis through control of cell proliferation and exclusion. Homeostasis of the uterus is closely related to apoptosis and involving many hormones and cyctokines [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Our results indicate that apoptosis might have crucial role in the regulation of the estrous cycle in Saidi sheep.\u003c/p\u003e \u003cp\u003eThe current study revealed PRA immunolocalization in the lamina epithelialis, stroma cells, endothelial cells, columnar epithelial cells of the uterine glands and in the smooth muscle fibers of the myometrium. Moreover, the endothelium and the smooth muscle fibers of the blood vessels of the stratum vascularis showed also PRA immunolocalization. On the other hand, there was PRA immunolocalization in the perimetrial mesothelial cells and in the perimetrial endothelial cells. Similar findings were obtained in rabbit uterus during psedopregnancy [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Progesterone, a critical steroid hormone in the reproductive system, plays vital roles during the follicular phase of the estrous cycle [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Although traditionally associated with the luteal phase, emerging research highlights its significant functions in the follicular phase [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Progesterone receptors (PRs), which exist in two main isoforms, PR-A and PR-B, are differentially expressed in ovarian follicles and uterus [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Their expression is dynamically regulated by fluctuating hormone levels throughout the cycle. During the follicular phase, PR-A predominantly mediates progesterone\u0026rsquo;s inhibitory effects on follicular atresia, promoting the survival of developing follicles. Conversely, PR-B is implicated in the regulation of ovulatory processes. Studies have shown that PR knockout models exhibit impaired follicular development and ovulation, underscoring the importance of these receptors. The PR-A is also essential for uterine decidualization and implantation [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. The interaction between PRs and other intracellular signaling pathways, such as the PI3K/AKT pathway, further illustrates the complexity of progesterone's role in folliculogenesis [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Progesterone, via acting through PR-A control the development and function of the endometrium and modifies cells essential for implantation and the establishment and maintenance of pregnancy. During pregnancy, progesterone via the PRs stimulats myometrial relaxation and cervical closure [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProgesterone modulates follicular development and ovulation through its interactions with progesterone receptors (PRs) [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. During the follicular phase, low levels of progesterone and its receptors are necessary to prime the ovarian follicles for growth and maturation. Progesterone (P4) works synergistically with estrogen to regulate the expression of genes involved in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) receptors, crucial for follicular responsiveness to gonadotropins [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt was discovered that the plane of nutrition, the estrous cycle phase, and/or FSH all had an impact on the percentage of PR-positive uterine cells and/or staining intensity [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Changes in endometrial functions in superovulated models may arise from direct or indirect FSH action pathways. FSH exerts its indirect effects through binding to ovarian receptors and stimulating the synthesis of the estrogen and P4 [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] which in turn control endometrial activities [\u003cspan additionalcitationids=\"CR71\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Remarkably, during the menstrual cycle, the endometrial lining has high expression of the functional FSH receptor (FSHR). Increased expression of FSHR in the human endometrium can shed light on the potential direct effects of FSH on endometrial regeneration, which is primarily sustained by tissue-resident endometrial stem cells [\u003cspan additionalcitationids=\"CR74 CR75\" citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Through a complicated paracrine signaling network, the progesterone receptor controls glandular growth, decidualization, implantation, and the maintenance of a healthy uterus [\u003cspan additionalcitationids=\"CR71\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe role of progesterone and its receptors during the follicular phase is crucial for maintaining the delicate hormonal balance required for successful ovulation. By modulating the expression of enzymes like matrix metalloproteinases (MMPs), which are involved in follicular rupture, progesterone ensures that the dominant follicle reaches full maturation and is capable of releasing a viable oocyte [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. We suggested that proper ovarian functions during follicular phase are necessary for proper uterine functions during both follicular phase and implantation. Additionally, other findings suggest that progesterone's actions extend beyond the ovaries and uterus, influencing the hypothalamic-pituitary-gonadal axis to fine-tune gonadotropin release, further demonstrating its integral role in reproductive physiology [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterestingly, tryptase-positive immunostaining mast cells were recruited in the deep lamina propria of the caruncles during the follicular phase of the estrous cycle. Some degranulated mast cells were seen close to the macrophage in the endometrial lamina propria. Mast cells were also observed in-between uterine glands and smooth muscle fibers of the myometrium. Moreover they were noticed around the blood vessels of the stratum vascularis. MCs during the follicular phase of the estrous cycle play a crucial role in secreting substances that promote tissue remodeling [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Histamine is one of these important substances released from uterine mast cells, influencing ovulation, embryo implantation, and myometrium contractility leading to successful implantation and ultimately to parturition [\u003cspan additionalcitationids=\"CR81\" citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. However, spatiotemporally expression of MCs in the female reproductive tract has been reported in other studies [\u003cspan additionalcitationids=\"CR83 CR84 CR85 CR86 CR87 CR88 CR89 CR90 CR91\" citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. In the uterus, they change in number and structure depending on the hormonal level variations during the menstrual or estrous cycle [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan additionalcitationids=\"CR94\" citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e]. An investigation under a light microscope has revealed alterations in the characteristics and location of MCs in mice, rats, hamsters, cows, and guinea pigs uterine tissues during pregnancy and estrous cycle [\u003cspan additionalcitationids=\"CR97 CR98\" citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e]. In human endometrium, MCs were decreased in the stromal tissue [\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e, \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e]. Notably, the number and activity of MCs are correlated with estrogen concentrations in the uterine tissue of sows and rats [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e, \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e], while they are correlated with progesterone concentrations in the canine uterus [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNevertheless, to preserve regular reproductive processes and create the best environment for potential implantation, all uterine cell types interact with one another through junctacrine, paracrine, or endocrine pathways [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides new insight of the uterine histomorphological and immunohistochemical picture of the Saidi sheep during the follicular phase of the estrus cycle. These findings have growing significance to understand the key mechanisms that is characteristic of successful reproduction in Saidi sheep in order to enhance fertility and reproductive health of this livestock species and help to use advanced reproductive techinques.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental protocol was approved by the Local Ethical Committee and by the Institutional Review Board of Molecular Biology Research and studies Institute, Assiut University (22-2023-0028) and was carried out in accordance with relevant guidelines and regulations. This research was done in compliance with the ARRIVE guidelines and regulations (https:// arriv eguid elines. org). All national and institutional guidelines for animal care and use have been followed throughout the study procedures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.A.: Conceptualization, Methodology, Investigation, Data curation, Writing\u0026mdash;original draft, Writing\u0026mdash;review \u0026amp; editing, and Formal analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eM.A.K.: Writing\u0026mdash;review \u0026amp; editing, Validation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eM.A.A.: Writing\u0026mdash;review \u0026amp; editing, Validation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eM.S.H.: Conceptualization, Methodology, Writing\u0026mdash;review\u0026amp; editing, Validation.\u003c/p\u003e\n\u003cp\u003eAll authors reviewed the manuscript and approved the final version for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang H, Feng X, Muhatai G, Wang L. 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Ank \u0026Uuml;niversitesi Vet Fak\u0026uuml;ltesi Derg. 2014;61:9\u0026ndash;14.\u003c/li\u003e\n\u003cli\u003eKaraca T, Y\u0026ouml;r\u0026uuml;k M, Uslu S, \u0026Ccedil;etin Y, Uslu BA. Distribution of eosinophil granulocytes and mast cells in the reproductive tract of female goats in the preimplantation phase. Vet Res Commun. 2009;33:545\u0026ndash;54.\u003c/li\u003e\n\u003cli\u003eValle GR, Castro ACS, Nogueira JC, V CM, Gra\u0026ccedil;a DS, Nascimento EF. Eosinophils and mast cells in the oviduct of heifers under natural and superovulated estrous cycles.\u003c/li\u003e\n\u003cli\u003eSch\u0026ouml;niger S, Schoon HA. The healthy and diseased equine endometrium: A review of morphological features and molecular analyses. Animals. 2020;10.\u003c/li\u003e\n\u003cli\u003eWalter J, Klein C, Wehrend A. Distribution of Mast Cells in Vaginal, Cervical and Uterine Tissue of Non-pregnant Mares: Investigations on Correlations with Ovarian Steroids. Reprod Domest Anim. 2012;47:e29\u0026ndash;31.\u003c/li\u003e\n\u003cli\u003eWehrend A, Huchzermeyer S, Bostedt H. Distribution of Eosinophils and Mast Cells in the Cervical Tissue of Non-Gravid Mares during Dioestrus. Reprod Domest Anim. 2005;40:562\u0026ndash;3.\u003c/li\u003e\n\u003cli\u003eWoidacki K, Jensen F, Zenclussen AC. Mast cells as novel mediators of reproductive processes. Front Immunol. 2013;4.\u003c/li\u003e\n\u003cli\u003eNorrby K. On Connective Tissue Mast Cells as Protectors of Life, Reproduction, and Progeny. Int J Mol Sci. 2024;25.\u003c/li\u003e\n\u003cli\u003eZierau O, Zenclussen AC, Jensen F. Role of female sex hormones, estradiol and progesterone, in mast cell behavior. Front Immunol. 2012;3.\u003c/li\u003e\n\u003cli\u003eHamouzova P, Cizek P, Jekl V, Gozdziewska-Harlajczuk K, Kleckowska-Nawrot J. Mast cells and Kurloff cells - Their detection throughout the oestrous cycle in normal guinea pig ovaries and in guinea pigs with cystic rete ovarii. Res Vet Sci. 2021;136:512\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eElieh Ali Komi D, Shafaghat F, Haidl G. Significance of mast cells in spermatogenesis, implantation, pregnancy, and abortion: Cross talk and molecular mechanisms. Am J Reprod Immunol N Y N 1989. 2020;83:e13228.\u003c/li\u003e\n\u003cli\u003eHamouzova P, Cizek P, Novotny R, Bartoskova A, Tichy F. Distribution of mast cells in the feline ovary in various phases of the oestrous cycle. Reprod Domest Anim. 2017;52:483\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eGlavaski M, Banovic P, Lalosevic D. Number and Distribution of Mast Cells in Reproductive Systems of Gravid and Non-Gravid Female Mice. Exp Appl Biomed Res EABR. 2022;23:67\u0026ndash;73.\u003c/li\u003e\n\u003cli\u003eSchmerse F, Woidacki K, Riek-Burchardt M, Reichardt P, Roers A, Tadokoro C. In vivo visualization of uterine mast cells by two-photon microscopy. Reproduction. 2014;147:781\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eMeyer N, Zenclussen AC. Mast cells\u0026mdash;Good guys with a bad image? Am J Reprod Immunol. 2018;80:e13002.\u003c/li\u003e\n\u003cli\u003eZaitsu M, Narita S-I, Lambert KC, Grady JJ, Estes DM, Curran EM. Estradiol activates mast cells via a non-genomic estrogen receptor-\u0026alpha; and calcium influx. Mol Immunol. 2007;44:1977\u0026ndash;85.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Saidi sheep, Uterus, Endometrium, Estrous cycle, SOD2, GR, PRA, Mast cells","lastPublishedDoi":"10.21203/rs.3.rs-4790328/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4790328/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSaidi sheep are one of the most important farm animals in Upper Egypt, particularly in the Assiut governorate. Since they can provide meat, milk, fiber, and skins from low-quality roughages, sheep are among the most economically valuable animals bred for food in Egypt. Regarding breeding, relatively little is known about the Saidi breed. The uterus is an important organ for reproduction in mammals. Therefore, the purpose of this work was to provide further details on the histological, histochemical, and immunohistochemical analyses of the uterus during the follicular phase of the estrous cycle. In order to examine the histological changes in the uterus, 11 healthy Saidi ewes (38.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03 kg weight) ranging in age from 2 to 5 years were used.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn Saidi sheep, the uterine histological and immunological picture during follicular phase of estrous cycle was characterized by epithelial and stromal proliferation and apoptosis. Leucocytic recruitment (lymphocytes, plasma cells and mast cells) was also observed. The most prominent features of the follicular phase were uterine gland adenogenesis, vascular angiogenesis, and oxidative marker expression, epithelial, stromal and muscular expression of PRA.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study provides new evidences of the uterine morphological and immunohistochemical picture of the Saidi sheep during the follicular phase of the estrus cycle. These findings have growing significance to understand the key mechanisms that is characteristic of successful reproduction in Saidi sheep in order to enhance fertility and reproductive health of this livestock species.\u003c/p\u003e","manuscriptTitle":"Uterine histomorphological and immunohistochemical investigation during follicular phase of estrous cycle in Saidi sheep","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-21 10:55:24","doi":"10.21203/rs.3.rs-4790328/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-27T05:36:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-09T18:25:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178429239828821088572769288429168515360","date":"2024-09-03T08:16:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3449254780654098120910724214671989651","date":"2024-08-30T01:04:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-20T12:00:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"111269935169234344167080212880866908444","date":"2024-08-07T12:27:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-07T12:06:37+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-25T15:24:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-25T02:41:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-25T02:41:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2024-07-23T16:35:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c200016d-192a-4016-af92-d3fff9d8147e","owner":[],"postedDate":"August 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-20T16:06:09+00:00","versionOfRecord":{"articleIdentity":"rs-4790328","link":"https://doi.org/10.1186/s12917-024-04456-3","journal":{"identity":"bmc-veterinary-research","isVorOnly":false,"title":"BMC Veterinary Research"},"publishedOn":"2025-01-13 15:57:27","publishedOnDateReadable":"January 13th, 2025"},"versionCreatedAt":"2024-08-21 10:55:24","video":"","vorDoi":"10.1186/s12917-024-04456-3","vorDoiUrl":"https://doi.org/10.1186/s12917-024-04456-3","workflowStages":[]},"version":"v1","identity":"rs-4790328","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4790328","identity":"rs-4790328","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}