Histological description of gonadal  development in a  neotropical  insectivorous bat  Eumops  patagonicus  (Chiroptera:  Molossidae)

Histological description of gonadal development in a neotropical insectivorous bat Eumops patagonicus (Chiroptera: Molossidae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Histological description of gonadal development in a neotropical insectivorous bat Eumops patagonicus (Chiroptera: Molossidae) Florencia Evelyn Rodriguez, Gabriela Beatriz Olea, María Victoria Aguirre, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4535388/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The order Chiroptera is one of the most diverse orders in terms of the number of species, however, very few studies have been conducted in this group regarding its embryonic development. In this area, studies have focused on staging through morphological characters, but those describing organogenesis are scarce. Therefore, this work describes the gonadogenesis of Eumops patagonicus a Sudamerican insectivorous bat, with an emphasis on ovarian development and determination of the migration stage of the primordial germ cells (PGCs) through immunostaining. Pregnant females of this species were captured to carry out the study, and the embryos were processed to obtain histological sections using the conventional histology technique and immunohistochemistry (OCT4, PCNA, Bax and Bcl-XL). Critical stages of gonadal differentiation were described, starting from the formation of the urogenital ridge and the identification to PCGs with the positive reaction for OCT4 in embryological development stadio 13. At stadio 17 to development, it is observed an undifferentiated gonad with active proliferation and then a defined testis (S. 21) and ovaries (S.23 and S.25). The embryo ovaries S.23 show a cortex formed for ovogonia cyst/nets that have a craniocaudal development whit difference in the immunolabeling to PCNA, Bax and Bcl-XL. The embryo ovaries S.25 show the primordial follicles in the ovary cortex. The results determined that there is a conserved pattern in embryonic development in this order when comparing E. patagonicus with frugivorous bat. This information is important to the establishment of relations in developmental in mammalian and into the order Chiroptera. Chiroptera embryonic development ovary testis Immunohistochemistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The Chiroptera order stands as one of the most diverse in terms of species numbers, attracting substantial research attention due to its reproductive characteristics stemming from its remarkable diversity. However, data pertaining to gonadogenesis within this order remain largely absent. Recently, early oogenesis has been delineated in Carollia perspicillata (Lechowska et al., 2012), where the formation of germ cell cysts consisting of 10 to 12 germ cells (oogonia) was observed in embryonic ovaries. Furthermore, the presence of transient cytoplasmic bridges between distinct cysts, an unusual feature, was noted. Subsequently, somatic cells infiltrate these cysts, with their cytoplasmic projections enveloping and separating the oocytes. This process leads to the establishment of primordial follicles, characterized by a single layer of enveloping cells encircling the oocyte. In three species from the Phillostomidae family, namely Artibeus jamaiquensis , Glossophaga soricina , and Sturnira lillium , germ cells were identified within the cortex of mature ovaries (Antonio-Rubio et al., 2013). The pluripotency of oogonia was confirmed by the continued expression of pluripotency markers (POUF1), suggesting their potential role as a reservoir within the adult ovary. Moreover, gonadogenesis in embryos and the establishment of the germ line have been elucidated in S. lillium , with a focus on testicular formation (Porras-Gómez et al., 2017). In S. lillium , it was determined that the formation of the gonadal crest and establishment of the bipotent gonad occur between developmental stages 11 and 14, with initial differentiation of the male gonad occurring at stage E.17. This underscores the similarity of gonadal development in this species to that described in mice, albeit with some differences in developmental timing relative to embryonic stages. On the other hand, in humans it has been observed that during the formation of the female gonad the formation of cysts is observed and these are organized in ovigerous cords and these are interconnected by cytoplasmic bridges (Lamothe et al. 2020), which differs from what is described in mice. Given the dearth of information concerning gonadal development within the Chiroptera order, particularly in the context of the prevailing focus on frugivorous species, as well as the importance of adding this information to improve and clarify the phylogeny of this group of mammals, the objective of this study is to "Detail the events of gonadal differentiation through histological analysis and the labeling of germ line and somatic cells in Eumops patagonicus, an insectivorous species." Materials and Methods Specimen Collection Gravid Females specimens of Eumops patagonicus (n = 15) were captured on the Campus of the Facultad de Ciencias Exactas y Naturales y Agrimensura (UNNE) in Corrientes, Argentina (27_28007“ S and 58_46054” W) 2,4 m mist nets activated in periods of high activity and near bat shelters during 2018 and 2019. Only adult individuals were collected, considering the relative age assigned in relation to the ossification of the phalanges (Wilkinson & Brunet-Rossinni, 2009), and juveniles were released. Sampling was approved by the Direction of Natural Resources of the Corrientes Province Government (Authorisation No. 845). The collected bats were intraperitoneally anesthetised with 0.012 mL/g lidocaine hydrochloride monohydrate (2%), The bats were sacrificed under deep anaesthesia following the guidelines of the AVMA for the Euthanasia of Animals (Underwood et al., 2013) and the American Society of Mammologists for the use of wild mammals in research and education (Sikes et al., 2016). In addition, these procedures were approved by the ethics committee of the FaCENA-UNNE (Res. 0756/18 CD), and all collected specimens were deposited in the Mastozoology Collection of FaCENA-UNNE (Res. 0768/14 CD). The collected individuals were dissected to isolate the female gonads and the oviduct of the females. Pregnant females that were collected were dissected to extract the uterus with the embryo. Embryos from stage 13 onwards (according to the developmental table proposed by Rodríguez et al. (2018) for E. patagonicus ) were isolated from the uterus and processed for conventional histology: stage 13 Complete embryo and in advanced stages (stages 17, 23, 24, and 25), only the gonads were used. All the material was fixed in Bouin's solution for 48 hours and subsequently preserved in 10% formalin. histological procedures In order to perform histological descriptions of the embryonic gonads, they were processed following the steps of conventional histological technique: dehydration, embedding in paraffin, and staining for the preparation of histological slides. The previously fixed material was dehydrated in increasing alcohol concentrations (70%, 80%, 96%, 100%), and then cleared with two consecutive baths of Xylene for 1 hour each. Subsequently, the material was embedded in paraffin through two successive baths lasting 2 hours each to create a histological block. Samples were oriented to obtain transverse histological sections of 2 to 5µm thickness. The sections were obtained using a Spenser Manual rotary microtome and placed on clean glass slides for conventional histology. The samples were stained for description using conventional Hematoxylin-Eosin staining and the histochemical PAS (Periodic Acid-Schiff) reaction. The preparations were observed and photographed using a system consisting of a optic microscope with epifluorescence and brightfield capabilities, a trinocular LEICA DM4000B LED® microscope, and a LEICA DFC310 FX® camera with digital support for image capture from LASZ LEICA Inc®. PCNA Immunodetection To assess the proliferative status of embryonic gonads, tissue sections of 3 – 5 µm thickness were selected and placed on previously silanized slides. These sections underwent immunohistochemical detection of PCNA (Proliferating Cell Nuclear Antigen). Histological cuts were deparaffinized in two xylene baths for 20 minutes each and rehydrated in decreasing concentrations of alcohol and distilled water (100%, 96% I, 96% II, 70%, and distilled water). After hydrating the samples, a wash followed by a 10-minute incubation in PBS Tween was conducted. Cell membranes were permeabilized using 1% Triton X-100 in PBS for 10 minutes, and endogenous peroxidases were blocked with 3% H2O2 in PBS for 20 minutes. Prior to incubation with the secondary antibody, samples were blocked for 20 minutes with Blocking Serum (normal serum) from the Vectastain® ABC Universal Kit peroxidase (Horse anti-mouse/Rabbit IgG). Samples were then incubated overnight in a humid chamber at 4°C with primary antibody PCNA anti-mouse (Santa Cruz Biotechnology PCNA Antibody, sc-7907, FL -261) at a dilution of 1:50, with PBS used as a negative control. Following a 10-minute wash with PBS Tween, samples were incubated for 30 minutes with Biotinylated Universal Antibody Vectastain® ABC Universal Kit, followed by Vectastain elite ABC reagent for an additional 30 minutes. Immunodetection was performed using DAB (diaminobenzidine) (DAKO K3468), and nuclei were counterstained with hematoxylin. OCT-4 Inmunodetection For the determination of OCT-4 expression, a polyclonal anti-OCT-4 antibody of human origin, anti-mouse, was used as the primary antibody at a standard working dilution. The detection was carried out using the "avidin/biotin" indirect protocol (Vectastain Elite ABC Universal Kit Peroxidase), as described in the preceding section. Inmunodetection of Bcl Proteis Family (Bax-BCL) The expression of Bax was inmunodetected using a mouse monoclonal antibody, Anti-Bax Clone 6A7 (Sigma Chemical Co, catalog number B 8429), at a working dilution of 1:300. The detection kit employed the "avidin/biotin" indirect protocol (Vectastain Elite ABC Universal Kit Peroxidase), as described in previous section. A negative control was conducted by incubating with DAKO secondary and tertiary reagents without primary anti-Bax antibody incubation. In the case of Bcl2, a rabbit polyclonal anti-Bcl2 antibody (C 21): sc-783 from Santa Cruz was employed, at a working dilution of 1:400 and overnight incubation at 4°C. Results Formation of Urogenital/Gonadal Ridge For the early embryonic development was analyzed the stage 13, n=4 (Fig. 1 a). In this stage, the initiation of gonadal formation becomes evident. The presence of urogenital/gonadal ridges can be observed, appearing as two paired rudiments originating from the intermediate mesoderm (Fig.1 b). These ridges are composed of ventral coelomic epithelium that proliferates into the underlying mesenchyme, situated laterally to the kidney primordia. Ventromedially, the mesentery connecting with the ridges is bordered by the epithelium covering them. Beyond the ridges, the large dorsal aorta is located, followed by the neural tube. Expression of OCT4, a marker of cellular pluripotency (detected in pluripotent cells and PGCs due to their mesenchymal behavior) was detected at this developmental stage, allowing us to visualize the location of CGPs. CGPs originate outside the gonad and migrate toward it during early development. These cells were found distributed along the mesentery, within its covering epithelium, as well as within the epithelium covering the developing urogenital/gonadal ridge, with some also present within its interior mesenchyme. (Fig.2 a-c). Bipotent or Indifferent Gonad In the intermediate embryonic development, stadio 17, (n= 4), the presence of bipotent or indifferent gonads is observed (Fig.3 a and b). These structures appear as two spherical formations parallel to the neural tube and lateral to the kidney primordial. Histologically, a mesenchyme of evenly distributed cells is enclosed by a layer of low cuboidal cells, adopting a spheroid shape with a tapered side. At this tapered end, the presence of a duct bordered by a layer of low cylindrical cells (precursor of the Müllerian duct) is identified (Fig.3 c). Within the medullary region of this gonad, small blood vessels with nucleated erythroblasts are observed, while in the central region, the presence of sexual cords is identified (Fig.3 b and d). The expression of PCNA, a reliable marker of proliferating cells, was observed in the central or medullary region of this bipotent gonad (Fig.3 e and f). Embryonic Testis In an embryo at stage 21 (n= 2) of E. patagonicus embryonic development, the testis appears as a solid structure surrounded by a stroma of dense connective tissue, lacking septa that delimit lobules. The parenchyma is distinguished by the presence of seminiferous tubules and interstitial tissue. The seminiferous tubules are observed as solid structures without luminal space, comprised of germ cells (spermatogonia) and supporting somatic cells. Spermatogonia appear as large, spherical cells, while supporting cells are smaller in size. Abundant interstitial tissue (between seminiferous tubules) consists of cells that will give rise to Leydig cells, characterized by elongated nuclei and limited intercellular material. Upon performing PCNA immunodetection, significant mitotic activity was observed in both the supporting somatic cells within the tubules and the interstitial cells (Fig.4). Embryonic Ovary and Oviduct In the late stage of embryonic development, at E 23 (n= 3), well-defined ovaries (left and right) are observed along with a forming embryonic oviduct (Figure 5a). The ovary presents itself as an ovoid structure with a volume of 190,681,368 µm³. The hilum is evident on the medio-lateral region, lined by a germinal epithelium of cuboidal cells (Fig.5 a). The ovary is observed as a solid structure, featuring a highly developed cortex where ovogonia cysts or nests are apparent, each containing 5 to 12 ovogonia (Ovogonia volume: 173,013.7 µm³), bounded by a basal lamina (Fig.5 b and c). In the hilum region, the formation of the future ovarian medulla is discernible. The ovary at this stage is encompassed by the forming oviduct, accompanied by a thin layer of mesenchyme that forms and ovarian bursa or sac (4 or 5 cell layers thick). The oviduct reveals the presence of a lining epithelium of columnar cells (Fig.5 d). In an advanced stage of development, stadio 25 (n =2), both ovaries (volume 154,216,850 µm³) exhibit a cortex formed by numerous primordial follicles comprising ovogonia (volume 4,422,556.3 µm³), delimited by the precursor cells of the follicular cells. The follicular cells are low in their basal-apical axis. The ovarian medulla is more developed around the hilum, featuring blood vessels and loose connective tissue (Fig.6). In this stage, the oviduct is observed with its characteristic cylindrical shape, and its mucosa bears characteristic folds lined by a columnar cell epithelium. The ovarian bursa accompanying the ovary has a few cell layers in thickness (1 or 2 cells thick). Both the left and right ovaries share similar characteristics. Regarding uterine development, at stage 23, a well-defined bicornuate uterus with a single vagina can be distinguished macroscopically (Fig.7 a). Histologically, a cavity is observed within the uterine horn, of moderate breadth, as is the case in the vaginal region (Fig.7 b). At stage 25, a well-defined and spacious uterine cavity is observed, connected with the vaginal cavity (Fig.7 c and d). In the cephalic region of the organ, the presence of large blood vessels within the walls is evident. Apoptosis and Proliferation in Embryonic Ovary: On the development of embryonic ovary, the expression of proteins from the Bcl family, involved in the apoptosis process, was detected at stage 23 of embryonic development. Moderate expression of anti-apoptotic Bcl-xL proteins was observed in the caudal region of the ovary (Fig.8 a and b), while the pro-apoptotic protein Bax was expressed in the same sector but with a broader range of expression (Fig.8 c and d). This suggests an increase in apoptotic cells, particularly oocytes, consistent with fetal ovarian physiology. Additionally, the expression of the transcription factor PCNA was detected in the nuclei of oocytes in the cephalic zone of the ovary, the opposite side to previous marks (Fig.8 e and f). In this stage, two events coincided: the proliferation and the apoptosis of the ovogonia in the cortex. Discussion and Conclusions In the Class of mammals, gonadal development has been extensively studied in the Murine model (order Rodentia), revealing differences compared to the human model according to the time of occurrence of events like sexual cord formation. However, information on gonadal development in wild mammals is scarce although new information shown interesting events that differ with rodents. Despite being the second-largest order in terms of species, studies related to gonadal development in Chiroptera are currently limited. This study describes the key events in gonadal formation, determining the embryonic developmental stage at which these events occur. Additionally, the histology of the gonad is detailed at each stage until the formation of the ovary and testis. In mammals, the formation of the urogenital crest is observed during early stages of gestation, giving rise to gonads derived from the intermediate mesoderm. This structure, known as the genital crest, differentiates into the undifferentiated gonad and the kidney (Birchmeier y Birchmeier, 1993; Polgar et al., 2007). This structure is initially composed of an epithelium that begins to proliferate inward and will subsequently be invaded by PGCs (De Falco, 2009). The results of this study reveal the formation of the genital crest in E. patagonicus embryos at the 13th developmental stage, as per the developmental table (Rodríguez et al. 2018). Similar findings have been reported for S. lilium (Phyllostomidae family) (Porras-Gómez et al., 2017), indicating the establishment of the bipotent gonad between developmental stages 11 and 14, aligning with our results, with stage 13 falling within this range for S. lilium. Furthermore, the histological structure of the gonadal crest shows no differences compared to what has been described for Mus musculus. Once the genital crest is formed, it is invaded by Primordial Germ Cells (PGCs) migrating through the posterior mesentery. These PGCs originate in early stages of development outside the embryo and then migrate along well-defined routes to colonize the gonadal crest and form part of the gonad (Soto-Suazo y Zorn 2005). In this study, PGCs were located using OCT4 protein immunodetection, OCT4 (POU class 5 homeobox gen 1, well well-known like POU5F1) is detected in germinal cells and pluripotent cells due to this protein is required to maintain the pluripotency of the cells ( Goto et al., 1999; Hansis et al., 2000; Looijenga et al., 2003). The OCT4 positive cells in stadio 13 of development revealing PCGs presence along the posterior mesentery, the epithelium of the gonadal crest, and within the crest itself at the 13th developmental stage. This suggests that PGCs initiate migration at earlier stages than analyzed, ultimately colonizing the gonad by the 13th stage. These events are consistent with the described timeline for S. lilium , reinforcing the conservation of these processes within the order Chiroptera. Advancing in gonadal development, once the genital crest is colonized by PGCs, the formation of a bipotent or undifferentiated gonad is described. This gonadal stage cannot be morphologically distinguished, and later, the formation of testicular cords is observed as the first characteristic of gonadal differentiation (Buehr et al., 1993). In E. patagonicus , the presence of some sexual cords confirms the establishment of the bipotent gonad at the 17th developmental stage and this event corresponds whit the observations did to S. lilium , a frugivorous bat, by Porras-Gomez et al., 2017. The similarity in the occurrence of morphogenetic events between species of different genera is useful to establish the monophyly of this group. Additionally, immunodetection of the PCNA protein revealed a marked number of proliferating cells in the medullary region of this gonad. Proliferation at the medullary level suggests the presence of a male gonad, considering that male gonadal evidence and proliferation typically begin in the medullary (Brenan et al., 2002). Moreover, in the undifferentiated gonad that becomes in the ovary, the primitive sex cord formed for the first time and secondly the epithelium continuously proliferation and forms de second generation of sex cords (Lamothe et al., 2020). This aligns with the initiation of gonadal differentiation at the 17 developmental stage, as described for S. lilium (Porras-Gomez et al., 2017) Studies in C. perspicillata (Lechowska et al., 2012) describe early oogenesis in prenatal ovaries, noting the presence of germ line PGCs forming germ cell cysts by the 12th developmental stage. In E. patagonicus , cysts are observed in an advanced stage of development, specifically at stage 23. The distinctive morphology of well-defined ovaries, including cortex, medulla, and hilum, along with the presence of cysts, suggests a potential difference in the timing of primordial follicle formation between families. Observations in E. patagonicus align with results from immunomarking of pro and antiapoptotic proteins Bcl-xl and Bax, demonstrating their influence on prenatal ovarian tissue remodeling and a higher proportion of proliferating cells, as evidenced by the PCNA marker. This expected result is similar to the described to formation of ovary in mammals, when the germ cell proliferation to form an ovogonia nest o cyst previously the formation of primordial follicle (Lamothe et al., 2020). Also, in a recent study of human development, evidenced the cranio-caudal progression of developmental events, such than organ development is consistently advanced in cranial versus caudal regions within the embryo (Himelreich Perić et al., 2023; Oppenheimer and Carroll, 2004). This cranio-caudal progression is evident in the ovaries of E. patagonicus showing differential immunomarkers to bax and Bcl-XL in the cranial end and PCNA in the caudal end of the embryo ovary to be described too for humans and rodents (mouse and rats). Regarding testicular formation, well-defined testes are observed in E. patagonicus at the 21 developmental stages, consistent with findings in S. lilium (Porras-Gomez et al., 2017) and PCNA immunodetection also reveals active proliferation in the testes and it is comparable to the results for human embryos described by Li et al., (2023) when its revels proliferation in somatic and sexual cells. In conclusion, this study provides valuable insights into the gonadal development of E. patagonicus an insectivorous bat and unpublished mammalian model. The results obtained so far demonstrate that the observed events during gonadal development in Chiroptera are conserved. Highlighting the identification of migrating PGCs in stadio 13 immunostaining OCT4. Additionally, the description of the development of the ovary and oviduct in this specie differs from another frugivorous bat. On another hand, it finds similarities between human and rodent development events. However, the study of the embryology of the order Chiropthera have a-holes that need more research help to develop the phylogeny origin of the group. Declarations Author contributions Rodríguez Florencia Evelyn: Writing – Original Draft, Writing – Review & Editing, Investigation, Visualization. Olea Gabriela Beatriz: Writing – Review & Editing, Investigation, Visualization. Aguirre María Victoria : Validation, Resources, Review & Editing. Lombardo Daniel Marcelo: Investigation, Validation, Resources, Visualization, Supervision, Project Administration. Fundings: This work was supported by grant PI N° 145 (2022-2025) from Secretary of investigation, science and technique - Universidad Nacional del Chaco Austral (UNCAUS) belonging to the research project: "Morphological characterization of the reproductive system in vertebrates from Northern Argentina" and PI 22B005 (2023-2026) from General Secretary of Science and Technique - Universidad Nacional del Nordeste (SGCyT-UNNE) ) belonging to the research project: "Biology of gonadal development in vertebrates from the autochthonous regions of Northeast Argentina: characterization of steroidogenesis, proliferation and cell death events." Ethical approval: Sampling was approved by the Direction of Natural Resources of the Corrientes Province Government (Authorisation No. 845). The procedures were approved by the ethics committee of the Facultad de Ciencias Exactas y Naturales y Aguimensura (FaCENA). Universidad Nacional del Nordeste (UNNE). Res. 0756/18 CD. Informed Consent: No applicable. 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Johns Hopkins University press: Baltimore. ISBN:9781284262391 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4535388","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":318892764,"identity":"56e869e0-36cb-430c-a6fd-48301a69ede8","order_by":0,"name":"Florencia Evelyn Rodriguez","email":"","orcid":"","institution":"Instituto de Química Básica y Aplicada del Nordeste (IQUIBA-NEA) CONICET","correspondingAuthor":false,"prefix":"","firstName":"Florencia","middleName":"Evelyn","lastName":"Rodriguez","suffix":""},{"id":318892765,"identity":"a56fba8e-e9ef-466e-8a76-b62176b5f5fb","order_by":1,"name":"Gabriela Beatriz Olea","email":"","orcid":"","institution":"Universidad Nacional del Noredeste, Facultad de Ciencias Veterinarias. Cátedra de Histología y Embriología","correspondingAuthor":false,"prefix":"","firstName":"Gabriela","middleName":"Beatriz","lastName":"Olea","suffix":""},{"id":318892767,"identity":"d56dbfe7-04ab-40e5-a602-2e6a0e3903f9","order_by":2,"name":"María Victoria Aguirre","email":"","orcid":"","institution":"Instituto de Química Básica y Aplicada del Nordeste (IQUIBA-NEA) CONICET","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Victoria","lastName":"Aguirre","suffix":""},{"id":318892769,"identity":"331dd9c9-8266-4c76-b729-531ad00ceab1","order_by":3,"name":"Daniel Marcelo Lombardo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYDCC4wwMzECKsZ/5AJifQFjLYaiWmW1AxQdI0rLhGLFa+A4zH91cUHNPdvMxHsPHHxgO5/E3MD/7gE+L5GG2tNszjhUbbzvGY2xwgOFwscQBNuMZ+LQYHOYxu83DlpC47X5bmgRQS2LDAQZjvA6DaPmXkLi5jS39B0jL/APsnwlr4W1LSNzAxnyMAaRlwwEe/LaA/TKzL8F4xjHmwxJnDNKLDQ/zFOPVwne8+djtgm8Jsv1tjI0fKiqs8+SOt2/GqwXdnQyQaBoFo2AUjIJRQBkAALRoT1ZRLHzSAAAAAElFTkSuQmCC","orcid":"","institution":"Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA)","correspondingAuthor":true,"prefix":"","firstName":"Daniel","middleName":"Marcelo","lastName":"Lombardo","suffix":""}],"badges":[],"createdAt":"2024-06-05 16:14:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4535388/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4535388/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59106496,"identity":"04203084-10af-41c9-88d3-fd9e3f92db78","added_by":"auto","created_at":"2024-06-26 12:15:48","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":688039,"visible":true,"origin":"","legend":"\u003cp\u003ea) \u003cem\u003eEumops patagonicus\u003c/em\u003e embryo at stage 13 of embryonic development according to the developmental table by Rodríguez et al., (2018) showing the presence of branchial arches (ba), hindlimb buds (h), forelimb buds (f) and yolks sac (ys). b) Histological microphotograph of the embryo at stage 13 showing the presence of urogenital/gonadal ridge (ur), intestinal mesentery (m), embryonic coelom (ec), dorsal aorta (ao), and neural tube (nt). Staining: Hematoxylin-eosin.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535388/v1/56572efd38326224d58ed954.jpg"},{"id":59106479,"identity":"1e3f4119-f42b-4404-bc96-c3ae797a10c5","added_by":"auto","created_at":"2024-06-26 12:15:46","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6186042,"visible":true,"origin":"","legend":"\u003cp\u003eEmbryo at stage 13 with immunostaining for OCT4. a) Panoramic view of embryonic histology with positive staining (brown) for OCT4. b) Detail at the mesentery level. c) Detail at the level of the developing gonadal/urogenital crest. References: a: dorsal aorta, CGPs: primordial germ cells, m: mesentery, ur: gonadal/urogenital ridge.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535388/v1/bd9c0c55cf2d3449f40750f3.jpg"},{"id":59107183,"identity":"2cf74d7e-ab0f-4ae9-b70b-d7d239b4e8bd","added_by":"auto","created_at":"2024-06-26 12:23:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4488125,"visible":true,"origin":"","legend":"\u003cp\u003ea) Embryonic stage 17 of \u003cem\u003eE. patagonicus\u003c/em\u003e and and external morphology of the bipotent gonad b) General view of the histological organization of the bipotent gonad. c) Detail of the Müllerian duct. d) Detail of the mesenchyme showing blood vessels with nucleated erythroblasts. e) PCNA immunodetection in bipotent gonad. f) Detail of the mesenchyme with PCNA immunostaining in the gonad. References: bv: blood vessels; ep: epithelium; f: forelimb; \u0026nbsp;g: gonad; h: hindlimb; k: kidney; md: Müllerian duct; p: patagium; sc: sexual cords; Black arrow: nucleated erythroblast; arrowhead: positive PCNA immunostaining.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535388/v1/3ba1ca34c02a5ff02774dff9.jpg"},{"id":59106494,"identity":"d7d5d253-276d-4d81-8e1e-403bc866c317","added_by":"auto","created_at":"2024-06-26 12:15:48","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2065122,"visible":true,"origin":"","legend":"\u003cp\u003eEmbryonic testis of \u003cem\u003eE. patagonicus\u003c/em\u003e, E. 21 of embryonic development. a) Immunodetection of proliferating cell antigen (PCNA) in embryonic testis. b) Negative control of the technique. References: yellow arrow: spermatogonia; black arrow: interstitial cells; red arrow: Sertoli cells (somatic cells).\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535388/v1/9ee8da1cd08692421e6875f1.jpg"},{"id":59106497,"identity":"b981e785-c97e-4407-a424-2465054560ae","added_by":"auto","created_at":"2024-06-26 12:15:49","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7054155,"visible":true,"origin":"","legend":"\u003cp\u003eHistology of the ovary at stadio 23. a) Panoramic histology of the embryonic ovary of \u003cem\u003eE. patagonicus\u003c/em\u003e at stadio 23. b) Detail of the ovarian cortex with cysts or nests of oogonia and detail of the germinal epithelium. c) Detail of oogonia in cysts. d) Detail of the embryonic oviduct and detail of the inner lining epithelium. References: bu: ovarian bursa/pouch; cy: cyst/nest of oogonia; eg: germinal epithelium; ep: lining epithelium; hi: hilus; me: ovarian medulla; ovd: oviduct. Asterisk: basal lamina of the cyst. Arrowhead: oogonia. Staining: HE (Hematoxylin and Eosin) a and d; PAS (Periodic Acid-Schiff) b and c.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535388/v1/c78ec185a7729f9f35ea67f1.jpg"},{"id":59106487,"identity":"3da90d3f-8646-4ff0-ab52-cfa90c7420f0","added_by":"auto","created_at":"2024-06-26 12:15:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":347543,"visible":true,"origin":"","legend":"\u003cp\u003eHistology of the ovary in stadio 25. \u0026nbsp;a) Panoramic histology of the embryonic ovary of \u003cem\u003eE. patagonicus \u003c/em\u003eat S.25. b) Detail of the ovarian cortex with primordial follicles. References: bu: ovarian bursa/pouch; cr: ovarian cortex; hi: hilus; me: ovarian medulla; ovd: oviduct; ov: oogonium. Asterisk: follicular cells. Arrowhead: nucleus of the oogonium. Dots circle: primordial follicle. Staining: HE (Hematoxylin and Eosin).\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535388/v1/b2afabf349431495f134b472.jpg"},{"id":59106490,"identity":"6cc31eea-3bd9-4634-9f85-daebb2ff1ba3","added_by":"auto","created_at":"2024-06-26 12:15:47","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5589495,"visible":true,"origin":"","legend":"\u003cp\u003eEmbryonic uterus at stadio 23 and 24. a) Morphology of the reproductive system of \u003cem\u003eE. patagonicus\u003c/em\u003e at embryonic development stage 23. b) Histological microphotograph of the reproductive system at S.23 (longitudinal section). c) Morphology of the reproductive system at S.25 (longitudinal section). d) Histology of the ovary and uterine horn at S.25. References: rh: right uterine horn; lh: left uterine horn; uh: uterine horn; ov: ovary; rov: right ovary; lov: left ovary; ovd: oviduct; vg: vagina; bv: blood vessel.\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535388/v1/eba8e2377184d831fd4c7f6e.jpg"},{"id":59106484,"identity":"d202cfb7-24c8-453a-aed6-be4c0f50a1e2","added_by":"auto","created_at":"2024-06-26 12:15:47","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":9887553,"visible":true,"origin":"","legend":"\u003cp\u003eEmbryonic Ovary of \u003cem\u003eE. patagonicus\u003c/em\u003e at stage 23. a) BCL-XL immunohistochemistry in embryonic ovary; b) closeup of cytoplasmic positive to BCL-XL in ovogonia(yellow arrowhead); c)Immunodetection of pro-apoptotic protein BAX in embryonic ovary; d) closeup of cytoplasmic positive to BAX in ovogonia (light-blue arrowhead) , e) Immunodetection of Nuclear Proliferation Antigen PCNA; f) closeup of nuclear positive to PCNA in ovogonia (red arrowhead); g) Negative control of the technique; h) closeup of negative control showing ovogonia in embryonc ovary from stadio 23 of development. Reference: Dashed circle encloses the region with positive staining. Dot circle: ovogonia net/cyst. Ov: ovogonia\u003c/p\u003e","description":"","filename":"Fig.8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4535388/v1/7189d63cab1b7b55599ea020.jpg"},{"id":63995847,"identity":"90732b20-8387-4440-99b9-5ca68984b6dc","added_by":"auto","created_at":"2024-09-04 16:30:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":36641747,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4535388/v1/d9eff105-f308-4b1e-aff1-0194b2bb9c8c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Histological description of gonadal development in a neotropical insectivorous bat Eumops patagonicus (Chiroptera: Molossidae)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Chiroptera order stands as one of the most diverse in terms of species numbers, attracting substantial research attention due to its reproductive characteristics stemming from its remarkable diversity. However, data pertaining to gonadogenesis within this order remain largely absent. Recently, early oogenesis has been delineated in \u003cem\u003eCarollia perspicillata\u003c/em\u003e\u0026nbsp; (Lechowska et al., 2012), where the formation of germ cell cysts consisting of 10 to 12 germ cells (oogonia) was observed in embryonic ovaries. Furthermore, the presence of transient cytoplasmic bridges between distinct cysts, an unusual feature, was noted. Subsequently, somatic cells infiltrate these cysts, with their cytoplasmic projections enveloping and separating the oocytes. This process leads to the establishment of primordial follicles, characterized by a single layer of enveloping cells encircling the oocyte.\u003c/p\u003e\n\u003cp\u003eIn three species from the Phillostomidae family, namely \u003cem\u003eArtibeus jamaiquensis\u003c/em\u003e, \u003cem\u003eGlossophaga soricina\u003c/em\u003e, and \u003cem\u003eSturnira lillium\u003c/em\u003e, germ cells were identified within the cortex of mature ovaries (Antonio-Rubio et al., 2013). The pluripotency of oogonia was confirmed by the continued expression of pluripotency markers (POUF1), suggesting their potential role as a reservoir within the adult ovary. Moreover, gonadogenesis in embryos and the establishment of the germ line have been elucidated in \u003cem\u003eS. lillium\u003c/em\u003e, with a focus on testicular formation (Porras-Gómez et al., 2017). In \u003cem\u003eS. lillium\u003c/em\u003e, it was determined that the formation of the gonadal crest and establishment of the bipotent gonad occur between developmental stages 11 and 14, with initial differentiation of the male gonad occurring at stage E.17. This underscores the similarity of gonadal development in this species to that described in mice, albeit with some differences in developmental timing relative to embryonic stages. On the other hand, in humans it has been observed that during the formation of the female gonad the formation of cysts is observed and these are organized in ovigerous cords and these are interconnected by cytoplasmic bridges (Lamothe et al.\u0026nbsp; 2020), which differs from what is described in mice.\u003c/p\u003e\n\u003cp\u003eGiven the dearth of information concerning gonadal development within the Chiroptera order, particularly in the context of the prevailing focus on frugivorous species, as well as the importance of adding this information to improve and clarify the phylogeny of this group of mammals, the objective of this study is to \"Detail the events of gonadal differentiation through histological analysis and the labeling of germ line and somatic cells in Eumops patagonicus, an insectivorous species.\"\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cu\u003eSpecimen Collection\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eGravid Females specimens of \u003cem\u003eEumops patagonicus\u003c/em\u003e\u0026nbsp; (n = 15) were captured on the Campus of the Facultad de Ciencias Exactas y Naturales y Agrimensura (UNNE) in Corrientes, Argentina (27_28007\u0026ldquo; S and 58_46054\u0026rdquo; W) 2,4 m mist nets activated in periods of high activity and near bat shelters during 2018 and 2019. Only adult individuals were collected, considering the relative age assigned in relation to the ossification of the phalanges (Wilkinson \u0026amp; Brunet-Rossinni, 2009), and juveniles were released. Sampling was approved by the Direction of Natural Resources of the Corrientes Province Government (Authorisation No. 845). The collected bats were intraperitoneally anesthetised with 0.012 mL/g lidocaine hydrochloride monohydrate (2%), The bats were sacrificed under deep anaesthesia following the guidelines of the AVMA for the Euthanasia of Animals (Underwood et al., 2013) and the American Society of Mammologists for the use of wild mammals in research and education (Sikes et al., 2016). In addition, these procedures were approved by the ethics committee of the FaCENA-UNNE (Res. 0756/18 CD), and all collected specimens were deposited in the Mastozoology Collection of FaCENA-UNNE (Res. 0768/14 CD). The collected individuals were dissected to isolate the female gonads and the oviduct of the females. Pregnant females that were collected were dissected to extract the uterus with the embryo. Embryos from stage 13 onwards (according to the developmental table proposed by Rodr\u0026iacute;guez et al. (2018) for \u003cem\u003eE. patagonicus\u003c/em\u003e) were isolated from the uterus and processed for conventional histology: stage 13 Complete embryo and in advanced stages (stages 17, 23, 24, and 25), only the gonads were used. All the material was fixed in Bouin\u0026apos;s solution for 48 hours and subsequently preserved in 10% formalin.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003ehistological procedures\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eIn order to perform histological descriptions of the embryonic gonads, they were processed following the steps of conventional histological technique: dehydration, embedding in paraffin, and staining for the preparation of histological slides. The previously fixed material was dehydrated in increasing alcohol concentrations (70%, 80%, 96%, 100%), and then cleared with two consecutive baths of Xylene for 1 hour each. Subsequently, the material was embedded in paraffin through two successive baths lasting 2 hours each to create a histological block. Samples were oriented to obtain transverse histological sections of 2 to 5\u0026micro;m thickness. The sections were obtained using a Spenser Manual rotary microtome and placed on clean glass slides for conventional histology. The samples were stained for description using conventional Hematoxylin-Eosin staining and the histochemical PAS (Periodic Acid-Schiff) reaction. The preparations were observed and photographed using a system consisting of a optic microscope with epifluorescence and brightfield capabilities, a trinocular LEICA DM4000B LED\u0026reg; microscope, and a LEICA DFC310 FX\u0026reg; camera with digital support for image capture from LASZ LEICA Inc\u0026reg;.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003ePCNA Immunodetection\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the proliferative status of embryonic gonads, tissue sections of 3 \u0026ndash; 5 \u0026micro;m thickness were selected and placed on previously silanized slides. These sections underwent immunohistochemical detection of PCNA (Proliferating Cell Nuclear Antigen). Histological cuts were deparaffinized in two xylene baths for 20 minutes each and rehydrated in decreasing concentrations of alcohol and distilled water (100%, 96% I, 96% II, 70%, and distilled water). After hydrating the samples, a wash followed by a 10-minute incubation in PBS Tween was conducted. Cell membranes were permeabilized using 1% Triton X-100 in PBS for 10 minutes, and endogenous peroxidases were blocked with 3% H2O2 in PBS for 20 minutes. Prior to incubation with the secondary antibody, samples were blocked for 20 minutes with Blocking Serum (normal serum) from the Vectastain\u0026reg; ABC Universal Kit peroxidase (Horse anti-mouse/Rabbit IgG). Samples were then incubated overnight in a humid chamber at 4\u0026deg;C with primary antibody PCNA anti-mouse (Santa Cruz Biotechnology PCNA Antibody, sc-7907, FL -261) at a dilution of 1:50, with PBS used as a negative control. Following a 10-minute wash with PBS Tween, samples were incubated for 30 minutes with Biotinylated Universal Antibody Vectastain\u0026reg; ABC Universal Kit, followed by Vectastain elite ABC reagent for an additional 30 minutes. Immunodetection was performed using DAB (diaminobenzidine) (DAKO K3468), and nuclei were counterstained with hematoxylin.\u003c/p\u003e\n\u003cp\u003eOCT-4 Inmunodetection\u003c/p\u003e\n\u003cp\u003eFor the determination of OCT-4 expression, a polyclonal anti-OCT-4 antibody of human origin, anti-mouse, was used as the primary antibody at a standard working dilution. The detection was carried out using the \u0026quot;avidin/biotin\u0026quot; indirect protocol (Vectastain Elite ABC Universal Kit Peroxidase), as described in the preceding section.\u003c/p\u003e\n\u003cp\u003eInmunodetection of Bcl Proteis Family (Bax-BCL)\u003c/p\u003e\n\u003cp\u003eThe expression of Bax was inmunodetected using a mouse monoclonal antibody, Anti-Bax Clone 6A7 (Sigma Chemical Co, catalog number B 8429), at a working dilution of 1:300. The detection kit employed the \u0026quot;avidin/biotin\u0026quot; indirect protocol (Vectastain Elite ABC Universal Kit Peroxidase), as described in previous section. A negative control was conducted by incubating with DAKO secondary and tertiary reagents without primary anti-Bax antibody incubation.\u003c/p\u003e\n\u003cp\u003eIn the case of Bcl2, a rabbit polyclonal anti-Bcl2 antibody (C 21): sc-783 from Santa Cruz was employed, at a working dilution of 1:400 and overnight incubation at 4\u0026deg;C.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFormation of Urogenital/Gonadal Ridge\u003c/p\u003e\n\u003cp\u003eFor the early embryonic development was analyzed the stage 13, n=4 (Fig. 1 a). In this stage, the initiation of gonadal formation becomes evident. The presence of urogenital/gonadal ridges can be observed, appearing as two paired rudiments originating from the intermediate mesoderm (Fig.1 b). These ridges are composed of ventral coelomic epithelium that proliferates into the underlying mesenchyme, situated laterally to the kidney primordia. Ventromedially, the mesentery connecting with the ridges is bordered by the epithelium covering them. Beyond the ridges, the large dorsal aorta is located, followed by the neural tube.\u003c/p\u003e\n\u003cp\u003eExpression of OCT4, a marker of cellular pluripotency (detected in pluripotent cells and PGCs due to their mesenchymal behavior) was detected at this developmental stage, allowing us to visualize the location of CGPs. CGPs originate outside the gonad and migrate toward it during early development. These cells were found distributed along the mesentery, within its covering epithelium, as well as within the epithelium covering the developing urogenital/gonadal ridge, with some also present within its interior mesenchyme. (Fig.2 a-c).\u003c/p\u003e\n\u003cp\u003eBipotent or Indifferent Gonad\u003c/p\u003e\n\u003cp\u003eIn the intermediate embryonic development, stadio 17, (n= 4), the presence of bipotent or indifferent gonads is observed (Fig.3 a and b). These structures appear as two spherical formations parallel to the neural tube and lateral to the kidney primordial. Histologically, a mesenchyme of evenly distributed cells is enclosed by a layer of low cuboidal cells, adopting a spheroid shape with a tapered side. At this tapered end, the presence of a duct bordered by a layer of low cylindrical cells (precursor of the M\u0026uuml;llerian duct) is identified (Fig.3 c). Within the medullary region of this gonad, small blood vessels with nucleated erythroblasts are observed, while in the central region, the presence of sexual cords is identified (Fig.3 b and d). The expression of PCNA, a reliable marker of proliferating cells, was observed in the central or medullary region of this bipotent gonad (Fig.3 e and f). \u003c/p\u003e\n\u003cp\u003eEmbryonic Testis\u003c/p\u003e\n\u003cp\u003eIn an embryo at stage 21 (n= 2) of \u003cem\u003eE. patagonicus\u003c/em\u003e\u0026nbsp; embryonic development, the testis appears as a solid structure surrounded by a stroma of dense connective tissue, lacking septa that delimit lobules. The parenchyma is distinguished by the presence of seminiferous tubules and interstitial tissue. The seminiferous tubules are observed as solid structures without luminal space, comprised of germ cells (spermatogonia) and supporting somatic cells. Spermatogonia appear as large, spherical cells, while supporting cells are smaller in size. Abundant interstitial tissue (between seminiferous tubules) consists of cells that will give rise to Leydig cells, characterized by elongated nuclei and limited intercellular material.\u003c/p\u003e\n\u003cp\u003eUpon performing PCNA immunodetection, significant mitotic activity was observed in both the supporting somatic cells within the tubules and the interstitial cells (Fig.4).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEmbryonic Ovary and Oviduct\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn the late stage of embryonic development, at E 23 (n= 3), well-defined ovaries (left and right) are observed along with a forming embryonic oviduct (Figure 5a). The ovary presents itself as an ovoid structure with a volume of 190,681,368 \u0026micro;m\u0026sup3;. The hilum is evident on the medio-lateral region, lined by a germinal epithelium of cuboidal cells (Fig.5 a). The ovary is observed as a solid structure, featuring a highly developed cortex where ovogonia cysts or nests are apparent, each containing 5 to 12 ovogonia (Ovogonia volume: 173,013.7 \u0026micro;m\u0026sup3;), bounded by a basal lamina (Fig.5 b and c). In the hilum region, the formation of the future ovarian medulla is discernible.\u003c/p\u003e\n\u003cp\u003eThe ovary at this stage is encompassed by the forming oviduct, accompanied by a thin layer of mesenchyme that forms and ovarian bursa or sac (4 or 5 cell layers thick). The oviduct reveals the presence of a lining epithelium of columnar cells (Fig.5 d).\u003c/p\u003e\n\u003cp\u003eIn an advanced stage of development, stadio 25 (n =2), both ovaries (volume 154,216,850 \u0026micro;m\u0026sup3;) exhibit a cortex formed by numerous primordial follicles comprising ovogonia (volume 4,422,556.3 \u0026micro;m\u0026sup3;), delimited by the precursor cells of the follicular cells. The follicular cells are low in their basal-apical axis. The ovarian medulla is more developed around the hilum, featuring blood vessels and loose connective tissue (Fig.6).\u003c/p\u003e\n\u003cp\u003eIn this stage, the oviduct is observed with its characteristic cylindrical shape, and its mucosa bears characteristic folds lined by a columnar cell epithelium. The ovarian bursa accompanying the ovary has a few cell layers in thickness (1 or 2 cells thick). Both the left and right ovaries share similar characteristics.\u003c/p\u003e\n\u003cp\u003eRegarding uterine development, at stage 23, a well-defined bicornuate uterus with a single vagina can be distinguished macroscopically (Fig.7 a). Histologically, a cavity is observed within the uterine horn, of moderate breadth, as is the case in the vaginal region (Fig.7 b). At stage 25, a well-defined and spacious uterine cavity is observed, connected with the vaginal cavity (Fig.7 c and d). In the cephalic region of the organ, the presence of large blood vessels within the walls is evident.\u003c/p\u003e\n\u003cp\u003eApoptosis and Proliferation in Embryonic Ovary:\u003c/p\u003e\n\u003cp\u003eOn the development of embryonic ovary, the expression of proteins from the Bcl family, involved in the apoptosis process, was detected at stage 23 of embryonic development. Moderate expression of anti-apoptotic Bcl-xL proteins was observed in the caudal region of the ovary (Fig.8 a and b), while the pro-apoptotic protein Bax was expressed in the same sector but with a broader range of expression (Fig.8 c and d). This suggests an increase in apoptotic cells, particularly oocytes, consistent with fetal ovarian physiology. Additionally, the expression of the transcription factor PCNA was detected in the nuclei of oocytes in the cephalic zone of the ovary, the opposite side to previous marks (Fig.8 e and f). In this stage, two events coincided: the proliferation and the apoptosis of the ovogonia in the cortex.\u003c/p\u003e"},{"header":"Discussion and Conclusions","content":"\u003cp\u003eIn the Class of mammals, gonadal development has been extensively studied in the Murine model (order Rodentia), revealing differences compared to the human model according to the time of occurrence of events like sexual cord formation. However, information on gonadal development in wild mammals is scarce although new information shown interesting events that differ with rodents. Despite being the second-largest order in terms of species, studies related to gonadal development in Chiroptera are currently limited.\u003c/p\u003e\n\u003cp\u003eThis study describes the key events in gonadal formation, determining the embryonic developmental stage at which these events occur. Additionally, the histology of the gonad is detailed at each stage until the formation of the ovary and testis.\u003c/p\u003e\n\u003cp\u003eIn mammals, the formation of the urogenital crest is observed during early stages of gestation, giving rise to gonads derived from the intermediate mesoderm. This structure, known as the genital crest, differentiates into the undifferentiated gonad and the kidney (Birchmeier y Birchmeier, 1993; Polgar et al., 2007). This structure is initially composed of an epithelium that begins to proliferate inward and will subsequently be invaded by PGCs (De Falco, 2009). The results of this study reveal the formation of the genital crest in \u003cem\u003eE. patagonicus\u003c/em\u003e embryos at the 13th developmental stage, as per the developmental table (Rodr\u0026iacute;guez et al. 2018). Similar findings have been reported for \u003cem\u003eS. lilium\u003c/em\u003e (Phyllostomidae family) (Porras-G\u0026oacute;mez et al., 2017), indicating the establishment of the bipotent gonad between developmental stages 11 and 14, aligning with our results, with stage 13 falling within this range for \u003cem\u003eS. lilium.\u003c/em\u003e Furthermore, the histological structure of the gonadal crest shows no differences compared to what has been described for Mus musculus.\u003c/p\u003e\n\u003cp\u003eOnce the genital crest is formed, it is invaded by Primordial Germ Cells (PGCs) migrating through the posterior mesentery. These PGCs originate in early stages of development outside the embryo and then migrate along well-defined routes to colonize the gonadal crest and form part of the gonad (Soto-Suazo y Zorn 2005). In this study, PGCs were located using OCT4 protein immunodetection, OCT4 (POU class 5 homeobox gen 1, well well-known like POU5F1) is detected in germinal cells and pluripotent cells due to this protein is required \u003cem\u003eto maintain the pluripotency of the cells (\u003c/em\u003eGoto et al., 1999; Hansis et al., 2000; Looijenga et al., 2003). The OCT4 positive cells in stadio 13 of development revealing PCGs presence along the posterior mesentery, the epithelium of the gonadal crest, and within the crest itself at the 13th developmental stage. This suggests that PGCs initiate migration at earlier stages than analyzed, ultimately colonizing the gonad by the 13th stage. These events are consistent with the described timeline for \u003cem\u003eS. lilium\u003c/em\u003e, reinforcing the conservation of these processes within the order Chiroptera.\u003c/p\u003e\n\u003cp\u003eAdvancing in gonadal development, once the genital crest is colonized by PGCs, the formation of a bipotent or undifferentiated gonad is described. This gonadal stage cannot be morphologically distinguished, and later, the formation of testicular cords is observed as the first characteristic of gonadal differentiation (Buehr et al., 1993). In \u003cem\u003eE. patagonicus\u003c/em\u003e, the presence of some sexual cords confirms the establishment of the bipotent gonad at the 17th developmental stage and this event corresponds whit the observations did to \u003cem\u003eS. lilium\u003c/em\u003e, a frugivorous bat, by Porras-Gomez et al., 2017. The similarity in the occurrence of morphogenetic events between species of different genera is useful to establish the monophyly of this group.\u003c/p\u003e\n\u003cp\u003eAdditionally, immunodetection of the PCNA protein revealed a marked number of proliferating cells in the medullary region of this gonad. Proliferation at the medullary level suggests the presence of a male gonad, considering that male gonadal evidence and proliferation typically begin in the medullary (Brenan et al., 2002). Moreover, in the undifferentiated gonad that becomes in the ovary, the primitive sex cord formed for the first time and secondly the epithelium continuously proliferation and forms de second generation of sex cords (Lamothe et al., 2020). This aligns with the initiation of gonadal differentiation at the 17 developmental stage, as described for \u003cem\u003eS. lilium\u003c/em\u003e (Porras-Gomez et al., 2017)\u003c/p\u003e\n\u003cp\u003eStudies in \u003cem\u003eC. perspicillata\u003c/em\u003e (Lechowska et al., 2012) describe early oogenesis in prenatal ovaries, noting the presence of germ line PGCs forming germ cell cysts by the 12th developmental stage. In \u003cem\u003eE. patagonicus\u003c/em\u003e, cysts are observed in an advanced stage of development, specifically at stage 23. The distinctive morphology of well-defined ovaries, including cortex, medulla, and hilum, along with the presence of cysts, suggests a potential difference in the timing of primordial follicle formation between families.\u003c/p\u003e\n\u003cp\u003eObservations in \u003cem\u003eE. patagonicus\u003c/em\u003e align with results from immunomarking of pro and antiapoptotic proteins Bcl-xl and Bax, demonstrating their influence on prenatal ovarian tissue remodeling and a higher proportion of proliferating cells, as evidenced by the PCNA marker. This expected result is similar to the described to formation of ovary in mammals, when the germ cell proliferation to form an ovogonia nest o cyst previously the formation of primordial follicle (Lamothe et al., 2020). Also, in a recent study of human development, evidenced the cranio-caudal progression of developmental events, such than organ development is consistently advanced in cranial versus caudal regions within the embryo (Himelreich Perić et al., 2023; Oppenheimer and Carroll, 2004). This cranio-caudal progression is evident in the ovaries of \u003cem\u003eE. patagonicus\u003c/em\u003e showing differential immunomarkers to bax and Bcl-XL in the cranial end and PCNA in the caudal end of the embryo ovary to be described too for humans and rodents (mouse and rats).\u003c/p\u003e\n\u003cp\u003eRegarding testicular formation, well-defined testes are observed in \u003cem\u003eE. patagonicus\u003c/em\u003e at the 21 developmental stages, consistent with findings in \u003cem\u003eS. lilium\u003c/em\u003e\u003cem\u003e \u003c/em\u003e(Porras-Gomez et al., 2017) and PCNA immunodetection also reveals active proliferation in the testes and it is comparable to the results for human embryos described by Li et al., (2023) when its revels proliferation in somatic and sexual cells.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study provides valuable insights into the gonadal development of E. patagonicus an insectivorous bat and unpublished mammalian model. The results obtained so far demonstrate that the observed events during gonadal development in Chiroptera are conserved. Highlighting the identification of migrating PGCs in stadio 13 immunostaining OCT4. Additionally, the description of the development of the ovary and oviduct in this specie differs from another frugivorous bat. On another hand, it finds similarities between human and rodent development events. However, the study of the embryology of the order Chiropthera have a-holes that need more research help to develop the phylogeny origin of the group.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRodr\u0026iacute;guez Florencia Evelyn:\u003c/strong\u003e Writing \u0026ndash; Original Draft, Writing \u0026ndash; Review \u0026amp; Editing, Investigation, Visualization. \u003cstrong\u003eOlea Gabriela Beatriz:\u003c/strong\u003e Writing \u0026ndash; Review \u0026amp; Editing, Investigation, Visualization. \u003cstrong\u003eAguirre Mar\u0026iacute;a Victoria\u003c/strong\u003e: Validation, Resources, Review \u0026amp; Editing. \u003cstrong\u003eLombardo Daniel Marcelo:\u003c/strong\u003e Investigation, Validation, Resources, Visualization, Supervision, Project Administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings: \u003c/strong\u003eThis work was supported by grant PI N\u0026deg; 145 (2022-2025) from Secretary of investigation, science and technique - Universidad Nacional del Chaco Austral (UNCAUS) belonging to the research project: \u0026quot;Morphological characterization of the reproductive system in vertebrates from Northern Argentina\u0026quot; and PI 22B005 (2023-2026) from General Secretary of Science and Technique - Universidad Nacional del Nordeste (SGCyT-UNNE) ) belonging to the research project: \u0026quot;Biology of gonadal development in vertebrates from the autochthonous regions of Northeast Argentina: characterization of steroidogenesis, proliferation and cell death events.\u0026quot;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval: \u003c/strong\u003eSampling was approved by the Direction of Natural Resources of the Corrientes Province Government (Authorisation No. 845). The procedures were approved by the ethics committee of the Facultad de Ciencias Exactas y Naturales y Aguimensura (FaCENA). Universidad Nacional del Nordeste (UNNE). Res. 0756/18 CD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent: \u003c/strong\u003eNo applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e: No potential conflicts of interest were disclosed\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAntonio-Rubio NR, Porras-G\u0026oacute;mez TJ, Moreno-Mendoza N (2013) Identification of cortical germ cells in adult ovaries from three phyllostomid bats: Artibeus jamaicensis, Glossophaga soricina and Sturnira lilium. Reprod fert develop. doi: 10.1071/RD12126\u003c/li\u003e\n\u003cli\u003eBirchmeier C, Birchmeier W (1993) Molecular aspects of mesenchymal- epithelial interactions. Annu. Rev. Cell. Biol. Dev. Doi: 10.1146/annurev.cb.09.110193.002455\u003c/li\u003e\n\u003cli\u003eBuehr M, Gu S, McLaren A (1993) Mesonephric contribution to testis differentiation in the fetal mouse. Development. Doi: 10.1242/dev.117.1.273\u003c/li\u003e\n\u003cli\u003eDeFalco T, Capel B (2009) Gonad morphogenesis in vertebrates: divergent means to a convergent end. Annu. Rev. Cell. Biol. Dev. Doi: 10.1146/annurev.cellbio.042308.13350\u003c/li\u003e\n\u003cli\u003eGoto T, Adjaye J, Rodeck CH, Monk M. (1999). Identification of genes expressed in human primordial germ cells at the time of entry of the female germ line into meiosis. Mol Hum Reprod. doi: 10.1093/molehr/5.9.851. \u003c/li\u003e\n\u003cli\u003eHansis C, Grifo JA, Krey L. (2000) Oct-4 expression in inner cell mass and trophectoderm of human blastocysts. Mol Hum Reprod. doi: 10.1093/molehr/6.11.999.\u003c/li\u003e\n\u003cli\u003eHimelreich Perić M, Takahashi M, Ježek D, Cunha GR (2023) Early development of the human embryonic testis. Differentiation. doi: 10.1016/j.diff.2022.07.001.\u003c/li\u003e\n\u003cli\u003eLamothe S, Bernard V, Christin-Maitre S (2020). Gonad differentiation toward ovary. In Anna. d\u0026apos;Endocrinol. Doi: 10.1016/j.ando.2020.04.004\u003c/li\u003e\n\u003cli\u003eLechowska A, Bilinski SM, Rasweiler IV JJ, Cretekos CJ, Behringer RR, Kloc (2012) Early oogenesis in the short‐tailed fruit bat Carollia perspicillata: Transient germ cell cysts and noncanonical intercellular bridges. Genesis. Doi: 10.1002/dvg.20780\u003c/li\u003e\n\u003cli\u003eLi Y, Overland M, Derpinghaus A, Aksel S, Cao M, Ladwig N. ... Baskin LS (2023) Development of the human fetal testis: Morphology and expression of cellular differentiation markers. Differentiation. Doi: 10.1016/j.diff.2022.03.002\u003c/li\u003e\n\u003cli\u003eLooijenga LH, Stoop H, de Leeuw HP, de Gouveia Brazao CA, Gillis AJ, van Roozendaal KE, van Zoelen EJ, Weber RF, Wolffenbuttel KP, van Dekken H, Honecker F, Bokemeyer C, Perlman EJ, Schneider DT, Kononen J, Sauter G, Oosterhuis JW (2003) POU5F1 (OCT3/4) identifies cells with pluripotent potential in human germ cell tumors. Cancer Res.\u003c/li\u003e\n\u003cli\u003eOppenheimer SB, Carroll EJ (2004) Introduction to Embryonic Development. Pearson Education, Upper Saddle River, New Jersey. Doi: 10.1016/j.diff.2022.07.001\u003c/li\u003e\n\u003cli\u003ePolgar K, Striker G, Elliott J, Hyink D, Weber O, Fehling HJ, Wilson P (2007) Mouse embryonic stem cell\u0026ndash;derived embryoid bodies generate progenitors that integrate long term into renal proximal tubules in vivo. J. Am Soc Neph. DOI: 10.1681/ASN.2006101078\u003c/li\u003e\n\u003cli\u003ePorras-G\u0026oacute;mez TJ, Mart\u0026iacute;nez-Ju\u0026aacute;rez A, Moreno-Mendoza N (2017) Gonadal morphogenesis and establishment of the germline in the phyllostomid bat Sturnira lilium. Acta Histochem. Doi: 10.1016/j.acthis.2017.08.002\u003c/li\u003e\n\u003cli\u003eRodriguez FE, Olea GB, Aguirre MV, Argoitia MA, Claver J, Lombardo DM (2023). Comparative study of the gular gland of three species of Molossidae bats Mammalia: Chiroptera) from South America. Anat. Rec. Doi: https://doi.org/10.1002/ar.25277\u003c/li\u003e\n\u003cli\u003eSikes RS Animal Care and Use Committee of the American Society of Mammalogists. (2016). Guidelines of the American Society of Mammalogists for the use of wild mammals in research and education. J. Mammal. DOI: 10.1093/jmammal/gyw078\u003c/li\u003e\n\u003cli\u003eSoto-Suazo M, Zorn, TM. (2018). Primordial germ cells migration: morphological and molecular aspects. Ani Rep.\u003c/li\u003e\n\u003cli\u003eUnderwood W, Anthony R, Gwaltney-Brant S, Poison ASPCA, Meyer R (2013) AVMA guidelines for the euthanasia of animals (2013th ed.). American Veterinary Medical Association.\u003c/li\u003e\n\u003cli\u003eWilkinson GS, Brunet-Rossinni AK (2009). Methods for age estimation and the study of senescence in bats. In T.H. Kunz \u0026amp; S. Parsons (Eds.), Ecological and Behavioral Methods for the Study of Bats. Johns Hopkins University press: Baltimore. ISBN:9781284262391\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chiroptera, embryonic development, ovary, testis, Immunohistochemistry","lastPublishedDoi":"10.21203/rs.3.rs-4535388/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4535388/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe order Chiroptera is one of the most diverse orders in terms of the number of species, however, very few studies have been conducted in this group regarding its embryonic development. In this area, studies have focused on staging through morphological characters, but those describing organogenesis are scarce. Therefore, this work describes the gonadogenesis of \u003cem\u003eEumops patagonicus\u003c/em\u003e a Sudamerican insectivorous bat, with an emphasis on ovarian development and determination of the migration stage of the primordial germ cells (PGCs) through immunostaining. Pregnant females of this species were captured to carry out the study, and the embryos were processed to obtain histological sections using the conventional histology technique and immunohistochemistry (OCT4, PCNA, Bax and Bcl-XL). Critical stages of gonadal differentiation were described, starting from the formation of the urogenital ridge and the identification to PCGs with the positive reaction for OCT4 in embryological development stadio 13. At stadio 17 to development, it is observed an undifferentiated gonad with active proliferation and then a defined testis (S. 21) and ovaries (S.23 and S.25). The embryo ovaries S.23 show a cortex formed for ovogonia cyst/nets that have a craniocaudal development whit difference in the immunolabeling to PCNA, Bax and Bcl-XL. The embryo ovaries S.25 show the primordial follicles in the ovary cortex. The results determined that there is a conserved pattern in embryonic development in this order when comparing \u003cem\u003eE. patagonicus\u003c/em\u003e with frugivorous bat. This information is important to the establishment of relations in developmental in mammalian and into the order Chiroptera.\u003c/p\u003e","manuscriptTitle":"Histological description of gonadal development in a neotropical insectivorous bat Eumops patagonicus (Chiroptera: Molossidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-26 12:15:38","doi":"10.21203/rs.3.rs-4535388/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3db7be9b-18e1-4a88-91c8-995780161e2d","owner":[],"postedDate":"June 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-04T16:22:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-26 12:15:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4535388","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4535388","identity":"rs-4535388","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}