Effect of heat exposure on prostaglandin production and expression of COX-2, PGES, PGFS, ITGAV and LGALS15 mRNAs in endometrial epithelial cells of buffalo (Bubalus bubalis)

Effect of heat exposure on prostaglandin production and expression of COX-2, PGES, PGFS, ITGAV and LGALS15 mRNAs in endometrial epithelial cells of buffalo (Bubalus bubalis) | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of heat exposure on prostaglandin production and expression of COX-2, PGES, PGFS, ITGAV and LGALS15 mRNAs in endometrial epithelial cells of buffalo (Bubalus bubalis) S. Mondal, A. Mor, I. J. Reddy, S. Nandi, P. S. P. Gupta This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3763933/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Mar, 2024 Read the published version in Molecular Biology Reports → Version 1 posted 7 You are reading this latest preprint version Abstract The present study aimed to investigate the influence of heat stress on prostaglandin production and the expression of key genes, including COX-2, PGES, PGFS, ITGAV, and LGALS15, in buffalo endometrial epithelial cells. Buffalo genitalia, containing ovaries with corpus luteum (CL), were collected immediately post-slaughter and transported to the laboratory. The stages of estrous cycle were determined based on macroscopic observations of the ovaries, and uteri from the mid luteal phase (days 6–10 of the estrous cycle) were utilized. Uterine lumens were washed and treated with trypsin to isolate epithelial cells, which were then cultured at control temperature (38.5°C for 24 hr) or exposed to elevated temperatures (38.5°C for 6 hr, 40.5°C for 18 hr; HS). The supernatant and endometrial epithelial cells were collected at various time points (0, 3, 6, 12, and 24 hr) from both control and treatment group. Despite the elevated temperature (40.5°C) not affecting the in vitro production of PGF 2α and PGE 2 in epithelial cells, there was a significant (P < 0.05) increase in COX-2, PGES, and PGFS transcripts in response to heat stress. Interestingly, the expression of ITGAV and LGALS15 mRNAs in endometrial epithelial cells remained unaltered under elevated temperature conditions. In conclusion, this study suggests that while elevated temperature did not directly modulate prostaglandin production, it promoted the expression of COX-2, PGES, and PGFS mRNA in buffalo endometrial epithelial cells. These findings contribute to our understanding of the molecular responses of buffalo reproductive cells to heat stress, potentially informing strategies for mitigating the impact of environmental factors on reproductive outcomes in this species. Heat stress Endometrial epithelial cells Buffalo Figures Figure 1 Figure 2 Introduction Heat stress stands out as a prominent factor adversely affecting fertility in livestock [ 1 , 2 , 3 ]. The consequential early embryonic mortality significantly contributes to reproductive failures, leading to diminished pregnancy rates, slower genetic enhancement, and substantial economic setbacks for farmers [ 4 , 5 , 6 ]. Buffalo, a key dairy animal in Asia's developing nations, contributes significantly to milk production, draught power, and meat production, with reports indicating a noteworthy 20% embryonic mortality during early pregnancy [ 7 ]. The challenges of silent heat, delayed maturity, suboptimal estrus expression, irregular estrous cycles, breeding seasonality, anoestrus, low conception rates, extended postpartum intervals, repeated breeding failures, and early embryonic mortality pose substantial constraints to buffalo productivity and improvement through artificial breeding [ 8 , 9 , 10 , 11 ]. The major reason for embryonic mortality lies in the failure of cellular and molecular dialogues at embryo-uterine interface [ 12 ]. The successful establishment of pregnancy in mammals hinges on a synchronized dialogue between the uterus and terminally differentiated embryos, culminating in maternal recognition of pregnancy (MRP). Disruptions in this communication, arising from compromised conceptus development or abnormal uterine receptivity, contribute significantly to the elevated incidence of embryonic mortality. A comprehensive understanding of heat-induced alterations in endometrial function could pave the way for enhancing reproductive efficiency by mitigating early embryonic losses in buffalo. Early embryonic development, including fetal implantation and pregnancy maintenance, is critically influenced by embryo-maternal crosstalk, where the uterine environment plays a pivotal role in mammalian embryonic growth [ 13 ]. In bovines, prostaglandins (PGs) produced by endometrial cells play crucial roles in maternal recognition of pregnancy, implantation, and parturition [ 14 ]. The rate-limiting enzymes, prostaglandin synthases − 1 and − 2 (PTGS2 -1, -2 or COX-1, -2), govern PG production by converting arachidonic acid into PGH 2 . Downstream enzymes, such as PGE synthase (PGES) and PGF synthase (PGFS), catalyze the conversion of PGH 2 into PGE 2 and PGF 2α , respectively. Previous reports have highlighted an increase in uterine PGF 2α secretion following heat stress in vitro in cows' endometrial explants at Day 17 of pregnancy [ 15 ]. Severe hyperthermia has been linked to altered embryonic development, contributing to pregnancy failure due to disruptions in the uterine endometrium's secretory activity. Integrin Subunit V (ITGAV) and Galectin 15 (LGALS15) play crucial roles in cellular differentiation and adhesion between the blastocyst and the endometrial luminal epithelium [ 16 ]. Despite these critical roles, there is a notable absence of reports delineating the effects of heat stress on prostaglandin production and the expression of genes regulating MRP in buffalo. Therefore, this study was designed to investigate whether in vitro heat stress induces alterations in prostaglandin production and endometrial mRNA levels of COX-2, PGES, PGFS, ITGAV, and LGALS15 in buffalo. Material and Methods Isolation of endometrial epithelial cells Uteri from healthy buffaloes devoid of uterine infections were procured immediately post-slaughter from a local abattoir and transported to the laboratory under chilled conditions. The stages of estrous cycle were determined based on the color, vasculature, size, and consistency of corpus luteum in ovaries [ 17 , 18 , 19 ]. Epithelial cells from buffalo uteri during the mid-luteal phase (days 6–10 of the estrous cycle) were isolated using previously described methods [ 20 , 21 ]. In summary, sterile HBSS containing 0.3% trypsin was infused into the uterine lumen after initial washing with HBSS supplemented with gentamycin and 0.1% BSA. Epithelial cells were isolated by incubation at 37°C for 60 min. The resulting cell suspension underwent filtration to remove undissociated tissue fragments, followed by washing with HBSS supplemented with gentamycin and BSA. Culture of endometrial epithelial cells The viable epithelial cells (1 X 10 5 ) were cultured in RPMI 1640 medium at 38.5°C in the presence of 5% CO 2 for 24 hr. The viability of the plated epithelial cells exceeded 90%. Exposure of heat stress to epithelial cells in vitro Following the isolation of epithelial cells, cultures were maintained in RPMI 1640 medium at 38.5°C with 5% CO 2 . Control cultures were incubated at 38.5°C for 24 hr, while heat-stressed cultures were acclimated at 38.5°C for 6 hr and then subjected to 40.5°C for 18 hr. Culture medium and endometrial epithelial cells were collected at 0, 3, 6, 12, and 24 hr after the initiation of culture from both control and heat-stressed groups. Samples were stored at -70°C until use. ELISA of PGF 2α and PGE 2 The concentrations of PGF 2α and PGE 2 were determined in 50 µl aliquots of culture medium after 10 fold dilution with extraction buffer using ELISA kits supplied by Neogen, USA. Sensitivities of PGF 2α and PGE 2 assays were 0.002 and 0.1 ng/ml, respectively, with intra- and inter-assay coefficients of variation less than 19% for PGF 2α and less than 14% for PGE 2 . The cross reactivity of the antisera against 6-keto prostaglandin F 1α , 13, 14 dihydro-15 keto-prostaglandin F 2α , prostaglandin D 2 and prostaglandin E 2 were 3.05%, 0.05%, 0.05% and < 0.01%, respectively. The cross reactivity of the antisera against 6-keto prostaglandin E 1 , 13, 14 dihydro-15 keto-prostaglandinF 2α and prostaglandin D 2 were 0.91%, 0.01%, 0.01%, respectively. Isolation of RNA Total RNA from cultured cell pellets in both treatment and control groups was extracted using the RNeasy Minikit (M/s Qiagen, Germany), quantified with a nanodrop spectrophotometer (Eppendorf, Germany), and checked for integrity through formaldehyde-agarose gel electrophoresis (1%) with UV light visualization after ethidium bromide staining. RNA quantity was further determined using the NanoDrop spectrophotometer. Reverse transcription Polymerase Chain Reaction (RT-PCR) cDNA synthesis was performed using the BIORAD iScript™ cDNA synthesis kit. The reaction included iScript reverse transcriptase (1µl), 5X iScript reaction mix (4µl), nuclease-free water (14 µl), and RNA (100 ng, 1 µl), resulting in a total volume of 20 µl. The reaction underwent incubation at 25°C for 5 min, 42°C for 30 min, followed by 85°C for 5 min, with the cDNA stored at -20°C. Real time PCR The quantification of specific gene expression in endometrial epithelial cells was conducted using real-time PCR (qPCR) with the StepOnePlus qPCR system (Applied Biosystem, Carlsbad, CA, USA). Gene-specific primers for both housekeeping and target genes (Table 1 ) were utilized in this analysis. Primers were designed with the Primer 3 program [ 22 ] with an annealing temperature of 59 0 C. To ensure primer efficiency, a 10-fold serial dilution of cDNAs was employed [ 23 ], yielding primer efficiencies within the range of 95–105%. β-actin served as the reference gene for this study. Thermal cycling adhered to the manufacturer's protocol, involving an initial step at 95°C for 20 sec, followed by 40 cycles of denaturation at 95°C for 3 sec and annealing/extension at 60°C for 25 sec. A melt curve analysis was performed at 95°C (15 sec), 60°C (1 min), and 95°C (15 sec). The specificity of each PCR product was verified through melt curve analysis, and amplicon size was confirmed by agarose gel electrophoresis (2%). Negative controls, encompassing all qRT-PCR mix components except cDNA, were employed for all primers. Ct (threshold cycle for target amplification) values were analysed using 2 _ΔΔCt (normalized expression ratio) method to ascertain the relative level of expression of each mRNA. Table 1 Gene specific primers for both housekeeping and target genes Gene Prime r sequence (5’- 3’) Amplicon size PGES Forward: 5’TGGTCATCAAAATGTACGTGGT-3’ Reverse: 5’AACAGGAAGGGGTAGATGGTCT-3’ 184 bp PGFS Forward: 5’-GATGGCTACTTCATTCCTGTCC-3’ Reverse: 5’-TGGCCTATCTGCTCTTCATTTT-3’ 155 bp PTGS-2 Forward:5’-TCCTGAGTACTGGAAGCCTAGC-3’ Reverse: 5’-GATATCGTCTAGTCCCGAGTGG-3’ 202 bp LGALS15 Forward:5’-GTGAAGATCAAGGCAAACCTTC-3’ Reverse: 5’-TTTCTCTTCCTTCTGCCAACTC-3’ 171 bp ITGAV Forward: 5’-CACCAGCAATCAGAGATGGATA-3’ Reverse: 5’-ACTTGGACCATTGTTTCTCAGC-3’ 261 bp β-actin Forward:5’-AATTCCATCATGAAGTGTGACG-3’ Reverse: 5’-GATCTTGATCTTCATCGTGCTG-3’ 150 bp Statistical Analysis The experiment underwent replication six times on different days, and the results are presented as Mean ± S.E.M. values derived from separate experiments. Variations in PGF 2α , PGE 2 concentrations, and transcript abundance were assessed through ANOVA, followed by Tukey’s multiple comparison tests, utilizing the statistical package of GraphPad Prism 5 (San Diego, USA). A significance level of P < 0.05 was considered statistically significanton of each mRNA. Results The impact of elevated temperature on PGE 2 and PGF 2α production in buffalo endometrial epithelial cells during different culture time periods is illustrated in Figs. 1 A & 1 B. PGF 2α concentrations exhibited no significant variation between the control and heat-stressed groups. The concentration of PGF 2α did not exhibit a significant (P > 0.05) increase in epithelial cells incubated at 40.5°C for 12 hr, compared to those incubated at 38.5°C for the same duration. PGFS mRNA expression was significantly higher (P < 0.05) in endometrial epithelial cells exposed to 40.5°C at 12 hr, in comparison to cells exposed to both 38.5°C and 40.5°C at 6 hr (Fig. 2 A). PGE 2 levels demonstrated no significant variation between the control and heat-stressed groups. PGE 2 levels did not exhibit a significant (P > 0.05) increase at both 12 hr and 24 hr in epithelial cells exposed to 40.5°C, compared to their respective control groups. Significant differences in PGES mRNA expression were observed among the various treatment groups (Fig. 2 B). PGES mRNA levels were significantly (P < 0.05) higher in epithelial cells incubated at both 38.5°C and 40.5°C during 24 hr as compared to those incubated at both temperatures during 0, 3, 6, and 12 hr of culture. Elevated temperature (40.5°C) enhanced the expression of COX-2 mRNA during 24 hr of culture in comparison to cells exposed to 40.5°C at 6 hr and the control (0 hr). A significant (P < 0.05) increase in COX-2 expression was observed in epithelial cells exposed to 38.5°C during 24 hr, compared to those incubated at 38.5°C and 40.5°C at 6 hr and the control (0 hr) (Fig. 2 C). No significant differences (P > 0.05) were noted in LGAL15 (Fig. 2 D) and ITGAV (Fig. 2 E) mRNA expression among the various groups. Discussion Elevated temperatures pose a significant challenge to fertility in farm animals, particularly in tropical regions. Heat stress exerts a multifaceted impact on reproductive performance, encompassing diminished estrous behavior expression, altered ovarian follicular development, hormonal profile changes, compromised oocyte competence, and inhibited embryonic development. Some studies have indicated an increase in PGF 2α secretion, both in vivo and in vitro, during early pregnancy from the endometrium under heat stress conditions [ 24 , 25 ]. This study delves into the impact of in vitro heat stress on prostaglandin production in buffalo endometrial epithelial cells, while also examining alterations in transcript abundance of key genes associated with maternal recognition of pregnancy (MRP), namely PTGS-2, PGES, and PGFS. To our knowledge, this is the initial report documenting heat stress-induced changes in the transcript levels of MRP-related genes in buffalo. Contrary to previous findings [ 15 ], our study indicates that in vitro production of PGF 2α and PGE 2 was not affected by elevated temperatures in buffalo epithelial cells. In particular, PGFS mRNA expression levels significantly increased at 40.5°C after 12 hours of culture compared to exposure at 6 hours, and elevated temperatures notably boosted PGES mRNA transcript abundance at 24 hours. This aligns with an earlier study reporting that elevated temperatures did not alter PGE 2 production in isthmic epithelial cells of cattle oviduct but stimulated the expression of mPGES-1 mRNA [ 26 ]. The discrepancy may be attributed to distinct cell populations (ciliated and secretory) in the isthmus. Our results deviate from the findings of Kobayashi et al. [ 26 ], who observed increased production of PGE 2 in ampullary epithelial cells of cattle oviduct and elevated expression of mPGES1 and cPGES mRNAs under heat stress. Similarly, while PGF 2α concentration did not significantly increase at 40.5°C in our study, contrary reports [ 27 , 28 ] observed increased release of PGF 2α following in vitro heat stress. The disparity might be due to alterations in endometrial membranes [ 27 , 29 ] or variations in the regulatory capacity of endometrial metabolic activity at higher temperatures [ 15 ]. Our findings reveal an elevated expression of COX-2 transcript at 40.5°C in buffalo endometrial epithelial cells compared to the control, aligning with studies associating excessive COX-2 mRNA expression with conditions such as uterine ectopic, inflammation, and tumorigenesis that impede embryonic implantation [ 30 ]. This parallels observations of heat stress-induced upregulation of COX-2 mRNA expression in human cells [ 31 ]. Intriguingly, the expression of ITGAV and LGALS 15 mRNAs in endometrial epithelial cells remained unaffected by elevated temperature, with no prior reports on their expression profiling in buffalo, sheep, or goat endometrium under heat stress. This study pioneers the documentation of heat stress-induced alterations in COX-2, PGES, PGFS, ITGAV, and LGALS15 mRNAs in buffalo endometrial epithelial cells. Conclusion In summary, the results from this study indicate that in vitro heat stress did not exert an impact on PGF 2α and PGE 2 production in buffalo endometrial epithelial cells. However, it did stimulate the expression of COX-2, PGES, and PGFS mRNAs. Declarations Acknowledgements We extend our gratitude to the Director of NIANP for providing the essential facilities for conducting this research. Special thanks are directed to Shiv Tripathi for his assistance in graph preparation, and the contributions of A. Jagannath are duly recognized. This work received support from the National Fund for Basic, Strategic, and Frontier Application Research in Agriculture, Ministry of Agriculture, ICAR, New Delhi, under Project No. NASF AS2002. Funding The authors express their appreciation to the National Fund for Basic, Strategic, and Frontier Application Research in Agriculture (Grant No. AS2002), Ministry of Agriculture, ICAR, New Delhi, for the financial backing that enabled the execution of this work. However, all authors affirm that they have no affiliations or involvement with any organization or entity that has financial or non-financial interests in the subject matter or materials discussed in this manuscript. Competing interests The authors declare that they have no competing interests. Ethical approval This study was performed in line with the principles of the Declaration of Helsinki. This work was approved by National Fund for Basic, Strategic and Frontier Application Research in Agriculture, ICAR, Ministry of Agriculture, New Delhi and Institutional Animal Ethics Committee (IAEC), ICAR-National Institute of Animal Nutrition and Physiology, Bangalore, Karnataka, India. Consent to participate and publish All the authors have read, approved the final manuscript, and given their consent to participate in this report and submit it to the journal. Data availability All data generated or analyzed during this study are included in this published manuscript. References Palta P, Mondal S, Prakash BS, Madan ML (1997) Peripheral inhibin levels in relation to climatic variations and stage of estrous cycle in buffaloes (Bubalus bubalis). Theriogenol 47:989–995 Hansen PJ (2009) Effects of heat stress on mammalian reproduction. 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Cite Share Download PDF Status: Published Journal Publication published 08 Mar, 2024 Read the published version in Molecular Biology Reports → Version 1 posted Editorial decision: Revision requested 02 Jan, 2024 Reviews received at journal 31 Dec, 2023 Reviewers agreed at journal 21 Dec, 2023 Reviewers invited by journal 21 Dec, 2023 Submission checks completed at journal 20 Dec, 2023 Editor assigned by journal 20 Dec, 2023 First submitted to journal 16 Dec, 2023 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 In Review Editorial Policies 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-3763933","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":261553015,"identity":"1f6aefda-479a-4bbe-887a-7bc64555486a","order_by":0,"name":"S. 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P.","lastName":"Gupta","suffix":""}],"badges":[],"createdAt":"2023-12-16 15:44:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3763933/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3763933/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-024-09361-4","type":"published","date":"2024-03-08T15:01:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":48608701,"identity":"93fbae2c-a843-4af9-bb00-d41eeeacd164","added_by":"auto","created_at":"2023-12-21 13:06:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":85681,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of elevated temperature on the productions of PGF\u003csub\u003e2α\u003c/sub\u003e (A) and PGE\u003csub\u003e2\u003c/sub\u003e (B) in buffalo endometrial epithelial cells during different time period of culture\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3763933/v1/bee91e9795fd6d752e54bb5a.png"},{"id":48608700,"identity":"2b046550-7005-45f6-9993-3830ab24820c","added_by":"auto","created_at":"2023-12-21 13:06:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":165210,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u0026amp;B \u003c/strong\u003eEffect of elevated temperature on the expressions of PGFS (A) and PGES (B) mRNAs in buffalo endometrial epithelial cells during different time period of culture. Different superscript letters indicate a significant difference (P\u0026lt;0.05; ANOVA)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u0026amp; D \u003c/strong\u003eEffect of elevated temperature on the expressions of PTGS-2 (C) and LGALS 15 (D) mRNAs in buffalo endometrial epithelial cells during different time period of culture. Different superscript letters indicate a significant difference (P\u0026lt;0.05; ANOVA)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE \u003c/strong\u003eEffect of elevated temperature on the expressions of ITGAV (E) mRNAs in buffalo endometrial epithelial cells during different time period of culture\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3763933/v1/736140db9df126d033917570.png"},{"id":52431925,"identity":"c75809a0-b8b4-489a-aeee-b71de8d03050","added_by":"auto","created_at":"2024-03-11 15:09:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":507789,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3763933/v1/97118024-7e70-4e69-ac20-325e09afae2d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of heat exposure on prostaglandin production and expression of COX-2, PGES, PGFS, ITGAV and LGALS15 mRNAs in endometrial epithelial cells of buffalo (Bubalus bubalis)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHeat stress stands out as a prominent factor adversely affecting fertility in livestock [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The consequential early embryonic mortality significantly contributes to reproductive failures, leading to diminished pregnancy rates, slower genetic enhancement, and substantial economic setbacks for farmers [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Buffalo, a key dairy animal in Asia's developing nations, contributes significantly to milk production, draught power, and meat production, with reports indicating a noteworthy 20% embryonic mortality during early pregnancy [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The challenges of silent heat, delayed maturity, suboptimal estrus expression, irregular estrous cycles, breeding seasonality, anoestrus, low conception rates, extended postpartum intervals, repeated breeding failures, and early embryonic mortality pose substantial constraints to buffalo productivity and improvement through artificial breeding [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The major reason for embryonic mortality lies in the failure of cellular and molecular dialogues at embryo-uterine interface [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The successful establishment of pregnancy in mammals hinges on a synchronized dialogue between the uterus and terminally differentiated embryos, culminating in maternal recognition of pregnancy (MRP). Disruptions in this communication, arising from compromised conceptus development or abnormal uterine receptivity, contribute significantly to the elevated incidence of embryonic mortality. A comprehensive understanding of heat-induced alterations in endometrial function could pave the way for enhancing reproductive efficiency by mitigating early embryonic losses in buffalo.\u003c/p\u003e \u003cp\u003eEarly embryonic development, including fetal implantation and pregnancy maintenance, is critically influenced by embryo-maternal crosstalk, where the uterine environment plays a pivotal role in mammalian embryonic growth [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In bovines, prostaglandins (PGs) produced by endometrial cells play crucial roles in maternal recognition of pregnancy, implantation, and parturition [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The rate-limiting enzymes, prostaglandin synthases \u0026minus;\u0026thinsp;1 and \u0026minus;\u0026thinsp;2 (PTGS2 -1, -2 or COX-1, -2), govern PG production by converting arachidonic acid into PGH\u003csub\u003e2\u003c/sub\u003e. Downstream enzymes, such as PGE synthase (PGES) and PGF synthase (PGFS), catalyze the conversion of PGH\u003csub\u003e2\u003c/sub\u003e into PGE\u003csub\u003e2\u003c/sub\u003e and PGF\u003csub\u003e2α\u003c/sub\u003e, respectively. Previous reports have highlighted an increase in uterine PGF\u003csub\u003e2α\u003c/sub\u003e secretion following heat stress in vitro in cows' endometrial explants at Day 17 of pregnancy [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Severe hyperthermia has been linked to altered embryonic development, contributing to pregnancy failure due to disruptions in the uterine endometrium's secretory activity. Integrin Subunit V (ITGAV) and Galectin 15 (LGALS15) play crucial roles in cellular differentiation and adhesion between the blastocyst and the endometrial luminal epithelium [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Despite these critical roles, there is a notable absence of reports delineating the effects of heat stress on prostaglandin production and the expression of genes regulating MRP in buffalo. Therefore, this study was designed to investigate whether in vitro heat stress induces alterations in prostaglandin production and endometrial mRNA levels of COX-2, PGES, PGFS, ITGAV, and LGALS15 in buffalo.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of endometrial epithelial cells\u003c/h2\u003e \u003cp\u003eUteri from healthy buffaloes devoid of uterine infections were procured immediately post-slaughter from a local abattoir and transported to the laboratory under chilled conditions. The stages of estrous cycle were determined based on the color, vasculature, size, and consistency of corpus luteum in ovaries [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Epithelial cells from buffalo uteri during the mid-luteal phase (days 6\u0026ndash;10 of the estrous cycle) were isolated using previously described methods [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In summary, sterile HBSS containing 0.3% trypsin was infused into the uterine lumen after initial washing with HBSS supplemented with gentamycin and 0.1% BSA. Epithelial cells were isolated by incubation at 37\u0026deg;C for 60 min. The resulting cell suspension underwent filtration to remove undissociated tissue fragments, followed by washing with HBSS supplemented with gentamycin and BSA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCulture of endometrial epithelial cells\u003c/h2\u003e \u003cp\u003eThe viable epithelial cells (1 X 10\u003csup\u003e5\u003c/sup\u003e) were cultured in RPMI 1640 medium at 38.5\u0026deg;C in the presence of 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 hr. The viability of the plated epithelial cells exceeded 90%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eExposure of heat stress to epithelial cells in vitro\u003c/h2\u003e \u003cp\u003eFollowing the isolation of epithelial cells, cultures were maintained in RPMI 1640 medium at 38.5\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Control cultures were incubated at 38.5\u0026deg;C for 24 hr, while heat-stressed cultures were acclimated at 38.5\u0026deg;C for 6 hr and then subjected to 40.5\u0026deg;C for 18 hr. Culture medium and endometrial epithelial cells were collected at 0, 3, 6, 12, and 24 hr after the initiation of culture from both control and heat-stressed groups. Samples were stored at -70\u0026deg;C until use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eELISA of PGF\u003csub\u003e2α\u003c/sub\u003e and PGE\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eThe concentrations of PGF\u003csub\u003e2α\u003c/sub\u003e and PGE\u003csub\u003e2\u003c/sub\u003e were determined in 50 \u0026micro;l aliquots of culture medium after 10 fold dilution with extraction buffer using ELISA kits supplied by Neogen, USA. Sensitivities of PGF\u003csub\u003e2α\u003c/sub\u003e and PGE\u003csub\u003e2\u003c/sub\u003e assays were 0.002 and 0.1 ng/ml, respectively, with intra- and inter-assay coefficients of variation less than 19% for PGF\u003csub\u003e2α\u003c/sub\u003e and less than 14% for PGE\u003csub\u003e2\u003c/sub\u003e. The cross reactivity of the antisera against 6-keto prostaglandin F\u003csub\u003e1α\u003c/sub\u003e, 13, 14 dihydro-15 keto-prostaglandin F\u003csub\u003e2α\u003c/sub\u003e, prostaglandin D\u003csub\u003e2\u003c/sub\u003e and prostaglandin E\u003csub\u003e2\u003c/sub\u003e were 3.05%, 0.05%, 0.05% and \u0026lt;\u0026thinsp;0.01%, respectively. The cross reactivity of the antisera against 6-keto prostaglandin E\u003csub\u003e1\u003c/sub\u003e, 13, 14 dihydro-15 keto-prostaglandinF\u003csub\u003e2α\u003c/sub\u003e and prostaglandin D\u003csub\u003e2\u003c/sub\u003e were 0.91%, 0.01%, 0.01%, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of RNA\u003c/h2\u003e \u003cp\u003eTotal RNA from cultured cell pellets in both treatment and control groups was extracted using the RNeasy Minikit (M/s Qiagen, Germany), quantified with a nanodrop spectrophotometer (Eppendorf, Germany), and checked for integrity through formaldehyde-agarose gel electrophoresis (1%) with UV light visualization after ethidium bromide staining. RNA quantity was further determined using the NanoDrop spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eReverse transcription Polymerase Chain Reaction (RT-PCR)\u003c/h2\u003e \u003cp\u003ecDNA synthesis was performed using the BIORAD iScript\u0026trade; cDNA synthesis kit. The reaction included iScript reverse transcriptase (1\u0026micro;l), 5X iScript reaction mix (4\u0026micro;l), nuclease-free water (14 \u0026micro;l), and RNA (100 ng, 1 \u0026micro;l), resulting in a total volume of 20 \u0026micro;l. The reaction underwent incubation at 25\u0026deg;C for 5 min, 42\u0026deg;C for 30 min, followed by 85\u0026deg;C for 5 min, with the cDNA stored at -20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eReal time PCR\u003c/h2\u003e \u003cp\u003eThe quantification of specific gene expression in endometrial epithelial cells was conducted using real-time PCR (qPCR) with the StepOnePlus qPCR system (Applied Biosystem, Carlsbad, CA, USA). Gene-specific primers for both housekeeping and target genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were utilized in this analysis. Primers were designed with the Primer 3 program [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] with an annealing temperature of 59\u003csup\u003e0\u003c/sup\u003eC. To ensure primer efficiency, a 10-fold serial dilution of cDNAs was employed [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], yielding primer efficiencies within the range of 95\u0026ndash;105%. β-actin served as the reference gene for this study. Thermal cycling adhered to the manufacturer's protocol, involving an initial step at 95\u0026deg;C for 20 sec, followed by 40 cycles of denaturation at 95\u0026deg;C for 3 sec and annealing/extension at 60\u0026deg;C for 25 sec. A melt curve analysis was performed at 95\u0026deg;C (15 sec), 60\u0026deg;C (1 min), and 95\u0026deg;C (15 sec). The specificity of each PCR product was verified through melt curve analysis, and amplicon size was confirmed by agarose gel electrophoresis (2%). Negative controls, encompassing all qRT-PCR mix components except cDNA, were employed for all primers. Ct (threshold cycle for target amplification) values were analysed using 2\u003csup\u003e_ΔΔCt\u003c/sup\u003e (normalized expression ratio) method to ascertain the relative level of expression of each mRNA.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGene specific primers for both housekeeping and target genes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrime\u003c/p\u003e \u003cp\u003er sequence (5\u0026rsquo;- 3\u0026rsquo;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmplicon size\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePGES\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward: 5\u0026rsquo;TGGTCATCAAAATGTACGTGGT-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse: 5\u0026rsquo;AACAGGAAGGGGTAGATGGTCT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e184 bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePGFS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward: 5\u0026rsquo;-GATGGCTACTTCATTCCTGTCC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse: 5\u0026rsquo;-TGGCCTATCTGCTCTTCATTTT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e155 bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePTGS-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward:5\u0026rsquo;-TCCTGAGTACTGGAAGCCTAGC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse: 5\u0026rsquo;-GATATCGTCTAGTCCCGAGTGG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e202 bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLGALS15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward:5\u0026rsquo;-GTGAAGATCAAGGCAAACCTTC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse: 5\u0026rsquo;-TTTCTCTTCCTTCTGCCAACTC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e171 bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eITGAV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward: 5\u0026rsquo;-CACCAGCAATCAGAGATGGATA-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse: 5\u0026rsquo;-ACTTGGACCATTGTTTCTCAGC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e261 bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward:5\u0026rsquo;-AATTCCATCATGAAGTGTGACG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003eReverse: 5\u0026rsquo;-GATCTTGATCTTCATCGTGCTG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e150 bp\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe experiment underwent replication six times on different days, and the results are presented as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. values derived from separate experiments. Variations in PGF\u003csub\u003e2α\u003c/sub\u003e, PGE\u003csub\u003e2\u003c/sub\u003e concentrations, and transcript abundance were assessed through ANOVA, followed by Tukey\u0026rsquo;s multiple comparison tests, utilizing the statistical package of GraphPad Prism 5 (San Diego, USA). A significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significanton of each mRNA.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe impact of elevated temperature on PGE\u003csub\u003e2\u003c/sub\u003e and PGF\u003csub\u003e2α\u003c/sub\u003e production in buffalo endometrial epithelial cells during different culture time periods is illustrated in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. PGF\u003csub\u003e2α\u003c/sub\u003e concentrations exhibited no significant variation between the control and heat-stressed groups. The concentration of PGF\u003csub\u003e2α\u003c/sub\u003e did not exhibit a significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) increase in epithelial cells incubated at 40.5\u0026deg;C for 12 hr, compared to those incubated at 38.5\u0026deg;C for the same duration. PGFS mRNA expression was significantly higher (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in endometrial epithelial cells exposed to 40.5\u0026deg;C at 12 hr, in comparison to cells exposed to both 38.5\u0026deg;C and 40.5\u0026deg;C at 6 hr (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). PGE\u003csub\u003e2\u003c/sub\u003e levels demonstrated no significant variation between the control and heat-stressed groups. PGE\u003csub\u003e2\u003c/sub\u003e levels did not exhibit a significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) increase at both 12 hr and 24 hr in epithelial cells exposed to 40.5\u0026deg;C, compared to their respective control groups. Significant differences in PGES mRNA expression were observed among the various treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). PGES mRNA levels were significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) higher in epithelial cells incubated at both 38.5\u0026deg;C and 40.5\u0026deg;C during 24 hr as compared to those incubated at both temperatures during 0, 3, 6, and 12 hr of culture. Elevated temperature (40.5\u0026deg;C) enhanced the expression of COX-2 mRNA during 24 hr of culture in comparison to cells exposed to 40.5\u0026deg;C at 6 hr and the control (0 hr). A significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase in COX-2 expression was observed in epithelial cells exposed to 38.5\u0026deg;C during 24 hr, compared to those incubated at 38.5\u0026deg;C and 40.5\u0026deg;C at 6 hr and the control (0 hr) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). No significant differences (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) were noted in LGAL15 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and ITGAV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) mRNA expression among the various groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eElevated temperatures pose a significant challenge to fertility in farm animals, particularly in tropical regions. Heat stress exerts a multifaceted impact on reproductive performance, encompassing diminished estrous behavior expression, altered ovarian follicular development, hormonal profile changes, compromised oocyte competence, and inhibited embryonic development. Some studies have indicated an increase in PGF\u003csub\u003e2α\u003c/sub\u003e secretion, both in vivo and in vitro, during early pregnancy from the endometrium under heat stress conditions [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This study delves into the impact of in vitro heat stress on prostaglandin production in buffalo endometrial epithelial cells, while also examining alterations in transcript abundance of key genes associated with maternal recognition of pregnancy (MRP), namely PTGS-2, PGES, and PGFS. To our knowledge, this is the initial report documenting heat stress-induced changes in the transcript levels of MRP-related genes in buffalo. Contrary to previous findings [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], our study indicates that in vitro production of PGF\u003csub\u003e2α\u003c/sub\u003e and PGE\u003csub\u003e2\u003c/sub\u003e was not affected by elevated temperatures in buffalo epithelial cells. In particular, PGFS mRNA expression levels significantly increased at 40.5\u0026deg;C after 12 hours of culture compared to exposure at 6 hours, and elevated temperatures notably boosted PGES mRNA transcript abundance at 24 hours. This aligns with an earlier study reporting that elevated temperatures did not alter PGE\u003csub\u003e2\u003c/sub\u003e production in isthmic epithelial cells of cattle oviduct but stimulated the expression of mPGES-1 mRNA [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The discrepancy may be attributed to distinct cell populations (ciliated and secretory) in the isthmus. Our results deviate from the findings of Kobayashi et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], who observed increased production of PGE\u003csub\u003e2\u003c/sub\u003e in ampullary epithelial cells of cattle oviduct and elevated expression of mPGES1 and cPGES mRNAs under heat stress. Similarly, while PGF\u003csub\u003e2α\u003c/sub\u003e concentration did not significantly increase at 40.5\u0026deg;C in our study, contrary reports [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] observed increased release of PGF\u003csub\u003e2α\u003c/sub\u003e following in vitro heat stress. The disparity might be due to alterations in endometrial membranes [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] or variations in the regulatory capacity of endometrial metabolic activity at higher temperatures [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur findings reveal an elevated expression of COX-2 transcript at 40.5\u0026deg;C in buffalo endometrial epithelial cells compared to the control, aligning with studies associating excessive COX-2 mRNA expression with conditions such as uterine ectopic, inflammation, and tumorigenesis that impede embryonic implantation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This parallels observations of heat stress-induced upregulation of COX-2 mRNA expression in human cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Intriguingly, the expression of ITGAV and LGALS 15 mRNAs in endometrial epithelial cells remained unaffected by elevated temperature, with no prior reports on their expression profiling in buffalo, sheep, or goat endometrium under heat stress. This study pioneers the documentation of heat stress-induced alterations in COX-2, PGES, PGFS, ITGAV, and LGALS15 mRNAs in buffalo endometrial epithelial cells.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, the results from this study indicate that in vitro heat stress did not exert an impact on PGF\u003csub\u003e2α\u003c/sub\u003e and PGE\u003csub\u003e2\u003c/sub\u003e production in buffalo endometrial epithelial cells. However, it did stimulate the expression of COX-2, PGES, and PGFS mRNAs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our gratitude to the Director of NIANP for providing the essential facilities for conducting this research. Special thanks are directed to Shiv Tripathi for his assistance in graph preparation, and the contributions of A. Jagannath are duly recognized. This work received support from the National Fund for Basic, Strategic, and Frontier Application Research in Agriculture, Ministry of Agriculture, ICAR, New Delhi, under Project No. NASF AS2002.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their appreciation to the National Fund for Basic, Strategic, and Frontier Application Research in Agriculture (Grant No. AS2002), Ministry of Agriculture, ICAR, New Delhi, for the financial backing that enabled the execution of this work. However, all authors affirm that they have no affiliations or involvement with any organization or entity that has financial or non-financial interests in the subject matter or materials discussed in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. This work was approved by National Fund for Basic, Strategic and Frontier Application Research in Agriculture, ICAR, Ministry of Agriculture, New Delhi and Institutional Animal Ethics Committee (IAEC), ICAR-National Institute of Animal Nutrition and Physiology, Bangalore, Karnataka, India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate and publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors have read, approved the final manuscript, and given their consent to participate in this report and submit it to the journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePalta P, Mondal S, Prakash BS, Madan ML (1997) Peripheral inhibin levels in relation to climatic variations and stage of estrous cycle in buffaloes (Bubalus bubalis). Theriogenol 47:989\u0026ndash;995\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHansen PJ (2009) Effects of heat stress on mammalian reproduction. Philosophical Trans Royal Soc Lond B Biology Soc 364:3341\u0026ndash;3350\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMondal S, Mor A, Reddy IJ, Nandi S (2017) Impact of in vitro heat shock (42.5\u003csup\u003e0\u003c/sup\u003eC) on prostaglandins, ionic and metabolic contents in sheep endometrial epithelial cells. Curr Trends Biomed Eng Biosci 3:CTBEB.MS.ID.555604\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMondal S, Prakash BS (2002) Comparison of luteal function between cows and buffaloes during estrous cycle. Ind J Dairy Sci 55:142\u0026ndash;144\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMondal S, Mor A, Reddy IJ, Nandi S, Gupta PSP (2015) Effect of Fibroblast Growth Factor 2 (FGF2) and Insulin Transferrin Selenium (ITS) on In Vitro Maturation, Fertilization and Embryo Development in Sheep. Braz Arch Biol Technol 58:521\u0026ndash;525\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMor A, Mondal S, Reddy IJ, Nandi S, Gupta PSP (2018) Molecular cloning and expression of FGF2 gene in pre-implantation developmental stages of in vitro produced sheep embryos. Reprod Domest Anim 53:895\u0026ndash;903\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVale WG, Ohasi OM, Sousa JS, Ribeiro HFL, Silva AOA, Nanba SY (1989) Morte embrionaria e fatal em bufalos, Bubalus bubalus Lin. 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Asian Australas J Anim Sci 23:169\u0026ndash;172\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMondal S, Mor A, Reddy IJ, Soumya NP (2015) Genes Regulating Maternal Recognition of Pregnancy in Domestic Animals: an Update. Braz Arch Biol Technol 58:854\u0026ndash;863\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarrett JE, Geisert RI, Zavy MT, Morgan GL (1988) Evidence for maternal regulation of early conceptus growth and development in beef cattle. J Reprod Fertil 84:437\u0026ndash;446\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoyser NL (1995) The control of prostaglandin production by the endometrium in relation to luteolysis and menstruation. 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J Dairy Sci 63:155\u0026ndash;160\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMondal S, Kumar V, Reddy IJ, Singh K (2004) Progesterone and nucleic acid contents of buffalo corpus luteum in relation to stages of estrous cycle. Ind J Dairy Sci 74:710\u0026ndash;712\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhosh J, Mondal S (2006) Nucleic acids and protein content in relation to growth and regression of buffalo corpora lutea. Anim Reprod Sci 93:316\u0026ndash;327\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMondal S, Nandi S, Reddy IJ, Suresh KP (2009) Isolation, culture and characterization of endometrial epithelial cells in buffalo (\u003cem\u003eBubalus bubalis\u003c/em\u003e). Buff Bull 28:101\u0026ndash;106\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNandi S, Mondal S, Reddy IJ (2012) Effect of prostaglandin producing modulators on in vitro growth characteristics in buffalo endometrial epithelial cells. Theriogenol 77:1014\u0026ndash;1020\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. In: Kravetz S, Minsener S (eds) Methods in Molecular Biology. Humana Press, Totown, NJ, pp 365\u0026ndash;386\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSvec D, Tichopad A, Novosadova V, Pfaffl MW, Kubista M (2015) How good is a PCR efficiency estimate: recommendations for precise and robust qPCR efficiency assessments. Biomol Detect Quantif 3:9\u0026ndash;16\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoagland TA, Wettemann RP (1984) Influence of elevated ambient temperature after breeding on plasma corticoids, estradiol and progesterone in gilts. 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Anim Reprod Sci 7:149\u0026ndash;176\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalayer JR, Hansen PJ, Gross TS, Thatcher WW (1990) Regulation of heat shock-induced alterations in the release of prostaglandins by the uterine endometrium of cows. Theriogenol 34:219\u0026ndash;230\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlint APF, Leat WMF, Sheldrick EL, Stewart HJ (1986) Stimulation of phosphoinositide hydrolysis by oxytocin and the mechanism by which oxytocin controls prostaglandin synthesis in the ovine endometrium. Biochem J 237:797\u0026ndash;805\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Q (2011) Cervical cancer and cervical intraepithelial neoplasia tissue expression of mPGES-1 and COX-2 and its correlation analysis. Prog Obstet Gyneco 20:358\u0026ndash;360\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRossi A, Coccia M, Trotta E, Angelini M, Santoro MG (2012) Regulation of Cyclooxygenase-2 Expression by Heat: A Novel Aspect of Heat Shock Factor 1 Function in Human Cells. PLoS ONE 7:e31304\u003c/span\u003e\u003c/li\u003e\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":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Heat stress, Endometrial epithelial cells, Buffalo","lastPublishedDoi":"10.21203/rs.3.rs-3763933/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3763933/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present study aimed to investigate the influence of heat stress on prostaglandin production and the expression of key genes, including COX-2, PGES, PGFS, ITGAV, and LGALS15, in buffalo endometrial epithelial cells. Buffalo genitalia, containing ovaries with corpus luteum (CL), were collected immediately post-slaughter and transported to the laboratory. The stages of estrous cycle were determined based on macroscopic observations of the ovaries, and uteri from the mid luteal phase (days 6\u0026ndash;10 of the estrous cycle) were utilized. Uterine lumens were washed and treated with trypsin to isolate epithelial cells, which were then cultured at control temperature (38.5\u0026deg;C for 24 hr) or exposed to elevated temperatures (38.5\u0026deg;C for 6 hr, 40.5\u0026deg;C for 18 hr; HS). The supernatant and endometrial epithelial cells were collected at various time points (0, 3, 6, 12, and 24 hr) from both control and treatment group. Despite the elevated temperature (40.5\u0026deg;C) not affecting the in vitro production of PGF\u003csub\u003e2α\u003c/sub\u003e and PGE\u003csub\u003e2\u003c/sub\u003e in epithelial cells, there was a significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase in COX-2, PGES, and PGFS transcripts in response to heat stress. Interestingly, the expression of ITGAV and LGALS15 mRNAs in endometrial epithelial cells remained unaltered under elevated temperature conditions. In conclusion, this study suggests that while elevated temperature did not directly modulate prostaglandin production, it promoted the expression of COX-2, PGES, and PGFS mRNA in buffalo endometrial epithelial cells. These findings contribute to our understanding of the molecular responses of buffalo reproductive cells to heat stress, potentially informing strategies for mitigating the impact of environmental factors on reproductive outcomes in this species.\u003c/p\u003e","manuscriptTitle":"Effect of heat exposure on prostaglandin production and expression of COX-2, PGES, PGFS, ITGAV and LGALS15 mRNAs in endometrial epithelial cells of buffalo (Bubalus bubalis)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-21 13:06:51","doi":"10.21203/rs.3.rs-3763933/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-01-02T12:30:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-12-31T07:00:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"abe5d074-48bb-40e0-9b60-83ee53fb2ae8","date":"2023-12-21T11:43:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-12-21T11:22:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-12-20T07:31:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-12-20T07:31:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2023-12-16T15:32:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fe78c70b-3220-482f-be54-9e229db2d010","owner":[],"postedDate":"December 21st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-03-11T15:05:07+00:00","versionOfRecord":{"articleIdentity":"rs-3763933","link":"https://doi.org/10.1007/s11033-024-09361-4","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2024-03-08 15:01:27","publishedOnDateReadable":"March 8th, 2024"},"versionCreatedAt":"2023-12-21 13:06:51","video":"","vorDoi":"10.1007/s11033-024-09361-4","vorDoiUrl":"https://doi.org/10.1007/s11033-024-09361-4","workflowStages":[]},"version":"v1","identity":"rs-3763933","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3763933","identity":"rs-3763933","version":["v1"]},"buildId":"i_I_jhV-QviBjPLXJga9V","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}