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Polycystic ovarian syndrome (PCOS) is the main cause of infertility in women. It is frequently associated with reduced progesterone production by human luteinised granulosa cells (hlGCs). However, the molecular mechanisms involved in these steroidogenesis alterations in PCOS patients are unclear. In a dihydrotestosterone-induced PCOS mouse model, steroid production is maintained in the setting of chemokine-like receptor 1 (Cmklr1) knockout. Thus, chemerin and chemerin receptors in terms of expression and progesterone regulation could be different in control and PCOS hlGCs. We first confirmed that progesterone levels in both plasma (P < 0.0001) and follicular fluid (FF) (P < 0.0001) were significantly reduced in PCOS normal weight women compared to control women. These data were associated with a lower STAR mRNA expression in both in vivo (P < 0.0001) and in vitro (P < 0.0001) hlGCs from PCOS women. Secondly, chemerin FF levels (P < 0.0001) and RARRES2 (P < 0.05) and CMKLR1 (P < 0.0001) mRNA levels in GCs were higher in PCOS normal weight patients. Thirdly, treatment of hlGCs with a specific nanobody (the VHH CA4910) targeting the human receptor for CMKLR1 leading to its inactivation abolished chemerin-induced progesterone inhibition, suggesting the involvement of CMKLR1 in this process. Furthermore, the inhibition of progesterone secretion induced by chemerin was two-fold higher in PCOS hlGCs (P < 0.05). Moreover, the VHH CA4910 reinstated a normal progesterone secretion with lower concentrations in PCOS hlGCs, suggesting a different chemerin sensitivity between PCOS and control hlGCs. Thus, chemerin, through CMKLR1, could be involved in the steroidogenesis alterations in PCOS hlGCs.

Inhibin subunit beta A (INHBA) participates in the synthesis of inhibin A, activin A and activin AB. Here we investigated the effect and molecular mechanism of INHBA on proliferation, apoptosis and hormone synthesis in sheep granulosa cells (GCs) using in vitro transfection. We first noticed that INHBA expression increased with follicle diameter and was widely distributed in ovarian tissue. The proliferation rate of GCs was significantly increased and decreased with overexpression and silence of INHBA, respectively, compared with the negative controls. INHBA transfection affected GC proliferation and apoptosis, regulating the expression of many cell cycle-related and apoptosis-related genes. INHBA overexpression significantly decreased activin and estradiol secretion while increasing inhibin and progesterone secretion. The expression of follicle-stimulating hormone beta subunit was significantly decreased and increased with INHBA overexpression and knockdown, respectively. Notably, silence of INHBA inhibited the expression of many transforming growth factor beta-related genes. Overall, the functional molecule of INHBA gene may be associated with follicular development via regulating proliferation, apoptosis and folliculogenesis-related hormone secretion of sheep GCs. In addition, our findings may contribute to a better understanding of the law of follicular development and thus improve the reproductive performance of female animals.

In the present study, we investigated the effect of the synthetic analog of prostaglandin F2α (PGF2α)—cloprostenol—on cultured steroidogenic luteal cells of selected felid species over a 2-day culture period. The changes induced by cloprostenol were measured based on progesterone concentration and mRNA expression analysis of selected genes. Cloprostenol significantly reduced concentration of progesterone in cell culture medium of small luteal cells isolated from domestic cat corpora lutea (CL) at the development/maintenance stage (P < 0.05), but did not influence progesterone production in cultured cells from the regression stage. A decrease or complete silencing of progesterone production was also measured in cultured luteal cells of African lion (formation stage) and Javan leopard (development/maintenance stage). Gene-expression analysis by real-time PCR revealed that treatment with cloprostenol did not have an influence on expression of selected genes coding for enzymes of steroidogenesis (StAR, HSD3B, CYP11A1) or prostaglandin synthesis (PTGS2, PGES), nor did it effect hormone receptors (AR, ESR1, PGR, PTGER2), an antioxidative enzyme (SOD1) or factors of cell apoptosis (FAS, CASP3, TNFRSF1B, BCL2) over the studied period. Significant changes were measured only for expressions of luteinizing hormone (P < 0.05), prolactin (P < 0.05) and PGF2α receptors (P < 0.005) (LHCGR, PRLR, and PTGFR). The obtained results confirm that PGF2α/cloprostenol is a luteolytic agent in CL of felids and its impact on progesterone production depends on the developmental stage of the CL. Cloprostenol short-term treatment on luteal cells was associated only with functional but not structural changes related to luteal regression.Summary sentenceIn cultured luteal cells of felids, cloprostenol can cause functional regression by reducing progesterone secretion and down-regulating expression of luteinizing hormone receptor, prolactin receptor, and prostaglandin F2α receptor.

It is well known that corpus luteum normal functioning is crucial for the luteal phase, which determines the embryo implantation and the progression of pregnancy. Luteal phase deficiency (LPD), associated with impaired progesterone secretion by the corpus luteum, is considered as a significant factor of infertility and early pregnancy loss, both in the natural cycle and in assisted reproductive technology (ART) programs. The LPD formation is associated with hypothalamic-pituitary-ovarian axis dysregulation, which leads to abnormal secretion of FSH, LH, ovulation and luteinization disorders, premature luteolysis. The most significant problem in the study of LPD is the lack of reliable and reproducible methods of its verification. This review summarizes the available data on the methods and issues of LPD diagnosing, including the duration of the luteal phase, the level of progesterone secretion, and endometrial biopsy. LPD is an important factor in reproductive failures during IVF, which is caused by suppression of the physiological FSH, LH secretion and requires mandatory progesterone support in the luteal phase of the cycle. It’s hard to define the contribution of LPD to miscarriage, however, empirical progestogen therapy may increase the live births rate in women with recurrent pregnancy loss. Currently, there is no evidence of the LPD role and progesterone support effectiveness in infertility management, so the diagnosis and therapy of LPD among these patients should not be considered.

SummaryTo study the role of orexin A in the reproductive regulation of Mongolian sheep, ovine ovarian granulosa cells were cultured in vitro. The cells were divided into groups after luteinization, the experimental group was given orexin A and the transcriptome was sequenced together with that of the control group. The different genes related to reproduction were screened out. qRT-PCR, western blot and enzyme-linked immunosorbent assay (ELISA) were used to verify the selected genes and detect the effect on progesterone secretion. In total, 123 differentially expressed genes were obtained by sequencing. Six genes with high expression related to reproduction (PRRT2, ABCG1, SOX4, TBX3, ID1 and ATP8) were screened. The results of qRT-PCR were consistent with those of sequencing; western blot and ELISA were used to verify the protein levels of steroidogenic acute regulatory protein (StAR) and its related PRRT2 and ABCG1, and to detect their effect on progesterone secretion. Validation results were consistent with those of qRT-PCR and sequencing. The experimental group was given orexin A and compared with the control group. Expression of PRRT2 protein was significantly increased (P < 0.05), ABCG1 protein expression was significantly decreased (P < 0.05), StAR expression was significantly increased (P < 0.05), and progesterone secretion was significantly increased (P < 0.05). The results showed that orexin A promoted the expression of StAR by upregulating PRRT2 and downregulating ABCG1, therefore affecting secretion of progesterone. Gene expression characteristics of orexin A affecting progesterone secretion were preliminarily explored; this study provides a theoretical basis for further study on signalling pathways and reproductive regulation in Mongolian sheep.

International audience; Dichlorodiphenyltrichloroethane (p,p ' DDT) is an endocrine-disrupting chemical (EDC). Several studies showed an association between p,p ' DDT exposure and reprotoxic effects. We showed that p,p ' DDT was a positive allosteric modulator of human follitropin receptor (FSHR). In contrast, we demonstrated that p,p ' DDT decreased the cyclic AMP (cAMP) production induced by human choriogonadotropin (hCG). This study evaluated further the effects of p,p ' DDT on Gs-, beta-arrestin 2- and steroidogenesis pathways induced by hCG or luteinizing hormone (LH). We used Chinese hamster ovary cells line stably expressing hCG/LHR. The effects of 10-100 mu M p,p ' DDT on cAMP production and on beta-arrestin 2 recruitment were measured using bioluminescence and time-resolved resonance energy transfer technology. The impact of 100 mu M of p,p ' DDT on steroid secretion was analysed in murine Leydig tumor cell line (mLTC-1). In cAMP assays, 100 mu M p,p ' DDT increased the EC50 by more than 300% and reduced the maximum response of the hCG/LHR to hCG and hLH by 30%. This inhibitory effect was also found in human granulosa cells line and in mLTC-1 cells. Likewise, 100 mu M p,p ' DDT decreased the hCG- and hLH-promoted beta-arrestin 2 recruitment down to 14.2 and 26.6%, respectively. Moreover, 100 mu M p,p ' DDT decreased by 30 and 47% the progesterone secretion induced by hCG or hLH, respectively, without affecting testosterone secretion. This negative effect of p,p'DDT was independent of cytotoxicity. p,p ' DDT acted as a negative allosteric modulator of the hCG/LHR signalling. This emphasizes the importance of analyzing all receptor-downstream pathways to fully understand the deleterious effects of EDC on human health.

Background Irisin, which is cleaved from fibronectin type III domain-containing protein 5 (Fndc5), plays an important role in energy homeostasis. The link between energy metabolism and reproduction is well known. However, the biological actions of irisin in reproduction remain largely unexplored. Methods In this study, we generated Fndc5 gene mutation to create irisin deficient mice. Female wild-type (WT) and Fndc5 mutant mice were fed with standard chow for 48 weeks. Firstly, the survival rate, body weight and fertility were described in mice. Secondly, the levels of steroid hormones in serum were measured by ELISA, and the estrus cycle and the appearance of follicles were determined by vaginal smears and ovarian continuous sections. Thirdly, mRNA-sequencing analysis was used to compare gene expression between the ovaries of Fndc5 mutant mice and those of WT mice. Finally, the effects of exogenous irisin on steroid hormone production was investigated in KGN cells. Results The mice lacking irisin presented increased mortality, reduced body weight and poor fertility. Analysis of sex hormones showed decreased levels of estradiol, follicle-stimulating hormone and luteinizing hormone, and elevated progesterone levels in Fndc5 mutant mice. Irisin deficiency in mice was associated with irregular estrus, reduced ratio of antral follicles. The expressions of Akr1c18, Mamld1, and Cyp19a1, which are involved in the synthesis of steroid hormones, were reduced in the ovaries of mutant mice. Exogenous irisin could promote the expression of Akr1c18, Mamld1, and Cyp19a1 in KGN cells, stimulating estradiol production and inhibiting progesterone secretion. Conclusions Irisin deficiency was related to disordered endocrinology metabolism in mice. The irisin deficient mice showed poor growth and development, and decreased fertility. Irisin likely have effects on the expressions of Akr1c18, Mamld1 and Cyp19a1 in ovary, regulating the steroid hormone production. This study provides novel insights into the potential role of irisin in mammalian growth and reproduction.

Article Details: Received: 2020-10-25 | Accepted: 2020-11-27 | Available online: 2021-01-31 https://doi.org/10.15414/afz.2021.24.mi-prap.58-61 The climate in the Czech Republic is mild, transient between oceanic and continental, with a typical alternation of 4-year periods. The continental climate is characterized by high temperatures in summer and low temperatures in winter. During the extreme fluctuation of microclimatic conditions in the stable, there is also a change in individual reproductive indicators due to disruption of hormonal secretion, which further affects individual physiological processes, thus worsening conception or milder manifestations of heat, which we currently try to detect by neck responders and other modern systems.The experiment analyzed the effect of the average monthly temperature and humidity in the stable on the percentage of pregnant cows, in 2017 without the use of neck responders and in 2019, when all cows already had a neck responder. Monitoring was performed in 2017 and 2019 on the private farm GenAgro Řicany, a.s. (GPS 49 ° 12'32.319 "N, 16 ° 23'42.666" V). The values of average temperatures and relative humidity were calculated from 15-minute interval recordings of HOBO brand data loggers (Onset), which were placed in the stable, at the height at the withers of the cows without the possibility of influencing the measurements by the cows themselves. The object of monitoring was a herd of approximately 700 cows of Czech Fleckvieh Simmental various lactations. Data on the conception of cows were obtained both from the monthly reports of the breeding organization and from the data of the breeder.Thermal stress can have a long-lasting effect observable for weeks or months after the summer. This is also evidenced by the results of the work, which shows the worsening conception of cows, especially in late summer and autumn. At the same time, the results show a positive effect of the used neck responders, which help with the detection of heat and thus improve the reproduction of the whole herd. Keywords: Czech Fleckvieh Simmental, smart farming, neck responders, reproduction, heat stress References Badinga, L., et al. (1985). Effects of climatic and management factors on conception rate of dairy cattle in subtropical environment. J Dairy Sci , 68, 78–85. Bertipaglia, E. C. A., et al. (2005). Fertility and hair coat characteristics of Holstein cows in a tropical environment . Anim Reprod , 2(3), 187–194. Bridges, P. J. et al. (2005). Elevated temperature (heat stress) in vitro reduces androstenedione and estradiol and increases progesterone secretion by follicular cells from bovine dominant follicles. Domestic Animal Endocrinology , 29, 508–522, https://doi.org/10.1016/j.domaniend.2005.02.017 . Funnell, A. (2015). Robots and the future of agriculture . [cited 2020-10-15]. Available from: http://www.abc.net.au/radionational/programs/futuretense/a-swarmof-agbots/6968940 Jelinek, P., Koudela, K. et al. (2003). Fyziologie hospodařských zviřat . Brno: Mendelova zemědělska a lesnicka univerzita. Rensis, F. D., Scaramuzzi, R. . (2003). Heat stress and seasonal effects on reproduction in the dairy cow – a review. Theriogenology, 60,1139–1151, https://doi.org/10.1016/S0093-691X(03)00126-2 . Schmitt, E. J. et al. (1996). Differential response of the luteal phase and fertility in cattle following ovulation of the first-wave follicle with human chorionic gonadotropin or an agonist of the gonadotropin-releasing hormone. J Anim Sci , 74, 1074–1083, DOI: 10.2527/1996.7451074x Schwab, K. (2016). The fourth industrial revolution . New York: Crown Business. Ustundag, A., Cevikcan, E. (2017). Industry 4.0: managing the digital transformation . New York: Springer Berlin Heidelberg. Velechovska, J. (2016). Monitoring přežvykovani pro efektivnějsi chov . Chov [online]., 8. cislo 18–20 str. [cited 2020-10-15]. Available from: https://www.eurofarm.cz/sites/www.eurofarm.cz/files/u2018/08-2016-chov-priloha_str18-20-eurofarm.pdf Wolfenson, D., Roth, Z., Meidan, R. (2000). Impaired reproduction in heat stressed: basic and applied aspects. Anim Reprod Sci , 60, 535–547, https://doi.org/10.1016/S0378-4320(00)00102-0 .

This study was aimed at evaluating the possible interference of formulated glyphosate to the endocrine control of ovarian growth exerted by progesterone, in the estuarine crab Neohelice granulata. The addition of Roundup Ultramax® (0.2 mg/L of glyphosate in the incubation medium) was able to potentiate the stimulating effect of progesterone on total vitellogenic protein (Vg) content of ovarian pieces from crabs. Moreover, the sole addition of mifepristone (antagonist of progesterone receptors) was able to produce a decrement of the Vg content, which was not reverted by the addition of Roundup. A similar result was confirmed by means of histological analysis, which showed that mifepristone, both alone and in combination with Roundup, inhibited ovarian maturation, while Roundup alone increased it, in terms of a higher proportion of vitellogenic oocytes. We conclude that Roundup could stimulate the progesterone secretion exerted by the ovary and/or could act as a partial agonist of this hormone in the same tissue.

STUDY QUESTION Are progesterone (P) levels on the day before natural cycle frozen embryo transfer (NC-FET) associated with live birth rate (LBR)? SUMMARY ANSWER Regular ovulatory women undergoing NC-FET with serum P levels 10 ng/ml. WHAT IS KNOWN ALREADY The importance of serum P levels around the time of embryo transfer in patients undergoing FET under artificial endometrial preparation has been well established. However, no study has analyzed the importance of serum P levels in patients undergoing FET under a true natural endometrial preparation cycle. STUDY DESIGN, SIZE, DURATION This was a retrospective cohort study including 294 frozen blastocyst transfers under natural cycle endometrial preparation at a university-affiliated fertility centre between January 2016 and January 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS All patients had regular menstrual cycles and underwent NC-FET with their own oocytes. Only patients who had undergone serum P measurement between 8 am and 11 am on the day before FET were included. Patients did not receive any external medication for endometrial preparation or luteal phase support. Patients were divided into two groups according to serum P levels below or above 10 ng/ml on the day before FET. Univariate analysis was carried out to describe and compare the cycle characteristics with reproductive outcomes. To evaluate the effect of P, a multivariable logistic model was fitted for each outcome after adjusting for confounding variables. MAIN RESULTS AND THE ROLE OF CHANCE Mean serum P levels on the day before FET were significantly higher in patients who had a live birth compared to those who did not (14.5 ± 7.0 vs 12.0 ± 6.6 ng/ml, 95% CI [0.83; 4.12]). The overall clinical pregnancy rate (CPR) and LBR were 42.9% and 35.4%, respectively. Patients in the higher P group (>10 ng/ml) had a higher LBR (41.1% vs 25.7%: risk difference (RD) 15.4%, 95% CI [5; 26]) and CPR (48.6% vs 33.0%: RD 15.6%, 95% CI [4; 27]). Patients with higher serum P levels on the day before FET (63% of patients) had an improved LBR (odds ratio: 1.05; 95% CI [1.02; 1.09]). Women with serum P levels 10 ng/ml. LIMITATIONS, REASONS FOR CAUTION The main limitation of our study is its retrospective design. Other potential limitations are the detection of LH surge through urine testing and the inclusion of patients who did and did not undergo preimplantation genetic testing for aneuploidies. The protocol used in our institution for monitoring NC-FET does not look for the onset of progesterone secretion by the corpus luteum, and a slow luteinisation process or delay of corpus luteum function cannot be ruled out. WIDER IMPLICATIONS OF THE FINDINGS We provide evidence that a minimum serum P threshold (P >10 ng/ml) might be required for improved reproductive outcomes in NC-FET. This result suggests that there are different mechanisms by which P is produced and/or distributed by each patient. This study also provides an excellent model to evaluate the impact of luteal phase defect through NC-FET. A prospective evaluation to assess whether P supplementation should be individualised according to patient’s needs is necessary to support our findings. STUDY FUNDING/COMPETING INTEREST(S) No external funding was used, and there are no competing interests.

Angiogenesis plays an integral role in follicular and luteal development and is positively regulated by several intra-ovarian factors including vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2). Various transforming growth factor-β (TGF-β) superfamily members function as intra-ovarian regulators of follicle and luteal function, but their potential roles in modulating ovarian angiogenesis have received little attention. In this study, we used a bovine theca interna culture model (exhibiting characteristics of luteinization) to examine the effects of TGF-β1 and bone morphogenetic protein 6 (BMP6) on angiogenesis and steroidogenesis. VEGFA/FGF2 treatment promoted endothelial cell network formation but had little or no effect on progesterone and androstenedione secretion or expression of key steroidogenesis-related genes. TGF-β1 suppressed basal and VEGFA/FGF2-induced endothelial cell network formation and progesterone secretion, effects that were reversed by an activin receptor-like kinase 5 (ALK5) inhibitor (SB-431542). The ALK5 inhibitor alone raised androstenedione secretion and expression of several transcripts including CYP17A1. BMP6 also suppressed endothelial cell network formation under VEGFA/FGF2-stimulated conditions and inhibited progesterone secretion and expression of several steroidogenesis-related genes under basal and VEGFA/FGF2-stimulated conditions. These effects were reversed by an ALK1/2 inhibitor (K02288). Moreover, the ALK1/2 inhibitor alone augmented endothelial network formation, progesterone secretion, androstenedione secretion and expression of several steroidogenesis-related genes. The results indicate dual suppressive actions of both TGF-β1 and BMP6 on follicular angiogenesis and steroidogenesis. Further experiments are needed to unravel the complex interactions between TGF-β superfamily signalling and other regulatory factors controlling ovarian angiogenesis and steroidogenesis.

Made available in DSpace on 2020-12-12T01:12:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-07-01 Follicle-like structures are three-dimensional matrices joint with living cells that allow the in vitro development of female gametes in more physiological conditions. They have been shown to be beneficial to fresh oocytes in different species, and in this study, domestic cat (Felis silvestris catus) granulosa cells were used to create a functional follicle-like structure aimed at supporting the in vitro maturation of conspecific vitrified oocytes, key players of fertility preservation programmes that usually struggle to acquire their full developmental competence after warming. Cat granulosa cells were cultured for up to 6 days in three-dimensional barium alginate microcapsules (i.e. follicle-like structures) or in two-dimensional monolayers, and their steroidogenic ability (estradiol and progesterone secretion) was assessed to confirm their functionality. The same systems were used (on day 2 or 6 of granulosa cells culture) for the in vitro maturation (IVM) of Cryotop® vitrified immature cat oocytes and compared with microdrops of IVM medium without cells (control). Granulosa cells were able to maintain their functionality in vitro in both the conditions, even if with a different extent of hormonal secretion along culture (p =.02). Vitrified oocytes resumed meiosis at higher rates when cultured with 2 days old granulosa cells (p =.03), but full maturation rates slightly raised when granulosa cells were cultured longer, albeit without differences with the control group. This study paved the road for the creation of enriched culture systems in the domestic cat, but innovations are strongly needed for vitrified oocytes that deserve better chances to develop in vitro. Dipartimento di Scienze Veterinarie per la Salute la Produzione Animale e la Sicurezza Alimentare “Carlo Cantoni” Università degli Studi di Milano Programa de Mestrado em Ciência Animal Universidade de Franca (UNIFRAN) Departamento de Medicina Veterinária Preventiva e Reprodução Animal Universidade Estadual Paulista Julio de Mesquita Filho (UNESP) Departamento de Medicina Veterinária Preventiva e Reprodução Animal Universidade Estadual Paulista Julio de Mesquita Filho (UNESP)

Follicle culture in vitro provides a method for investigating stages of folliculogenesis that can lead to preserving fertility through cryopreservation techniques. This study aims to assess the effects of various concentrations of human follicular fluid (hFF) on growth, development, and expression of the proliferating cell nuclear antigen (PCNA) gene in mouse ovarian follicles in vitro. Preantral follicles were isolated from 14-day NMRI mouse ovaries. The follicles were cultured in basic media enriched with FBS, FSH, and insulin-transferrin-selenium, and supplemented with different concentrations of hFF (10, 20, and 30%) for 12 days. During the culture period, survival rate and follicular maturation, follicular diameter, levels of estrogen and progesterone secretion, and PCNA gene expression rate were evaluated. Survival rate, maturation, and antrum formation were significantly higher in the 10% hFF group than in the 20 and 30% hFF groups. On day 4, follicle diameter in the 10% hFF group was also higher than in the 20 and the 30% hFF group. In comparison with other groups, significantly higher estrogen and progesterone production levels were measured in the 10% hFF group. PCNA gene expression was also higher with 10 than 20 and 30% hFF concentrations. The present study suggests that addition of 10% hFF to mice ovarian preantral follicle culture media enhances follicle growth and oocyte maturation.

Background: Cumulus cells, as oocyte nurse cells, provide a suitable microenvironment with growth factors and cellular interactions required for oocyte maturation. Thus, these cells may serve as a natural niche for in vitro studies of female germ cell development. Cumulus cells may help attain a better understanding of the causes of infertility in women and eventually improve the outcomes of cases that respond poorly to standard infertility treatment. Objective: The aim of this study was to isolate, culture, and investigate the biological characteristics of human cumulus cells. Materials and Methods: In this experimental study, cumulus cells were isolated, cultured, and characterized using reverse transcription-polymerase chain reaction analyses of specific genes including FOXL2, CYP19A1, FSHR, AMHR, and LHR. The presence of vimentin, a structural protein, was examined via immunofluorescent staining. Moreover, levels of anti-mullerian hormone (AMH) and progesterone secretion by cumulus cells were measured with ELISA after 2, 4, 12, 24, and 48 hr of culture. Results: In adherent culture, human cumulus cells expressed specific genes and markers as well as secreted AMH and progesterone into the medium. Conclusion: Cumulus cells secrete AMH and progesterone in an adherent culture and might be applicable for in vitro maturation (IVM) and in vitro gametogenesis (IVG) studies. Key words: Cumulus cells, Conditioned medium, In vitro maturation, In vitro gametogenesis, Niche.

Recently, we observed that lipopolysaccharide (LPS) suppresses corpus luteum (CL) function in isolated perfused ovaries. It remained unclear if this suppression was due to increased luteal PGF2α secretion or LPS-induced apoptosis. Therefore, possible impacts of PGF2α and LPS were inhibited by a non-steroidal anti-inflammatory drug (flunixin) and an endotoxin-binding agent (polymyxin B), respectively. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and perfused for 240 min. After 50 min of equilibration, either flunixin or polymyxin B (5 μg/ml of each) were added to the perfusion medium of six ovaries, respectively. All ovaries (n = 12) were treated with E. coli LPS (0.5 μg/ml) 60 min after the onset of perfusion, and received 500 I.U. of hCG after 210 min of perfusion. Progesterone and PGF2α were measured in the effluent perfusate every 10 and 30 min, respectively. Biopsies of the CL were collected every 60 min to determine the mRNA expression of the cytokine TNFA and factors of apoptosis (CASP3, -8). Flunixin-treatment inhibited the increase of PGF2α after LPS-challenge that was observed in the polymyxin B-treated (PX-LPS) ovaries. After hCG-stimulation, progesterone secretion increased (P < 0.05) in group PX-LPS but not in the flunixin-treated (F-LPS) ovaries. Compared to initial values before LPS-challenge, luteal mRNA expression of TNFA and CASP3 was increased (P < 0.05) in group F-LPS at 120 and 180 min, respectively, and those of CASP8 was decreased (P < 0.05) in PX-LPS at 60 and 120 min after LPS-treatment. In conclusion, although flunixin managed to inhibit PGF2α, it did not suffice to successfully prevent LPS-induced apoptosis. However, endotoxin-binding polymyxin B resulted in luteal responsiveness to hCG after LPS-challenge., Journal of reproduction and development, 68 (1), ISSN:0916-8818, ISSN:1348-4400

Polycyctic Ovarian Syndrome (PCOS) adalah penyebab paling sering terjadinya anovulasi dan infertilitas anovulasi. PCOS dapat dikatakan endokrinopati yang sering terjadi pada wanita dengan usia subur dan berkaitan dengan kelainan metabolik dan disfungsi reproduksi. Apabila tidak dilakukan manajemen terapi pada wanita dengan PCOS dapat terjadi komplikasi. Beberapa studi percontohan menilai efikasi terapeutik vitamin D dalam parameter metabolik terkait PCOS. Vitamin D adalah hormon steroid esensial yang berperan dalam menjaga hemostasis kalsium dan fosfat. Konsentrasi vitamin D dalam serum dinyatakan kurang jika kadar 25(OH)D 21-29 ng/mL dan dinyatakan defisiensi vitamin D jika kadar 25(OH)D

Purpose of the study. To study the fetoplacental system (FPS) status in women with obstetric pathology and a burdened reproductive history. Materials and methods. The study included 500 pregnant women who were divided into four groups: I – 150 women with an uterus scar, II – 150 women after assisted reproductive technologies, III – 150 women with anomalies in labor, 0 – 50 healthy pregnant women without obstetric pathology and without burdened reproductive history. The level of PPS hormones was determined in serum, depending on type of obstetric pathology and characteristics of the reproductive history. The study of hormonal indicators was carried out in the pregnancy dynamics. Fetus state was evaluated with a cardiotocographic study. Results. Increase in placental lactogen level was observed in the pregnancy dynamics in women with obstetric pathology and a burdened reproductive history, but its growth in 21–30 and 31–40 weeks was less than in pregnant women without obstetric pathology and without a burdened reproductive history. Dynamics of the estriol and estradiol levels in the blood in all groups in the gestational age of 21–30 weeks was characterized by approximately the same levels of decrease compared with healthy pregnant women without obstetric complications. Decrease in progesterone secretion was observed in all pregnant women with obstetric pathology and burdened reproductive history. Dynamics of it level in the blood serum during pregnancy up to 20 weeks, 21–30 weeks and 31–40 weeks showed a decrease. This is a reflection of changes in the PPS state in general and leads to fetus deterioration. Conclusions. There is a feto-placental insufficiency in pregnant women with obstetric pathology and with a burdened reproductive history that is manifested by inhibition of the hormonal function of the feto-placental complex and proceeds against the background of fetal heart rate changes. Identified changes require the preventive and therapeutic measures aimed at correcting the fetus impaired condition and changes in FPS

Deoxynivalenol (DON) is a type-B trichothecene mycotoxin produced by Fusarium species, reported to be the most common mycotoxin present in food and feed products. DON is known to affect the production of testosterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH) in male rats, consequently affecting reproductive endpoints. Our previous study showed that DON induces oxidative stress in prostate cancer (PCa) cells, however the effect of DON on the intratumor steroidogenesis in PCa and normal prostate cells was not investigated. In this study human normal (PNT1A) and prostate cancer cell lines with different hormonal sensitivity (PC-3, DU-145, LNCaP) were exposed to DON treatment alone or in combination with dehydroepiandrosterone (DHEA) for 48 h. The results of the study demonstrated that exposure to DON alone or in combination with DHEA had a stimulatory effect on the release of estradiol and testosterone and also affected progesterone secretion. Moreover, significant changes were observed in the expression of genes related to steroidogenesis. Taken together, these results indicate that DON might affect the process of steroidogenesis in the prostate, demonstrating potential reproductive effects in humans.

Mancozeb is classified as an endocrine disruptor; thus the present study was carried out to investigate the impact of mancozeb on mammalian ovarian functions using in vitro caprine oocyte maturation and granulosa cell culture models. Caprine cumulus oocyte complexes (COCs) and granulosa cells were cultured under standard culture conditions and treated with mancozeb concentrations of 0.3, 3, and 30 μg/ml along with a control for 24 h and assessed. Granulosa cell viability and progesterone concentration in spent culture media after treatments were also assessed. Mancozeb significantly decreased (P

Ιnformation on the role of adiponectin in human ovarian steroidogenesis is limited. The present study aimed to investigate the effect of different doses of adiponectin on the secretion of estradiol and progesterone by human luteinized granulosa cells in culture. Granulosa cells, obtained from women undergoing in vitro fertilization (IVF) treatment, were pre-incubated for 24 h and then cultured for 48 h. Adiponectin was used in 3 doses, i.e., 5, 10, and 100 μg/ml alone and in combinations with FSH (10 and 100 ng/ml). Estradiol and progesterone were measured by radioimmunoassays in culture supernatants at 24 h and 48 h. Adiponectin after 48 h of culture stimulated the secretion of estradiol and, to a lesser extent, progesterone in a dose-dependent manner. FSH showed a variable effect on steroidogenesis. However, when the low dose FSH was combined with adiponectin, estradiol, and progesterone secretion were increased disproportionally to the dose of adiponectin. With the high dose FSH, the positive effect of adiponectin on FSH-induced estradiol secretion was less pronounced, while the effect on progesterone secretion was negligible. This study shows for the first time a stimulatory effect of adiponectin on the secretion of estradiol and progesterone by human luteinized granulosa cells

The Association of Zoos and Aquariums Reproductive Management Center (RMC) in the US and the European Association of Zoos and Aquaria Reproductive Management Group (RMG) in Europe monitor efficacy of contraceptive products in participating institutions and use those results to inform contraceptive recommendations. This study used the joint RMC-RMG Contraception Database to analyze efficacy of deslorelin implants (Suprelorin®), a contraceptive used in a wide range of mammalian taxa. More recently its use has increased in birds and in some reptiles and fish. Deslorelin, a gonadotropin-releasing hormone (GnRH) agonist, stimulates the reproductive system before downregulating receptors on pituitary cells that produce hormones that stimulate gonadal steroids in both males (testosterone) and females (estradiol and progesterone), interrupting sperm production and ovulation, respectively. Nevertheless, it has been used mostly in females. Efficacy has been high in mammals, with failures resulting in offspring in only 1.3% of treated individuals and 0.5% of treatment bouts. The failure rate has been higher in birds, with 14.7% of individuals in 7.2% of bouts producing eggs, perhaps reflecting differences in avian GnRH molecules. Too few reptiles and fish have been treated for meaningful analysis. Although deslorelin appears very safe, a possible exception exists in carnivores, because the stimulatory phase can result in ovulation and subsequent sustained progesterone secretion that may cause endometrial pathology. However, the stimulatory phase can be prevented by treatment with megestrol acetate for 7 d before and 7 d after implant insertion. The two current formulations of Suprelorin are effective for minimums of 6 (4.7 mg) or 12 mo (9.4 mg). The data indicate that Suprelorin is an effective and safe contraceptive option for female mammals, although it may not be effective in males of some mammalian species. Further research is needed to ascertain its usefulness in nonmammalian taxa.

Simple Summary The first month of gestation in the pig is characterised by many changes, such as entry of the embryos into the uterus, increased secretion of the pregnancy hormone, progesterone, and the implantation of the embryos into the uterus. Before implantation, embryos have not yet attached to the uterus, and are reliant on nutrients and signals in the uterine fluids, which are controlled by progesterone, among others. High feed intake has a positive effect on progesterone secretion and supports the establishment and development of pregnancy. Very low feed intakes, such as in sows competing for feed in group housing, can be detrimental to embryo survival and even the maintenance of pregnancy. After implantation, specific nutrients that improve the vascularisation of the placenta, such as arginine, can support the capacity of the uterus to support a litter of developing embryos since in this stage competition between embryos starts to play a role. Abstract In the pig, the establishment and maintenance of luteal function in early gestation is crucial to endometrial function, embryo development, and survival. The level of feed intake has a positive effect on formation of luteal tissue and progesterone secretion by the ovaries in the pre-implantation period, which is important for endometrial remodeling and secretion. These effects are independent of luteinising hormone (LH) and probably driven by metabolic cues, such as insulin and insulin-like growth factor (IGF-1), and seem to support progesterone secretion and delivery to the endometrium, the latter which occurs directly, bypassing the systemic circulation. Even after implantation, a high feed intake seems to improve embryo survival and the maintenance of pregnancy. In this stage, luteal function is LH-dependent, although normal variations in energy intake may not result in pregnancy failure, but may contribute to nutrient supply to the embryos, since in this phase uterine capacity becomes limiting. Feed incidents, however, such as unintended fasting of animals or severe competition for feed, may result in embryo or even pregnancy loss, especially in periods of seasonal infertility. Specific nutrients such as arginine have a role in the vascularisation of the placenta and can improve the uterine capacity in the period after implantation.

Polybrominated Diphenyl Ethers (PBDEs) have been extensively applied as flame retardants in different polymeric materials since the 1970s, which have become a group of long-lasting environmental pollutants. They have been reported from previous studies to accumulate and then disrupt the endocrine system in humans. However, the mechanisms are still little known. In the present study, mouse Leydig tumor cells were utilized to investigate steroidogenic activity influenced by deca-brominated diphenyl ether (BDE-209). Our data showed that BDE-209 did not change intracellular cAMP level in the presence of human Chorionic Gonadotropin (hCG), cholera toxin (CT), and forskolin, which indicated that reduction of progesterone may not be related to the hCG-cAMP signal pathway in MLTC-1 cells. Furthermore, the reduction of progesterone generation was not shifted by 8-Br-cAMP, an analog of cAMP, indicating that BDE-209 may inhibit post-cAMP sites. In addition, mRNA expression levels of P450 side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) presented a concentration-dependent decrease. In conclusion, this study suggested that BDE-209 may attenuate the progesterone secretion mainly through lowering the expression of these two enzymes.

Bovine theca cells are thought to differentiate from cortical stromal cells, and ovary-derived Wilms' tumor 1+ (WT1+ ) cells are the primary source of mouse theca cells. However, it is not known whether the differentiation of cortical stromal cells is regulated by WT1. Here, we identified WT1 in the cortical stroma and theca layer of the bovine ovary and analyzed the theca cell functional markers in cortical stromal cells and theca cells; in addition, we determined the effects of this gene on the secretion of androstenedione and progesterone by cortical stromal cells and the responsiveness of cortical stromal cells to luteinizing hormone (LH) in vitro. We used quantitative reverse-transcription polymerase chain reaction (RT-qPCR), western blot analysis, and immunohistochemistry to discover that the cortical stroma had higher WT1 expression than the theca layer. We used RT-qPCR and ELISA analyses to determine that the cortical stromal cells had lower levels of androstenedione and progesterone secretion and LHR messenger RNA expression than the levels of the theca cells. In cultured bovine cortical stromal cells, we found that WT1 downregulation increased androstenedione and progesterone secretion but had no effect on the LH responsiveness. Notably, the increase in androstenedione and progesterone secretion was associated with an increase in 3-β-hydroxysteroid dehydrogenase expression. In conclusion, the results suggest that WT1 is involved in the differentiation of cortical stromal cells into cells with characteristics similar to theca cells of antral follicles in adult bovine ovaries.

Granulosa cells play important roles in the regulation of ovarian functions. Phospholipase C is crucial in several signalling pathways and could participate in the molecular mechanisms of cell proliferation, differentiation and ageing. The objective of this study was to identify the effects of phospholipase C on the steroidogenesis of oestradiol and progesterone in porcine granulosa cells cultured in vitro. Inhibitor U73122 or activator m-3M3FBS of phospholipase C was added to the in vitro medium of porcine granulosa cells, respectively. The secretion of oestradiol decreased after 2 hr, 8 hr, 12 hr, 24 hr and 48 hr of treatment with 500 nM U73122 (p < .05) and decreased after 2 hr of treatment in the 500 nM m-3M3FBS addition group (p < .05). The secretion of progesterone increased after 4 hr of treatment with 500 nM U73122 (p < .05) and increased after 2 hr and 8 hr of treatment in the 500 nM m-3M3FBS addition group (p < .05). The ratio of oestradiol to progesterone decreased at each time point, except 8 hr after the addition of 500 nM U73122 (p < .05). The ratio of oestradiol to progesterone decreased after 2 hr (p < .05) of treatment with 500 nM m-3M3FBS. In genes that regulate the synthesis of oestradiol or progesterone, the mRNA expression of CYP11A1 was markedly increased (p < .05), and the mRNA expression of other genes did not change significantly in the U73122 treatment group, while the addition of m-3M3FBS did not change those genes significantly despite the contrary trend. Our results demonstrated that phospholipase C can be a potential target to stimulate the secretion of oestradiol and suppress progesterone secretion in porcine granulosa cells cultured in vitro, which shed light on a novel biological function of phospholipase C in porcine granulosa cells.

Purpose A clinical case presenting secondary amenorrhea accompanied by an adrenal adenoma and hyperprogesteronemia is described in this study. Methods Selective catheterization and sampling of adrenal and ovarian veins were performed. Results The source of hyperprogesteronemia was located in the right adrenal gland. A progesterone-producing tumor in the right adrenal gland was diagnosed and removed. Twenty-six days after tumor resection, menstruation occurred. Conclusions Progesterone-producing tumors should be considered with the presence of an adrenal mass and hyperprogesteronemia. Combined adrenal and ovarian venous sampling may help to identify the source of progesterone secretion.

Prior studies, mainly on rodents demonstrated that orexin A (OXA) plays an important role in regulating the hypothalamo-pituitary-gonadal (HPG) axis. However, the results of those works have indicated that the central impact of OXA on the HPG axis may vary depending on the species or sex of the animal, the phase of the estrous cycle, or the experimental model system (in vivo vs. in vitro). The aim of the present study was to further investigate the role of OXA in the regulation of reproduction in seasonally breeding, intact, non-steroid-primed ewes during the luteal phase of the estrous cycle and during anestrus. We examined the effect of fourth-ventricle injections of OXA (0.3 μg/kg body weight) on plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH) and progesterone concentrations and on the level of gonadotropin-releasing hormone receptor (GnRH-R) protein in the anterior pituitary (AP). Radioimmunoassays revealed that OXA treatments inhibited (P

Background Nerve growth factor (β-NGF) from llama seminal plasma has been described as a potent ovulatory and luteotrophic molecule after intramuscular or intrauterine infusion in llamas and alpacas. We tested the hypothesis that systemic administration of purified β-Nerve Growth Factor (β-NGF) during the preovulatory stage will up-regulate steroidogenic enzymes and Vascular Endothelial Growth Factor (VEGF) gene expression in granulosa cells inducing a change in the progesterone/estradiol ratio in the follicular fluid in llamas. Methods Experiment I: Female llamas (n = 64) were randomly assigned to receive an intramuscular administration of: a) 50 μg gonadorelin acetate (GnRH, Ovalyse, Pfizer Chile SA, Santiago, Chile, n = 16), b) 1.0 mg of purified llama β-NGF (n = 16), or c) 1 ml phosphate buffered saline (PBS, negative control group, n = 16). An additional group of llamas (n = 16) were mated with a fertile male. Follicular fluid and granulosa cells were collected from the preovulatory follicle at 10 or 20 h after treatment (Time 0 = administration of treatment, n = 8/treatment/time point) to determine progesterone/estradiol concentration and steroidogenic enzymes and VEGF gene expression at both time points. Experiment II: Granulosa cells were collected from preovulatory follicles from llamas (n = 24) using ultrasound-guided transvaginal follicle aspiration for in vitro culture to determine mRNA relative expression of Steroidogenic Acute Regulatory Protein (StAR) and VEGF at 10 or 20 h (n = 4 replicates) and progesterone secretion at 48 h (n = 4 replicates) after LH or β-NGF treatment. Results Experiment I: There was a significant increase in the progesterone/estradiol ratio in mated llamas or treated with GnRH or purified β-NGF. There was a significant downregulation in the mRNA expression of Aromatase (CYP19A1/P450 Arom) for both time points in llamas mated or treated with GnRH or llama purified β-NGF with respect to the control group. All treatments except β-NGF (20 h) significantly up regulated the mRNA expression of 3-beta-hydroxysteroid dehydrogenase (HSD3B) whereas the expression of StAR and Side-Chain cleavage enzyme (CYP11A1/P450scc) where significantly up regulated only by mating (20 h), or β-NGF at 10 or 20 h after treatment. VEGF was up regulated only in those llamas submitted to mating (10 h) or treated with purified β-NGF (10 and 20 h). Experiment II: Only β-NGF treatment induced an increase of mRNA abundance of StAR from llama granulosa cells at 20 h of in vitro culture. There was a significant increase on mRNA abundance of VEGF at 10 and 20 h of in vitro culture from granulosa cells treated with β-NGF whereas LH treatment increases VEGF mRNA abundance only at 20 h of in vitro culture. In addition, there was a significant increase on progesterone secretion from llama granulosa cells 48 h after LH or β-NGF treatment. Conclusions Systemic administration of purified β-NGF from llama seminal fluid induced a rapid shift from estradiol to progesterone production in the preovulatory follicle. Differences in gene expression patterns of steroidogenic enzymes between GnRH and mated or β-NGF-treated llamas suggest local effects of seminal components on the preovulatory follicle.

Genistein, a biologically active isoflavone, exists in many soy products. It is well known that genistein binds to both oestrogen receptor alpha (ERα) and oestrogen receptor beta (ERβ), but it has a higher affinity to ERβ. Genistein can also bind to the G protein-coupled receptor 30 (GPR30, also known as G protein-coupled oestrogen receptor 1 or GPER). Furthermore, weak oestrogenic activity has been found in genistein, but the mechanism of action remains unknown. The aim of this study was to investigate the in vitro effects of genistein on the secretion of progesterone (P4) and oestradiol (E2) in chicken granulosa cells harvested from follicles, as well as the mRNA expression of ERs in these cells. In addition, we examined the expression of key enzymes including steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) in the process of P4 synthesis. The results showed that genistein did not affect the viability of granulosa cells, nor was the proliferating cell nuclear antigen (PCNA) protein changed. Among the 1-, 10-, 100-, and 1,000-nM concentrations tested, treatment with 1 nM genistein for 48 h significantly increased P4 but did not affect E2 secretion. Real-time PCR results showed that the ERβ gene expression in granulosa cells was markedly upregulated by 1 nM genistein treatment for 48 h, but there was no significant difference in ERα and GPR30 expression. Genistein also increased the gene expression of StAR, P450scc and 3β-HSD in the cultured granulosa cells. These results indicate that genistein acts directly on chicken granulosa cells to increase P4 production by upregulating the gene expression of key enzymes through binding in ERβ. It may exert positive effects on the reproduction of late-laying hens and act as an effective and safe feed additive for animals.

Ovarian follicular development and ovulation in mammals is a highly-regulated process. Most of the current knowledge of ovarian processes was obtained from the studies of non-human models. Molecular studies on human ovarian processes suffer from lack of material and appropriate research tools. Mural granulosa cells (MGCs) culture is a major tool for studying the effect of different substances but a major problem for using these primary MGCs is their unresponsiveness to hCG stimulation at the time of oocyte retrieval. It is acceptable that MGCs regain responsiveness during days in culture but when the best time is and how to accelerate the regenerative process are unknown. The aim of the current study was to establish an optimized protocol which will provide a practical and efficient tool to examine the effect of LH/hCG on different downstream targets in luteinized MGCs. hCG effects were examined according to days in culture and hCG stimulation time. As read-out, we analyzed the gene expression of known hCG targets, protein production, and progesterone secretion. Our results show that with a daily medium exchange, the strongest effect was achieved already 4 days after seeding. On day 4, hCG stimulation triggers two major patterns of gene expression. Early induced genes were highly expressed 6–8 h after hCG, while 24 h of hCG stimulation was needed for late induced genes. Based on our results, we suggest daily medium exchange for 4 days before adding hCG and examine its effect 6 and 24 h later.

Melatonin is present in mammalian follicular fluid and plays an important role in regulating steroidogenesis in follicular cells. In this study, we report the effect of melatonin on steroidogenesis in the theca interna (TI) in small bovine follicles and theca cells (TCs) cultured in vitro. Treatment with melatonin significantly increased the expression of steroidogenic acute regulatory protein (STAR) and the production of progesterone in both TI and in TCs. Melatonin stimulated the phosphorylation of AKT but not ERK1/2, and the addition of luzindole (a nonspecific MT1 and MT2 inhibitor) or 4P-PDOT (specific MT2 inhibitor) reduced melatonin-induced STAR expression, progesterone secretion, and PI3K/AKT pathway activation. The effect of melatonin on the TI in follicles was more obvious than on the TCs in vitro. Results indicate that melatonin stimulates the steroidogenesis of TCs mainly via the activation of the PI3K/AKT pathway by MT1 and MT2.

Follicular cells from various species secrete steroids and prostaglandins, which are crucial for reproduction, in response to gonadotropins. Here, we examined prostaglandin E2 (PGE2) secretion from immortalized rat granulosa cells derived from preovulaotry follicles expressing the rat follicle stimulating hormone receptor (denoted as FSHR cells) that produce progesterone in response to gonadotropins. The cells were stimulated with a) pregnant mare’s serum gonadotropin (PMSG; a rat FSH receptor agonist), b) activators of the protein kinase A (PKA) pathway (forskolin and a cell permeable cAMP analog Dibutyryl-cAMP (DB-cAMP)) and c) protein kinase C (PKC) (12-O-tetradecanoylphorbol 13-acetate; TPA), alone and in combination for 24 h. Thereafter, PGE2 and progesterone levels in the culture media were determined. In accordance with previous studies, while PMSG and the PKA pathway activators induced progesterone accumulation in the media, TPA did not. In contrast, our data indicate that TPA, but neither PMSG, forskolin and DB-cAMP evoked PGE2 accumulation in the media. Western Blot analysis of cell lysate showed a drastic TPA induced increase of COX-2 levels, which was not seen with neither PMSG nor forskolin treatment. This association between the COX-2 and PGE2 levels suggests that the enzyme activity is the likely factor that determines the synthesis and levels of the prostaglandin in the culture media of the granulosa-derived cells. The addition of the PKA inhibitor H-89 to the FSHR cultures suppressed the gonadotropin and forskolin induction of progesterone secretion. Incubation in the presence of GF109203X (a PKC inhibitor) attenuated the TPA induced PGE2 accumulation in the culture media of the cells (a dose dependent reduction of 40–70%). In addition, while TPA inhibited the PMSG and forskolin induced-accumulation of progesterone in the media, the gonadotropin and forskolin inhibited the elevation of PGE2 levels evoked by TPA (a dose dependent decrease of 35–55%). These data suggest that cAMP/PKA and PKC signaling have opposite effects on PGE2 and progesterone synthesis in FSHR cells. We propose that this PKA and PKC interplay on progesterone and PGE2 may be advantageous for the coordination of these key mediators for successful ovulation and luteinization.

Disruption of granulosa cells (GCs), the main functional cells in the ovary, is associated with impaired female fertility. Epidemiological studies demonstrated that women have detectable levels of organic pollutants (e.g., perfluorooctanoate, perfluorooctane sulfonate, 2,2-dichlorodiphenyldichloroethylene, polychlorinated biphenyl 153, and hexachlorobenzene) in their follicular fluid (FF), and thus these compounds may directly affect the function of GCs in the ovary. Considering that humans are exposed to multiple pollutants simultaneously, we elucidated the effects of a mixture of endocrine-disrupting chemicals (EDCs) on human granulosa HGrC1 cells. The EDC mixture directly increased progesterone secretion by upregulating 3β-hydroxysteroid dehydrogenase (3βHSD) expression. Furthermore, the EDC mixture increased activity of mitochondria, which are the central sites for steroid hormone biosynthesis, and the ATP content. Unexpectedly, the EDC mixture reduced glucose transporter 4 (GLUT4) expression and perturbed glucose uptake; however, this did not affect the glycolytic rate. Moreover, inhibition of GLUT1 by STF-31 did not alter the effects of the EDC mixture on steroid secretion but decreased basal estradiol secretion. Taken together, our results demonstrate that the mixture of EDCs present in FF can alter the functions of human GCs by disrupting steroidogenesis and may thus adversely affect female reproductive health. This study highlights that the EDC mixture elicits its effects by targeting mitochondria and increases mitochondrial network formation, mitochondrial activity, and expression of 3βHSD, which is associated with the inner mitochondrial membrane.

Aquaporins (AQPs) are integral membrane proteins, which play an important role in water homeostasis in the uterus. According to the literature, the expression of aquaporins in reproductive structures depends on the local hormonal milieu. The current study investigated the effect of selected PKA kinase inhibitor H89 and MAPK kinase inhibitor PD98059, on the expression of AQP1, 2, 5, and 7, and steroid hormones (E2), progesterone (P4), and arachidonic acid (AA) in the porcine endometrium on days 18–20 and 2–4 of the estrous cycle (the follicular phase where estrogen and follicle-stimulating hormone (FSH) are secreted increasingly in preparation for estrus and the luteal phase where the ovarian follicles begin the process of luteinization with the formation of the corpus luteum and progesterone secretion, respectively). The luminal epithelial cells were incubated in vitro in the presence of the aforementioned factors. The expression of mRNA was determined by the quantitative real-time PCR technique. In general, in Experiment 1, steroid hormones significantly increased expression of AQP1, 2, and 5 while arachidonic acid increased expression of AQP2 and AQP7. On the other hand, MAPK kinase inhibitor significantly decreased the expression of AQP1 and 5. In Experiment 2, E2, P4, or AA combined with kinase inhibitors differentially affected on AQPs expression. E2 in combination with PKA inhibitor significantly decreased expression of AQP1 but E2 or P4 combined with this inhibitor increased the expression of AQP5 and 7. On the contrary, E2 with PD98059 significantly increased AQP5 and AQP7 expression. Progesterone in combination with MAPK kinase inhibitor significantly downregulated the expression of AQP5 and upregulated AQP7. Arachidonic acid mixed with H89 or PD98059 caused a decrease in the expression of AQP5 and an increase of AQP7. The obtained results indicate that estradiol, progesterone, and arachidonic acid through PKA and MAPK signaling pathways regulate the expression of AQP1 and AQP5 in the porcine luminal epithelial cells in the periovulatory period.

OBJECTIVES: To evaluates the effect of different modes of final follicular maturation triggering on the degree of apoptosis of granulosa cells (GCs) and the potential effect on progesterone secretion. METHODS: Thirty patients undergoing controlled ovarian hyperstimulation for IVF who received hCG, GnRH agonist, or dual trigger for final follicular maturation were included in the study. Granulosa cells were obtained at the time of oocyte retrieval. The proportion of apoptotic cells was evaluated via TUNEL and immunohistochemistry. RESULTS: The proportion of apoptotic cells was significantly higher in the GnRH agonist–alone group compared to hCG-alone and the dual trigger groups (13.5 ± 1.5% vs. 7.8% ± 1.8 vs. 10.1% ± 2, respectively, P < 0.01). Moreover, the expression of active-caspase-3 was also significantly increased in the GnRH agonist–alone group compared with the hCG-alone and the dual trigger groups (15.5% ± 2.9 vs. 8.4% ± 1.6 vs. 12.7% ± 2.6, respectively, P < 0.01). The progesterone levels measured in the granulosa-luteal cell culture medium after 24 h of incubation were similar between the three groups. CONCLUSIONS: The levels of apoptosis are increased after GnRH agonist/dual trigger. The increased apoptosis might be one of the culprit of the subsequent premature demise of the corpus luteum post GnRH agonist trigger.

Problem The miRNAs show placenta-specific expression patterns, which alter during pregnancy-related complications. In present study, the role of miR-27b-5p during forskolin-mediated BeWo cells fusion has been investigated. Method of study The fusion of BeWo cells in response to forskolin treatment (25 µM) was studied by desmoplakin I+II staining. Expression profile of miR-27b-5p by qRT-PCR and its targets HSD3β1 and WNT2B by qRT-PCR and in Western blot were studied. The effect of overexpression of miR-27b-5p and silencing of HSD3β1 & WNT2B by siRNA on forskolin-mediated BeWo cells fusion and secretion of hCG and progesterone by ELISA was investigated. Results Time-dependent down-regulation in the expression of miR-27b-5p in forskolin-treated BeWo cells has been confirmed by qRT-PCR. Overexpression of miR-27b-5p significantly inhibits forskolin-mediated BeWo cells fusion as well as hCG & progesterone secretion. HSD3β1 and WNT2B were identified as targets of miR-27b-5p and are up-regulated in forskolin-treated BeWo cells. Overexpression of miR-27b-5p in BeWo cells downregulates their expression. Further, luciferase reporter assay revealed that miR-27b-5p directly target expression of both HSD3β1 and WNT2B. Silencing of both HSD3β1 and WNT2B leads to a significant reduction in forskolin-mediated BeWo cells fusion with concomitant decrease in the secretion of progesterone or/and hCG. Decrease in forskolin-mediated cells fusion observed in miR-27b-5p mimic transfected BeWo cells could be rescued by the overexpression of both HSD3β1 and WNT2B. Conclusion These observations suggest that reduced miR-27b-5p in forskolin-treated BeWo cells leads to increased secretion of progesterone and hCG due to loss of repressional control on HSD3β1 and WNT2B.