Increased Progesterone Secretion Research Articles

Luteinizing Hormone Surge-Induced Krüppel-like Factor 4 Inhibits Cyp17A1 Expression in Preovulatory Granulosa Cells.

Previous in vivo and in vitro studies have demonstrated a dramatic up-regulation of Krüppel-like factor 4 (Klf4) in rat preovulatory granulosa cells (GCs) after LH/hCG treatment and its role in regulating Cyp19A1 expression during the luteal shift in steroidogenesis. In this study, we examined whether Klf4 also mediates the LH-induced repression of Cyp17A1 expression in primary rat preovulatory GCs. In response to LH treatment of GCs in vitro, Cyp17A1 expression declined to less than half of its initial value by 1 h, remaining low for 24 h of culture. Overexpression of Klf4 decreased basal and Sf1-induced Cyp17A1 expressions and increased progesterone secretion. Reduction of endogenous Klf4 by siRNA elevated basal Cyp17A1 expression but did not affect LH-stimulated progesterone production. Overexpression of Klf4 also significantly attenuated Sf1-induced Cyp17A1 promoter activity. On the other hand, mutation of the conserved Sp1/Klf binding motif in the promoter revealed that this motif is not required for Klf4-mediated repression. Taken together, these data indicate that the Cyp17A1 gene may be one of the downstream targets of Klf4, which is induced by LH in preovulatory GCs. This information may help in identifying potential targets for preventing the molecular changes occurring in hyperandrogenic disorders.

FRI392 Functional Interrelationship Between Oxytocin And Bone Morphogenetic Proteins In Ovarian Steroidogenesis By Granulosa Cells

Abstract Disclosure: K. Yamamoto: None. Y. Nakano: None. N. Iwata: None. Y. Soejima: None. A. Suyama: None. T. Hasegawa: None. F. Otsuka: None. Oxytocin (OXT) is a neuropeptide that is produced in the hypothalamus and is secreted from the posterior pituitary. OXT has diverse physiological roles mediated by the OXT receptor (OXTR), including not only stimulation of uterine contractions during labor and peripheral functions of milk ejection during lactation but also functions in pair bonding, maternal nurturing, and social behavior related to the action of OXT on the brain. It has been shown that OXT and OXTR are expressed in ovarian granulosa cells (GCs) in several species including humans. Secretion of OXT by bovine GCs is reported to be increased after the preovulatory luteinizing hormone surge, which is coincident with an increase in progesterone secretion and a decrease in secretion of estradiol and androgen, suggesting a functional interrelationship between OXT and ovarian steroidogenesis. It has been demonstrated that application of OXT to cultures of GCs from preovulatory follicles in bovines, marmoset monkeys, and humans led to increases in progesterone production. However, the precise molecular mechanism by which OXT modulates ovarian steroidogenesis in GCs remains unknown. Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor beta (TGF-β) superfamily, play important roles in steroidogenesis and cellular proliferation in ovarian follicles via autocrine/paracrine fashion. BMP ligands and receptors are expressed in a cell-dependent manner in the ovary and have a pivotal role in female fertility. Throughout folliculogenesis, BMPs commonly inhibit follicle-stimulating hormone (FSH)-induced progesterone production, and oocyte-derived factors BMP-6 and BMP-15 are known to act on GCs through BMP receptors and suppress FSH-induced progesterone synthesis. To clarify the reproductive role of OXT in ovarian steroidogenesis and its functional interaction with BMPs, the effects of OXT on ovarian steroidogenesis were investigated by using primary culture of rat GCs and human granulosa KGN cells. Here we found that the OXT receptor was expressed in both rat GCs and human KGN cells and that OXT treatment significantly increased FSH- and forskolin (FSK)-induced progesterone production, but not estradiol production, by rat GCs and KGN cells, respectively. In accordance with the effects of OXT on progesterone production, OXT enhanced mRNA levels of CYP11A1 and HSD3B2 induced by FSK in KGN cells. Of note, OXT enhanced the phosphorylation of SMAD1/5/9 and the mRNA level of ID1 induced by BMP-15, but not those induced by BMP-6, in KGN cells. It was also revealed that OXT application upregulated the expression of BMPR2, a crucial type-II receptor of BMP-15, and enhanced the BMP-15-induced expression of inhibitory SMAD6 by KGN cells. Taken together, it was shown that OXT enhances ovarian progesterone production with upregulation of BMP-15 activity, leading to a fine-tuning of ovarian steroidogenesis. Presentation: Friday, June 16, 2023

P-227 Exposure to triclosan modifies oocyte development and embryo metabolism

Abstract Study question Does the endocrine disruptor, triclosan, affect the function of cumulus-oocyte-complexes (COCs), and if so, do these effects persist in early embryo developmental competence and metabolism? Summary answer Triclosan caused COCs to i) increase progesterone secretion, ii) switch from releasing to consuming pyruvate, and iii) produce fewer blastocysts that were iv) metabolically compromised. What is known already Triclosan (TCS) is used widely as an antimicrobial agent in a range of consumer healthcare products, including hand sanitiser, toothpastes, soaps, and shampoos. Its prevalent use has led to accumulation in the environment and human exposure. Indeed, TCS has been identified in a range of bodily fluids. It is classed as an endocrine-disrupting chemical and there are reports of its detrimental impact on the vertebrate reproductive system. However, the direct effects of TCS on mammalian Cumulus-Oocyte-Complexes’ (COCs) function and subsequent development of the embryo remains unknown. Study design, size, duration COCs, isolated from abattoir-derived bovine ovaries on 5 independent occasions, were exposed of two environmentally-relevant doses of TCS (10nM and 1nM) during in vitro maturation. The TCS-exposed COCs were subsequently fertilised in vitro without further TCS addition, and the cleavage and blastocyst rate were recorded on Day 2 and Day 7/Day 8, respectively. The resulting blastocysts were collected and further cultured individually for 24 hours, and depletion/release of key-metabolites quantified. Participants/materials, setting, methods In vitro production was performed for the maturation of the bovine oocytes and the embryo culture. The production of steroid hormones (17b-estradiol and progesterone) during IVM was determined by ELISA. The concentrations of glucose, pyruvate, lactate during IVM and blastocyst formation were quantified in the spent media using microfluorometric assays. Main results and the role of chance TCS at 1nM had a significant impact on steroidogenesis during COCs in vitro maturation; progesterone production was significantly higher compared to control group (4.784 ±1.69 vs 2.85 ±0.83 pg/hour/COCs, p = 0.035; n = 5), although oestrogen release was not affected. In addition, there was a dramatic shift in pyruvate metabolism. Over the course of in vitro maturation, COCs exposed to TCS depleted pyruvate from the culture media regardless of the administrated dose whilst net pyruvate concentration increased in the medium of COCs in the control groups. The cleavage rate of bovine embryos produced from TCS-exposed COCs was not significantly different to control group. However, COCs-exposed to 1nM TCS produced significantly fewer blastocysts (12.22% vs 29.11% n = 5; p = 0.02). Those blastocysts that did originate from COCs treated with TCS depleted significantly more pyruvate from the culture media compared to controls, regardless of TCS dose (19.86 ±10.54 vs 10.12 ±12.5 pmol/hour/embryo, p = 0.011 and 21.41 ±10.08 vs 10.12 ±12.5 pmol/hour/embryo, p = 0.002, TCS-10nM and TCS-1nM compared to Control, respectively). Additionally, while glucose depletion did not differ, the lactate release from blastocysts derived from COCs exposed to 10nM TCS was approximately halved compared to controls (15.20 ±14.59 vs 31.83 ±22.73 pmol/hour/embryo p = 0.0054). Limitations, reasons for caution The origin of the ovaries may be a limitation since they came from animals at different stages of estrous cycle. Furthermore, pre-exposure to TCS prior to animal slaughter cannot be discounted. Finally, these observations were made in an animal model in a laboratory setting which may differ from natural exposure. Wider implications of the findings TCS modifies oocyte maturation, developmental competence, and metabolic activity of resulting embryos. The differences in pyruvate metabolism and steroidogenesis point to altered mitochondrial function. These data indicate that TCS directly modulates oocyte and embryo physiology which may relate to fertility in domestic livestock species and may be extrapolated to humans. Trial registration number not applicable

Females with Diabetes Mellitus Increased the Incidence of Premenstrual Syndrome.

Background: Premenstrual syndrome (PMS) is a multifactorial disorder caused by hormone and autonomic imbalance. In our study, hyperglycemia-induced insulin secretion increased progesterone secretion and progressive autonomic imbalance. The young patients with diabetes mellitus (DM) revealed hypo-parasympathetic function and hypersympathetic function compared with nondiabetic controls. Young female patients with DM with higher blood sugar and autonomic malfunction may be associated with PMS. However, there is a lack of evidence about DM in females related to PMS. We evaluated female patients with DM who subsequently followed PMS in a retrospective cohort study. Methods: We retrieved data from the National Health Insurance Research Database in Taiwan. Female patients with DM between 20 and 50 years old were assessed by the International Classification of Disease, 9 Revision, Clinical Modification (ICD-9-CM) disease code of 250. Patients who were DM-free females were fourfold matched to the control group by age and disease index date. The ICD-9-CM disease code of 625.4 identified the incidence of PMS followed by the index date as events. The possible risk factors associated with PMS were detected with a Cox proportional regression. Results: DM was a significant risk factor for PMS incidence with an adjusted hazard ratio of 1.683 (95% confidence interval: 1.104–2.124, p < 0.001) in females after adjusting for age, other comorbidities, season, urbanization status of patients and the hospital status of visiting. Conclusions: This study noted an association between DM and PMS in female patients. Healthcare providers and female patients with DM must be aware of possible complications of PMS, aggressive glycemic control, decreased hyperglycemia and autonomic dysfunction to prevent this bothersome disorder.

Does combining estradiol cypionate and GnRH for ovulation induction in recipient cows increase pregnancy rate after timed embryo transfer?

Estradiol cypionate (EC) or GnRH have been widely used for ovulation induction in timed embryo transfer (TET). EC administration increases the proportion of cows that show estrus, whereas GnRH promotes more synchronized ovulations. The aim of the present study was to evaluate the potential beneficial effects of combining EC and GnRH in TET. In experiment 1, no difference was observed on serum progesterone concentrations on Day 6 and 13 after GnRH treatment between GnRH and EC+GnRH groups. In experiment 2, pregnancy per embryo transfer (P/ET) did not differ (p = 0.69) between GnRH (62.8%) and EC+GnRH (58.7%) groups. In conclusion, combining EC and GnRH for ovulation induction does not increase progesterone secretion and pregnancy rate after TET in cattle.

A mixture of persistent organic pollutants detected in human follicular fluid increases progesterone secretion and mitochondrial activity in human granulosa HGrC1 cells

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.

Prognostic impact of neck responders on conception rate of Czech Fleckvieh Simmental cows

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 .

Urban PM2.5 Induces Cellular Toxicity, Hormone Dysregulation, Oxidative Damage, Inflammation, and Mitochondrial Interference

Epidemiological studies have found air pollution to be a driver of adverse pregnancy outcomes but biological mechanisms are not clear. A first trimester trophoblast cell line (HTR-8/SVneo) was exposed to various concentrations of PM2.5 in order to elucidate the effect of urban particulate matter (PM) of size &lt;2.5 μm on placental function. PM2.5 were collected at a site representative of urban traffic and dispersed in cell media by indirect and direct sonication. The HTR-8 cells were grown under standard conditions. Cellular uptake was studied after 24 and 48 h of exposure by transmission electron microscopy (TEM). The secretion of human chorionic gonadotropin (hCG), progesterone, and Interleukin-6 (IL-6) was measured by ELISA. Changes in membrane integrity and H2O2 production were analyzed using the CellToxTM Green Cytotoxicity and ROSGloTM assays. Protease activity was evaluated by MitoToxTM assay. Mitochondrial function was assessed through high resolution respirometry in an Oroboros O2k-FluoRespirometer, and mitochondrial content was quantified by citrate synthase activity. Results: TEM analysis depicted PM2.5 cellular uptake and localization of the PM2.5 to the mitochondria after 24 h. The cells showed aggregated cytoskeleton and generalized necrotic appearance, such as chromatin condensation, organelle swelling and signs of lost membrane integrity. The mitochondria displayed vacuolization and disruption of cristae morphology. At 48 h exposure, a significant drop in hCG secretion and a significant increase in progesterone secretion and IL-6 production occurred. At 48 h exposure, a five-fold increase in protease activity and a significant alteration of H2O2 production was observed. The HTR-8 cells exhibited evidence of increased cytotoxicity with increasing exposure time and dose of PM2.5. Conclusion: PM may contribute to the placental dysfunction associated with pregnancy outcomes, such as preeclampsia and intrauterine growth restriction, through their direct and indirect effects on trophoblast protein secretion, hormone regulation, inflammatory response, and mitochondrial interference.

Effect of nervous stimulation on ovarian steroid secretion in amphibians.

In the present study, the effect of nerve stimulation on the secretory activity of the ovary of adult females was analyzed for the first time in amphibians. Results revealed that in Rhinella arenarum the stimulation of nerves that supply the gonad induced an increase in estradiol and progesterone secretion, this response showing differences during the reproductive cycle of the species. During the postreproductive period, an increase in estradiol secretion was observed while, in the reproductive period, progesterone secretion increased. Our results suggest that the sympathetic division of the autonomic nervous system would be responsible for this increase, taking into account that, under our experimental conditions, acetylcholine did not affect the endocrine activity of the gonad, while adrenaline (epinephrine) was effective in inducing steroid secretion an effect that could be due to interaction with β receptors. On the other hand, our data show that the association of adrenaline with follicle-stimulating hormoneincreased estradiol secretion during the postreproductive period, while the association of catecholamine with LH or hCG increased progesterone secretion during the reproductive period. Our results would suggest that nerve stimulation, mediated by the release of adrenaline, would act synergistically with gonadotrophins to stimulate steroid secretion.

Knockdown of CYP19A1 in Buffalo Follicular Granulosa Cells Results in Increased Progesterone Secretion and Promotes Cell Proliferation.

Cytochrome P450 aromatase 19A1 (CYP19A1) is a critical enzyme in estrogen synthesis. However, the effect of CYP19A1 on cell growth and hormone secretion of buffalo follicular granulosa cells (BFGCs) is poorly understood. The objective of this study was to assess the role of CYP19A1 in cell proliferation and hormone secretion of BFGCs by knocking down CYP19A1 mRNA expression. The mRNA expression level of CYP19A1 gene was knocked down in BFGCs using the siCYP19A1-296 fragment with the best interference efficiency of 72.63%, as affirmed by real-time quantitative PCR (qPCR) and cell morphology analysis. The CYP19A1 knockdown promoted the proliferation of BFGCs through upregulating the mRNA expression levels of six proliferation-related genes (CCND1, CCNE1, CCNB1, CDK2, CDKN1A, and CDKN1B). Moreover, CYP19A1 knockdown increased (P < 0.05) the concentrations of progesterone secretion (P4) in BFGCs through increasing the mRNA expression levels of three steroidogenic genes (HSD17B1, HSD17B7, and CYP17A1). Our data further found that the FSH could inhibit the mRNA expression level of CYP19A1 in BFGCs, while LH obtains the opposite effect. These findings showed that the CYP19A1 knockdown had a regulatory role in the hormone secretion and cell proliferation in BFGCs.

Urban PM2.5 Induces Cellular Toxicity, Hormone Dysregulation, Oxidative Damage, Inflammation, and Mitochondrial Interference in the HRT8 Trophoblast Cell Line.

Objective: Epidemiological studies have found air pollution to be a driver of adverse pregnancy outcomes, including gestational diabetes, low term birth weight and preeclampsia. It is unknown what biological mechanisms are involved in this process. A first trimester trophoblast cell line (HTR-8/SVneo) was exposed to various concentrations of PM2.5 (PM2.5) in order to elucidate the effect of urban particulate matter (PM) of size <2.5 μm on placental function.Methods: PM2.5 were collected at a site representative of urban traffic and dispersed in cell media by indirect and direct sonication. The HTR-8 cells were grown under standard conditions. Cellular uptake was studied after 24 and 48 h of exposure by transmission electron microscopy (TEM). The secretion of human chorionic gonadotropin (hCG), progesterone, and Interleukin-6 (IL-6) was measured by ELISA. Changes in membrane integrity and H2O2 production were analyzed using the CellToxTM Green Cytotoxicity and ROSGloTM assays. Protease activity was evaluated by MitoToxTM assay. Mitochondrial function was assessed through high resolution respirometry in an Oroboros O2k-FluoRespirometer, and mitochondrial content was quantified by citrate synthase activity.Results: TEM analysis depicted PM2.5 cellular uptake and localization of the PM2.5 to the mitochondria after 24 h. The cells showed aggregated cytoskeleton and generalized necrotic appearance, such as chromatin condensation, organelle swelling and signs of lost membrane integrity. The mitochondria displayed vacuolization and disruption of cristae morphology. At 48 h exposure, a significant drop in hCG secretion and a significant increase in progesterone secretion and IL-6 production occurred. At 48 h exposure, a five-fold increase in protease activity and a significant alteration of H2O2 production was observed. The HTR-8 cells exhibited evidence of increased cytotoxicity with increasing exposure time and dose of PM2.5. No significant difference in mitochondrial respiration or mitochondrial mass could be demonstrated.Conclusion: Following exposure to air pollution, intracellular accumulation of PM may contribute to the placental dysfunction associated with pregnancy outcomes, such as preeclampsia and intrauterine growth restriction, through their direct and indirect effects on trophoblast protein secretion, hormone regulation, inflammatory response, and mitochondrial interference.

Suppression of Notch Signaling Stimulates Progesterone Synthesis by Enhancing the Expression of NR5A2 and NR2F2 in Porcine Granulosa Cells

The conserved Notch pathway is reported to be involved in progesterone synthesis and secretion; however, the exact effects remain controversial. To determine the role and potential mechanisms of the Notch signaling pathway in progesterone biosynthesis in porcine granulosa cells (pGCs), we first used a pharmacological γ-secretase inhibitor, N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT), to block the Notch pathway in cultured pGCs and then evaluated the expression of genes in the progesterone biosynthesis pathway and key transcription factors (TFs) regulating steroidogenesis. We found that DAPT dose- and time-dependently increased progesterone secretion. The expression of steroidogenic proteins NPC1 and StAR and two TFs, NR5A2 and NR2F2, was significantly upregulated, while the expression of HSD3B was significantly downregulated. Furthermore, knockdown of both NR5A2 and NR2F2 with specific siRNAs blocked the upregulatory effects of DAPT on progesterone secretion and reversed the effects of DAPT on the expression of NPC1, StAR, and HSD3B. Moreover, knockdown of NR5A2 and NR2F2 stimulated the expression of Notch3. In conclusion, the inhibition of Notch signaling stimulated progesterone secretion by enhancing the expression of NPC1 and StAR, and the two TFs NR5A2 and NR2F2 acted as downstream TFs of Notch signaling in regulating progesterone synthesis.

Prolyl oligopeptidase regulates progesterone secretion via the ERK signaling pathway in murine luteal cells.

Prolyl oligopeptidase (POP), one of the most widely distributed serine endopeptidases, is highly expressed in the ovaries. However, the physiological role of POP in the ovaries is not clear. In this study, we investigated the significance of POP in the corpus luteum. Murine luteal cells were cultured in vitro and treated with a POP selective inhibitor, (2S)-1[[(2 S)-1-(1-oxo-4-phenylbutyl)-2-pyrrolidinyl carbonyl]-2-pyrrolidinecarbonitrile (KYP-2047). We found that KYP-2047 treatment decreased progesterone secretion. In contrast, POP overexpression increased progesterone secretion. Three essential steroidogenic enzymes, including p450 cholesterol side-chain cleavage enzyme (CYP11A), 3β-hydroxysteroid dehydrogenase (3β-HSD), and the steroidogenic acute regulatory protein (StAR), were regulated by POP. Further studies showed that POP overexpression increased ERK1/2 phosphorylation and increased the expression of steroidogenic factor 1 (SF1), while KYP-2047 treatment decreased ERK1/2 phosphorylation and SF1 expression. To clarify the role of ERK1/2 signaling in POP-regulated progesterone synthesis, U0126-EtOH, an inhibitor of the ERK signaling pathway, was used to treat luteal cells. We found that U0126-EtOH decreased progesterone production and the expression of steroidogenic enzymes and SF1. POP overexpression did not reverse the effects of U0126-EtOH. Overall, POP regulates progesterone secretion by stimulating the expression of CYP11A, 3β-HSD, and StAR in luteal cells. ERK signaling and downstream SF1 expression contribute to this process.

How does nutrition influence luteal function and early embryo survival

The pre-ovulatory LH surge triggers luteinisation of follicle tissue, but subsequent development of corpora lutea to full size is independent of LH up to around day 12 of pregnancy. Thereafter, severe (pharmacological) inhibition of LH secretion for 3 to 5 days will result in luteal failure and loss of pregnancy. It is unlikely that nutritional circumstances will have a similar effect, although scenarios with severe undernutrition have hardly been studied during early pregnancy. Milder levels of pre- and postmating undernutrition (around maintenance requirements), do affect luteal tissue development, but whether this is related to variation in LH is not clear as studies are equivocal, and there are indications that other nutrition related factors, like IGF-1 and insulin, may mediate these effects. A high plane of nutrition seems to increase progesterone secretion by the ovaries, even though systemic progesterone is reduced at the same time. Since there is direct transfer of progesterone from ovarian veins to the uterus, this may explain why a high plane of nutrition may actually benefit embryo survival and pregnancy, although very early during luteal formation (first days after mating), secretion by the ovaries may be overridden by systemic clearance of progesterone on a high feed level. Direct measurement of progesterone secretion by the ovaries is poorly understood as is the transfer of progesterone to the uterus and effects of specific nutrition related mediators such as IGF-1 and insulin on these processes.

Genistein increases progesterone secretion by elevating related enzymes in chicken granulosa cells

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.

Docosahexaenoic acid (DHA) effects on proliferation and steroidogenesis of bovine granulosa cells

BackgroundDocosahexaenoic acid (DHA) is a n-3 polyunsaturated fatty acid (PUFA) belonging to a family of biologically active fatty acids (FA), which are known to have numerous health benefits. N-3 PUFAs affect reproduction in cattle, and notably directly affect follicular cells. In terms of reproduction in cattle, n-3 PUFA-enriched diets lead to increased follicle size or numbers.MethodsThe objective of the present study was to analyze the effects of DHA (1, 10, 20 and 50 μM) on proliferation and steroidogenesis (parametric and/or non parametric (permutational) ANOVA) of bovine granulosa cells in vitro and mechanisms of action through protein expression (Kruskal-Wallis) and signaling pathways (non parametric ANOVA) and to investigate whether DHA could exert part of its action through the free fatty acid receptor 4 (FFAR4).ResultsDHA (10 and 50 μM) increased granulosa cell proliferation and DHA 10 μM led to a corresponding increase in proliferating cell nuclear antigen (PCNA) expression level. DHA also increased progesterone secretion at 1, 20 and 50 μM, and estradiol secretion at 1, 10 and 20 μM. Consistent increases in protein levels were also reported for the steroidogenic enzymes, cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), and of the cholesterol transporter steroidogenic acute regulatory protein (StAR), which are necessary for production of progesterone or androstenedione. FFAR4 was expressed in all cellular types of bovine ovarian follicles, and in granulosa cells it was localized close to the cellular membrane. TUG-891 treatment (1 and 50 μM), a FFAR4 agonist, increased granulosa cell proliferation and MAPK14 phosphorylation in a similar way to that observed with DHA treatment. However, TUG-891 treatment (1, 10 and 50 μM) showed no effect on progesterone or estradiol secretion.ConclusionsThese data show that DHA stimulated proliferation and steroidogenesis of bovine granulosa cells and led to MAPK14 phosphorylation. FFAR4 involvement in DHA effects requires further investigation, even if our data might suggest FFAR4 role in DHA effects on granulosa cell proliferation. Other mechanisms of DHA action should be investigated as the steroidogenic effects seemed to be independent of FFAR4 activation.

Uteroplacental secretion of progesterone and estradiol-17β in an ovine model of intrauterine growth restriction

Using a mid to late gestation model of intrauterine growth restriction, uteroplacental secretion of progesterone and estradiol-17β were examined. From day 50 to 130 of gestation, 31 ewe lambs were allocated to receive 100% (ADQ) or 60% (RES) of nutrient requirements. At day 130, umbilical and uterine artery blood flows were determined and blood samples were collected from maternal saphenous artery, gravid uterine vein, umbilical vein, and umbilical artery. Uteroplacental secretion of progesterone was increased in RES compared to ADQ fed dams. There was a net secretion and net metabolism of estradiol-17β in RES, and ADQ fed dams, respectively. In relation to steroid synthesis, cotyledonary abundance of steroidogenic acute regulatory protein was greater in RES compared with ADQ fed dams, while abundance of aromatase was not different between dietary treatments. Caruncular aldo-keto reductase 1C abundance was less in RES compared to ADQ fed dams. The increase in progesterone secretion, therefore, is due in part to an increase in synthesis and a decrease in placental catabolism. Caruncular cytochrome P450 3A, which catalyzes the conversion of estrogens to catechol-estrogens, was in lesser abundance in RES compared to ADQ fed dams. Opposite responses in estradiol-17β uteroplacental secretion compared with metabolism may be mediated through placental estrogen metabolism via cytochrome P450 enzymes.

Apelin (APLN) regulates progesterone secretion and oocyte maturation in bovine ovarian cells.

APLN and its G-protein coupled receptor APLNR are expressed in the bovine ovary. However their role in granulosa cells and oocytes is unknown. Here, we studied their expression in bovine ovarian cells and investigated their regulation in cultured luteinizing granulosa cells in response to IGF1 and FSH. We determined the effect and the molecular mechanism of APLN (isoforms 17 and 13) on bovine granulosa cell progesterone secretion and on oocyte maturation. By RT-qPCR and immunoblot, we showed that the expression of both APLN and APLNR in granulosa and oocytes significantly increased with ovarian follicles size whereas it was similar in theca interstitial cells. In vitro, in unstimulated luteinizing bovine granulosa cells and in response to IGF1 (10-8 M) but not to FSH (10-8 M), we observed that APLN (-17 and -13) (10-9 M) increased progesterone production; this was abolished in response to the APLNR antagonist ML221. These latter effects were dependent on the MAPK ERK1/2 kinase. Furthermore, we showed that APLN (-17 and -13) (10-9 M) increased cell proliferation through AKT signaling. Conversely, the addition of APLN-13 and APLN-17 to in vitro maturation medium containing IGF1 (10-8 M) but not FSH (10-8 M) arrested most oocytes at the germinal vesicle stage, which was associated with a decrease in progesterone secretion, an inhibition in MAPK ERK1/2 phosphorylation and an increase in PRKA phosphorylation in oocytes. Thus, APLN can increase progesterone secretion and cell proliferation in bovine luteinizing granulosa cells in vitro, while it blocks meiotic progression at the germinal vesicle stage during bovine oocyte in vitro maturation.

The role of adrenergic activation on murine luteal cell viability and progesterone production

Sympathetic innervations exist in mammalian CL. The action of catecholaminergic system on luteal cells has been the focus of a variety of studies. Norepinephrine (NE) increased progesterone secretion of cattle luteal cells by activating β-adrenoceptors. In this study, murine luteal cells were treated with NE and isoprenaline (ISO). We found that NE increased the viability of murine luteal cells and ISO decreased the viability of luteal cells. Both NE and ISO promoted the progesterone production. Nonselective β-adrenergic antagonist, propranolol reversed the effect of ISO on cell viability but did not reverse the effect of NE on cell viability. Propranolol blocked the influence of NE and ISO on progesterone production. These results reveal that the increase of luteal cell viability induced by NE is not dependent on β-adrenergic activation. α-Adrenergic activation possibly contributes to it. Both NE and ISO increased progesterone production through activating β-adrenergic receptor. Further study showed that CyclinD2 is involved in the increase of luteal cell induced by NE. 3β-Hydroxysteroid dehydrogenase, LHR, steroidogenic acute regulatory protein (StAR), and PGF2α contribute to the progesterone production induced by NE and ISO.

Ellagitannins--compounds from pomegranate as possible effector in steroidogenesis of rabbit ovaries.

This study has observed possible effect of ellagitannins - compounds from pomegranate on process of steroidogenesis in ovaries. The aim of the study was to investigate the possible effect of punicalagin on secretion of steroid hormones - progesterone, androstenedione, testosterone and 17beta-estradiol by ovarian fragments of rabbits in vitro. Ovarian fragments from sexually mature female New Zealand white rabbits (n=20) were incubated without (control group) or with punicalagin at various doses 1, 10 and 100 microg.ml(-1) for 24 h. Hormones were evaluated by ELISA (The Enzyme-Linked Immunosorbent Assay). Data showed that progesterone and 17beta-estradiol (but not androstenedione and testosterone) release by rabbit ovarian fragments was significantly affected by punicalagin addition at various doses. Punicalagin (at 100 microg.ml(-1)) significantly (P<0.05) increased progesterone secretion. On the other hand, the release of 17beta-estradiol was significantly (P<0.005) decreased by punicalagin addition (at 10 microg.ml(-1)). Our results suggest that punicalagin could have dose-dependent impact on secretion of steroid hormones progesterone and 17beta-estradiol by rabbit ovarian fragments and it may be effector in process of ovarian steroidogenesis.