Stimulated Progesterone Secretion Research Articles

Exposure to atrazine stimulates progesterone secretion and induces oxidative stress, inflammation, and apoptosis in the ovary of pseudopregnant rats

Atrazine (ATR) is one of the most commonly used herbicides worldwide. As an endocrine disruptor, it causes ovarian dysfunction, but the mechanism is unclear. We hypothesized that ATR could affect ovarian steroidogenesis, oxidative stress, inflammation, and apoptosis. In the current study, rats aged 28 days were treated with PMSG and HCG to obtain amounts of corpora lutea. Then, rats were injected with ATR (50 mg/kg/day) or saline (0.9%) for 7 days. Sera were collected to detect biochemical indices and progesterone (P4) level, ovaries were collected for antioxidant status, HE, qPCR, and WB analysis. Results showed that ATR exposure affected growth performance as well as serum TP, GLB, and ALB levels, increased serum P4 level and ovarian mRNA and protein levels of StAR, CYP11A1, and HSD3B. ATR treatment increased ovarian mRNA and protein levels of CREB but not PKA expression. ATR treatment increased ovarian mRNA abundances of Nrf-2 and Nqo1, MDA level, and decreased SOD, GST, and T-AOC levels. ATR exposure increased the mRNA abundances of pro-inflammatory cytokines including Tnf-α, Il-1β, Il-6, Il-18, and Inos. ATR exposure increased the mRNA and protein level of Caspase 3 and the ratio of BAX/BCL-2. In conclusion, NRF-2/NQO1 signaling pathway and CREB might be involved in the regulation of ATR in luteal steroidogenesis, oxidative stress, inflammation, and apoptosis in rat ovary.

FUNCTIONAL STATE OF THE PINEAL GLAND IN WOMEN WITH THREATENED ABORTION IN THE FIRST TRIMESTER – FURTHER PROSPECTS OF PREGNANCY

Melatonin is an important hormone for normal pregnancy development, but the infl uence of serotonin remains poorlyunderstood. Low melatonin production can lead to spontaneous abortion by stimulating progesterone secretion and inhibiting prostaglandin synthesis. Melatonin has an eff ect on the induction and blocking of apoptosis of the villous cytotrophoblast and promotes the development of the syncytiotrophoblast. Analysis of the frequency of positive pregnancy showed a signifi cant impact on the course of pregnancy and childbirth, compliance with sleep and rest also contribute to a positive eff ect, but there is no general concept, which makes the subject relevant.The aim of the study. To evaluate the functional state of the pineal gland, in particular, the amount of melatonin and serotonin in the blood of pregnant women at 10-12 weeks of gestation with a threat of abortion, which was manifested by bloody discharge or formation of retrochorionic hematoma, and the consequences of pregnancy.Materials and methods. Two groups were formed: I main group – 40 pregnant women with a threat of abortion and formation of retrochorionic hematoma, II control group – 44 healthy pregnant women. Obstetric examination, enzyme- linked immunosorbent assay for determination of melatonin, serotonin, chorionic gonadotropin, and ultrasound were performed. Informed consent of pregnant women was obtained with adequate explanation of the purpose, objectives, methods and scope of laboratory and instrumental research methods. The study protocol was approved by the Commission on Biomedical Ethics of the Bukovinian State Medical University, 2015 (minutes of the Commission meeting No 2 from the 19.10.2023 year). The study was conducted in accordance with the basic provisions of GCP (1996), the Convention of the Council of Europe on Human Rights and Biomedicine (April 4, 1997), the Declaration of Helsinki of the World Medical Association for the Ethical Principles of Scientifi c Medical Research Involving Human Subjects (1964-2008), the Order of the Ministry of Health of Ukraine 690 of September 23, 2009 (as amended by the Order of the Ministry of Health of Ukraine 523 of July 12, 2012). Statistical analysis was performed using generally accepted methods of variance statistics. Reliability was assessed by Student’s t-test. Diff erences were considered signifi cant if the signifi cance level was p≤0.05. We also performed correlation analysis in Microsoft Excel, using the method of squares (Pearson’s method) to determine the correlation coeffi cient. The work is a fragment of the research work of the Department of Obstetrics and Gynecology, UDC 618.1-053.34 + 618.1-0536 /.8]. –07-084-08 «Prevention, diagnosis and treatment of disorders of the perinatal period and reproductive system of women and adolescent girls» (No. 201110Н, state registration number 0111U006499. The term of implementation is 02.2011-12.2015).Results of the study. According to the obtained results, retrochorionic hematoma up to 1 cm is detected three times more often than over 1 cm, while the level of melatonin is not signifi cantly lower, but with retrochorionic hematoma over 1 cm a signifi cant decrease is observed in cases of further normal pregnancy and childbirth. With further complications of pregnancy in retrochoroidal hematoma up to 1 cm, there is a signifi cant signifi cant decrease in melatonin (p ˂ 0.001), in contrast to retrochoroidal hematoma over 1 cm, where no signifi cant diff erence was found in the complicated course of pregnancy (p > 0.05). Serotonin levels were elevated in all subgroups regardless of hematoma size and subsequent pregnancy outcome. Conclusions. In case of threatened abortion in the fi rst trimester with formation of retrochorionic hematoma, the risk ofcomplicated pregnancy remains in 60.87 % of cases. Determination of pineal hormones revealed a signifi cant increase inserotonin with a simultaneous signifi cant decrease in melatonin, while maintaining a positive relationship between the hormones, indicating a disruption of melatonin synthesis in the central nervous system. This is confi rmed by the normal level of chorionic gonadotropin and the preservation of embryonic viability. Since the disruption of hormone formation in the pineal gland has been established, adherence to the recommendations for adequate rest may positively aff ect the further outcome of pregnancy and childbirth.

Differential actions of diacylglycerol acyltransferase (DGAT) 1 and 2 in regulating lipid metabolism and progesterone secretion of goose granulosa cells

Accumulating evidence shows that granulosa cells within both mammalian and avian ovaries have the ability to synthesize fatty acids through de novo lipogenesis and to accumulate triglycerides essential for oocyte and ovarian development. However, very little is known about the exact roles of key genes involved in the lipid metabolic pathway in granulosa cells. The goal of this study was to investigate the differential actions of diacylglycerol acyltransferase (DGAT) 1 and 2, which are recognized as the rate-limiting enzymes catalyzing the last step of triglyceride biosynthesis, in regulating lipid metabolism and steroidogenesis in granulosa cells of goose follicles at different developmental stages. It was observed that the mRNAs encoding DGAT1 and DGAT2 were ubiquitous in all examined granulosa cell layers but exhibited distinct expression profiles during follicle development. Notably, the mRNA levels of DGAT1, DGAT2, FSHR, LHR, STAR, CYP11A1, and 3βHSD remained almost constant in all except for 1–2 follicles within the 8−10 mm cohort, followed by an acute increase/decrease in the F5 follicles. At the cellular level, siRNA-mediated downregulation of DGAT1 or DGAT2 did not change the amount of lipids accumulated in both undifferentiated- and differentiated granulosa cells, while overexpression of DGAT2 promoted lipid accumulation and expression of lipogenic-related genes in these cells. Meanwhile, we found that interfering DGAT2 had no effect but interfering DGAT1 or overexpressing DGAT2 stimulated progesterone secretion in undifferentiated granulosa cells; in contrast, interference or overexpression of DGAT1/2 failed to change progesterone levels in differentiated granulosa cells but differently modulated expression of steroidogenic-related genes. Therefore, it could be concluded that DGAT1 is less efficient than DGAT2 in promoting lipid accumulation in both undifferentiated- and differentiated granulosa cells and that DGAT1 negatively while DGAT2 positively regulates progesterone production in undifferentiated granulosa cells.

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.

Differential actions of second messenger systems in the corpus luteum

Our current working hypothesis for the intracellular mechanism of action for LH and PGF-2 alpha is shown in Fig. 8. Luteinizing hormone appears to act primarily on the small luteal cell through the cAMP/protein kinase A effector system and thereby stimulates secretion of progesterone. Activation of the protein kinase C effector pathway is inhibitory to progesterone secretion from stimulated small luteal cells but it is not clear which hormones, if any, activate this effector system. Results from studies in whole animals suggest that LH may also stimulate differentiation of small luteal cells into large luteal cells (Donaldson & Hansel, 1965; Farin et al., 1988). Although there are LH receptors on large luteal cells, LH treatment does not stimulate progesterone secretion and does not appear to activate any of the second messenger pathways which we have examined. Prostaglandin F-2 alpha appears to act on the large luteal cell through free intracellular calcium and protein kinase C effector systems. Apparently, PGF-2 alpha-induced activation of protein kinase C results in the acute inhibition of progesterone production seen in the first 8 h after PGF-2 alpha treatment. The cytotoxic effects of PGF-2 alpha on the large luteal cell (Fitz et al., 1984; Braden et al., 1988) may be caused by a sustained elevation in free intracellular calcium concentrations. No direct effects of PGF-2 alpha on small luteal cells have been detected (no inhibition of progesterone production, no activation of protein kinase C, no increase in free intracellular calcium), which is consistent with an absence of high affinity PGF-2 alpha receptors on this cell type. The cytotoxic effects of PGF-2 alpha on small luteal cells and endothelial cells (Braden et al., 1988) may be caused by decreases in luteal blood flow (Niswender et al., 1975; Wiltbank et al., 1990b), actions of cytotoxic agents released by large luteal cells, or increases in cytotoxic white blood cells (Murdoch, 1987; Bagavandoss et al., 1988).

Regulated and Functional Expression of the Corepressor MTA3 in Rodent Testis.

Metastasis-associated protein (MTA)3 is a subunit of the Mi-2/nucleosome remodeling and deacetylase protein complex, with relevant roles in the regulation of cancerous epithelial to mesenchymal transition in an estrogen-dependent manner, recently involved in the modulation of different physiological processes. Although these findings connect MTA3 expression with hormonal signaling in various systems, little is known about whether this relationship is conserved in testis where hormonal action is intensive. We, therefore, document here for the first time the expression of Mta3/MTA3 in mammalian testes, where MTA3 protein was identified mainly in interstitial Leydig cells. Testicular levels of Mta3/MTA3 were overtly modulated by pituitary gonadotropins, as well as metabolic signals, such as dexamethasone, T4, and rosiglitazone. In addition, ablation of endogenous Mta3 by short hairpin RNA significantly inhibited human choriogonadotropin/dibutyryl-cAMP (db-cAMP)-stimulated progesterone secretion in MA-10 Leydig cells, whereas overexpression of exogenous MTA3 effectively reversed Mta3 deficiency damaged progesterone production. Moreover, attenuated Mta3 expression positively correlated to the deregulated level of serum testosterone in murine type 2 diabetes mellitus. From a functional standpoint, MTA3 deficiency was involved in insulin-mediated inhibition of testicular steroidogenesis. Our data are the first to disclose the presence and functional role of MTA3 in the testis, where its expression is regulated by developmental, metabolic, and hormonal cues and is closely associated with steroidogenic dysfunction. The current study expands the reproductive dimension of MTA3, which may operate directly at the testicular level to modulate steroidogenic function.

Human chorionic gonadotrophin in early gestation induces growth of estrogenic ovarian follicles and improves primiparous sow fertility during summer

Reduced summer farrowing rates may be due to inadequate corpora luteal (CL) support. Porcine CL become dependent on LH from 12 d of pregnancy and the embryonic estrogen signal for maternal recognition of pregnancy (MRP) is initiated at about 11–12 d after insemination. We hypothesised that injection of the LH analogue human chorionic gonadotropin (hCG) would induce growth of estrogenic follicles and, by mimicking the signal for MRP and stimulating progesterone secretion, increase primiparous sow fertility. In Experiment 1, during a 28 d lactation 53 mixed parity sows were full-fed either throughout lactation (n=16) or until 18 d and then feed restricted during the last 10 d of lactation (n=36). At 12 d after mating restrict-fed sows were injected with 1000IU hCG (n=17) or were not injected (n=19); the full-fed sows acted as non-treated positive controls. Transrectal ovarian ultrasound exams were performed on days 12, 16, 20, 24, and 28; blood samples were obtained on days 12, 14, and 15 for estradiol and progesterone assay. For Experiment 2, during the summer months primiparous sows received 1000IU hCG 12 d after mating (n=28) or were non-injected controls (n=27). Pregnancy status was determined at 28 d and sows allowed to go to term to determine farrowing rates and litter sizes. In Experiment 1, injection of hCG increased (P<0.001) follicle diameter and serum concentrations of estradiol (P<0.01) and progesterone (P<0.05). There were no effects of lactation feeding level on wean-estrus interval, farrowing rate or subsequent litter size. In Experiment 2, hCG injection was associated with a higher pregnancy rate (P<0.05) and farrowing rate (P<0.08). There was no effect on litter size. These data confirm that hCG stimulates growth of estrogenic follicles and CL function, and improves primiparous sow fertility during the summer months.

Human chorionic gonadotropin: Different glycoforms and biological activity depending on its source of production

Human chorionic gonadotropin (hCG) is the first hormonal message from the placenta to the mother. It is detectable in maternal blood two days after implantation and behaves like a super LH agonist stimulating progesterone secretion by the corpus luteum. In addition to maintaining the production of progesterone until the placenta itself produces it, hCG also has a role in myometrial quiescence and local immune tolerance. Specific to humans, hCG is a complex glycoprotein composed of two highly glycosylated subunits. The α-subunit is identical to the pituitary gonadotropin hormones (LH, FSH, TSH), contains two N-glycosylation sites, and is encoded by a single gene (CGA). By contrast, the β-subunits are distinct for each hormones and confer both receptor and biological specificity, although LH and hCG bind to the same receptor (LH/CG-R). The hCG ß-subunit is encoded by a cluster of genes (CGB) and contains two sites of N-glycosylation and four sites of O-glycosylation. The hCG glycosylation state varies with the stage of pregnancy, its source of production and in the pathology. It is well established that hCG is mainly secreted into maternal blood, where it peaks at 8–10weeks of gestation (WG), by the syncytiotrophoblast (ST), which represents the endocrine tissue of the human placenta. The invasive extravillous trophoblast (iEVT) also secretes hCG, and in particular hyperglycosylated forms of hCG (hCG-H) also produced by choriocarcinoma cells. In maternal blood, hCG-H is elevated during early first trimester corresponding to the trophoblastic cell invasion process and then decreases. In addition to its endocrine role, hCG has autocrine and paracrine roles. It promotes formation of the ST and angiogenesis through LH/CG-R but has no effect on trophoblast invasion in vitro. By contrast, hCG-H stimulates trophoblast invasion and angiogenesis by interacting with the TGFß receptor in a LH/CG-R independent signalling pathway. hCG is largely used in antenatal screening and hCG-H might represent a serum marker of implantation and early trophoblast invasion. In conclusion, hCG is the major pregnancy glycoprotein hormone, whose maternal concentration and glycan structure change all along pregnancy. Depending on its source of production, glycoforms of hCG display different biological activities and functions that are essential for pregnancy outcome.

Notch Signaling Pathway Regulates Progesterone Secretion in Murine Luteal Cells.

Notch signaling is an evolutionarily conserved pathway, which involves in various cell life activities. Other studies and our report showed that the Notch signaling plays very important role in follicle development in mammalian ovaries. In luteal cells, Notch ligand, delta-like ligand 4, is involved in normal luteal vasculature. In this study, murine luteal cells were cultured in vitro and treated with Notch signaling inhibitors, L-658,458 and N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester (DAPT). We found that L-658,458 and DAPT treatment decrease basal and human chorionic gonadotropin (hCG)-stimulated progesterone secretion. On the contrary, overexpression of intracellular domain of Notch3 increased basal and hCG-stimulated progesterone secretion. Further studies demonstrated that Notch signaling regulated the expression of steroidogenic acute regulatory protein and CYP11A, 2 key enzymes for progesterone synthesis. In conclusion, Notch signaling plays important role in regulating progesterone secretion in murine luteal cells.

Effect of growth hormone on basal and LH-stimulated steroid secretion by chicken yellow ovarian follicles. An in vitro study.

The aim of the present study was to determine the effect of growth hormone (GH) on basal and LH-regulated steroid secretion by yellow hierarchical follicles before and after maturation, and the granulosa and theca layers of the largest preovulatory follicles during the ovulatory cycle in the chicken. In the first experiment, whole yellow follicles (8-12 mm, 12-18 mm, 18-24 mm and 24-30 mm) isolated from 15 and 17-18 week-old chickens were used. In the second experiment, the granulosa and theca layers of the 3 largest yellow preovulatory follicles (F3<F2<F1) were isolated from the ovary of the laying hen (age 21 weeks) at stages 22 h and 3 h before F1 ovulation. The collected follicles and fragments of the granulosa and theca layers were randomly assigned to Eagle's medium containing 0 (control), chicken GH (cGH; 10 ng/ml), ovine LH (oLH; 10 ng/ml) or cGH+oLH (10 ng/ml+10 ng/ml) and incubated for 24 h at 38 degrees C. Following incubation, the progesterone and estradiol levels were determined in collected medias by RIA. It was found that cGH (1) elevated basal progesterone secretion by all classes of yellow follicles, (2) decreased estradiol secretion by the largest (18-30 mm) follicles and (3) attenuated to the control level the LH-stimulated estradiol secretion by the theca layer of F3 and F2 follicles at 22 h before ovulation. These results suggest that GH directly stimulates progesterone secretion and inhibits estradiol secretion by yellow hierarchical ovarian follicles in chicken. However, the effect of GH on separated granulosa and theca layers of the preovulatory follicles is not as pronounced as on whole follicles. It cannot be excluded that GH temporarily modulates LH action in estradiol production by the theca cells of chicken preovulatory ovarian follicles.

Everything you ever wanted to know about hCG

s / Placenta 35 (2014) A1eA112 A4 hormones. The placenta, therefore, has an important role in integrating the various endocrine signals involved in regulating resource allocation to fetal growth. PL2.2. EVERYTHING YOU EVER WANTED TO KNOW ABOUT HCG Thierry Fournier a,b UMR-S 1139, INSERM, University of Paris Descartes, Paris, France; b PremUp Foundation, Paris, France Human chorionic gonadotrophin (hCG) is the first hormonal message from the placenta to themother. It is detectable inmaternal blood two days after implantation and behaves like a super agonist of LH stimulating progesterone secretion by the corpus luteum. hCG has also a role in quiescence of the myometrium and local immune tolerance. Specific to the primate superior, hCG is a complex glycoprotein composed of two glycosylated subunits. The a-subunit is identical to the pituitary gonadotrophin hormones (LH, FSH, TSH), contains two N-glycosylation sites, and is encoded by a single gene, whereas the b-subunits in each of the hormones are distinct and confer receptor and biological specificity. The hCG b-subunit contains two sites of N-glycosylation and 4 sites of O-glycosylation. It is encoded by a cluster of genes that have evolved by duplication from LHB. CGA and CGB expressions are controlled by transcription factors such as AP2 and SP1 that recognize specific response elements in their promoters. hCG expression is regulated by growth factors and cytokines such as EGF, TNFa, activators of the cAMP signalling pathways and by ligands of the nuclear receptor PPARg. In this talk we will stress the importance of hCG glycosylation state, which varies with the stage of pregnancy, its trophoblastic source of production and in the pathology (choriocarcinoma, foeto-placental trisomy 21), modifying its biological activity and clearance. It is well established that hCG is mainly secreted by the endocrine syncytiotrophoblast into the maternal serumwhere it peaks around 10WG. In addition, we have reported that invasive extravillous trophoblasts also secrete hCG, and like choriocarcinoma cells, hyperglycosylated forms of hCG (hCG-H). In addition to its endocrine role, hCG has autocrine and paracrine role. It promotes formation of the syncytiotrophoblast and angiogenesis through LHCG receptor and activation of cAMP and phospholipase C signalling. In contrast, hCG-H does not signal through LHCGR but stimulates trophoblast invasion and angiogenesis by interacting with the TGFs receptor 2. hCG is largely used in antenatal screening and hCG-H may present a good serum marker to screen pregnancy diseases from placental origin. In conclusion, hCG is the major pregnancy glycoprotein hormone, whose maternal concentration and glycan structure change all along pregnancy. Depending on its source of production, the different glycoforms of hCG display different biological activities and functions that are essential for pregnancy outcome. PL2.3. PROFILE OF PLACENTAL GENE EXPRESSION AND PREGNANCY OUTCOME Maris Laan University of Tartu, Tartu, Estonia Placenta is a mammalian pregnancy-specific temporary organ encoded by the fetal genome. Normal implantation process and placental function throughout pregnancy have critical contributions to guarantee the intrauterine development of the offspring and modulation of the maternal gestational metabolism. Also, the following temporal dynamics of placental transcriptional regulation across three trimesters of gestation is of high importance. Aberrant placental gene expression profile and its temporal dynamics may affect the normal pregnancy course. And vice versa, perturbed placental transcriptome may reflect a consequence of impaired implantation, placental development and function. There are numerous reports addressing expression levels of biological candidate genes in placenta in relation to pregnancy complications. Past 10 years have brought along an abundance of studies addressing differential expression of placental genes in pregnancy complications using microarray approach. These analyses have provided novel insights into placental biology and served as discovery studies to identify biomarkers in order to predict pregnancy complications. Next-generation sequencing approach enables to obtain the most detailed coverage of transcriptomes. In order to bring novel insights into the placental transcriptional landscape in normal gestation and study differential gene expression in complicated pregnancies we performed total RNA and small RNA sequencing in 40 samples from term placentas. Samples were selected from the REPROMETA sample collection (Tartu, Estonia) and represent normal pregnancies along with the most clearly defined cases of preeclampsia and gestational diabetes, as well as small (SGA) and large (LGA) for gestational age births. This dataset represents a high quality resource to detect differences in placental transcription over diverse pregnancy courses, to investigation of key molecular changes in placentas from complicated pregnancies and to suggest novel potential biomarkers for monitoring the risk to develop pregnancy complications. PL3.1. INTERCELLULAR COMMUNICATION BY EXOSOMES IN PLACENTA Michel Record INSERM UMR 1037 CRCT Toulouse-Oncopole, TOULOUSE,

Insulin stimulates progesterone secretion to a greater extent than LH in early pregnant buffalo luteal cells cultured in vitro

In the present study, effect of insulin alone or in combination with LH on modulation of progesterone production by early pregnant buffalo luteal cells was reported. Luteal cells were isolated using collagenase and subsequently cultured in Ham’F-12 at 37°C in an atmosphere of 5% CO2 and 95% humidified air. Small luteal cells (SLC, 12–23μ) appeared as spindle shaped with eccentrically placed irregular nucleus, however, large luteal cells (LLC, 25–55μ) were polyhedral or spherical in shape with centrally placed large round nucleus having one or two nucleoli. There was an abundance of cytoplasmic lipid droplets and a greater cytoplasmic to nuclear ratio as compared to SLC. Both small and large luteal cells were positive to 3β-HSD, a marker for steroidogenic capacity. Luteal cells attached to surface within 24h of culture and appeared typical of epithelial cells with numerous cytoplasmic lipid droplets within the cytoplasm. These cells maintained the morphological characteristics throughout the culture period. Luteal cells were treated with insulin (0.05IU/ml) and LH (10ng/ml) alone or in combination for 7 days to study the effect on progesterone production. Morphology of luteal cells did not change with the addition of LH and insulin. Addition of insulin enhanced (P<0.01) basal as well as LH stimulated progesterone production and also minimized loss of cell number by maintaining greater cell populations throughout the culture period as compared to control and LH treatment. In the absence of tropic stimulation, progesterone secretion decreased rapidly in the control group while addition of insulin greatly decreased the rate of decline. The findings of the present study reveal insulin enhances progesterone secretion by the luteal cells indicating its possible role to modulate corpus luteum function in buffalo.

Kisspeptin stimulates progesterone secretion via the Erk1/2 mitogen-activated protein kinase signaling pathway in rat luteal cells

To observe the effect of kisspeptin on the endocrine function of rat luteal cells. Experimental animal study. Research institute laboratory. Immature Sprague-Dawley rats. The expression of kisspeptin and its receptor, GPR54, in immature rat ovaries treated with gonadotropin was observed via immunohistochemistry and real-time polymerase chain reaction. Then recombinant kisspeptin was used to examine the effect on the endocrine function of rat luteal cells. Expression and localization of kisspeptin, localization of GPR54, P and E2 secretion, expression of steroidogenic enzymes, and phosphorylation of Erk1/2. Real-time polymerase chain reaction indicated that ovarian KiSS-1 mRNA levels increased significantly, showing a peak at the luteal period in gonadotropin-primed immature rats. Immunostaining analysis showed that after gonadotropin treatment, kisspeptin was strongly localized in theca cells, the interstitial compartment, and the corpus luteum and that GPR54 protein was clearly detected in the corpus luteum of rat ovaries. In cultured luteal cells, kisspeptin treatment augmented basal and hCG-induced P levels but not E2 production, with concomitant increases detected in the transcript levels of key steroidogenic enzymes (StAR, CYP11A, and 3β-HSD). Furthermore, treatment with kisspeptin increased the phosphorylation of Erk1/2 mitogen-activated protein kinase in cultured luteal cells. The kisspeptin/GPR54 signaling system could stimulate P secretion in rat luteal cells via the Erk1/2 mitogen-activated protein kinase signaling pathway, suggesting an important role for the function of the corpus luteum.

Possible Role of Insulin-Like Factor 3 in the Bovine Corpus Luteum

Insulin-like factor 3 (INSL3) is a local regulator in mammalian gonads, but little is known of its function in bovine corpus luteum (CL). Here, we show that RXFP2 protein, the receptor of INSL3, was expressed throughout the estrous cycle and significantly high at the early luteal stage compared to the regressed luteal stage. INSL3 stimulated progesterone secretion, but not prostaglandin F2α and viability in cultured luteal cells. Together, these results suggest that INSL3 plays a luteotropic role as a local regulator in the bovine CL.

Progesterone Profiles in the Caudal Vena Cava and Jugular Vein in Response to Pulsatile Luteinizing Hormone Stimulation Induced by GnRH Treatment During the Mid-luteal Phase in Lactating Dairy Cows

The aim of this study was to examine whether increased frequency of luteinizing hormone (LH) pulses influences luteal progesterone (P4) secretion by measuring progesterone concentrations at the secreted (caudal vena cava) and circulating levels (jugular vein) in lactating dairy cows. Cows received six intravenous administrations of 2.5 μg of GnRH (gonadorelin acetate, n=4) or 2 ml saline (n=3) at 1-h intervals on 12.4 ± 0.4 (mean ± SE) days after ovulation. Blood samples were collected from the caudal vena cava and jugular vein every 12 min for 12 h (6 h before and after treatment). During the 6 h after treatment, frequency of LH pulses (5.3 ± 0.3 and 3.0 ± 0.0 pulses/6 h) and mean LH concentration (0.50 ± 0.06 and 0.38 ± 0.05 ng/ml) were greater (P<0.05) in GnRH-treated cows than in saline-treated cows. Mean P4 concentration and amplitude of P4 pulses in the caudal vena cava during the 6 h after treatment were greater (P<0.05) in GnRH-treated cows than in saline-treated cows, but the frequency of P4 pulses was not different between the groups. Mean P4 concentration in the jugular vein during the 6 h after treatment was also higher (P<0.05) in GnRH-treated cows than in saline-treated cows (7.0 ± 1.3 and 5.4 ± 0.9 ng/ml). These results indicate that the increased frequency of LH pulses stimulates progesterone secretion from the functional corpus luteum and brings about higher P4 concentrations in the circulating blood in lactating dairy cows.

Preovulatory progesterone concentration associates significantly to follicle number and LH concentration but not to pregnancy rate

Using data from a large prospective randomized controlled trial that evaluated the effect of recombinant LH (rLH) co-administration for ovarian stimulation, the present study assessed whether progesterone concentration on the day of human chorionic gonadotrophin (HCG) administration was associated with pregnancy outcome. Progesterone concentration was measured on stimulation day 1 and on the day of HCG administration in 475 patients who underwent IVF/intracytoplasmic sperm injection treatment following ovarian stimulation with gonadotrophin-releasing hormone (GnRH) agonist and recombinant FSH with or without rLH administration from day 6 of stimulation. There was no significant association between the late-follicular-phase progesterone concentration and the clinical pregnancy rate. However, progesterone concentration was strongly associated with the number of follicles and retrieved oocytes. Late-follicular-phase LH concentration also showed a significant positive association with progesterone concentration ( P = 0.018). Administration of rLH during ovarian stimulation did not affect progesterone concentration. The present study does not support an association between progesterone concentration on the day of HCG administration and the probability of clinical pregnancy in women undergoing ovarian stimulation with GnRH agonists and gonadotrophins for assisted reproduction treatment. Instead, late-follicular-phase progesterone concentration appears to be governed by the number of preovulatory follicles and LH concentration. The present study evaluates whether the sex steroid progesterone produced by the granulosa cells of the developing follicle reflects the likelihood of whether a woman undergoing stimulation with exogenous hormone preparations will achieve pregnancy. The study was unable to demonstrate any association between the progesterone concentration close to ovulation and the chance of conceiving. However, the study found a close association between the number of follicles that developed and the concentration of progesterone at the end of the stimulation period, suggesting that each individual follicle contributed to the circulating progesterone concentration around the time of ovulation. A significant association was found between the pituitary-derived hormone LH and the progesterone concentration, suggesting that LH stimulated progesterone secretion by the follicle.

Influence of nitric oxide onin vitrogrowth, survival, steroidogenesis, and apoptosis of follicle stimulating hormone stimulated buffalo (Bubalus bubalis) preantral follicles

Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, steroidogenesis, and apoptosis of buffalo preantral follicles (PFs) was investigated. PFs (200~250 µm) were isolated by micro-dissection and cultured in 0 (control), 10-3, 10-5, 10-7, and 10-9 M SNP. To examine the reversible effect of SNP, PFs were cultured with 10-5 M SNP + 1 mM Nω-nitro-L-arginine methyl ester (L-NAME) or 1.0 µg hemoglobin (Hb). The results showed that greater concentrations of SNP (10-3, 10-5, 10-7 M) inhibited (p < 0.05) FSH-induced survival, growth, antrum formation, estradiol production, and oocyte apoptosis in a dose-dependent manner. However, a lower dose of SNP (10-9 M) significantly stimulated (p < 0.05) the survival, growth, antrum formation, follicular oocyte maturation, and stimulated progesterone secretion compared to the control. A combination of SNP + L-NAME promoted the inhibitor effect of SNP while a SNP + Hb combination reversed this effect. Nitrate and nitrite concentrations in the culture medium increased (p < 0.05) in a dose-dependent manner according to SNP concentration in the culture medium. At higher concentrations, SNP had a cytotoxic effect leading to follicular oocyte apoptosis whereas lower concentrations have stimulatory effects. In conclusion, NO exerts a dual effect on its development of buffalo PFs depending on the concentration in the culture medium.

Macrophages Are Essential for Maintenance of Corpus Luteum Function in Early Pregnancy.

Macrophages are abundant within the ovary, and have been identified in the corpus luteum (CL) of most species studied, including in rodents and human. Through their secretory products, macrophages are thought to be involved in ovarian tissue remodelling, including luteinization, and in regulating steroidogenesis. Macrophages co-cultured with granulosa or luteal cells act to stimulate progesterone secretion in vitro. To determine the impact of macrophage ablation during early pregnancy, we utilised CD11b-DTR transgenic mice to elicit transient systemic ablation of macrophages by administration of diphtheria toxin (DT). The effects of macrophage ablation during the pre-implantation phase of pregnancy were evaluated. Ablation of macrophages on day 1 pc or day 4 pc caused complete pregnancy loss in all DT-treated CD11b-DTR mice, while DT-treated wild-type mice or PBS-treated CD11b-DTR mice maintained viable pregnancies. Macrophage ablation on day 3 pc significantly reduced serum progesterone (P4) levels to 40% of control levels when measured 24 h later. Administration of exogenous P4 on each of day 4-7 pc prevented fetal loss in DT-treated CD11b-DTR mice, and pregnancy progressed with viable pups at late gestation, while no pregnancies remained viable in DT-treated mice administered vehicle only. Immunohistochemical evaluation with anti-CD31 mAb showed that macrophage ablation in the CD11b-DTR mice resulted in disappearance of endothelial cells from within the CL. This suggests that macrophages are essential for support of endothelial cells within the CL, either by provision of trophic support and/or by macrophage transdifferentiation into endothelial cells. In conclusion, these data indicate a critical role for macrophages in corpus luteum development and steroidogenic function in early pregnancy. Given that various nutritional and immune stressors can profoundly affect macrophage numbers and behaviour, it seems reasonable to speculate that macrophage-regulated CL development is a vulnerable event and may contribute to some forms of unexplained infertility. (platform)

Luteinizing Hormone Stimulates Mammalian Target of Rapamycin Signaling in Bovine Luteal Cells via Pathways Independent of AKT and Mitogen-Activated Protein Kinase: Modulation of Glycogen Synthase Kinase 3 and AMP-Activated Protein Kinase

LH stimulates the production of cAMP in luteal cells, which leads to the production of progesterone, a hormone critical for the maintenance of pregnancy. The mammalian target of rapamycin (MTOR) signaling cascade has recently been examined in ovarian follicles where it regulates granulosa cell proliferation and differentiation. This study examined the actions of LH on the regulation and possible role of the MTOR signaling pathway in primary cultures of bovine corpus luteum cells. Herein, we demonstrate that activation of the LH receptor stimulates the phosphorylation of the MTOR substrates ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1. The actions of LH were mimicked by forskolin and 8-bromo-cAMP. LH did not increase AKT or MAPK1/3 phosphorylation. Studies with pathway-specific inhibitors demonstrated that the MAPK kinase 1 (MAP2K1)/MAPK or phosphatidylinositol 3-kinase/AKT signaling pathways were not required for LH-stimulated MTOR/S6K1 activity. However, LH decreased the activity of glycogen synthase kinase 3Beta (GSK3B) and AMP-activated protein kinase (AMPK). The actions of LH on MTOR/S6K1 were mimicked by agents that modulated GSK3B and AMPK activity. The ability of LH to stimulate progesterone secretion was not prevented by rapamycin, a MTOR inhibitor. In contrast, activation of AMPK inhibited LH-stimulated MTOR/S6K1 signaling and progesterone secretion. In summary, the LH receptor stimulates a unique series of intracellular signals to activate MTOR/S6K1 signaling. Furthermore, LH-directed changes in AMPK and GSK3B phosphorylation appear to exert a greater impact on progesterone synthesis in the corpus luteum than rapamycin-sensitive MTOR-mediated events.

Effects of unsaturated fatty acids on progesterone secretion and selected protein kinases in goat granulosa cells

Previous studies in cattle have shown influences of dietary unsaturated fatty acid (UFA) supplementation on ovarian function. However, it is unclear whether these UFA exert direct or indirect effects on ovarian steroid production or their mechanisms of action. We have recently shown that 5′AMP-activated protein kinase (AMPK) regulates progesterone secretion through mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK ERK1/2) in rodent granulosa cells. Here, we investigated the effects of 3 UFAs, oleic acid (OA), linoleic acid (LA), and α-linolenic acid (ALA) on progesterone secretion in goat granulosa cells. Finally, we examined the effects of UFAs on MAPK ERK1/2 and AMPK phosphorylation in these granulosa cells. Oleic acid and LA (10 μM each), but not ALA (100 μM), increased progesterone secretion ( P < 0.05) in the presence or absence of insulin-like growth factor (IGF)-1 (10 -8 M) or FSH (5 × 10 −8 M). The different AMPK subunits, except for γ3, are present in the goat ovary. Treatment with metformin (10 mM), an activator of AMPK, increased AMPK phosphorylation ( P < 0.05) and reduced progesterone secretion by 50% ( P < 0.05) in the basal state and in response to IGF-1 or FSH in goat granulosa cells. Oleic acid and LA had no effect on AMPK phosphorylation, whereas they rapidly increased MAPK ERK1/2 phosphorylation ( P < 0.05). Finally, U0126, a MAPK ERK1/2 inhibitor, decreased OA- and LA-induced progesterone secretion ( P < 0.05), suggesting that these UFAs could stimulate progesterone secretion partly through MAPK ERK1/2 in the absence of IGF-1 and FSH in goat granulosa cells. The involvement of AMPK in this process remains to be demonstrated. Taken together, some fatty acids could improve ovarian steroidogenesis through the MAPK ERK1/2 signaling pathway and, consequently, have beneficial effects on goat fertility.