Induction of right open reading frame kinase 3 (RIOK3) during ovulation and luteinisation in rat ovary.

Atypical protein serine kinase RIOK3 is involved in cellular invasion and survival. The spatiotemporal expression pattern and regulatory mechanisms controlling expression of Riok3 were investigated in the rat ovary during the periovulatory period. Immature female rats (22-23 days old) were treated with pregnant mare's serum gonadotropin (PMSG) to stimulate follicular development, followed 48h later by injection with human chorionic gonadotrophin (hCG). Ovaries, granulosa cells, or theca-interstitial cells were collected at various times after hCG administration. Both real-time polymerase chain reaction (PCR) and in situ hybridisation analysis revealed that Riok3 was highly induced in both granulosa cells and theca-interstitial cells by hCG. Riok3 expression was induced in theca-interstitial cells at 4h after hCG. However, the expression of Riok3 mRNA was stimulated in granulosa cells at 8h. Both protein kinase C inhibitor (GF109203) and the protein kinase A inhibitor (H89) could block the stimulation of Riok3 mRNA by hCG. Furthermore, Riok3 induction is dependent on new protein synthesis. Inhibition of prostaglandin synthesis or progesterone action did not alter Riok3 mRNA expression, whereas inhibition of the epidermal growth factor (EGF) pathway downregulated Riok3 expression. In conclusion, our findings suggest that the induction of the RIOK3 may be important for ovulation and luteinisation.

LH-induced Transcriptional Regulation of Klf4 Expression in Granulosa Cells Occurs via the cAMP/PKA Pathway and Requires a Putative Sp1 Binding Site.

Krüppel-like factor 4 (Klf4) plays an important role in the transition from proliferation to differentiation in a wide variety of cells. Previous studies demonstrated its critical role in the luteal transition of preovulatory granulosa cells (GCs). This study used cultured rat preovulatory GCs to investigate the mechanism by which luteinizing hormone (LH) regulates Klf4 gene expression. Klf4 mRNA and protein were rapidly and transiently induced by LH treatment, reaching peak levels after 45 min and declining to basal levels by 3 h. Pretreatment with the protein synthesis inhibitor cycloheximide had no effect on LH-stimulated Klf4 expression, indicating that Klf4 is an immediate early gene in response to LH. To investigate the signaling pathway involved in LH-induced Klf4 regulation, the protein kinase A (PKA) and protein kinase C (PKC) pathways were evaluated. A-kinase agonists, but not a C-kinase agonist, mimicked LH in inducing Klf4 transcription. In addition, specific inhibitors of A-kinase abolished the stimulatory effect of LH on Klf4 expression. Truncation of a Klf4 expression construct to -715 bp (pKlf4-715/luc) had no effect on transcriptional activity, whereas deletion to -402 bp (pKlf4-402/luc) dramatically reduced it. ChIP analysis revealed in vivo binding of endogenous Sp1 to the -715/-500 bp region and maximal transcriptional responsiveness to LH required the Sp1 binding element at -698/-688 bp, which is highly conserved in mice, rats, and humans. These findings demonstrate that Klf4 is activated by LH via the cAMP/PKA pathway and a putative Sp1 binding element at -698/-688 bp is indispensable for activation and suggest that Klf4 could be a target for strategies for treating luteal phase insufficiency induced by an aberrant response to the LH surge.

MRL/MpJ mice produce more oocytes and exhibit impaired fertilisation and accelerated luteinisation after superovulation treatment.

MRL/MpJ mice exhibit distinct phenotypes in several biological processes, including wound healing. Herein we report two unique phenotypes in the female reproductive system of MRL/MpJ mice that affect ovulation and luteinisation. We found that superovulation treatment resulted in the production of significantly more oocytes in MRL/MpJ than C57BL/6 mice (71.0±13.4 vs 26.8±2.8 respectively). However, no exon mutations were detected in genes coding for female reproductive hormones or their receptors in MRL/MpJ mice. In addition, the fertilisation rate was lower for ovulated oocytes from MRL/MpJ than C57BL/6 mice, with most of the fertilised oocytes showing abnormal morphology, characterised by deformation and cytolysis. Histological tracing of luteinisation showed that MRL/MpJ mice formed corpora lutea within 36h after ovulation, whereas C57BL/6 mice were still at the corpora haemorrhagica formation stage after 36h. The balance between the expression of matrix metalloproteinases and their tissue inhibitors shifted towards the former earlier after ovulation in MRL/MpJ than C57BL/6 mice. This result indicates a possible link between accelerated extracellular matrix remodelling in the ovulated or ruptured follicles and luteinisation in MRL/MpJ mice. Together, these findings reveal novel phenotypes in MRL/MpJ mice that provide novel insights into reproductive biology.

Expression and regulation of high mobility group AT-hook 1 (HMGA1) during ovulation and luteinisation in rat ovary.

High mobility group AT-hook 1 (HMGA1) is able to regulate gene expression and function as a tumour suppressor. The spatiotemporal expression pattern of HMGA1 was investigated in this study. Immature female rats (22-23 days old) were treated with 10IU, s.c., pregnant mare's serum gonadotrophin to stimulate follicular development, followed 48h later by injection with 5IU, s.c., human chorionic gonadotrophin (hCG). Whole ovaries or granulosa cells were collected at various times after hCG administration (n=3 per time point). Real-time polymerase chain reaction and western blot analysis revealed that HMGA1 was highly stimulated in the ovary by 4-12h after hCG treatment. In situ hybridisation analysis demonstrated that Hmga1 mRNA expression was induced in granulosa cells between 8 and 12h after hCG treatment. There was negligible Hmga1 mRNA signal observed in newly forming corpora lutea. In addition, the data indicated that both the protein kinase (PK) A and PKC pathways regulated Hmga1 expression in rat granulosa cells. In rat granulosa cell cultures, upregulation of Hmga1 was dependent on new protein synthesis because Hmga1 was inhibited by cycloheximide. Furthermore, Hmga1 mRNA expression in rat granulosa cell cultures was inhibited by AG1478, whereas NS398 and RU486 had no effect, suggesting that Hmga1 expression was regulated, in part, by the epidermal growth factor pathway. In summary, the findings of this study suggest that induction of Hmga1 may be important for theca and granulosa cell differentiation into luteal cells.

Evaluating the electrical impedance and mucus-related gene expression of uterine endometrial tissues in mares.

We investigated the electrical impedance of the reproductive tracts (vagina and uterine endometrial tissues) and the expression of mucus-related genes to identify the stage of the estrous cycle in mares. We first examined vaginal impedance in native Hokkaido mares during their estrous cycle and found no significant differences. However, impedance levels tended to decrease towards ovulation. Furthermore, we investigated the estrous cycle by measuring the electrical impedance of the uterine endometrial tissues obtained from carcasses of mares. We found that impedance levels in the endometrial tissues decreased in the regressed phase of the corpus luteum (CL). Expression of mucus-related genes (ATP1A1, CFTR, AQP3, and AQP5) varied at different stages of the estrous cycle. Among them, AQP3 expression was consistent with previous reports. We concluded that electrical impedance in the uterine endometrial tissues of mares could be potentially used to verify the presence of active CL in horses for experimental purposes. However, further studies are needed to determine the reference value and to identify the day of the estrous cycle in mares.

Oxidized low-density lipoprotein suppresses mouse granulosa cell differentiation through disruption of the hypoxia-inducible factor 1 pathway.

Obesity predisposes women to reproductive disorders. One symptom of obesity in women is higher levels of oxidized Low-density lipoprotein (oxLDL) in serum and preovulatory follicles. The present study was designed to test the hypothesis that oxLDL might impair follicle differentiation and luteinization. Given that Hypoxia-inducible factor 1 (HIF1) plays crucial roles in supporting follicle differentiation and luteinization in mammals, we focused on oxLDL-mediated events that may affect the HIF1 pathway. We report that exposure to oxLDL diminished the expression of HIF1α and its target genes and suppressed the differentiation of mouse luteinized granulosa cells following induction by human Chorionic gonadotophin (hCG) under hypoxic conditions (1% oxygen). Significantly, the proteasome inhibitor MG-132 prevented this oxLDL-attenuation differentiation phenotype by blocking HIF1α degradation. Together, these findings suggest that suppression of granulosa cell differentiation by oxLDL, via HIF1α down-regulation, may contribute the negative effects of obesity on female fertility.

Temporal effects of human chorionic gonadotropin on expression of the circadian genes and steroidogenesis-related genes in human luteinized granulosa cells.

OBJECTIVE: It has been shown in animal models that circadian clock exists in corpora luteum which is essential for maintaining pregnancy. However, it is unknown whether circadian clock exists in corpora luteum and its relation with steroidogenesis in human ovary. STUDY DESIGN: Human luteinized granulosa cells from patients who underwent in vitro fertilization treatment were purified and cultured in vitro. Accumulation patterns of circadian gene and steroidogenesis-related gene mRNAs in human luteinized granulosa cells were observed during the 48 hours after treatment with human chorionic gonadotropin (hCG) by quantitative PCR. RESULTS: We found that the circadian genes CLOCK, PER2, and BMAL1 were expressed in cultured human luteinized granulosa cells. Among these genes, only expression of PER2 displayed oscillating patterns with a 16-h period in these cells after stimulation by hCG. Expression of CLOCK and BMAL1 did not show significant oscillating patterns. Expression of the steroidal acute regulatory protein (STAR) gene showed an oscillating pattern that was similar to that of PER2. Expression of CYP11A1, HSD3B2, and CYP19A1 increased significantly after hCG stimulation; however, none of these genes displayed significant oscillating patterns. CONCLUSIONS: Molecular circadian clock exists in human luteinized granulosa cells and may be related with steroidogenesis in human ovary.

Growth Arrest Specific-1 (GAS1) Is a C/EBP Target Gene That Functions in Ovulation and Corpus Luteum Formation in Mice.

Ovulation and luteinization are initiated in preovulatory follicles by the luteinizing hormone (LH) surge; however, the signaling events that mediate LH actions in these follicles remain incompletely defined. Two key transcription factors that are targets of LH surge are C/EBPalpha and C/EBPbeta, and their depletion in granulosa cells results in complete infertility. Microarray analyses of these mutant mice revealed altered expression of a number of genes, including growth arrest specific-1 (Gas1). To investigate functions of Gas1 in ovulation- and luteinization-related processes, we crossed Cyp19a1-Cre and Gas1(flox/flox) mice to conditionally delete Gas1 in granulosa and cumulus cells. While expression of Gas1 is dramatically increased in granulosa and cumulus cells around 12-16 h post-human chorionic gonadotropin (hCG) stimulation in wild-type mice, this increase is abolished in Cebpa/b double mutant and in Gas1 mutant mice. GAS1 is also dynamically expressed in stromal cells of the ovary independent of C/EBPalpha/beta. Female Gas1 mutant mice are fertile, exhibit enhanced rates of ovulation, increased fertility, and higher levels of Areg and Lhcgr mRNA in granulosa cells. The morphological appearance and vascularization of corpora lutea appeared normal in these mutant females. Interestingly, levels of mRNA for a number of genes (Cyp11a1, Star, Wnt4, Prlr, Cd52, and Sema3a) associated with luteinization are decreased in corpora lutea of Gas1 mutant mice as compared with controls at 24 h post-hCG; these differences were no longer detectable by 48 h post-hCG. The C/EBP target Gas1 is induced in granulosa cells and is associated with ovulation and luteinization.

Apelin (APLN) and Apelin Receptor (APLNR) in Human Ovary: Expression, Signaling, and Regulation of Steroidogenesis in Primary Human Luteinized Granulosa Cells.

Apelin (APLN) is a recently discovered adipokine involved in the regulation of various metabolic functions. Its receptor, APLNR, is expressed in reproductive tissues, however, its role in human ovarian cells is unknown. In this study, we identified APLN and APLNR in human ovarian follicles and analyzed their expression in granulosa cells and follicular fluid obtained from obese and nonobese patients, with or without polycystic ovary syndrome (PCOS). We also investigated the effect of APLN on steroidogenesis in cultured human luteinized granulosa cells (hGCs) from nonobese patients without PCOS. Using RT-PCR and immunoblotting, we found that APLN and APLNR were expressed in hGCs and cumulus and theca cells. We confirmed these data immunohistochemically and observed that APLNR and APLN are present in human oocytes at different stages of follicular development. In patients with PCOS, we observed that follicular fluid APLN concentration and granulosa cell APLN and APLNR mRNA expression was higher than that observed in control patients. In cultured hGCs from nonobese patients without PCOS, insulin-like growth factor 1 (IGF1) increased APLNR expression, and recombinant human APLN (APLN-13 and APLN-17) increased both basal and IGF1-induced steroid secretion. These effects on steroid production were reversed when cultured in the presence of ML221, an APLNR antagonist, which was associated with an increased 3beta-hydrosteroid dehydrogenase (HSD3B) protein concentration. We showed that these effects were dependent on the activation of the AKT and MAPK3/1 pathways using pharmacological inhibitors. Our results show that APLN and APLNR are present in human ovarian cells and APLN increases IGF1-induced steroidogenesis in granulosa cells through an increase in HSD3B protein expression and activation of the MAPK3/1 and Akt pathways. Therefore, APLN and APLNR may play a role in human follicular development and the pathogenesis of PCOS.

Expressions of aquaporin family in human luteinized granulosa cells and their correlations with IVF outcomes.

STUDY QUESTION: Are mRNAs for specific aquaporins (AQPs) expressed in human luteinized granulosa cells (GCs) and are their expression levels correlated with in vitro fertilization (IVF) outcomes? SUMMARY ANSWER: The mRNAs of AQP1-7, 9, 11, and 12 were expressed in human luteinized GCs; the level of AQP1 mRNA was negatively associated with retrieved oocyte number and the level of AQP7 mRNA was positively associated with fertilization rate. WHAT IS KNOWN ALREADY: mRNAs of AQP1-4 and AQP9 have been detected in human GCs. STUDY DESIGN, SIZE, DURATION: Prospective observational study involving 111 women undergoing a stimulated IVF cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS: In a preliminary experiment on 27 women, the mRNA expression of AQP0-12 in GCs was explored by RT-PCR. In the main experiment, luteinized GCs were obtained from 111 women at the time of oocyte retrieval of whom 102 had an embryo replacement. Real-time quantitative RT-PCR (qRT-PCR) was performed to quantify the mRNA level of AQP1-7, 9, 11, and 12. The mRNA for luteinizing hormone receptor (LHR) and steroidogenic acute regulatory protein (StAR) were also quantified by qRT-PCR. MAIN RESULTS AND THE ROLE OF CHANCE: mRNAs for AQP0, 8 and 10 were not detected in the preliminary experiment. In samples from 111 women, retrieved oocyte number was negatively associated with the mRNA levels of AQP1, 4, 6, and 11 and LHR (r = -0.311, r = -0.233, r = -0.203, r = -0.194, and r = -0.202, respectively, P < 0.05 for each), however, after adjustment for woman's age and serum anti-Müllerian hormone (AMH) levels, only correlation with AQP1 was found (r = -0.299, P < 0.05). BMI was negatively associated (after adjustment for age) with the mRNA level of AQP7 (r = -0.259, P < 0.05). Fertilization rate was positively associated with the mRNA level of AQP7 (r = 0.269, P < 0.05). The number or quality of embryos or clinical pregnancy was not associated with the mRNA levels of any of ten AQP subtypes. The mRNA levels for the ten AQP subtypes were correlated positively with LHR expression but negatively with StAR expression. Amongst high responders (oocyte number ≥14), the mRNA levels of AQP11 (1.4 ± 0.7 versus 1.7 ± 0.6) and LHR (1.3 ± 0.7 versus 1.7 ± 0.7) were significantly lower in the group with PCOS than in the non-PCOS group. LIMITATIONS, REASONS FOR CAUTION: A relative small number of subjects in PCOS group is the main limitation of our study. P-values were not corrected for multiple comparisons. WIDER IMPLICATION OF THE FINDINGS: AQP1 may be one of the factors that modulate individual ovarian response to exogenous gonadotrophin. The mRNA level of AQP7 was positively associated with fertilization rate, which is a surrogate marker of oocyte competence, thus expression of AQP7 could be a marker for adequate folliculogenesis and healthy oocytes. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grant no. A120043 from the Korea Health Care Technology R&D Project, Ministry of Health and Welfare, Korea. None of the authors has any conflict of interest to declare.

GATA4 and GATA6 Knockdown During Luteinization Inhibits Progesterone Production and Gonadotropin Responsiveness in the Corpus Luteum of Female Mice.

The surge of luteinizing hormone triggers the genomic reprogramming, cell differentiation, and tissue remodeling of the ovulated follicle, leading to the formation of the corpus luteum. During this process, called luteinization, follicular granulosa cells begin expressing a new set of genes that allow the resulting luteal cells to survive in a vastly different hormonal environment and to produce the extremely high amounts of progesterone (P4) needed to sustain pregnancy. To better understand the molecular mechanisms involved in the regulation of luteal P4 production in vivo, the transcription factors GATA4 and GATA6 were knocked down in the corpus luteum by crossing mice carrying Gata4 and Gata6 floxed genes with mice carrying Cre recombinase fused to the progesterone receptor. This receptor is expressed exclusively in granulosa cells after the luteinizing hormone surge, leading to recombination of floxed genes during follicle luteinization. The findings demonstrated that GATA4 and GATA6 are essential for female fertility, whereas targeting either factor alone causes subfertility. When compared to control mice, serum P4 levels and luteal expression of key steroidogenic genes were significantly lower in conditional knockdown mice. The results also showed that GATA4 and GATA6 are required for the expression of the receptors for prolactin and luteinizing hormone, the main luteotropic hormones in mice. The findings demonstrate that GATA4 and GATA6 are crucial regulators of luteal steroidogenesis and are required for the normal response of luteal cells to luteotropins.

Augmentation of Metastin/Kisspeptin mRNA Expression by the Proestrous Luteinizing Hormone Surge in Granulosa Cells of Rats: Implications for Luteinization.

Variations in mRNA levels and sources of metastin/kisspeptin, neurokinin B (NKB), dynorphin, and kisspeptin receptor GPR54 were examined in the ovaries of cycling rats. Kisspeptin and dynorphin mRNAs dramatically increased at 2000 h of the proestrous day. NKB mRNA also increased, but the peak was delayed by 6 h. GPR54 mRNA declined inversely with kisspeptin. Whole-ovary expressions of kisspeptin and dynorphin mRNAs, but not of NKB mRNA, were augmented by the administration of human chorionic gonadotropin (hCG). By means of laser-capture microdissection, kisspeptin mRNA was shown mostly in follicles at 2000 h of proestrus, whereas NKB and dynorphin were expressed mainly in interstitial tissues. GPR54 mRNA was detected equally in follicles, corpora lutea, and interstitial tissues. The hCG stimulated the follicular expression of kisspeptin and interstitial tissue expression of dynorphin mRNA. In primary cultures of granulosa cells prepared from equine chorionic gonadotropin-pretreated immature rats, hCG stimulated the expression of kisspeptin, dynorphin, and NKB mRNAs. Distortion of the corpus luteum and surrounding tissue borders was sometimes seen after intra-ovarian bursa administration of kisspeptin antagonist p234 for 3 days from proestrus. Progesterone production stimulated by hCG in granulosa cell culture was suppressed by p234. These data demonstrate that significant amounts of kisspeptin are synthesized in granulosa cells and dynorphin in interstitial tissues, in response to the proestrous luteinizing hormone surge, whereas granulosa cells also contain dynorphin and NKB, suggesting at least a role for kisspeptin in the luteinization of granulosa cells.

Atrazine enhances progesterone production through activation of multiple signaling pathways in FSH-stimulated rat granulosa cells: evidence for premature luteinization.

Premature luteinization is a possible cause of infertility in women. It is currently unknown whether environmental chemicals can induce changes associated with premature luteinization. Using rat granulosa cells (GC) in vitro, we demonstrated that exposure to atrazine (ATR), a widely used herbicide, causes GC phenotype that resembles that of human premature luteinization. At the end of the 48-h stimulation with FSH, ATR-exposed GC showed (1) higher levels of progesterone, (2) overexpression of luteal markers (Star and Cyp11a1), and (3) an increase in progesterone:estradiol ratio above 1. Mechanistic experiments were conducted to understand the signaling events engaged by ATR that lead to this phenotype. Western blot analysis revealed prolonged phosphorylation of protein kinase B (AKT) and cAMP response element-binding protein (CREB) in ATR- and FSH-exposed GC. An increased level of ERK1/2-dependent transcriptional factor CCATT/enhancer-binding protein beta (CEBPB) was observed after 4 h of ATR exposure. Inhibitors of PI3K (wortmannin) and MEK (U0126) prevented ATR-induced rise in progesterone level and expression of luteal markers in FSH-stimulated GC. Atrazine intensified AKT and CEBPB signaling and caused Star overexpression in forskolin-stimulated GC but not in epidermal growth factor (EGF)-stimulated GC. In the presence of rolipram, a specific inhibitor of phosphodiesterase 4 (PDE4), ATR was not able to further elevate AKT phosphorylation, CEBPB protein level, and Star mRNA in FSH-stimulated GC, suggesting that ATR inhibits PDE4. Overall, this study showed that ATR acts as a FSH sensitizer leading to enhanced cAMP, AKT, and CEBPB signaling and progesterone biosynthesis, which promotes premature luteinization phenotype in GC.

Localization of luteinizing hormone receptor protein in the human ovary.

The luteinizing hormone receptor (LHR) plays a pivotal role during follicular development. Consequently, its expression pattern is of major importance for research and has clinical implications. Despite the accumulated information regarding LHR expression patterns, our understanding of its expression in the human ovary, specifically at the protein level, is incomplete. Therefore, our aim was to determine the LHR protein localization and expression pattern in the human ovary. We examined the presence of LHR by immunohistochemical staining of human ovaries and western blots of mural granulosa and cumulus cells aspirated during IVF treatments. We were not able to detect LHR protein staining in primordial or primary follicles. We observed equivocal positive staining in granulosa cells and theca cells of secondary follicles. The first appearance of a clear signal of LHR protein was observed in granulosa cells and theca cells of small antral follicles, and there was evidence of increasing LHR production as the follicles mature to the pre-ovulatory stage. After ovulation, LHR protein was ubiquitously produced in the corpus luteum. To confirm the expression pattern in granulosa cells and cumulus cells, we performed western blots and found that LHR expression was stronger in granulosa cells than in cumulus cells, with the later demonstrating low, but still significant, amounts of LHR protein. In summary, we conclude that LHR protein starts to appear on granulosa cells and theca cells of early antral follicles, and low but significant expression of LHR exists also in the cumulus cells. These results may have implications for the future design of clinical protocols and culture mediums for in vitro fertilization and especially in vitro maturation of oocytes.

Hypoxia promotes progesterone synthesis during luteinization in bovine granulosa cells.

To determine whether hypoxia has an effect on luteinization, we examined the influence of hypoxia on a model of bovine luteinizing and non-luteinizing granulosa cell culture. The granulosa cells were obtained from small antral follicles (≤ 6 mm in diameter). To induce luteinization, the cells were treated for 24 h with insulin (2 µg/ml), forskolin (10 µM) or insulin in combination with forskolin at 20% O2. After 24 h, progesterone (P4) production was higher in the treated cells, which we defined as luteinizing granulosa cells, than in non-treated cells, which we defined as non-luteinizing granulosa cells. P4 production by non-luteinizing granulosa cells was not affected by hypoxia (24 h at 10% and 5% O2), while P4 production by granulosa cells treated with insulin in combination with forskolin was significantly increased under hypoxia (24 h at 10% and 5% O2). Because hypoxia affected P4 production by the luteinizing granulosa cells but not by the non-luteinizing granulosa cells, hypoxia seems to promote P4 production during, rather than before, luteinization. In the cells treated with insulin in combination with forskolin, mRNA and protein expression of steroidogenic acute regulatory protein (StAR) and protein expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) increased under 10% O2, while mRNA and protein expressions of key protein and enzymes in P4 biosynthesis did not increase under 5% O2. The overall results suggest that hypoxia plays a role in progressing and completing the luteinization by enhancing P4 production through StAR as well as 3ß-HSD expressions in the early time of establishing the corpus luteum.

Progesterone antagonist, RU486, represses LHCGR expression and LH/hCG signaling in cultured luteinized human mural granulosa cells.

Progesterone, the main steroid synthesized by the corpus luteum (CL), prepares the uterus for implantation, maintains the CL survival, and induces progesterone auto-secretion. However, the molecular mechanisms involving the progesterone auto-secretion pathways at the luteal phase are not fully understood, especially in humans. We aim to study the molecular mechanism of the progesterone pathway in human granulosa cells. Our model system consists of luteinized human-mural-granulosa-cells (hmGCs) obtained from follicles aspirated during in vitro fertilization (IVF) procedures. hmGCs were seeded in culture and were subjected to different hormonal treatments. mRNA levels were analyzed by quantitative real-time PCR (qRT-PCR). Progesterone levels were measured by enzyme immunoassay (EIA). We show that exposure of luteinized hmGCs to the progesterone receptor antagonist, RU486 (mifepristone), resulted in inhibition of LHCGR, LH/hCG target genes and progesterone secretion. Exposure of hmGCs to medium that was incubated with hmGCs for 4 d - conditioned medium (CM), which contain 150 ± 7.5 nM progesterone, resulted in induction of LHCGR and LH/hCG target genes, which was blocked by RU486. In addition, RU486 inhibited some of the progesterone biosynthesis pathway genes. Our results revealed a novel mechanism of the progesterone antagonist pathway in the luteal granulosa cells and emphasis the fundamental role of progesterone in the early luteal phase.

Misregulated progesterone secretion and impaired pregnancy in Cyp11a1 transgenic mice.

Normal pregnancy is supported by increased levels of progesterone (P4), which is secreted from ovarian luteal cells via enzymatic steps catalyzed by P450scc (CYP11A1) and HSD3B. The development and maintenance of corpora lutea during pregnancy, however, are less well understood. Here we used Cyp11a1 transgenic mice to delineate the steps of luteal cell differentiation during pregnancy. Cyp11a1 in a bacterial artificial chromosome was injected into mouse embryos to generate transgenic mice with transgene expression that recapitulated endogenous Cyp11a1 expression. Cyp11a1 transgenic females displayed reduced pregnancy rate, impaired implantation and placentation, and decreased litter size in utero, although they produced comparable numbers of blastocysts. The differentiation of transgenic luteal cells was delayed during early pregnancy as shown by the delayed activation of genes involved in steroidogenesis and cholesterol availability. Luteal cell mitochondria were elongated, and their numbers were reduced, with morphology and numbers similar to those observed in granulosa cells. Transgenic luteal cells accumulated lipid droplets and secreted less progesterone during early pregnancy. The progesterone level returned to normal on gestation day 9 but was not properly withdrawn at term, leading to delayed stillbirth. P4 supplementation rescued the implantation rates but not the ovarian defects. Thus, overexpression of Cyp11a1 disrupts normal development of the corpus luteum, leading to progesterone insufficiency during early pregnancy. Misregulation of the progesterone production in Cyp11a1 transgenic mice during pregnancy resulted in aberrant implantation, anomalous placentation, and delayed parturition.

Effect of feeding level on luteal function and progesterone concentration in the vena cava during early pregnancy in gilts.

This study assessed the effect of feeding level on progesterone concentration in the caudal vena cava during early pregnancy in gilts. Twenty-four Landrace gilts were allocated to either a high (2.8±0.02) or a low (1.5±0.01 kg day⁻¹) feeding level at Day 0 of pregnancy. Serial blood samples were collected every 15 min for 3 h before and 3 h after feeding on Days 6 and 9 of pregnancy. Embryo survival and development as well as in vitro luteal progesterone production were assessed at Day 10 of pregnancy. Progesterone concentration in the vena cava was pulsatile with gilts on the high feeding level having more pulses compared with Low gilts on Day 9 of pregnancy (P<0.05). On Day 6 the number of pulses did not differ significantly between treatments; however, the average progesterone concentration in the vena cava tended to be higher in the gilts on the high feeding level (P<0.10). Embryo survival at Day 10 was 92±3% for High gilts compared with 77±3% for Low gilts (P<0.05). No difference in embryo development between the treatments was seen. There was no difference between treatments in in vitro secretion of progesterone by luteal tissue. In conclusion, a high plane of nutrition positively affects progesterone secretion by the ovaries in early pregnancy.

The concentration of human follicular fluid stromal cell-derived factor-1 is correlated with luteinization in follicles.

Stromal cell-derived factor-1 (SDF-1/CXCL12) and vascular endothelial growth factor (VEGF) are angiogenic factors that have possible roles in ovarian function. The objectives of this study were to investigate the association between the individual concentrations of SDF-1 and VEGF and sex steroid hormones in human preovulatory follicles and to verify the SDF-1 expression in ovarian follicles. Follicular fluid (FF) and luteinizing granulosa cells (LGCs) were collected from follicles at the time of oocyte retrieval. The concentrations of SDF-1, VEGF, estradiol (E2) and progesterone (P4) were determined by biochemical assay. The expression levels of SDF-1 mRNA and protein were analyzed by RT-PCR and immunohistochemical analysis, respectively. A total of 177 follicles were analyzed. The FF concentrations of SDF-1 and VEGF positively correlated with P4 concentrations (r = 0.457 and p < 0.01, r = 0.698 and p < 0.01, respectively), but did not correlate with E2 concentrations in FF. Furthermore, we confirmed that SDF-1 mRNA was expressed in LGCs and SDF-1 protein is present in the granulosa cells of the human ovary. Our findings suggest that SDF-1, as well as VEGF, may play important modulatory roles in early luteinization of human preovulatory follicles.

Effect of the metabolic environment at key stages of follicle development in cattle: focus on steroid biosynthesis.

Cellular mechanisms that contribute to low estradiol concentrations produced by the preovulatory ovarian follicle in cattle with a compromised metabolic status are largely unknown. To gain insight into the main metabolic mechanisms affecting preovulatory follicle function, two different animal models were used. Experiment 1 compared Holstein-Friesian nonlactating heifers (n = 17) and lactating cows (n = 16) at three stages of preovulatory follicle development: 1) newly selected dominant follicle in the luteal phase (Selection), 2) follicular phase before the LH surge (Differentiation), and 3) preovulatory phase after the LH surge (Luteinization). Experiment 2 compared newly selected dominant follicles in the luteal phase in beef heifers fed a diet of 1.2 times maintenance (M, n = 8) or 0.4 M (n = 11). Lactating cows and 0.4 M beef heifers had higher concentrations of ß-hydroxybutyrate, and lower concentrations of glucose, insulin, and IGF-I compared with dairy heifers and 1.2 M beef heifers, respectively. In lactating cows this altered metabolic environment was associated with reduced dominant follicle estradiol and progesterone synthesis during Differentiation and Luteinization, respectively, and in 0.4 M beef heifers with reduced dominant follicle estradiol synthesis. Using a combination of RNA sequencing, Ingenuity Pathway Analysis, and qRT-PCR validation, we identified several important molecular markers involved in steroid biosynthesis, such as the expression of steroidogenic acute regulatory protein (STAR) within developing dominant follicles, to be downregulated by the catabolic state. Based on this, we propose that the adverse metabolic environment caused by lactation or nutritional restriction decreases preovulatory follicle function mainly by affecting cholesterol transport into the mitochondria to initiate steroidogenesis.