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.

Integrated Analysis of Transcriptome and Histone Modifications in Granulosa Cells During Ovulation in Female Mice.

The ovulatory luteinizing hormone (LH) surge induces rapid changes of gene expression and cellular functions in granulosa cells (GCs) undergoing luteinization. However, it remains unclear how the changes in genome-wide gene expression are regulated. H3K4me3 histone modifications are involved in the rapid alteration of gene expression. In this study, we investigated genome-wide changes of transcriptome and H3K4me3 status in mouse GCs undergoing luteinization. GCs were obtained from mice treated with equine chorionic gonadotropin (hCG) before, 4 hours, and 12 hours after human chorionic gonadotropin injection. RNA-sequencing identified a number of upregulated and downregulated genes, which could be classified into 8 patterns according to the time-course changes of gene expression. Many genes were transiently upregulated or downregulated at 4 hours after hCG stimulation. Gene Ontology terms associated with these genes included steroidogenesis, ovulation, cumulus-oocyte complex (COC) expansion, angiogenesis, immune system, reactive oxygen species (ROS) metabolism, inflammatory response, metabolism, and autophagy. The cellular functions of DNA repair and cell growth were newly identified as being activated during ovulation. Chromatin immunoprecipitation-sequencing revealed a genome-wide and rapid change in H3K4me3 during ovulation. Integration of transcriptome and H3K4me3 data identified many H3K4me3-associated genes that are involved in steroidogenesis, ovulation, COC expansion, angiogenesis, inflammatory response, immune system, ROS metabolism, lipid and glucose metabolism, autophagy, and regulation of cell size. The present results suggest that genome-wide changes in H3K4me3 after the LH surge are associated with rapid changes in gene expression in GCs, which enables GCs to acquire a lot of cellular functions within a short time that are required for ovulation and luteinization.

The adiponectin agonist, AdipoRon, inhibits steroidogenesis and cell proliferation in human luteinized granulosa cells.

During obesity, excess body weight is not only associated with an increased risk of type 2-diabetes, but also several other pathological processes, such as infertility. Adipose tissue is the largest endocrine organ of the body that produces adipokines, including adiponectin. Adiponectin has been reported to control fertility through the hypothalamic-pituitary-gonadal axis, and folliculogenesis in the ovaries. In this study, we focused on a recent adiponectin-like synthetic agonist called AdipoRon, and its action in human luteinized granulosa cells. We demonstrated that AdipoRon activated the adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor alpha (PPAR) signalling pathways in human luteinized granulosa cells. A 25 µM AdipoRon stimulation reduced granulosa cell proliferation by inducing cell cycle arrest in G1, associated with PTEN and p53 pathway activation. In addition, AdipoRon perturbed cell metabolism by decreasing mitochondrial activity and ATP production. In human luteinized granulosa cells, AdipoRon increased phosphodiesterase activity, leading to a drop in cyclic adenosine monophosphate (cAMP) production, aromatase expression and oestrogens secretion. In conclusion, AdipoRon impacted folliculogenesis by altering human luteinized granulosa cell function, via steroid production and cell proliferation. This agonist may have applications for improving ovarian function in metabolic disorders or granulosa cancers.

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.

Placental Growth Factor Is Required for Ovulation, Luteinization, and Angiogenesis in Primate Ovulatory Follicles.

Placental growth factor (PGF) is member of the vascular endothelial growth factor (VEGF) family of angiogenesis regulators. VEGFA is an established regulator of ovulation and formation of the corpus luteum. To determine whether PGF also mediates aspects of ovulation and luteinization, macaques received gonadotropins to stimulate multiple follicular development. Ovarian biopsies and whole ovaries were collected before (0 hours) and up to 36 hours after human chorionic gonadotropin (hCG) administration to span the ovulatory interval. PGF and VEGFA were expressed by both granulosa cells and theca cells. In follicular fluid, PGF and VEGFA levels were lowest before hCG. PGF levels remained low until 36 hours after hCG administration, when PGF increased sevenfold to reach peak levels. Follicular fluid VEGFA increased threefold to reach peak levels at 12 hours after hCG, then dropped to intermediate levels. To explore the roles of PGF and VEGFA in ovulation, luteinization, and follicular angiogenesis in vivo, antibodies were injected into the follicular fluid of naturally developed monkey follicles; ovariectomy was performed 48 hours after hCG, with ovulation expected about 40 hours after hCG. Intrafollicular injection of control immunoglobulin G resulted in no retained oocytes, follicle rupture, and structural luteinization, including granulosa cell hypertrophy and capillary formation in the granulosa cell layer. PGF antibody injection resulted in oocyte retention, abnormal rupture, and incomplete luteinization, with limited and disorganized angiogenesis. Injection of a VEGFA antibody resulted in oocyte retention and very limited follicle rupture or structural luteinization. These studies demonstrate that PGF, in addition to VEGFA, is required for ovulation, luteinization, and follicular angiogenesis in primates.

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.

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.

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.

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.

Precision-cut luteal slices: a promising approach for studying luteal function in pigs.

The study was aimed to validate the precision-cut luteal slices to investigate porcine luteal function. Corpora lutea (CLs) were cut into 180-µm thick slices using Krumdick Tissue Slicer. The viability, tissue structure and steroidogenic acute regulatory protein (STAR) expression in the luteal slices did not differ between the beginning and the end of the 24-h incubation period. The luteal progesterone secretion showed a time- and dose-dependent response to porcine luteinizing hormone. The effects of prostaglandin F(2α) and 17ß-estradiol on progesterone secretion by porcine luteal slices were comparable to the previously reported in vivo results of the CL microdialysis system in the pig.

Lgr4 gene regulates corpus luteum maturation through modulation of the WNT-mediated EGFR-ERK signaling pathway.

Luteal-phase insufficiency is one of the major causes of female infertility, but the molecular mechanisms are still largely unknown. Here we found that disruption of Lgr4/Gpr48, the newly identified receptor for R-spondins, greatly reduced female fertility in mice. The expression of Lgr4 was induced specifically in granulosa-lutein cells during luteinization. In Lgr4-deficient female mice, the estrous cycle was prolonged and serum progesterone levels were dramatically downregulated. In Lgr4(-/-) corpora lutea, the expression of key enzymes for steroidogenesis as well as common luteal marker genes was significantly decreased. Additionally, the activity of epidermal growth factor receptor (EGFR)-ERK signaling was attenuated in Lgr4(-/-) granulosa-lutein cells. We found that the maturation of Lgr4(-/-) cells was impaired in cultured primary granulosa cells, but the defect was partially rescued by reactivation of EGFR signaling by heparin-binding EGF-like growth factor treatment. We found that the expression of wingless-type MMTV integration site family (WNT)/catenin (cadherin associated protein), beta 1 (CTNNB1) downstream targets, including matrix metalloproteinase 9, which is a critical matrix metalloproteinase for activation of EGF-like factors, was significantly downregulated in Lgr4(-/-) ovaries. Matrix metalloproteinase 9 inhibitor treatment attenuated human chorionic gonadotropin- but not heparin-binding EGF-like growth factor-induced ERK activation and luteinization in primary granulosa cells. Together, we report that Lgr4 modulates WNT-mediated EGFR-ERK signaling to facilitate corpus luteum maturation and ovarian steroidogenesis to maintain female reproduction.

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.