Periovulatory Follicles Research Articles

Efficacy of the synergic action of myoinositol, tyrosine, selenium and chromium in women with PCOS.

The aim of the study is to investigate the efficacy of a treatment with myoinositol plus L-tyrosine, selenium, and chromium in women with polycystic ovarian syndrome (PCOS). One hundred and eighty-six women, with diagnosis of PCOS, were divided in four groups according to their clinical features. Phenotype A: androgen excess + ovulatory dysfunction + polycystic ovarian morphology. Phenotype B: androgen excess + ovulatory dysfunction. Phenotype C: androgen excess + polycystic ovarian morphology. Phenotype D: ovulatory dysfunction + polycystic ovarian morphology. All patients were given daily for six months a compound with 2 g myo-inositol, 0.5 mg L-Tyrosine, 0.2 mg folic acid, 55 mcg selenium, 40 mcg chromium. Hormonal assessment, BMI, Ferriman-Gallway Gallway score, HOMA index, and follicular monitoring were reported before starting the therapy, three months and six months after. Phenotype A showed an improvement, consistent with restored ovulation: more regular length of the menstrual cycle, detection of periovulatory follicle at ultrasound, and rising of progesterone in the luteal phase. A total of 45 patients (65.2%) ovulated after six months. In the same period glucose and HOMA index decreased. In the phenotype B, 80% of patients ovulated after six months. An improvement of the clinical and biochemical sign of hyperandrogenism was also reported. In the phenotype C, after BMI had followed the treatment for six months, it decreased in a statistically significant manner. In the phenotype D, 49 out of 82 women (59.7%) restored their regular menstrual period and ovulated. Our study reported how the synergistic action of myoinositol, L-tyrosine, selenium, and chromium could restore normal menstrual cycle, ovulation, and decrease weight in these patients.

Chronically elevated androgen and/or consumption of a Western-style diet impairs oocyte quality and granulosa cell function in the nonhuman primate periovulatory follicle.

To investigate the impact of chronically elevated androgens in the presence and absence of an obesogenic diet on oocyte quality in the naturally selected primate periovulatory follicle. Rhesus macaques were treated using a 2-by-2 factorial design (n = 10/treatment) near the onset of menarche with implants containing either cholesterol (C) or testosterone (T, 4-5-fold increase above C) and a standard or "Western-style" diet alone (WSD) or in combination (T+WSD). Following ~ 3.5years of treatment, females underwent controlled ovulation (COv, n = 7-10/treatment) cycles, and contents of the naturally selected periovulatory follicle were aspirated. Follicular fluid (FF) was analyzed for cytokines, chemokines, growth factors, and steroids. RNA was extracted from luteinizing granulosa cells (LGCs) and assessed by RNA-seq. Only healthy, metaphase (M) I/II-stage oocytes (100%) were retrieved in the C group, whereas several degenerated oocytes were recovered in other groups (33-43% of T, WSD, and T+WSD samples). Levels of two chemokines and one growth factor were reduced (p < 0.04) in FF of follicles with a MI/MII oocyte in WSD+T (CCL11) or T and WSD+T groups (CCL2 and FGF2) compared to C and/or WSD. Intrafollicular cortisol was elevated in T compared to C follicles (p < 0.02). Changes in the expression pattern of 640+ gene products were detected in LGC samples from follicles with degenerated versus MI/MII-stage oocytes. Pathway analysis on RNAs altered by T and/or WSD found enrichment of genes mapping to steroidogenic and immune cell pathways. Female primates experiencing hyperandrogenemia and/or consuming a WSD exhibit an altered intrafollicular microenvironment and reduced oocyte quality/competency, despite displaying menstrual cyclicity.

Contribution of the immune system to follicle differentiation, ovulation and early corpus luteum formation.

Much of what we know about the involvement of the immune system in periovulatory follicle differentiation, ovulation and subsequent formation of the corpus luteum in cattle is drawn from the findings of studies in several mammalian livestock species. By integrating published histological data from cattle, sheep and pigs and referring back to the more comprehensive knowledge bank that exists for mouse and humans we can sketch out the key cells of the immune system and the cytokines and growth factors that they produce that are involved in follicle differentiation and luteinization, ovulation and early follicle development. These contributions are reviewed and the key findings, discussed.

The Influence of Pentraxin 3 on the Ovarian Function and Its Impact on Fertility.

Follicular development is a highly coordinated process that in humans takes more than 6 months. Pituitary gonadotropins and a variety of locally produced growth factors and cytokines are involved in determining a precise sequence of changes in cell metabolism, proliferation, vascularization, and matrix remodeling in order to obtain a follicle with full ovulatory and steroidogenic capability. A low-grade inflammation can alter such processes leading to premature arrest of follicular growth and female reproductive failure. On the other hand, factors that are involved in inflammatory response as well as in innate immunity are physiologically upregulated in the follicle at the final stage of maturation and play an essential role in ovulation and fertilization. The generation of pentraxin 3 (PTX3) deficient mice provided the first evidence that this humoral pattern recognition molecule of the innate immunity has a non-redundant role in female fertility. The expression, localization, and molecular interactions of PTX3 in the periovulatory follicle have been extensively studied in the last 10 years. In this review, we summarize findings demonstrating that PTX3 is synthesized before ovulation by cells surrounding the oocyte and actively participates in the organization of the hyaluronan-rich provisional matrix required for successful fertilization. Data in humans tend to confirm these findings, indicating PTX3 as a biomarker of oocyte quality. Moreover, we discuss the emerging evidence that in humans altered PTX3 systemic levels, determined by genetic variations and/or low-grade chronic inflammation, can also impact the growth and development of the follicle and affect the incidence of ovarian disorders.

FOS, a Critical Downstream Mediator of PGR and EGF Signaling Necessary for Ovulatory Prostaglandins in the Human Ovary.

Fos null mice failed to ovulate and form a corpus luteum (CL) even when given exogenous gonadotropins, suggesting that ovarian Fos expression is critical for successful ovulation and CL formation. However, little is known about FOS in the human ovary. To determine the expression, regulation, and function of FOS in human periovulatory follicles. Timed periovulatory follicles were obtained from normally cycling women. Granulosa/lutein cells were collected from in vitro fertilization patients. The in vivo expression after human chorionic gonadotropin (hCG) administration and in vitro regulation of FOS, JUN, JUNB, and JUND was evaluated at the mRNA and protein level. Binding of progesterone receptor (PGR) and FOS to their target genes was assessed by chromatin immunoprecipitation analyses. Prostaglandin E2 (PGE2) and progesterone were measured. The expression of FOS, JUNB, and JUND drastically increased in ovulatory follicles after hCG administration. In human granulosa/lutein cell cultures, hCG increased the expression of FOS and JUN proteins. Inhibitors of PGR and epidermal growth factor (EGF) receptors reduced hCG-induced increases in the expression and phosphorylation of FOS. PGR bound to the FOS gene. A selective FOS inhibitor blocked hCG-induced increases in PGE2 and the expression of prostaglandin (PG) synthases and transporters (PTGES, SLCO2A1, and ABCC1). FOS bound to the promoter regions of these genes. The increase of FOS/activator protein 1 in human periovulatory follicles after hCG administration is mediated by collaborative actions of PGR and EGF signaling and critical for the upregulated expression of key ovulatory genes required for the rise in ovulatory PG in human granulosa cells.

PGRMC1/2 promotes luteal vascularization and maintains the primordial follicles of mice

To determine whether conditional depletion of progesterone receptor membrane component (PGRMC) 1 and PGRMC2 affected ovarian follicle development, follicle distribution was assessed in ovaries of young (≈3-month-old) and middle-aged (≈6-month-old) control (Pgrmc1/2fl/fl) and double conditional PGRMC1/2-knockout (Pgrmc1/2d/d) mice. This study revealed that the distribution of primary, preantral and antral follicles was not altered in Pgrmc1/2d/d mice, regardless of the age. Although the number of primordial follicles was similar at ≈3 months of age, their numbers were reduced by ≈80% in 6-month-old Pgrmc1/2d/d mice compared to age-matched Pgrmc1/2fl/fl mice. The Pgrmc1/2d/d mice were generated using Pgr-cre mice, so ablation of Pgrmc1 and Pgrmc2 in the ovary was restricted to peri-ovulatory follicles and subsequent corpora lutea (CL). In addition, the vascularization of CL was attenuated in Pgrmc1/2d/d mice, although mRNA levels of vascular endothelial growth factor A (Vegfa) were elevated. Moreover, depletion of Pgrmc1 and Pgrmc2 altered the gene expression profile in the non-luteal component of the ovary such that Vegfa expression, a stimulator of primordial follicle growth, was elevated; Kit Ligand expression, another stimulator of primordial follicle growth, was suppressed and anti-Mullerian hormone, an inhibitor of primordial follicle growth, was enhanced compared to Pgrmc1/2fl/fl mice. These data reveal that luteal cell depletion of Pgrmc1 and 2 alters the expression of growth factors within the non-luteal component of the ovary, which could account for the premature demise of the adult population of primordial follicles. In summary, the survival of adult primordial follicles is dependent in part on progesterone receptor membrane component 1 and 2.

Core Binding Factor β Expression in Ovarian Granulosa Cells Is Essential for Female Fertility.

Core binding factor β (CBFβ) is a non-DNA-binding partner of all RUNX proteins and critical for transcription activity of CBF transcription factors (RUNXs/CBFβ). In the ovary, the expression of Runx1 and Runx2 is highly induced by the luteinizing hormone (LH) surge in ovulatory follicles, whereas Cbfb is constitutively expressed. To investigate the physiological significance of CBFs in the ovary, the current study generated two different conditional mutant mouse models in which granulosa cell expression of Cbfb and Runx2 was reduced by Cre recombinase driven by an Esr2 promoter. Cbfbgc-/- and Cbfbgc-/- × Runx2gc+/- mice exhibited severe subfertility and infertility, respectively. In the ovaries of both mutant mice, follicles develop normally, but the majority of preovulatory follicles failed to ovulate either in response to human chorionic gonadotropin administration in pregnant mare serum gonadotropin-primed immature animals or after the LH surge at 5 months of age. Morphological and physiological changes in the corpus luteum of these mutant mice revealed the reduced size, progesterone production, and vascularization, as well as excessive lipid accumulation. In granulosa cells of periovulatory follicles and corpora lutea of these mice, the expression of Edn2, Ptgs1, Lhcgr, Sfrp4, Wnt4, Ccrl2, Lipg, Saa3, and Ptgfr was also drastically reduced. In conclusion, the current study provided in vivo evidence that CBFβ plays an essential role in female fertility by acting as a critical cofactor of CBF transcription factor complexes, which regulate the expression of specific key ovulatory and luteal genes, thus coordinating the ovulatory process and luteal development/function in mice.

Inductions of granulosa cell luteinization and cumulus expansion are dependent on the fibronectin-integrin pathway during ovulation process in mice

It has been known that EGF-like factor secreted from LH-stimulated granuloma cells acts on granulosa cells and cumulus cells to induce ovulation process. Granulosa cells are changed the morphology with differentiating cell functions to produce progesterone. Cumulus cells are detached to make a space between the cells to accumulate hyaluronan rich matrix. LH also changes extracellular matrix (ECM) components including fibronectin in the follicular walls and granulosa cell layers. EGF like factor and fibronectin synergistically play important roles in numerous cell functions, especially cancer cell migration, estimating that fibronectin would impact on granulosa cells and cumulus cells. To clear this hypothesis, the localizations of fibronectin and its receptor integrin were observed by immunofluorescence technique. The functions were monitored by the detection of downstream signaling pathway, focal adhesion kinase (FAK). The pharmacological approach in both in vivo and in vitro were used for analyzing the physiological roles of FAK during ovulation process. The immunofluorescence staining revealed that fibronectin and integrin were observed in granulosa cells, cumulus cells and the space between cumulus cells and oocyte at 4 and 8 h after hCG injection. Concomitantly with the changes of fibronectin-integrin localization, FAK was phosphorylated in periovulatory follicles. The injection of FAK inhibitor suppressed not only ovulation but also luteinization of granulosa cells and cumulus expansion. In cultured-granulosa cells, fibronectin-integrin synergistically activated FAK with amphiregulin (AREG). Such cooperative stimulations induced a morphological change in granulosa cells, which resulted in the maximum level of progesterone production via the induction of Hsd3b. When cumulus-oocyte complexes (COCs) were cultured with AREG in the presence of serum, the maximum level of cumulus expansion was observed. The AREG-induced cumulus expansion was also suppressed by FAK inhibitor. Thus, it is concluded that fibronectin and AREG synergistically activate FAK not only in granulosa cells and cumulus cells to induce successful ovulation process.

Ovulation is associated with the LH-dependent induction of pla2g4aa in zebrafish.

The effects of the preovulatory luteinizing hormone (LH) surge on the ovulatory process are mediated by prostaglandins (PGs), the synthesis of which involves prostaglandin synthetase and cytosolic phospholipase A2 (cPLA2). In our previous study, we systematically investigated the function of prostaglandin endoperoxide synthase (ptgs) genes on ovulation in zebrafish. However, the role of cPLA2 in ovulation was not determined in zebrafish. In this study, we investigated the function of cpla2α in PGs production and ovulation in periovulatory follicles. Our data showed that the expression of pla2g4aa increased during zebrafish folliculogenesis and the follicular layer was the primary region with expression of pla2g4aa. In addition, the expression of pla2g4aa was regulated by LH invitro and invivo. Furthermore, injection of AACOCF3, a specific inhibitor of cPLA2, significantly reduced ovarian PGs level and blocked hCG-induced ovulation. Collectively, these findings suggest that pla2g4aa is related to the ovulation process in zebrafish.

Granulosa cell endothelin-2 expression is fundamental for ovulatory follicle rupture

Ovulation is dependent upon numerous factors mediating follicular growth, vascularization, and ultimately oocyte release via follicle rupture. Endothelin-2 (EDN2) is a potent vasoconstrictor that is transiently produced prior to follicle rupture by granulosa cells of periovulatory follicles and induces ovarian contraction. To determine the role of Edn2 expression, surgical transplant and novel conditional knockout mice were super-ovulated and analyzed. Conditional knockout mice utilized a new iCre driven by the Esr2 promoter to selectively remove Edn2. Follicle rupture and fertility were significantly impaired in the absence of ovarian Edn2 expression. When ovaries of Edn2KO mice were transplanted in wild type recipients, significantly more corpora lutea containing un-ovulated oocytes were present after hormonal stimulation (1.0 vs. 5.4, p = 0.010). Following selective ablation of Edn2 in granulosa cells, Esr2-Edn2KO dams had reduced oocytes ovulated (3.8 vs. 16.4 oocytes/ovary) and smaller litters (4.29 ± l.02 vs. 8.50 pups/dam). However, the number of pregnancies per pairing was not different and the reproductive axis remained intact. Esr2-Edn2KO ovaries had a higher percentage of antral follicles and fewer corpora lutea; follicles progressed to the antral stage but many were unable to rupture. Conditional loss of endothelin receptor A in granulosa cells also decreased ovulation but did not affect fecundity. These data demonstrate that EDN2-induced intraovarian contraction is a critical trigger of normal ovulation and subsequent fecundity.

Coordinated Regulation Among Progesterone, Prostaglandins, and EGF-Like Factors in Human Ovulatory Follicles.

Context:In animal models, the luteinizing hormone surge increases progesterone (P4) and progesterone receptor (PGR), prostaglandins (PTGs), and epidermal growth factor (EGF)–like factors that play essential roles in ovulation. However, little is known about the expression, regulation, and function of these key ovulatory mediators in humans.Objective:To determine when and how these key ovulatory mediators are induced after the luteinizing hormone surge in human ovaries.Design and Participants:Timed periovulatory follicles were obtained from cycling women. Granulosa/lutein cells were collected from in vitro fertilization patients.Main Outcome Measures:The in vivo and in vitro expression of PGR, PTG synthases and transporters, and EGF-like factors were examined at the level of messenger RNA and protein. PGR binding to specific genes was assessed. P4 and PTGs in conditioned media were measured.Results:PGR, PTGS2, and AREG expressions dramatically increased in ovulatory follicles at 12 to 18 hours after human chorionic gonadotropin (hCG). In human granulosa/lutein cell cultures, hCG increased P4 and PTG production and the expression of PGR, specific PTG synthases and transporters, and EGF-like factors, mimicking in vivo expression patterns. Inhibitors for P4/PGR and EGF-signaling pathways reduced hCG-induced increases in PTG production and the expression of EGF-like factors. PGR bound to the PTGS2, PTGES, and SLCO2A1 genes.Conclusions:This report demonstrated the time-dependent induction of PGR, AREG, and PTGS2 in human periovulatory follicles. In vitro studies indicated that collaborative actions of P4/PGR and EGF signaling are required for hCG-induced increases in PTG production and potentiation of EGF signaling in human periovulatory granulosa cells.

Progesterone receptor and prostaglandins mediate luteinizing hormone‐induced changes in messenger RNAs for ADAMTS proteases in theca cells of bovine periovulatory follicles

Little is known about the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of extracellular proteases in ovarian follicles of non-rodent species, particularly in theca cells. In the present study, temporal changes in the abundance of mRNA encoding four ADAMTS subtypes and hormonal regulation of mRNA encoding two subtypes were investigated in theca interna cells during the periovulatory period in cattle. Gonadotropin-releasing hormone (GnRH) was injected into animals to induce a luteinizing hormone (LH)/follicle-stimulating hormone (FSH) surge, and follicles were obtained at 0 hr post-GnRH (preovulatory) or at 6, 12, 18, or 24 hr (periovulatory). ADAMTS1, -2, -7, and -9 transcript abundance was then determined in the isolated theca interna. ADAMTS1 and -9 mRNA levels were up-regulated at 24 hr post-GnRH, whereas ADAMTS2 mRNA was higher at 12-24 hr post-GnRH and ADAMTS7 mRNA increased transiently at 12 hr post-GnRH compared to other time points. Subsequent in vitro experiments using preovulatory theca interna (0 hr post-GnRH) showed that application of LH in vitro can mimic the effects of the gonadotropin surge on mRNAs encoding ADAMTS1 and -9 and that progesterone/progesterone receptor and/or prostaglandins may regulate the levels of mRNA encoding ADAMTS1 and -9 in theca interna, downstream of the LH surge. Time- and subtype-specific changes in ADAMTS mRNA abundance in vivo, and their regulation in vitro by hormones, indicate that ADAMTS family members produced by theca cells may play important roles in follicle rupture and the accompanying tissue remodeling in cattle. Mol. Reprod. Dev. 84: 55-66, 2017. © 2016 Wiley Periodicals, Inc.

Core Binding Factor-β Knockdown Alters Ovarian Gene Expression and Function in the Mouse.

Core binding factor (CBF) is a heterodimeric transcription factor complex composed of a DNA-binding subunit, one of three runt-related transcription factor (RUNX) factors, and a non-DNA binding subunit, CBFβ. CBFβ is critical for DNA binding and stability of the CBF transcription factor complex. In the ovary, the LH surge increases the expression of Runx1 and Runx2 in periovulatory follicles, implicating a role for CBFs in the periovulatory process. The present study investigated the functional significance of CBFs (RUNX1/CBFβ and RUNX2/CBFβ) in the ovary by examining the ovarian phenotype of granulosa cell-specific CBFβ knockdown mice; CBFβ f/f * Cyp19 cre. The mutant female mice exhibited significant reductions in fertility, with smaller litter sizes, decreased progesterone during gestation, and fewer cumulus oocyte complexes collected after an induced superovulation. RNA sequencing and transcriptome assembly revealed altered expression of more than 200 mRNA transcripts in the granulosa cells of Cbfb knockdown mice after human chorionic gonadotropin stimulation in vitro. Among the affected transcripts are known regulators of ovulation and luteinization including Sfrp4, Sgk1, Lhcgr, Prlr, Wnt4, and Edn2 as well as many genes not yet characterized in the ovary. Cbfβ knockdown mice also exhibited decreased expression of key genes within the corpora lutea and morphological changes in the ovarian structure, including the presence of large antral follicles well into the luteal phase. Overall, these data suggest a role for CBFs as significant regulators of gene expression, ovulatory processes, and luteal development in the ovary.

In vitro maturation affects chromosome segregation, spindle morphology and acetylation of lysine 16 on histone H4 in horse oocytes.

Implantation failure and genetic developmental disabilities in mammals are caused by errors in chromosome segregation originating mainly in the oocyte during meiosis I. Some conditions, like maternal ageing or in vitro maturation (IVM), increase the incidence of oocyte aneuploidy. Here oocytes from adult mares were used to investigate oocyte maturation in a monovulatory species. Experiments were conducted to compare: (1) the incidence of aneuploidy, (2) the morphology of the spindle, (3) the acetylation of lysine 16 on histone H4 (H4K16) and (4) the relative amount of histone acetyltransferase 1 (HAT1), K(lysine) acetyltransferase 8 (KAT8, also known as MYST1), histone deacetylase 1 (HDAC1) and NAD-dependent protein deacetylase sirtuin 1 (SIRT1) mRNA in metaphase II stage oocytes that were in vitro matured or collected from peri-ovulatory follicles. The frequency of aneuploidy and anomalies in spindle morphology was increased following IVM, along with a decrease in H4K16 acetylation that was in agreement with our previous observations. However, differences in the amount of the transcripts investigated were not detected. These results suggest that the degradation of transcripts encoding for histone deacetylases and acetyltransferases is not involved in the changes of H4K16 acetylation observed following IVM, while translational or post-translational mechanisms might have a role. Our study also suggests that epigenetic instabilities introduced by IVM may affect the oocyte and embryo genetic stability.

X-linked lymphocyte regulated gene 5c-like (Xlr5c-like) is a novel target of progesterone action in granulosa cells of periovulatory rat ovaries

Progesterone (P4), acting through its nuclear receptor (PGR), plays an essential role in ovulation by mediating the expression of genes involved in ovulation and/or luteal formation. To identify ovulatory specific PGR-regulated genes, a preliminary microarray analysis was performed using rat granulosa cells treated with hCG ± RU486 (PGR antagonist). The transcript most highly down-regulated by RU486 was an EST (expressed sequence tag) sequence (gb: BI289578.1) that matches with predicted sequence for Xlr5c-like mRNA. Since nothing is known about Xlr5c-like, we first characterized the expression pattern of Xlr5c-like mRNA in the rat ovary. The level of mRNA for Xlr5c-like is transiently up-regulated in granulosa cells of periovulatory follicles after hCG stimulation in PMSG-primed rat ovaries. The transient induction of Xlr5c-like mRNA was mimicked by hCG treatment in cultured granulosa cells from preovulatory ovaries. We further demonstrated that the LH-activated PKA, MEK, PI3K, and p38 signaling is involved in the increase in Xlr5c-like mRNA. The increase in Xlr5c-like mRNA was abolished by RU486. The inhibitory effect of RU486 was reversed by MPA (synthetic progestin), but not by dexamethasone (synthetic glucocorticoid). Furthermore, mutation of SP1/SP3 and PGR response element sites in the promoter region of Xlr5c-like decreased Xlr5c-like reporter activity. RU486 also inhibited Xlr5c-like reporter activity. ChIP assay verified the binding of PGR and SP3 to the Xlr5c-like promoter in periovulatory granulosa cells. Functionally, siRNA-mediated Xlr5c-like knockdown in granulosa cell cultures resulted in reduced levels of mRNA for Snap25, Cxcr4, and Adamts1. Recombinant Xlr5c-like protein expressed using an adenoviral approach was localized predominantly to the nucleus and to a lesser extent to the cytoplasm of rat granulosa cells. In conclusion, this is the first report showing the spatiotemporally regulated expression of Xlr5c-like mRNA by hCG in rat periovulatory ovaries. P4/PGR mediates the LH-induced increase in Xlr5c-like mRNA. In turn, Xlr5c-like is involved in regulating the expression of specific ovulatory genes such as Snap25, Cxcr4, and Adamts1, possibly acting in the nucleus of periovulatory granulosa cells.

The contribution of the maternal immune system to the establishment of pregnancy in cattle.

Immune cells play an integral role in affecting successful reproductive function. Indeed, disturbed or aberrant immune function has been identified as primary mechanisms behind infertility. In contrast to the extensive body of literature that exists for human and mouse, studies detailing the immunological interaction between the embryo and the maternal endometrium are quite few in cattle. Nevertheless, by reviewing the existing studies and extrapolating from sheep, pig, mouse, and human data, we can draw a reasonably comprehensive picture. Key contributions of immune cell populations include granulocyte involvement in follicle differentiation and gamete transfer, monocyte invasion of the peri-ovulatory follicle and their subsequent role in corpus luteum formation and the pivotal roles of maternal macrophage and dendritic cells in key steps of the establishment of pregnancy, particularly, the maternal immune response to the embryo. These contributions are reviewed in detail below and key findings are discussed.

99 GLUCOCORTICOID RECEPTORS ARE EXPRESSED IN OVARIES OF NEWBORN AND ADULT FEMALE HORSES

In contrast to other domestic animal species, in vitro maturation (IVM) of oocytes in the horse is still not successful. Oocytes for IVM are obtained either from slaughterhouse ovaries or via ovum pick-up from living mares. Both situations may be associated with a stress-induced glucocorticoid release. So far, neither an involvement of glucocorticoids in follicle and oocyte maturation nor the presence of glucocorticoid receptors (GCR) in ovarian tissue has been investigated in the horse. We hypothesised that GCR are expressed in equine ovarian tissue independent of the animal's age and stage of the oestrous cycle. Ovaries (n = 40) were collected from killed newborn female foals (n = 10) and killed or slaughtered adult mares (n = 10). For assessment of GCR mRNA expression, ovarian samples were fixed in Tissue-Tek O.C.T. Compound (Sakura Finetek, Zoeterwoude, the Netherlands) and stored at –80°C. Various cell populations were isolated using laser capture microdissection on cryosections. After RNA extraction, samples were analysed by qualitative RT-PCR and real time-PCR. For analysis of GCR protein, tissue was fixed in Bouin's solution and histological slides immunostained using a monoclonal antibody for GCR (Ab2768, Abcam, Cambridge, UK), followed by visualisation with diaminobenzidine. One tertiary follicle per slide (40×; light microscopy) was analysed and percentages of cells staining positive for GCR calculated. Statistical analysis was done with the SPSS Statistics 21 software (SPSS Inc., Chicago, IL, USA). Expression of mRNA for GCR was detected in oocytes, cumulus cells, granulosa, and theca cells, independent of age and stage of the oestrous cycle. In both neonates and adults, nuclei of the oocytes and cumulus cells stained positive for GCR regardless of stage of folliculogenesis. Also, GCR were constantly expressed in granulosa cells from both preantral and antral follicles. Percentage of granulosa cells staining positive for GCR (adult: 73.6 ± 3.2, fillies: 72.4 ± 1.9%) was higher (P &lt; 0.001) than of theca cells (adult: 56.8 ± 3.9, fillies: 57.2 ± 1.9%), but not affected by age. GCR were lacking in ovarian stroma of adults but not of neonates. In periovulatory follicles from adult mares, GCR were abundant in developing luteal cells. GCR were also detected in the nuclei of luteal cells in corpora haemorrhagica and corpora lutea. Follicular atresia was associated with a decrease of GCR independent of cell type and age. This study describes for the first time the expression of GCR in horse ovaries, which are present independent of age of the animal, stage of folliculogenesis, and oestrous cycle stage. Results suggest that glucocorticoids are involved in follicular and oocyte maturation, ovulation, and luteal function in the horse. Presence of GCR in the ovaries of newborn horses suggests a role of glucocorticoids in ovarian tissue maturation. Nevertheless, detrimental effects of excess glucocorticoid secretion due to stress on follicular development, oocyte maturation, and luteal function cannot be excluded in the mare.

Cortisol may promote cumulus cell (CC) lipid utilization as an energy source during oocyte meiotic resumption in women undergoing ovarian stimulation for in vitro fertilization (IVF)

Cortisol is produced by the periovulatory follicle from cortisone via LH/hCG-induction of type 1, 11β-hydroxysteroid dehydrogenase (11βHSD1) (cortisol synthesis) vs. type 2, 11βHSD (11βHSD2) (cortisone synthesis) (1). Follicle fluid (FF) cortisol may promote cumulus cell (CC) lipid metabolism to provide fatty acid as an energy source during oocyte meiosis (2). This study examines if FF cortisol levels correlate with CC lipid content during oocyte development, and if CCs express mRNA for glucocorticoid receptor (NR3C1), 11βHSD1/2 and the lipolytic enzymes, lipoprotein lipase [LPL] and hormone sensitive lipase [HSL].

Determination of the Granulosa Cell-Specific Endothelin Receptor A Deletion on Ovarian Function

Endothelin 2 (EDN2) induces follicular rupture by constricting periovulatory follicles. In this study, it was investigated the mechanisms of EDN2 action on follicular rupture with respect of receptor using the conditionally granulosa cell specific EDN2 receptor type A (ETa) KO mice (gcETaKO; ETaflox/-․Amhr2Cre). It was generated the gcETaKO mice by breeding with ETaflox/- mice after mono-alleic ETa knockout by ZP3Cre and Amhr2Cre mice. Fertility, ovulation and maturation rates of ovulated oocytes after super ovulation were investigated in the gcETaKO mice compared with wild-type mice (ETaflox/flox and ETaflox/-) as a control group. In the gcETaKO mice, normal fertility after breeding with male mice was shown compared with wild-type mice. And, there was no significant differences in ovulation rates after super ovulation, however its maturation rates was lower than that of wild type mice. These findings show that EDN2 in follicular rupture for ovulation is related with an other ETa not in granulosa cells. Further studies are needed to investigate how EDN2 is acted in ovarian follicular rupture for ovulation.

Loss of Ntrk2/Kiss1r signaling in oocytes causes premature ovarian failure.

Neurotrophins (NTs), once believed to be neural-specific trophic factors, are now known to also provide developmental cues to non-neural cells. In the ovary, NTs contribute to both the formation and development of follicles. Here we show that oocyte-specific deletion of the Ntrk2 gene that encodes the NTRK2 receptor (NTRK2) for neurotrophin-4/5 and brain-derived neurotrophic factor (BDNF) results in post-pubertal oocyte death, loss of follicular organization, and early adulthood infertility. Oocytes lacking NTRK2 do not respond to gonadotropins with activation of phosphatidylinositol 3-kinase (PI3K)-AKT-mediated signaling. Before puberty, oocytes only express a truncated NTRK2 form (NTRK2.T1), but at puberty full-length (NTRK2.FL) receptors are rapidly induced by the preovulatory gonadotropin surge. A cell line expressing both NTRK2.T1 and the kisspeptin receptor (KISS1R) responds to BDNF stimulation with activation of Ntrk2 expression only if kisspeptin is present. This suggests that BDNF and kisspeptin that are produced by granulosa cells (GCs) of periovulatory follicles act in concert to mediate the effect of gonadotropins on Ntrk2 expression in oocytes. In keeping with this finding, the oocytes of NTRK2-intact mice fail to respond to gonadotropins with increased Ntrk2 expression in the absence of KISS1R. Our results demonstrate that the preovulatory gonadotropin surge promotes oocyte survival at the onset of reproductive cyclicity by inducing oocyte expression of NTRK2.FL receptors that set in motion an AKT-mediated survival pathway. They also suggest that gonadotropins activate NTRK2.FL expression via a dual communication pathway involving BDNF and kisspeptin produced in GCs and their respective receptors NTRK2.T1 and KISS1R expressed in oocytes.