Current Insights in Prolactin Signaling and Ovulatory Function.

Prolactin (PRL) is a pleiotropic hormone released from lactotrophic cells of the anterior pituitary gland that also originates from extrapituitary sources and plays an important role in regulating lactation in mammals, as well as other actions. Acting in an endocrine and paracrine/autocrine manner, PRL regulates the hypothalamic-pituitary-ovarian axis, thus influencing the maturation of ovarian follicles and ovulation. This review provides a detailed discussion of the current knowledge on the role of PRL in the context of ovulation and ovulatory disorders, particularly with regard to hyperprolactinemia, which is one of the most common causes of infertility in women. Much attention has been given to the PRL structure and the PRL receptor (PRLR), as well as the diverse functions of PRLR signaling under normal and pathological conditions. The hormonal regulation of the menstrual cycle in connection with folliculogenesis and ovulation, as well as the current classifications of ovulation disorders, are also described. Finally, the state of knowledge regarding the importance of TIDA (tuberoinfundibular dopamine), KNDγ (kisspeptin/neurokinin B/dynorphin), and GnRH (gonadotropin-releasing hormone) neurons in PRL- and kisspeptin (KP)-dependent regulation of the hypothalamic-pituitary-gonadal (HPG) axis in women is reviewed. Based on this review, a rationale for influencing PRL signaling pathways in therapeutic activities accompanying ovulation disorders is presented.

Transcriptome analyses of potential regulators of pre- and post-ovulatory follicles in the pigeon (Columba livia).

To identify the dominant genes controlling follicular maturation, ovulation and regression for pigeon, we used RNA-seq to explore the gene expression profiles of pre- and post-ovulatory follicles of pigeon. We obtained total of 4.73million (96% of the raw data) high-quality clean reads, which could be aligned with 20282 genes. Gene expression profile analysis identified 1461 differentially expressed genes (DEGs) between the pre- (P4) and post-ovulatory follicles (P5). Of these, 843 genes were upregulated, and 618 genes were down-regulated. Furthermore, many DEGs were significantly enriched in some pathways closely related to follicle maturation, ovulation and regression, such as ECM-receptor interaction, vascular smooth muscle contraction, progesterone-mediated oocyte maturation, phagosome. Importantly, the DGEs in ECM-receptor interaction pathway included COL1A1 , COL1A2 , COL4A1 , COL4A2 , ITGA11 , ITGB3 and SDC3 , in the progesterone-mediated oocyte maturation pathway involved CDK1 , CDC25A , CCNB3 , CDC20 and Plk1 , and in the vascular smooth muscle contraction covered CALD1 , KCNMA1 , KCNMB1 , CACNA1 , ACTA2 , MYH10 , MYL3 , MYL6 , MYL9 , closely related to promoting follicular maturation and ovulation in pre-ovulatory follicles. Moreover, it seems that the lysosomal cathepsin family has a decisive role in the regression of early stage of post-ovulatory follicle. Taken together, these data enrich the research of molecular mechanisms of pigeon follicular activities at the transcriptional level and provide novel insight of breeding-related physiology for birds.

Progesterone receptor-Src kinase signaling pathway mediates neuroprogesterone induction of the luteinizing hormone surge in female rats.

Neural circuits in female rats are exposed to sequential estradiol and progesterone to regulate the release of luteinizing hormone (LH) and ultimately ovulation. Estradiol induces progesterone receptors (PGRs) in anteroventral periventricular nucleus (AVPV) kisspeptin neurons, and as estradiol reaches peak concentrations, neuroprogesterone (neuroP) synthesis is induced in hypothalamic astrocytes. This local neuroP signals to PGRs expressed in kisspeptin neurons to trigger the LH surge. We tested the hypothesis that neuroP-PGR signaling through Src family kinase (Src) underlies the LH surge. As observed in vitro, PGR and Src are co-expressed in AVPV neurons. Estradiol treatment increased the number of PGR immunopositive cells and PGR and Src colocalization. Furthermore, estradiol treatment increased the number of AVPV cells that had extranuclear PGR and Src in close proximity (< 40 nm). Infusion of the Src inhibitor (PP2) into the AVPV region of ovariectomized/adrenalectomized (ovx/adx) rats attenuated the LH surge in trunk blood collected 53 h post-estradiol (50 µg) injection that induced neuroP synthesis. Although PP2 reduced the LH surge in estradiol benzoate treated ovx/adx rats, activation of either AVPV PGR or Src in 2 µg estradiol-primed animals significantly elevated LH concentrations compared to dimethyl sulfoxide infused rats. Finally, antagonism of either AVPV PGR or Src blocked the ability of PGR or Src activation to induce an LH surge in estradiol-primed ovx/adx rats. These results indicate that neuroP, which triggers the LH surge, signals through an extranuclear PGR-Src signaling pathway.

Ovulatory upregulation of angiotensin-converting enzyme 2, a receptor for SARS-CoV-2, in dominant follicles of the human ovary.

OBJECTIVE: To determine the temporal expression of angiotensin-converting enzyme 2 (ACE2), a receptor for SARS-CoV-2, in dominant follicles throughout the periovulatory period in women and the regulatory mechanisms underlying ACE2 expression in human granulosa/lutein cells (hGLC). DESIGN: Experimental prospective clinical study and laboratory-based investigation. SETTING: University Medical Center and private in vitro fertilization center. PATIENT(S): Thirty premenopausal women undergoing surgery for tubal ligation and 16 premenopausal women undergoing in vitro fertilization. INTERVENTION(S): Administration of human chorionic gonadotropin (hCG) and harvesting of preovulatory/ovulatory follicles by timed laparoscopy, and collection of granulosa/lutein cells and cumulus cells at the time of oocyte retrieval. MAIN OUTCOME MEASURE(S): Expression and localization of ACE2 in granulosa cells and dominant follicles collected throughout the periovulatory period of the menstrual cycle and in hGLC using quantitative polymerase chain reaction, immunoblotting, and immunohistochemistry. RESULT(S): ACE2 expression (mRNA and protein) is up-regulated in human ovulatory follicles after administration of hCG. ACE2 expression was higher in cumulus cells than in granulosa cells. hCG increased the expression of ACE2 in primary hGLC cultures; the increase was inhibited by RU486 (an antagonist for progesterone receptor and glucocorticoid receptor) and CORT125281 (a selective glucocorticoid receptor antagonist), but not by AG1478 (an EGF receptor tyrosine kinase inhibitor) or by dexamethasone. CONCLUSION(S): The hormone-regulated expression of ACE2 in granulosa cells suggests a potential role of ACE2 in the ovulatory process. These data also imply the possible impact of COVID-19 on a vital cyclic event of ovarian function and thus on women's overall reproductive health. However, SAR-CoV-2 infection in ovarian cells in vivo or in vitro has yet to be determined.

Orphan nuclear receptors in angiogenesis and follicular development.

Orphan nuclear receptors (ONRs) are a subset of the nuclear receptor family that lacks known endogenous ligands. Among 48 nuclear receptors identified in humans, 25 are classified as ONRs. They function as transcription factors and control the expression of a wide range of genes to regulate metabolism, fertility, immunity, angiogenesis, and many other functions. Angiogenic factors are essential during ovarian follicle development, including follicle growth and ovulation. The correct development of blood vessels contributes to preantral and antral follicular development, selection of the dominant follicle or follicles, follicular atresia, and ovulation. Although progress has been made in understanding the molecular mechanisms that regulate follicular angiogenesis, the role of ONRs as regulators is not clear. Based on their functions in other tissues, the ONRs NR1D1 (REV-ERBß), NR2C2 (TR4), NR2F2 (COUP-TF-II) and NR3B1, 2, and 3 (ERRα, ERRß and ERRγ) may modulate angiogenesis during antral follicle development. We hypothesize that this is achieved by effects on the expression and function of VEGFA, ANGPT1, THBS1, and soluble VEGFR1. Further, angiogenesis during ovulation is expected to be influenced by ONRs. NR5A2 (LRH-1), which is required for ovulation, regulates angiogenic genes in the ovary, including VEGFA and the upstream regulator of angiogenesis, PGE2. These angiogenic molecules may also be regulated by NR5A1 (SF-1). Evidence from outside the reproductive tract suggests that NR2F2 and NR4A1(NUR77) promote VEGFC and PGF, respectively, and NR4As (NUR77, NOR1) seem to be necessary for the angiogenic effects of VEGFA and PGE2. Together, the data suggest that ONRs are important regulators of follicular angiogenesis.

The FOS/AP-1 Regulates Metabolic Changes and Cholesterol Synthesis in Human Periovulatory Granulosa Cells.

FOS, a subunit of the activator protein-1 (AP-1) transcription factor, has been implicated in various cellular changes. In the human ovary, the expression of FOS and its heterodimeric binding partners JUN, JUNB, and JUND increases in periovulatory follicles. However, the specific role of the FOS/AP-1 remains elusive. The present study determined the regulatory mechanisms driving the expression of FOS and its partners and functions of FOS using primary human granulosa/lutein cells (hGLCs). Human chorionic gonadotropin (hCG) induced a biphasic increase in the expression of FOS, peaking at 1 to 3 hours and 12 hours. The levels of JUN proteins were also increased by hCG, with varying expression patterns. Coimmunoprecipitation analyses revealed that FOS is present as heterodimers with all JUN proteins. hCG immediately activated protein kinase A and p42/44MAPK signaling pathways, and inhibitors for these pathways abolished hCG-induced increases in the levels of FOS, JUN, and JUNB. To identify the genes regulated by FOS, high-throughput RNA sequencing was performed using hGLC treated with hCG ± T-5224 (FOS inhibitor). Sequencing data analysis revealed that FOS inhibition affects the expression of numerous genes, including a cluster of genes involved in the periovulatory process such as matrix remodeling, prostaglandin synthesis, glycolysis, and cholesterol biosynthesis. Quantitative PCR analysis verified hCG-induced, T-5224-regulated expression of a selection of genes involved in these processes. Consistently, hCG-induced increases in metabolic activities and cholesterol levels were suppressed by T-5224. This study unveiled potential downstream target genes of and a role for the FOS/AP-1 complex in metabolic changes and cholesterol biosynthesis in granulosa/lutein cells of human periovulatory follicles.

In vitro follicle culture shows that progestagens are the maturation-inducing hormones (MIH) and possible regulators of the ovulation-mediating hormone PGE2 in female Eurasian perch Perca fluviatilis.

In European aquaculture, Eurasian perch, Perca fluviatilis L., is perceived as one of the most highly valuable freshwater fish species and a strong candidate for the development of freshwater aquaculture. In the pursuit of improving the quality of reproduction in this domesticated species, investigating the hormones mediating the final oocyte maturation (FOM) is therefore indispensable. But, the exact nature of the maturation-inducing hormone (MIH) in Eurasian perch is unknown. To further validate the existence of a maturation-inducing activity behind potential hormonal candidates in this species, we in vitro tested a group of nine hormones: cortisol (Co), 11-deoxycortisol (11-D), corticosterone (coS), 11-deoxycorticosterone (DOC), 17α,20ßdihydroxy-4-pregnen-3-one (DHP) and 17α,20ß,21 trihydroxy-4-pregnen-3-one (THP), prostaglandin E2 (PGE2), estradiol-17ß (E2) and testosterone (T), in their ability to trigger FOM advancement and the production of sex steroids potentially involved in FOM. Using mature female perch, two in vitro experiments were conducted with oocytes at the start of the FOM. The follicles were incubated for 62 h in Cortland media with and without human chorionic gonadotropin (hCG). By the end of the incubation, only DHP and THP triggered the full advancement in FOM even at low doses with the effect of DHP being in vivo validated. However, the de novo productions of E2 and DHP were not shown to be regulated by either of the MIH candidates. Progestagens are hence more credible candidates as MIH than corticosteroids in Eurasian perch. Our in vitro study also revealed that both PGE2 and DHP are strongly associated with ovulation and that PGE2 might have slightly contributed to such DHP activity.

Nerve Growth Factor: A Dual Activator of Noradrenergic and Cholinergic Systems of the Rat Ovary.

The functioning of the ovary is influenced by the autonomic system (sympathetic and cholinergic intraovarian system) which contributes to the regulation of steroid secretion, follicular development, and ovulation. There is no information on the primary signal that activates both systems. The nerve growth factor (NGF) was the first neurotrophic factor found to regulate ovarian noradrenergic neurons and the cholinergic neurons in the central nervous system. The aim of this study was to determine whether NGF is one of the participating neurotrophic factors in the activation of the sympathetic and cholinergic system of the ovary in vivo and its role in follicular development during normal or pathological states. The administration of estradiol valerate (a polycystic ovary [PCO] phenotype model) increased norepinephrine (NE) (through an NGF-dependent mechanism) and acetylcholine (ACh) levels. Intraovarian exposure of rats for 28 days to NGF (by means of an osmotic minipump) increased the expression of tyrosine hydroxylase and acetylcholinesterase (AChE, the enzyme that degrades ACh) without affecting enzyme activity but reduced ovarian ACh levels. In vitro exposure of the ovary to NGF (100 ng/ml for 3 h) increased both choline acetyl transferase and vesicular ACh transporter expression in the ovary, with no effect in ACh level. In vivo NGF led to an anovulatory condition with the appearance of follicular cysts and decreased number of corpora lutea (corresponding to noradrenergic activation). To determine whether the predominance of a NE-induced polycystic condition after NGF is responsible for the PCO phenotype, rats were exposed to an intraovarian administration of carbachol (100 µM), a muscarinic cholinergic agonist not degraded by AChE. Decreased the number of follicular cysts and increased the number of corpora lutea, reinforcing that cholinergic activity of the ovary participates in controlling its functions. Although NGF increased the biosynthetic capacity for ACh, it was not available to act in the ovary. Hence, NGF also regulates the ovarian cholinergic system, implying that NGF is the main regulator of the dual autonomic control. These findings highlight the need for research in the treatment of PCO syndrome by modification of locally produced ACh as an in vivo regulator of follicular development.

Cellular and molecular features of EDC exposure: consequences for the GnRH network.

The onset of puberty and the female ovulatory cycle are important developmental milestones of the reproductive system. These processes are controlled by a tightly organized network of neurotransmitters and neuropeptides, as well as genetic, epigenetic and hormonal factors, which ultimately drive the pulsatile secretion of gonadotropin-releasing hormone. They also strongly depend on organizational processes that take place during fetal and early postnatal life. Therefore, exposure to environmental pollutants such as endocrine-disrupting chemicals (EDCs) during critical periods of development can result in altered brain development, delayed or advanced puberty and long-term reproductive consequences, such as impaired fertility. The gonads and peripheral organs are targets of EDCs, and research from the past few years suggests that the organization of the neuroendocrine control of reproduction is also sensitive to environmental cues and disruption. Among other mechanisms, EDCs interfere with the action of steroidal and non-steroidal receptors, and alter enzymatic, metabolic and epigenetic pathways during development. In this Review, we discuss the cellular and molecular consequences of perinatal exposure (mostly in rodents) to representative EDCs with a focus on the neuroendocrine control of reproduction, pubertal timing and the female ovulatory cycle.

The potential of volatile organic compound analysis in cervicovaginal mucus to predict estrus and ovulation in estrus-synchronized heifers.

Cervicovaginal mucus is a mixture of mucins, ions, salts, and water, the proportions of which change during the reproductive cycle. It is suspected that this mucus emits an important volatile signal indicative of the reproductive state of the female. The objective of this study was to identify volatile organic compounds (VOC) in bovine cervicovaginal mucus that are modulated during the estrous cycle and could potentially be used as biomarkers of estrus and ovulation. Cervicovaginal mucus was collected from crossbred beef heifers (n = 8), which were synchronized using an 8-d controlled internal drug release (CIDR) protocol and in which onset of estrus and time of ovulation were determined by visual observation and ultrasonography, respectively. Mucus samples were collected between 0 and 96 h after CIDR removal (estrus onset occurred at 49.1 ± 3.3 h after CIDR removal). A validation study was performed on an independent group of 15 heifers from which cervicovaginal mucus samples were collected every 8 h from 40 to 80 h after CIDR removal. The VOC in mucus were identified using gas chromatography-mass spectrometry and selected compounds were quantified using selected-ion flow-tube mass spectrometry. The presence of 47 VOC was detected in mucus samples by gas chromatography-mass spectrometry with those exhibiting highest abundance including 2-butanone, acetone, 2-pentanone, 4-methyl-2-pentanone, 1-(1-methylethoxy)-2-propanone, ethanol, 2-methyl-2-propanol, and 2-butanol. All VOC peaked between 24 to 47 h after the onset of estrus (ovulation occurred 26.6 ± 5.6 h after estrus onset). Two VOC, 2-pentanone and 4-methyl-2-pentanone, exhibited a significant increase at the onset of estrus, whereas concentration of 2-butanone increased significantly just after estrus onset, indicating that these VOC may be used as putative biomarkers of estrus. The results of our study may contribute to the development of a sensor device based on VOC to aid the detection of estrus and ovulation in cattle, with particular relevance for the dairy industry where the majority of females are bred by artificial insemination.

Hypoxia-inducible factor-1alpha and nitric oxide synthases in bovine follicles close to ovulation and early luteal angiogenesis.

The objective of the study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A), inducible nitric oxide synthase (iNOS) and endothelial (eNOS) isoforms in time-defined follicle classes before and after GnRH application in the cow. Ovaries containing pre-ovulatory follicles or corpora lutea were collected by transvaginal ovariectomy (n = 5 cows/group) as follow: (I) before GnRH administration; (II) 4h after GnRH; (III) 10h after GnRH; (IV) 20h after GnRH; (V) 25h after GnRH; and (VI) 60h after GnRH (early corpus luteum). The mRNA abundance of HIF1A in the follicle group before GnRH was high, followed by a significant down regulation afterwards with a minimum level 25h after GnRH (close to ovulation) and significant increase only after ovulation. The mRNA abundance of iNOS before GnRH was high, decreased significantly during LH surge, with minimum levels afterwards. In contrast, the mRNA of eNOS decreased in the follicle group 20h after GnRH, followed by a rapid and significant upregulation just after ovulation. Immunohistochemically, the granulosa cells of antral follicles and the eosinophils of the theca tissue as well of the early corpus luteum showed a strong staining for HIF1A. The location of the eosinophils could be clearly demonstrated by immunostaining with an eosinophil-specific antibody (EMBP) and transmission electron microscopy. In conclusion, the parallel and acute regulated expression patterns of HIF1A and NOS isoforms, specifically during the interval between the LH surge and ovulation, indicate that these paracrine factors are involved in the local mechanisms, regulating final follicle maturation, ovulation and early luteal angiogenesis.

Beta-nerve growth factor stimulates spontaneous electrical activity of in vitro embryonic mouse GnRH neurons through a P75 mediated-mechanism.

The control of ovulation helps guarantee the success of reproduction and as such, contributes to the fitness of a species. In mammals, two types of ovulation are observed: induced and spontaneous ovulation. Recent work on camelids, that are induced ovulators, highlighted the role of a factor present in seminal plasma, beta Nerve Growth Factor (ß-NGF), as the factor that triggers ovulation in a GnRH dependent manner. In the present work, we characterized alpaca ß-NGF (aß-NGF) and its 3D structure and compared it with human recombinant ß-NGF (hß-NGF). We showed that the ß-NGF enriched fraction of alpaca semen and the human recombinant protein, both stimulated spontaneous electrical activity of primary GnRH neurons derived from mouse embryonic olfactory placodes. This effect was dose-dependent and mediated by p75 receptor signaling. P75 receptors were found expressed in vitro by olfactory ensheathing cells (OEC) in close association with GnRH neurons and in vivo by tanycytes in close vicinity to GnRH fibers in adult mouse. Altogether, these results suggested that ß-NGF induced ovulation through an increase in GnRH secretion provoked by a glial dependent P75 mediated mechanism.

Progesterone Receptor Serves the Ovary as a Trigger of Ovulation and a Terminator of Inflammation.

Ovulation is triggered by the gonadotropin surge that induces the expression of two key genes, progesterone receptor (Pgr) and prostaglandin-endoperoxide synthase 2 (Ptgs2), in the granulosa cells of preovulatory follicles. Their gene products PGR and PTGS2 activate two separate pathways that are both essential for successful ovulation. Here, we show that the PGR plays an additional essential role: it attenuates ovulatory inflammation by diminishing the gonadotropin surge-induced Ptgs2 expression. PGR indirectly terminates Ptgs2 expression and PGE2 synthesis in granulosa cells by inhibiting the nuclear factor κB (NF-κB), a transcription factor required for Ptgs2 expression. When the expression of PGR is ablated in granulosa cells, the ovary undergoes a hyperinflammatory condition manifested by excessive PGE2 synthesis, immune cell infiltration, oxidative damage, and neoplastic transformation of ovarian cells. The PGR-driven termination of PTGS2 expression may protect the ovary from ovulatory inflammation.

Mitofusin 1 is required for oocyte growth and communication with follicular somatic cells.

Mitochondrial function, largely regulated by the dynamics of this organelle, is inextricably linked to the oocyte health. In comparison with most somatic cells, mitochondria in oocytes are smaller and rounder in appearance, suggesting limited fusion. The functional implications of this distinct morphology, and how changes in the mitochondrial shape translate to mitochondrial function in oogenesis is little understood. We, therefore, asked whether the pro-fusion proteins mitofusins 1 (MFN1) and 2 (MFN2) are required for the oocyte development. Here we show that oocyte-specific deletion of Mfn1, but not Mfn2, prevents the oocyte growth and ovulation due to a block in folliculogenesis. We pinpoint the loss of oocyte growth and ovulation to impaired PI3K-Akt signaling and disrupted oocyte-somatic cell communication. In support, the double loss of Mfn1 and Mfn2 partially rescues the impaired PI3K-Akt signaling and defects in oocyte development secondary to the single loss of Mfn1. Together, this work demonstrates that the mitochondrial function influences the cellular signaling during the oocyte development, and highlights the importance of distinct, nonredundant roles of MFN1 and MFN2 in oogenesis.

Association of raised serum progesterone and gene polymorphisms with risk of failure pregnancy and miscarriage.

Balanced cytokine required for a healthy pregnancy to avoid miscarriage. There is yet no accord on the effect of raised progesterone on the endometrium, recurrent miscarriage and association with cytokines. The present study see the effect of raised serum progesterone level on ovulation, miscarriage, and association of selected Cytokines polymorphisms with recurrent miscarriage. In a controlled prospective study patients undergoing COS under controlled ovarian hyper stimulation were evaluated. On the day of trigger progesterone levels were measured and serum hormonal estimation assay was done on the day of ovulation trigger by automated immunoassay. Genotyping analysis using allelic discrimination method was conducted which detects SNPs base pair differences by comparing allele-specific fluorescence signal. There was no significant different between cases and controls in age, smoking habit and alcohol consumption habit. The ovulation trigger yielded >6 oocytes retrieval in majority of the patients. The mean stromal day were found to be statistically significant whereas the mean day of glands were insignificant. There is no significant difference observed between two groups for three studied polymorphisms. None of the polymorphisms deviated significantly from the Hardy Weinberg equilibrium, suggesting that the distribution in our subjects was representative of the actual population. The level of cytokines is guarded by various parameters, which are essential for a successful pregnancy. It is very complicated to predict the effect on endometrium and corresponding pregnancy rates due to increased progesterone.

The role of semen and seminal plasma in inducing large-scale genomic changes in the female porcine peri-ovulatory tract.

Semen modifies the expression of genes related to immune function along the porcine female internal genital tract. Whether other pathways are induced by the deposition of spermatozoa and/or seminal plasma (SP), is yet undocumented. Here, to determine their relative impact on the uterine and tubal transcriptomes, microarray analyses were performed on the endocervix, endometrium and endosalpinx collected from pre-ovulatory sows 24 h after either mating or artificial insemination (AI) with specific ejaculate fractions containing spermatozoa or sperm-free SP. After enrichment analysis, we found an overrepresentation of genes and pathways associated with sperm transport and binding, oxidative stress and cell-to-cell recognition, such as PI3K-Akt, FoxO signaling, glycosaminoglycan biosynthesis and cAMP-related transcripts, among others. Although semen (either after mating or AI) seemed to have the highest impact along the entire genital tract, our results demonstrate that the SP itself also modifies the transcriptome. The detected modifications of the molecular profiles of the pre/peri-ovulatory endometrium and endosalpinx suggest an interplay for the survival, transport and binding of spermatozoa through, for instance the up-regulation of the Estrogen signaling pathway associated with attachment and release from the oviductal reservoir.

The FSH-HIF-1α-VEGF Pathway Is Critical for Ovulation and Oocyte Health but Not Necessary for Follicular Growth in Mice.

Follicle-stimulating hormone (FSH)-induced growth of ovarian follicles is independent of follicular vascularization. Recent evidence has indicated that follicular vascularization is critical to ovarian follicle development and survival. FSH, a gonadotropin that induces follicular growth and development, also acts as the major survival factor for antral follicles. FSH has been reported to stimulate angiogenesis in the theca layers mediated in part by the vascular endothelial growth factor A (VEGFA) and the transcription factor hypoxia inducible factor 1α (HIF-1α). However, it remains largely undetermined whether FSH-dependent growth and survival of antral follicles relies on FSH-induced vascularization. Here, we first demonstrated that induction of angiogenesis through the FSH-HIF-1α-VEGFA axis is not required for FSH-stimulated follicular growth in mouse ovary. FSH increased the total number of blood vessels in mouse ovarian follicles, which was correlated with elevated expression of VEGFA and HIF-1α in granulosa cells. In contrast, blocking of follicular angiogenesis using inhibitors against the HIF-1α-VEGFA pathway repressed vasculature formation in follicles despite FSH administration. Interestingly, by measuring follicular size and ovarian weight, we found that the suppression of angiogenesis via HIF-1α-VEGFA pathway did not influence FSH-mediated follicular growth. However, inhibition of FSH-induced follicular vascularization by PX-478, a small-molecule inhibitor that suppresses HIF-1α activity, blocked ovulation and triggered atresia in large follicles. On the other hand, PX-478 injection reduced oocyte quality via impairing the meiotic apparatus, showing a prominently defective spindle assembly and actin dynamics. Collectively, our findings unveiled a vascularization-independent effect of FSH on follicular growth, whereas follicular survival, ovulation, and oocyte development relies on FSH-mediated angiogenesis in the follicles.

Proteomic analysis of follicular fluid during human ovulation.

INTRODUCTION: Human ovulation is a biologically complex process that involves several biochemical factors, promoting follicular rupture and release of a fertilizable oocyte. Proteins which are present in follicular fluid at high concentrations during ovulation are likely to be active participants in the biochemical pathways of ovulation. The aim of the study was to identify, by use of a modern proteomic technique, proteins of human follicular fluid which are differentially regulated during ovulation of the natural menstrual cycle. MATERIAL AND METHODS: This prospective experimental study over 3 years included women planned for laparoscopic sterilization. During surgery, retrieval of the dominant follicle was performed either at the preovulatory stage or during ovulation. Four women of preovulatory phase and four women of ovulatory phase met the predetermined criteria of hormone levels for respective phases, and samples of these were finally included out of the 15 women operated. Follicular fluid was aspirated from the excised follicle and subjected to mass spectrometry with the isobaric tags for relative and absolute quantification (iTRAQ) technology for isobaric tagging of peptides. This enables simultaneous identification and quantification of proteins. The protein profiles of the follicular fluid of the preovulatory phase and the ovulatory phase were analyzed, and proteins that were present were identified. RESULTS: A total of 502 proteins were identified, several of which previously have not been identified in human follicular fluid. Of the 115 proteins that were found in all samples, 20 proteins were at higher levels during ovulation. These were inflammatory-related proteins, coagulation factors, proteins in lipid metabolism, complement factors and antioxidants. Five proteins were present in lower levels during ovulation, with three being enzymes and the other two proteins of lipid metabolism and iron transport. CONCLUSIONS: Twenty-five follicular fluid proteins, with differential regulation during ovulation, were identified in human follicular fluid of the natural menstrual cycle. These proteins may have essential roles in the ovulatory cascade.

Plasticity of Anterior Pituitary Gonadotrope Cells Facilitates the Pre-Ovulatory LH Surge.

Gonadotropes cells located in the anterior pituitary gland are critical for reproductive fitness. A rapid surge in the serum concentration of luteinizing hormone (LH) secreted by anterior pituitary gonadotropes is essential for stimulating ovulation and is thus required for a successful pregnancy. To meet the requirements to mount the LH surge, gonadotrope cells display plasticity at the cellular, molecular and morphological level. First, gonadotrope cells heighten their sensitivity to an increasing frequency of hypothalamic GnRH pulses by dynamically elevating the expression of the GnRH receptor (GnRHR). Following ligand binding, GnRH initiates highly organized intracellular signaling cascades that ultimately promote the synthesis of LH and the trafficking of LH vesicles to the cell periphery. Lastly, gonadotrope cells display morphological plasticity, where there is directed mobilization of cytoskeletal processes towards vascular elements to facilitate rapid LH secretion into peripheral circulation. This mini review discusses the functional and organizational plasticity in gonadotrope cells including changes in sensitivity to GnRH, composition of the GnRHR signaling platform within the plasma membrane, and changes in cellular morphology. Ultimately, multimodal plasticity changes elicited by gonadotropes are critical for the generation of the LH surge, which is required for ovulation.

Differential Roles of Hypothalamic AVPV and Arcuate Kisspeptin Neurons in Estradiol Feedback Regulation of Female Reproduction.

Mammalian reproductive function includes puberty onset and completion, reproductive cyclicity, steroidogenesis, gametogenesis, fertilization, pregnancy, and lactation; all are indispensable to perpetuate species. Reproductive cycles are critical for providing the hormonal milieu needed for follicular development and maturation of eggs, but cycles, in and of themselves, do not guarantee ovulation will occur. Here, we review the roles in female reproductive neuroendocrine function of two hypothalamic populations that produce the neuropeptide kisspeptin, demonstrating distinct roles in maintaining cycles and ovulation.