Preovulatory Stage Research Articles

Maturation Environment and Impact on Subsequent Developmental Competence of Bovine Oocytes

In vitro production (IVP) of bovine embryos has been improved immensely throughout the last decades. Nevertheless, embryos generated in vitro still differ from their in vivo-produced counterparts. It is possible to achieve blastocyst rates of up to 70% if in vivo-matured oocytes are used. In contrast, if oocytes are matured in vitro, blastocyst rates are only half that of those matured in vivo. This rather limited success may be attributed to the heterogeneous population of oocytes which are normally retrieved from follicles of 3-8 mm rather than from preovulatory follicles. In contrast to the in vivo-ovulated oocyte, these oocytes lack development up to the preovulatory stage and are matured in vitro. Therefore, much effort has been devoted to the establishment of non-invasive and non-perturbing means for selecting the most competent oocytes, for example the extensiveness and compactness of the cumulus-corona investment and the granulation of the ooplasm. In vitro culture (IVC) conditions have been enhanced in the last few years, mainly by adjustment of media formulations, whereas the in vitro maturation (IVM) protocols stay invariable. Consequently, maintaining or mimicking the in vivo situation in vitro will aid to improve the quality and developmental competence of the resulting matured oocyte. The scope of this review is to give an overview of the current situation of in vitro maturation of mammalian oocytes with emphasis on the bovine species. Special attention has been paid to the in vivo situation in the follicle and how a better understanding of these intrafollicular factors will aid to improve the in vitro maturation conditions.

Mycotoxins and female reproduction: in vitro approaches

Exposure to mycotoxins has been linked to adverse effects on female reproduction by interfering with the synthesis, metabolism or degradation of steroid hormones, interaction with steroid receptors or impairing oocyte maturation and competence. To assess such effects, many studies initially focussed on possible endocrine actions of mycotoxins using specific cell lines known to express key enzymes involved in the synthesis of steroid hormones. Using these models, zearalenone, deoxynivalenol, ochratoxin A, T-2 and HT-2 toxins, and aflatoxin B1 were claimed to be endocrine active substances. As yet, zearalenone is the only mycotoxin for which a direct interaction with oestrogen receptors could be demonstrated, classifying this mycotoxin as an endocrine disruptor. Mycotoxin exposure of complex cell systems like ovarian follicles at the earliest (primordial) to most advanced (pre-ovulatory) stages can serve not only as the first indication of the potential of a mycotoxin to affect female reproduction, but also provides insight in specific mechanisms involved in such an effect and identifies vulnerable phases in follicle development. Zearalenone is the most widely studied mycotoxin regarding female reproduction, but effects on oocyte maturation have also been demonstrated for deoxynivalenol. Exposure to zearalenone impairs the formation of primordial, while its metabolite ?-zearalenol is more harmful to fertilised oocytes than zearalenone itself. This short overview aims to provide an introduction into the different models, such as cell lines and oocytes, commonly used to assess the potential adverse effects of mycotoxins on female reproduction.

Phosphoinositide 3-Kinase p110δ Mediates Estrogen- and FSH-Stimulated Ovarian Follicle Growth

In the mammalian ovary, primordial follicles are generated early in life and remain dormant for prolonged periods. Their growth resumes via primordial follicle activation, and they continue to grow until the preovulatory stage under the regulation of hormones and growth factors, such as estrogen, FSH, and IGF-1. Both FSH and IGF-1 activate the phosphatidylinositol-3 kinase (PI3K)/Akt (acute transforming retrovirus thymoma protein kinase) signaling pathway in granulosa cells (GCs), yet it remains inconclusive whether the PI3K pathway is crucial for follicle growth. In this study, we investigated the p110δ isoform (encoded by the Pik3cd gene) of PI3K catalytic subunit expression in the mouse ovary and its function in fertility. Pik3cd-null females were subfertile, exhibited fewer growing follicles and more atretic antral follicles in the ovary, and responded poorly to exogenous gonadotropins compared with controls. Ovary transplantation showed that Pik3cd-null ovaries responded poorly to FSH stimulation in vitro; this confirmed that the follicle growth defect was intrinsically ovarian. In addition, estradiol (E2)-stimulated follicle growth and GC proliferation in preantral follicles was impaired in Pik3cd-null ovaries. FSH and E2 substantially activated the PI3K/Akt pathway in GCs of control mice but not in those of Pik3cd-null mice. However, primordial follicle activation and oocyte meiotic maturation were not affected by Pik3cd knockout. Taken together, our findings indicate that the p110δ isoform of the PI3K catalytic subunit is a key component of the PI3K pathway for both FSH and E2-stimulated follicle growth in ovarian GCs; however, it is not required for primordial follicle activation and oocyte development.

Na+/H+ exchange is inactivated during mouse oocyte meiosis, facilitating glycine accumulation that maintains embryo cell volume

The coupled action of the Na(+)/H(+) exchanger NHE1 and the HCO3(-)/Cl(-) exchanger AE2 constitutes the principal mechanism for acute correction of decreased cell volume in mammalian somatic cells, while, when acting separately, they regulate intracellular pH. It was previously found that AE2 becomes inactivated during meiosis in mouse oocytes. Similarly, NHE1 activity stimulated by intracellular acidosis was present in preovulatory germinal vesicle stage (GV) mouse oocytes and then decreased during meiotic maturation. In contrast, NHE1 activity stimulated by decreased cell volume was low in GV oocytes but became active during meiotic maturation as the oocyte detached from the zona pellucida. It then decreased again in mature eggs similar to activity stimulated by acidosis. The subcellular localization of NHE1 was investigated with YFP-tagged NHE1. Exogenous NHE1 expressed in GV oocytes localized to the plasma membrane and resulted in increased Na(+)/H(+) exchanger activity, but only when co-expressed with calcineurin homologous protein 1 (CHP1). When oocytes expressing functional NHE1 were matured to eggs, however, membrane localization of NHE1 and Na(+)/H(+) exchanger activity were lost. It was unknown why NHE1 and AE2 activities are suppressed during meiotic maturation. Maintenance of cell volume in preimplantation embryos requires glycine accumulation via the GLYT1 transporter, a process unique to eggs and early embryos that is initiated during meiotic maturation. When NHE1 and AE2 activities were maintained in GV oocytes by exogenous expression, glycine accumulation was inhibited. We propose that NHE1-mediated acute cell volume regulation is inactivated during meiotic maturation to allow preferential accumulation of glycine in eggs.

Detection of Glycosylated Proteins in Rabbit Oviductal Isthmus and Uterine Endometrium During Early Embryo Development

In this study, lectin histochemistry was performed on paraffin sections to compare carbohydrate expression of oviductal isthmus and uterine endometrium in rabbits during early embryo development. Rabbit embryos are surrounded not only by the zona pellucida but also by tubal secretion-derived mucinous coat material, the mucin coat. Twenty sexually mature females were euthanized at 0 (pre-ovulatory group) and 24, 72 and 96h after insemination (pseudopregnancy group). The following lectin-binding agents were used: Arachis hypogaea, Peanut (PNA) to label galactosyl (β-1,3)N- acetyl-galactosamine, Dolichos biflorus Agglutinin (DBA) to label galactosyl (β-1,4)N- acetyl-galactosamine, Lens curinaris (LCA) to label α--mannose, α-d-glucose and Pisum sativum agglutinin (PSA) to label α-d-mannose, α-d-glucose. Blood was collected by cardiac puncture, and direct enzyme immunoassay technique was used to measure progesterone concentration. A significant increase in total plasma progesterone concentrations was detected at 96h post-ovulation when compared with 0, 24 and 72h post-ovulation (2.9±0.5 vs 0.5±0.15, 1.6±0.5 and 1.5±0.4ng/ml, at 96h vs 0, 24 and 72h post-ovulation, respectively). No differences between pre-ovulatory and pseudopregnant females were observed for glycoprotein localization in isthmus. In contrast, in the endometrium, differences in the glycoprotein detection between pre-ovulatory and pseudopregnant stages were detected. PNA to label galactosyl (β-1,3)N- acetyl-galactosamine was not detected at the pre-ovulatory stage, but its presence was detected at 24h after ovulation. Both PSA and LCA to label α-d-mannose, α-d-glucose were only detected at 72h after ovulation. DBA detection was similar for all stages of the reproductive cycle. Therefore, N-acetyl-galactosamine secreted from isthmus could be involved in the formation of the embryonic mucin coat. d-galactose (PNA), d-glucose and d-mannose (PSA and LCA) might be crucial for the implantation period.

Lhcgr Expression in Granulosa Cells: Roles for PKA-Phosphorylated β-Catenin, TCF3, and FOXO1

Ovarian follicles lacking FSH or FSH receptors fail to progress to a preovulatory stage, resulting in infertility. One hallmark of the preovulatory follicle is the presence of luteinizing hormone/choriogonadotropin receptors (LHCGR) on granulosa cells (GCs). However, the mechanisms by which FSH induces Lhcgr gene expression are poorly understood. Our results show that protein kinase A (PKA) and phosphoinositide 3-kinase (PI3K)/AKT pathways are required for FSH to activate both the murine Lhcgr-luciferase reporter and expression of Lhcgr mRNA in rat GCs. Based on results showing that an adenovirus (Ad) expressing a steroidogenic factor 1 (SF1) mutant that cannot bind β-catenin abolished FSH-induced Lhcgr mRNA, we evaluated the role of β-catenin in the regulation of Lhcgr gene expression. FSH promoted the PKA-dependent, PI3K-independent phosphorylation of β-catenin on Ser552 and Ser665. FSH activated the β-catenin/T-cell factor (TCF) artificial promoter-reporter TOPFlash via a PKA-dependent, PI3K-independent pathway, and dominant-negative (DN) TCF abolished FSH-activated Lhcgr-luciferase reporter and induction of Lhcgr mRNA. Microarray analysis of GCs treated with Ad-DN-TCF and FSH identified the Lhcgr as the most down-regulated gene. Chromatin immunoprecipitation results placed β-catenin phosphorylated on Ser552 and Ser675 and SF1 on the Lhcgr promoter in FSH-treated GCs; TCF3 was constitutively associated with the Lhcgr promoter. Transduction with an Ad-phospho-β-catenin mutant (Ser552/665/Asp) enhanced Lhcgr mRNA expression in FSH-treated cells greater than 3-fold. Finally, we identified a recognized PI3K/AKT target, forkhead box O1, as a negative regulator of Lhcgr mRNA expression. These results provide new understanding of the complex regulation of Lhcgr gene expression in GCs.

Intraovarian regulation of gonadotropin-dependent folliculogenesis depends on notch receptor signaling pathways not involving Delta-like ligand 4 (Dll4)

BackgroundIn-situ hybridisation studies demonstrate that Notch receptors and ligands are expressed in granulosa cells (GCs) and in the theca layer vasculature of growing follicles. Notch signaling involves cell-to-cell interaction mediated by transmembrane receptors and ligands. This signaling pathway may represent a novel intraovarian regulator of gonadotropin-dependent follicular development to the preovulatory stage. We hypothesized that blocking Notch pathways would disrupt follicular maturation in the mouse ovary.MethodsHypophysectomized CD21 female mice were administered pregnant mare serum gonadotropin (PMSG) for 3 days to stimulate follicular development. In one experiment, a pan-notch inhibitor, compound E, was initiated 2 days prior to and throughout stimulation (n = 10), while in a second experiment, a humanized phage Dll4 blocking antibody, YW152F, was used (n = 5). After sacrifice, ovarian histology, serum estradiol levels and uterine weights were compared to controls. The ovarian morphology was evaluated with hematoxylin/eosin staining and immunohistochemistry was performed for Notch1, Notch2, Notch3, Notch4, Jagged1, Dll4, platelet endothelial cell adhesion molecule (PECAM) and alpha-smooth muscle actin (α-SMA) detection.ResultsWe localized specific Notch ligands and receptors in the following structures: Dll4 is specific to theca layer endothelial cells (ECs); Notch1/Notch4 and Jagged1 are expressed in theca layer ECs and vascular smooth muscle cells (VSMCs), whereas Notch3 is restricted to VSMCs; Notch2 is expressed mostly on GCs of small follicles. Administration of a pan-Notch inhibitor, compound E, inhibits follicular development to the preovulatory stage (8.5 preovulatory follicles in treatment vs. 3.4 preovulatory follicles in control, p < 0.01; average number per ovary) with significant secondary effects on ovarian and uterine weight and estradiol secretion in a setting of uninhibited vascular proliferation, but disorganized appearance of ECs and VSMCs. Inhibition of endothelial Notch1 function through the inactivation of its ligand Dll4 with the blocking antibody YW152F induces mild disorganisation of follicular vasculature, but has no significant effect on gonadotropin-dependent folliculogenesis.ConclusionsOur experiments suggest that the complete blockage of the Notch signaling pathway with compound E impairs folliculogenesis and induces disruption of gonadotropin stimulated angiogenesis. It seems the mechanism involves Notch1 and Notch3, specifically, causing the improper assembly of ECs and VSMCs in the theca layer, although the potential role of non-angiogenic Notch signaling, such as Jagged2 to Notch2 in GCs, remains to be elucidated.

Modulation of microvascular permeability in the preovulatory rat ovary by an ovulatory gonadotropin stimulus

To characterize the size selectivity of the rat ovarian vasculature and its changes after gonadotropin induction of ovulation. Experimental study. Obstetrics and Gynecology Department. Immature, female Sprague-Dawley rats. Rats were pretreated with equine chorionic gonadotropin, and ovaries were retrieved either 48 hours later or at any of several time points during ovulation induced by hCG. Fluorescein isothiocyanate-labeled Ficoll was injected 10 minutes before ovarian sampling, and the distribution of Ficoll was measured in plasma and ovarian extracts. The Ficoll data were analyzed according to a two-pore model to acquire information on small (rS) and large (rL) pore radii as well as the number of large pores reflected by the large pore fraction of the hydraulic conductance (LpS%) at each periovulatory time interval. Before hCG, rS and rL were 54.7 ± 1.2 Å (mean ± SEM) and 149.3 ± 5.3 Å, respectively. At this preovulatory stage, LpS% was 7.1% ± 3.2%. Stimulation with hCG caused close to a three-fold increase in LpS% at 2 and 4 hours (20.9% ± 1.8% and 20.7% ± 2.5%, respectively) and approximately 15% enlargements of rS and rL. Thus, the change in LpS% represents a dramatic increase in the number of large pores and not an increased size of preexisting large pores, since the small and large pore radii changed in parallel. These results indicate that capillary permeability of the ovarian blood-follicle barrier is modulated by gonadotropin, mainly through increased numbers of large pores, similar to a classical inflammatory response.

Folliculogenesis, Ovulation and Endocrine Control of Oocytes and Embryos in the Dog

Reproductive physiology in dogs is quite unusual compared with that in other mammalian species. The peculiarities include the presence of numerous polyoocyte follicles, the ovulation of an immature oocyte (GV stage, non-fertilizable) and a peri-ovulatory period during which concentrations of circulating progesterone are particularly high. The aim of this review is to examine the unusual aspects of the reproductive physiology of dogs and how this relates to the clinical biology of this species.

Stage-specific expression and effect of bone morphogenetic protein 2 on bovine granulosa cell estradiol production: regulation by cocaine and amphetamine regulated transcript

Members of the bone morphogenetic protein (BMP) family regulate follicular development and granulosa cell function. However, changes in expression of BMP2 and its receptors during follicular waves in cattle and ability of BMP2 to modulate bovine granulosa cell estradiol production are not well understood. The objectives of this study were to determine temporal regulation of mRNA for BMP2 and its type I and II receptors (BMPR1A and BMPR2) in bovine follicles collected at specific stages of a follicular wave (predeviation, early dominance, mid dominance, preovulatory), ability of BMP2 to modulate bovine granulosa cell steroidogenesis, and whether effects of BMP2 on granulosa cell estradiol production are influenced by cotreatment with cocaine- and amphetamine-regulated transcript (CART), an intrafollicular regulatory peptide shown to inhibit estradiol production in response to other trophic hormones (FSH and IGF1). Relative abundance of mRNAs for Bmp2 and Bmpr2 was elevated at the mid dominance stage relative to earlier stages of the follicular wave and further increased at the preovulatory stage. Abundance of mRNA for Bmpr1a was lowest at early dominance stage and highest at preovulatory stage relative to other stages of the follicular wave examined. Treatment of bovine granulosa cells in vitro with BMP2 increased estradiol but decreased progesterone concentrations. Co-incubation with CART reduced the BMP2-stimulated increase in granulosa cell estradiol production. Results suggest that BMP2 may play a regulatory role in development of bovine follicles to the preovulatory stage and that CART can inhibit granulosa cell estradiol production in response to multiple hormones/growth factors, including BMP2.

Effects of menstrual periods on postural stability in eumenorrheic female group

High rate of injuries detected during certain periods of menstrual cycle, raised the suggestion of hormonal impact on soft tissue and neuromuscular control with subsequent lost in joint stability, deterioration of postural balance and injury development. The aim of present study was to investigate possible differences in static postural balance during the preovulatory and postovulatory stages of menstrual cycle. Twenty one randomly selected eumenorrheic female university students accepted to participate in this study. Postural stability index (PSI) and sway distances were measured on a computer-based static stability force-platform with eyes open and eyes closed trials. Height, weight, waist and hip circumferences of the subjects were also measured. Evaluation of body fat percentage (BF) was made through biceps-triceps-subscapular-suprailiac skin-fold thickness measurements. Analysis of balance scores demonstrated better postovulatory comparing to preovulatory postural stability in terms of PSI (93.29 ± 4.26 versus 91.05 ± 6.21, p = 0.035), antero-posterior (0.99 ± 0.72 versus 0.77 ± 0.51 cm, p = 0.05) and medio-lateral (0.77 ± 0.64 versus 0.50 ± 0.42 cm, p = 0.04) sway distances. There was also statistically significant correlation between anthopometric and balance parameters with better postural balance in thinner participants. Our findings demonstrated that menstrual cycle does indeed influence postural stability, which also is the function of body composition in young females. Key words: Postural balance, menstrual cycle, females, periods.

Usefulness of Finasteride to induce ovulation in a non responder hyperandrogenic woman: birth of a healthy male infant

We report a case for the first time describing a new use of Finasteride. A 33-year-old woman participated with other hyperandrogenic anovulatory volunteers in our study on the efficacy of Finasteride in inducing ovulation [1]. She consulted her gynaecologist, because she was hoping to become pregnant. She reported oligomenorrhea, since menarche. Body mass index and a hirsutism score were evaluated according to the modified Ferriman–Gallway scoring system of 21 and 22, respectively. Pelvic examination showed micropolycystic enlarged ovaries. Serum levels of FSH, LH, T, DHT, androstenedione (A) and estradiol (E2)were measured on the 1st day of menses, using a recombinant immunoassay; T, DHT, A and E2 were repeated on the HCG day and DHT even on the 12th day after Finasteride discontinuance. Initial hormone levels were: FSH 4.1 mIU, LH 16.2 mIU, T 77 ng/dL, DHT 43 ng/dL, E2 55.9 pg/mL, and A 3.68 ng/mL. Initially, the patient underwent three consecutive cycles of controlled gonadotropin ovarian stimulation with recombinant FSH (b FSH, Organon, The Netherlands) starting at a daily dose of 150 IU on the 3rd day of cycle. Ovulation did not occur and after 21 days of stimulation, the cycles discontinued. On the 4th cycle, Finasteride was added. The patient was treated with recombinant FSH as described above for precedent stimulation cycles, plus 5 mg of Finasteride (Finastid; Neopharmed, Rome, Italy) daily, from day 1. On the 16th day of treatment, ultrasound examination showed one follicle of 20 mm; E2 was 180 pg/ml, human chorionic gonadotropin (HCG 10.000 UI; Gonasi; Amsa, Rome, Italy) was administered and Finasteride discontinued. On the same day, a significant decrease in DHT was achieved. After 14 days from Finasteride discontinuance, DHT was above basal level. Ultrasound examination revealed an intrauterine pregnancy. On the 6th week of amenorrhea, b hCG was 12.250 m UI/ml. The pregnancy evolved physiologically and after 40 weeks, a normal male infant of 3,600 g was delivered. Although follicular development is a complex interplay between many autocrine and paracrine factors, an important role is claimed by the competition between the enzymes aromatase and 5aR and their respective products E2 and DHT [2]. In PCOS, there is a raised activity of 5aR, responsible of higher level of intrafollicular DHT. The majority of ovarian follicles, in this syndrome, undergo atresia. Raised intrafollicular DHT concentrations are able to lead to follicular atresia that occurs at all stages of follicle development. Intrafollicular production of estrogens is indispensable for the growth of follicles. Atresic follicles exhibit decreased estrogen production and a lower estrogen/androgen ratio in the follicular fluid. In this case, sufficient exposure of antral follicles to FSH, increasing aromatase activity joined with low intrafollicular DHT level, due to Finasteride effect could be the critical stimulus for the follicle to escape atresia and reach the preovulatory follicle stage [3, 4]. In our patient, Finasteride not only favoured follicular growth (failure to achieved ovulation in 3 previous cycles), but also did not affect male genitalia differentiation. Finasteride seems not M. Tartagni G. R. Damiani (&) M. V. Tartagni G. Loverro Department of Obstetric and Gynecology, University of Bari, Policlinico, Piazza Giulio Cesare, 70124 Bari, Italy e-mail: damiani14@alice.it

Defective folliculogenesis in female mice lacking Vaccinia-related kinase 1

The Vaccinia-related kinase 1(VRK1), which is generally implicated in modulating cell cycle, plays important roles in mammalian gametogenesis. Female infertility in VRK1-deficient mice was reported to be caused by defective meiotic progression in oocyte at postovulatory stage. VRK1 roles in folliculogenesis, however, remain largely unknown. Here, accurate quantification of folliculogenesis is performed by a direct visualization of ‘intact’ ovary in 3-dimensions (3-D) using a synchrotron X-ray microtomography. In VRK1-deficient ovaries, the numbers of pre-antral and antral follicles are significantly reduced by 38% and 46%, respectively, comparing to control. The oocytes volumes in antral and Graffian follicles also decrease by 42% and 37% in the mutants, respectively, indicating defects in oocyte quality at preovulatory stage. Genetic analysis shows that gene expressions related to folliculogenesis are down-regulated in VRK1-deficient ovaries, implying defects in folliculogenesis. We suggest that VRK1 is required for both follicle development and oocyte growth in mammalian female reproduction system.

C-Type Natriuretic Peptide Stimulates Ovarian Follicle Development

C-type natriuretic peptide (CNP) encoded by the NPPC (Natriuretic Peptide Precursor C) gene expressed in ovarian granulosa cells inhibits oocyte maturation by activating the natriuretic peptide receptor (NPR)B (NPRB) in cumulus cells. RT-PCR analyses indicated increased NPPC and NPRB expression during ovarian development and follicle growth, associated with increases in ovarian CNP peptides in mice. In cultured somatic cells from infantile ovaries and granulosa cells from prepubertal animals, treatment with CNP stimulated cGMP production. Also, treatment of cultured preantral follicles with CNP stimulated follicle growth whereas treatment of cultured ovarian explants from infantile mice with CNP, similar to FSH, increased ovarian weight gain that was associated with the development of primary and early secondary follicles to the late secondary stage. Of interest, treatment with FSH increased levels of NPPC, but not NPRB, transcripts in ovarian explants. In vivo studies further indicated that daily injections of infantile mice with CNP for 4 d promoted ovarian growth, allowing successful ovulation induction by gonadotropins. In prepubertal mice, CNP treatment alone also promoted early antral follicle growth to the preovulatory stage, leading to efficient ovulation induction by LH/human chorionic gonadotropin. Mature oocytes retrieved after CNP treatment could be fertilized in vitro and developed into blastocysts, allowing the delivery of viable offspring. Thus, CNP secreted by growing follicles is capable of stimulating preantral and antral follicle growth. In place of FSH, CNP treatment could provide an alternative therapy for female infertility.

Androstenedione induces abnormalities in morphology and function of developing oocytes, which impairs oocyte meiotic competence

To obtain insight into the effects of androstenedione on ovarian folliculogenesis and oogenesis. Experimental study. St. Marianna University School of Medicine. Prepubertal (14-day-old) BDF1 female mice. Early secondary follicles were isolated from the ovaries and were cultured individually in vitro with or without androstenedione (10(-11) to 10(-5) M) for 12 days. Thereafter, the follicles were treated with hCG and epidermal growth factor (EGF). Diameters and morphology of follicles and oocytes; E(2) and P secretion; and chromatin configuration and expression of growth differentiation factor 9 (GDF9) in oocytes were examined. Early secondary follicles developed to the preovulatory stage. Androstenedione treatments increased the follicle diameters, reduced survival rates of follicles, and promoted the formation of follicles with abnormal morphology, including misshapen oocyte. The secretion of E(2) and P was significantly higher in androstenedione-exposed follicles. Androstenedione prevented the alteration in chromatin configuration and reduced oocyte GDF9 expression. When follicles cultured with androstenedione were treated with hCG and EGF, the first polar body exclusion, chromosome alignment on metaphase plate, and spindle assembly were inhibited in the oocytes. These results demonstrate that excess androgen induces abnormalities in the morphology and function of developing oocytes, which impairs oocyte meiotic competence.

Hyal‐1 but not hyal‐3 deficiency has an impact on ovarian folliculogenesis and female fertility by altering the follistatin/activin/Smad3 pathway and the apoptotic process

Ovarian follicle development is a process regulated by various endocrine, paracrine and autocrine factors that act coordinately to promote follicle growth. However, the vast majority of follicles does not reach the pre-ovulatory stage but instead, undergo atresia by apoptosis. We have recently described a role for the somatic hyaluronidases (Hyal-1, Hyal-2, and Hyal-3) in ovarian follicular atresia and induction of granulosa cell apoptosis. Herein, we show that Hyal-1 but not Hyal-3 null mice have decreased apoptotic granulosa cells after the induction of atresia and an increased number of retrieved oocytes after stimulation of ovulation. Furthermore, young Hyal-1 null mice had a significantly higher number of primordial follicles than age matched wild-type animals. Recruitment of these follicles at puberty resulted in an increased number of primary and healthy preantral follicles in Hyal-1 null mice. Consequently, older Hyal-1 deficient female mice have prolonged fertility. At the molecular level, immature Hyal-1 null mice have decreased mRNA expression of follistatin and higher levels of phospho-Smad3 protein, resulting in increased levels of phospho-Akt in pubertal mice. Hyal-1 null ovarian follicles did not exhibit hyaluronan accumulation. For Hyal-3 null mice, compensation by Hyal-1 or Hyal-2 might be related to the lack of an ovarian phenotype. In conclusion, our results demonstrate that Hyal-1 plays a key role in the early phases of folliculogenesis by negatively regulating ovarian follicle growth and survival. Our findings add Hyal-1 as an ovarian regulator factor for follicle development, showing for the first time an interrelationship between this enzyme and the follistatin/activin/Smad3 pathway.

High expression of luteinizing hormone receptors messenger RNA by human cumulus granulosa cells is in correlation with decreased fertilization

To elucidate the LH receptor (LHR) expression patterns in human granulosa cells (GCs) from antral to preovulatory stages, and to investigate a correlation to oocyte function. Luteinized preovulatory GCs were obtained from preovulatory follicles aspirated during IVF (≥ 17 mm). The GCs from small- (<10 mm) and medium-sized (10-15 mm) follicles were obtained during in vitro maturation (IVM) procedures. Cumulus GCs were obtained during oocyte denudation for intracytoplasmatic sperm injection (ICSI) procedures (IVF). Referral center. Seventy IVF patients and 20 IVM patients. GC collection. The LHR expression levels in mural and cumulus GCs of different follicular sizes and their correlation to oocyte outcome. The LHR expression increased with follicle size and was higher in mural GCs compared with cumulus cells. The LHR expression in cumulus GCs from preovulatory follicles was higher in metaphase II (MII) oocytes than in metaphase I or germinal vesicle oocytes (IVF). Unexpectedly, higher expression of LHR in cumulus GCs of MII oocytes correlated with decreased fertilization rates. The LHR expression in small follicles obtained in IVM suggests a role for hCG administration during IVM procedures. Overexpression of LHR in cumulus GCs of MII oocytes may signal malfunction of oocytes and low fertilization capacity.

Expression of Extracellular Matrix Components Is Disrupted in the Immature and Adult Estrogen Receptor β-Null Mouse Ovary

Within the ovary, Estrogen Receptor β (ERβ) is localized to the granulosa cells of growing follicles. 17β-estradiol (E2) acting via ERβ augments the actions of follicle stimulating hormone in granulosa cells, leading to granulosa cell differentiation and formation of a preovulatory follicle. Adult ERβ-null females are subfertile and possess ovaries with reduced numbers of growing follicles and corpora lutea. Because the majority of E2 production by granulosa cells occurs once puberty is reached, a role for ERβ in the ovary prior to puberty has not been well examined. We now provide evidence that lack of ERβ disrupts gene expression as early as post-natal day (PND) 13, and in particular, we identify a number of genes of the extracellular matrix (ECM) that are significantly higher in ERβ-null follicles than in wildtype (WT) follicles. Considerable changes occur to the ECM occur during normal folliculogenesis to allow for the dramatic growth, cellular differentiation, and reorganization of the follicle from the primary to preovulatory stage. Using quantitative PCR and immunofluorescence, we now show that several ECM genes are aberrantly overexpressed in ERβ-null follicles. We find that Collagen11a1, a protein highly expressed in cartilage, is significantly higher in ERβ-null follicles than WT follicles as early as PND 13, and this heightened expression continues through PND 23–29 into adulthood. Similarly, Nidogen 2, a highly conserved basement membrane glycoprotein, is elevated in ERβ-null follicles at PND 13 into adulthood, and is elevated specifically in the ERβ-null focimatrix, a basal lamina-like matrix located between granulosa cells. Focimatrix laminin and Collagen IV expression were also higher in ERβ-null ovaries than in WT ovaries at various ages. Our findings suggest two novel observations: a) that ERβ regulates granulosa cell gene expression ovary prior to puberty, and b) that ERβ regulates expression of ECM components in the mouse ovary.

A new classification of a preovulatory oocyte maturation stage suitable for the synchronization of ovulation in controlled reproduction of Eurasian perch, Perca fluviatilis L

To improve controlled reproduction of Eurasian perch Perca fluviatilis, the criteria for the evaluation of final oocyte maturation stages were revised. The new classification covers six preovulatory maturational stages (I -VI) from the end of vitellogenesis to germinal vesicle breakdown (GVBD) and was based on macroscopic changes of preovulatory oocytes (position of the germinal vesicle, GVBD, oil droplets coalescence). The observation was performed during out-of-season artificial reproduction with the use of a single hCG injection (500 IU/kg). The classification was subsequently verified with the controlled reproduction of wild female perch with the use of hormonal stimulation (500 IU hCG/kg of body weight at 12°C). The females were at different maturational stages and constituted respective experimental groups (I-VI). During the experiment, ovulation appeared to be considerably synchronized within particular groups. Statistical differences in latency time (time between hormonal treatment and ovulation) were found between experimental groups (mean latency time: 110, 92, 68, 49, 29 and 18 h in groups representing VI, V, IV, III, II and I stage of the proposed classification, respectively). The proposed classification and the results presented in the study allowed for effective synchronisation of ovulation. The use of our new oocyte maturation classification may positively influence the effectiveness of Eurasian perch production.

A proteomic analysis of human follicular fluid in women undergoing IVF cycles

A proteomic analysis of human follicular fluid in women undergoing IVF cycles