Follicle Oocyte Research Articles

Murine follicular development requires oocyte DICER, but not DROSHA.

Both DICER and DROSHA are RNase III enzymes involved in the biogenesis of small noncoding RNAs. DROSHA cleaves the stem-loop portion of the primary miRNAs and produces precursor miRNAs in the nucleus, whereas DICER processes double-stranded RNA precursors into mature miRNAs and endogenous small interference RNAs in the cytoplasm. Selective inactivation of Dicer in growing oocytes of primary follicles leads to female infertility due to oocyte spindle defects. However, it remains unknown if oocyte Dicer expression in the fetal ovary is required for proper follicular development in the postnatal ovary. Moreover, the role of Drosha in folliculogenesis has never been investigated. Here, we report that conditional knockout of Dicer in prophase I oocytes of the fetal ovary led to compromised folliculogenesis, premature ovarian failure, and female infertility in the adult ovary, whereas selective inactivation of Drosha in oocytes of either the fetal or the developing ovary had no effects on normal folliculogenesis and female fertility in adulthood. Our data indicate that oocyte DICER expression in the fetal ovary is required, and oocyte DROSHA is dispensable, for postnatal follicular development and female fertility in adulthood.

Transactivation of MicroRNA-320 by MicroRNA-383 Regulates Granulosa Cell Functions by Targeting E2F1 and SF-1 Proteins

Our previous studies have shown that microRNA-320 (miR-320) is one of the most down-regulated microRNAs (miRNA) in mouse ovarian granulosa cells (GCs) after TGF-β1 treatment. However, the underlying mechanisms of miR-320 involved in GC function during follicular development remain unknown. In this study, we found that pregnant mare serum gonadotropin treatment resulted in the suppression of miR-320 expression in a time-dependent manner. miR-320 was mainly expressed in GCs and oocytes of mouse ovarian follicles in follicular development. Overexpression of miR-320 inhibited estradiol synthesis and proliferation of GCs through targeting E2F1 and SF-1. E2F1/SF-1 mediated miR-320-induced suppression of GC proliferation and of GC steroidogenesis. FSH down-regulated the expression of miR-320 and regulated the function of miR-320 in mouse GCs. miR-383 promoted the expression of miR-320 and enhanced miR-320-mediated suppression of GC proliferation. Injection of miR-320 into the ovaries of mice partially promoted the production of testosterone and progesterone but inhibited estradiol release in vivo. Moreover, the expression of miR-320 and miR-383 was up-regulated in the follicular fluid of polycystic ovarian syndrome patients, although the expression of E2F1 and SF-1 was down-regulated in GCs. These data demonstrated that miR-320 regulates the proliferation and steroid production by targeting E2F1 and SF-1 in the follicular development. Understanding the regulation of miRNA biogenesis and function in the follicular development will potentiate the usefulness of miRNA in the treatment of reproduction and some steroid-related disorders.

Histological and histometrical study of the protective role of α-tocopherol against sodium arsenite toxicity in rat ovaries.

This study examines histometrical changes induced by sodium arsenite (SA), as an environmental pollutant, and investigates the protective effect of α-tocopherol on ovaries of SA-treated rats during the prenatal stage until sexual maturity. Rats were classified into groups: control, SA (8 ppm/day), α-tocopherol (100 ppm/day), and SA+α-tocopherol. Treatment was performed from pregnancy until maturation when the rats and ovaries were weighed. The Cavalieri method was used to estimate volume of the ovaries, cortex, medulla, and corpus luteum. The mean diameter of oocytes, granulosa cells, and nuclei were measured and volume was estimated using the Nucleator method. The number of oocytes and thickness of the zona pellucida (ZP) were determined using an optical dissector and orthogonal intercept method, respectively. SA reduced the body and ovary weight, the number of secondary, antral and Graafian oocytes, volume of the ovaries, cortex, medulla and corpus luteum, mean diameter and volume of oocytes in primordial and primary follicles, mean diameter and volume of oocyte nuclei in all types of follicles, and mean thickness of the ZP in secondary and antral follicles. Also, the mean diameter and volume of granulosa cells and their nuclei in antral and Graafian follicles decreased significantly. Vacuolization and vascular congestion in the corpus luteum and an increase in the number of atretic oocytes were seen in the SA group. Most of these parameters were unchanged from the control level in the SA+α-tocopherol group. It was concluded that α-tocopherol supplementation reduced the toxic effects of SA exposure on ovarian tissue in rats.

The role of androgen hormones in early follicular development.

Background. Although chronic hyperandrogenism, a typical feature of polycystic ovary syndrome, is often associated with disturbed reproductive performance, androgens have been shown to promote ovarian follicle growth in shorter exposures. Here, we review the main effects of androgens on the regulation of early folliculogenesis and the potential of their application in improving follicular in vitro growth. Review. Androgens may affect folliculogenesis directly via androgen receptors (ARs) or indirectly through aromatization to estrogen. ARs are highly expressed in the granulosa and theca cells of early stage follicles and slightly expressed in mature follicles. Short-term androgen exposure augments FSH receptor expression in the granulosa cells of developing follicles and enhances the FSH-induced cAMP formation necessary for the transcription of genes involved in the control of follicular cell proliferation and differentiation. AR activation also increases insulin-like growth factor (IGF-1) and its receptor gene expression in the granulosa and theca cells of growing follicles and in the oocytes of primordial follicles, thus facilitating IGF-1 actions in both follicular recruitment and subsequent development. Conclusion. During the early and intermediate stages of follicular maturation, locally produced androgens facilitate the transition of follicles from the dormant to the growing pool as well as their further development.

Human Amniotic Fluid Stem Cells Possess the Potential to Differentiate into Primordial Follicle Oocytes In Vitro1

Previous reports have demonstrated that embryonic stem cells were capable of differentiating into primordial germ cells through the formation of embryoid bodies that subsequently generated oocyte-like cells (OLCs). Such a process could facilitate studies of primordial follicle oocyte development in vitro and regenerative medicine. To investigate the pluripotency of human amniotic fluid stem cells (hAFSCs) and their ability to differentiate into germ cells, we isolated a CD117(+)/CD44(+) hAFSC line that showed fibroblastoid morphology and intrinsically expressed both stem cell markers (OCT4, NANOG, SOX2) and germ cell markers (DAZL, STELLA). To encourage differentiation into OLCs, the hAFSCs were first cultured in a medium supplemented with 5% porcine follicular fluid for 10 days. During the induction period, cell aggregates formed and syntheses of steroid hormones were detected; some OLCs and granulosa cell-like cells could be loosened from the surface of the culture dish. Cell aggregates were collected and replated in oocyte culture medium for an additional 7-10 days. OLCs ranging from 50 to 120 μm presenting zona pellucida were observed in cumulus-oocyte complexes; some OLCs developed spontaneously into multicell structures similar to preimplantation embryos. Approximately 2% of the hAFSCs differentiated to meiotic germ cells that expressed folliculogenesis- and oogenesis-associated markers. Although the in vitro maturation and fertilization potentials are as yet unproven, short-term (<25 days) and high-efficiency (>2%) derivation of OLCs from hAFSCs might provide a new approach to the study of human germ cell development in vitro.

α-endosulfine (ENSA) regulates exit from prophase I arrest in mouse oocytes

Mammalian oocytes in ovarian follicles are arrested in meiosis at prophase I. This arrest is maintained until ovulation, upon which the oocyte exits from this arrest, progresses through meiosis I and to metaphase of meiosis II. The progression from prophase I to metaphase II, known as meiotic maturation, is mediated by signals that coordinate these transitions in the life of the oocyte. ENSA (α-endosulfine) and ARPP19 (cAMP-regulated phosphoprotein-19) have emerged as regulators of M-phase, with function in inhibition of protein phosphatase 2A (PP2A) activity. Inhibition of PP2A maintains the phosphorylated state of CDK1 substrates, thus allowing progression into and/or maintenance of an M-phase state. We show here ENSA in mouse oocytes plays a key role in the progression from prophase I arrest into M-phase of meiosis I. The majority of ENSA-deficient oocytes fail to exit from prophase I arrest. This function of ENSA in oocytes is dependent on PP2A, and specifically on the regulatory subunit PPP2R2D (also known as B55δ). Treatment of ENSA-deficient oocytes with Okadaic acid to inhibit PP2A rescues the defect in meiotic progression, with Okadaic acid-treated, ENSA-deficient oocytes being able to exit from prophase I arrest. Similarly, oocytes deficient in both ENSA and PPP2R2D are able to exit from prophase I arrest to an extent similar to wild-type oocytes. These data are evidence of a role for ENSA in regulating meiotic maturation in mammalian oocytes, and also have potential relevance to human oocyte biology, as mouse and human have genes encoding both Arpp19 and Ensa.

Concentrations of bone morphogenetic protein-15 (BMP-15) and growth differentiation factor-9 (GDF-9) in follicular cysts, mono - and polyoocyte follicles in gilts

Abstract The objective of the study was to determine the concentration of BMP-15 and GDF-9 in the fluid of follicular cysts and ovarian follicles, and to compare their concentrations in mono- and polyoocyte follicles in gilts. The study involved two experiments conducted on the ovaries collected post-slaughter from gilts (7-8 months old). The first experiment covered 31 follicular single cyst gilts (15-25 mm in diameter) and 41 gilts without cysts. Follicular fluid from follicles of 8-10 mm in diameter (n=41) and 5-8 mm in diameter (n=41), and cystic fluid (n=31) were collected for analysis. The second experiment involved collecting follicular fluid from poly- (n=19) and monooocyte (n=22) follicles. The concentration of BMP-15 and GDF-9 was then determined in the samples using specimen-specific ELISA kits. The differences in the concentration of these factors were calculated by means of analysis of variance and a posthoc test. Duncan’s multiple range test was used to verify the significance of differences at P&lt;0.05 and P&lt;0.01. In addition, correlations between the factors were calculated. BMP-15 and GDF-9 levels in the cystic fluid were significantly higher than those in the follicular fluid (P&lt;0.01). However, no differences were observed between various size follicles or between mono- and polyoocyte follicles. BMP-15 and GDF-9 concentrations were found to be positively correlated (P&lt;0.01). Differences in BMP-15 and GDF-9 concentrations in ovarian follicles and follicular cysts, as evidenced by our study, indicate that these factors may be related to folliculogenesis disorders in gilts. What is more, the number of oocytes in ovarian follicles does not influence the intrafollicular concentration of BMP-15 and GDF-9.

Transforming Growth Factor-β Signaling Participates in the Maintenance of the Primordial Follicle Pool in the Mouse Ovary

Physiologically, only a few primordial follicles are activated to enter the growing follicle pool each wave. Recent studies in knock-out mice show that early follicular activation depends on signaling from the tuberous sclerosis complex, the mammalian target of rapamycin complex 1 (mTORC1), phosphatase and tensin homolog deleted on chromosome 10, and phosphatidylinositol 3-kinase (PI3K) pathways. However, the manner in which these pathways are normally regulated, and whether or not TGF-β acts on them are poorly understood. So, this study aims to identify whether or not TGF-β acts on the process. Ovary organ culture experiments showed that the culture of 18.5 days post-coitus (dpc) ovaries with TGF-β1 reduced the total population of oocytes and activated follicles, accelerated oocyte growth was observed in ovaries treated with TGF-βR1 inhibitor 2-(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-yl-amine (SD208) compared with control ovaries, the down-regulation of TGF-βR1 gene expression also activated early primordial follicle oocyte growth. We further showed that there was dramatically more proliferation of granulosa cells in SD208-treated ovaries and less proliferation in TGF-β1-treated ovaries. Western blot and morphological analyses indicated that TGF-β signaling manipulated primordial follicle growth through tuberous sclerosis complex/mTORC1 signaling in oocytes, and the mTORC1-specific inhibitor rapamycin could partially reverse the stimulated effect of SD208 on the oocyte growth and decreased the numbers of growing follicles. In conclusion, our results suggest that TGF-β signaling plays an important physiological role in the maintenance of the dormant pool of primordial follicles, which functions through activation of p70 S6 kinase 1 (S6K1)/ribosomal protein S6 (rpS6) signaling in mouse ovaries.

An oocyte-specific ELAVL2 isoform is a translational repressor ablated from meiotically competent antral oocytes

At the end of the growth phase, mouse antral follicle oocytes acquire full developmental competence. In the mouse, this event is marked by the transition from the so-called non-surrounded nucleolus (NSN) chromatin configuration into the transcriptionally quiescent surrounded nucleolus (SN) configuration, which is named after a prominent perinucleolar condensed chromatin ring. However, the SN chromatin configuration alone is not sufficient for determining the developmental competence of the SN oocyte. There are additional nuclear and cytoplamic factors involved, while a little is known about the changes occurring in the cytoplasm during the NSN/SN transition. Here, we report functional analysis of maternal ELAVL2 an AU-rich element binding protein. Elavl2 gene encodes an oocyte-specific protein isoform (denoted ELAVL2°), which acts as a translational repressor. ELAVL2° is abundant in fully grown NSN oocytes, is ablated during the NSN/SN transition and remains low during the oocyte-to-embryo transition (OET). ELAVL2° overexpression during meiotic maturation causes errors in chromosome segregation, indicating the significance of naturally reduced ELAVL2° levels in SN oocytes. On the other hand, during oocyte growth, prematurely reduced Elavl2 expression results in lower yields of fully grown and meiotically matured oocytes, suggesting that Elavl2 is necessary for proper oocyte maturation. Moreover, Elavl2 knockdown showed stimulating effects on translation in fully grown oocytes. We propose that ELAVL2 has an ambivalent role in oocytes: it functions as a pleiotropic translational repressor in efficient production of fully grown oocytes, while its disposal during the NSN/SN transition contributes to the acquisition of full developmental competence.

Primordial follicle assembly was regulated by notch signaling pathway in the mice

Notch signaling pathway, a highly conserved cell signaling system, exists in most multicellular organisms. The objective of this study was to examine Notch signaling pathway in germ cell cyst breakdown and primordial follicle formation. The receptor and ligand genes of Notch pathway (Notch1, Notch2, Jagged1, Jagged2 and Hes1) were extremely down-regulated after newborn mouse ovaries were cultured then exposed to DAPT or L-685,458 in vitro (P<0.01). Since DAPT or L-685,548 inhibits Notch signaling pathway, the expression of protein LHX8 and NOBOX was significantly reduced during the formation of the primordial follicles. Down-regulated mRNA expression of specific genes including Lhx8, Figla, Sohlh2 and Nobox, were also observed. The percentages of female germ cells in germ cell cysts and primordial follicles were counted after culture of newborn ovaries for 3days in vitro. The result showed female germ cells in cysts was remarkably up-regulated while as the oocytes in primordial follicles was significantly down-regulated (P<0.05). In conclusion, Notch signaling pathway may regulate the formation of primordial follicle in mice.

176 IN VITRO MATURATION OF DOG OOCYTES IN CANINE FOLLICULAR FLUID

In the canine species, assisted reproductive technologies, especially in vitro maturation (IVM) and IVF, are still ineffective. The main limiting factor remains the immaturity of the oocytes collected from anestrus ovaries. The ability of an oocyte to reach the MII stage in vitro is linked to the diameter of its follicle and anestrus oocytes, collected from small (&lt;1 mm) follicles, are profoundly immature (De Lesegno et al. 2008). The objective of this study was to improve cytoplasmic quality by mimicking in vivo conditions; that is, to test the effect of pure preovulatory follicular fluid (FF) on survival and IVM rates of anestrus dog oocytes, in order to improve the nuclear and cytoplasmic maturation of these immature oocytes. Follicular fluids samples were collected from 54 Beagle bitches at 2 stages: before the LH peak (n = 23 bitches) and after the LH peak (n = 31 bitches). Only follicular fluid samples from large (&gt;4 mm) follicles were collected and pooled by stage. Control oocytes were matured in 20% FCS/M199 medium. Groups of 5 oocytes were in vitro matured in 30 μL of follicular fluid, in half-area 96-well plates (5% CO2, 38°C). After 72 h of IVM, oocytes were denuded, fixed, and stained for DNA and tubulin before observation by confocal microscopy, and nuclear stages were classified as GV-A to GV-E, MI, and MII (Reynaud et al. 2012). A total of 460 oocytes were collected from 13 anestrus bitches and allocated to either the control medium (n = 155), the Pre-LH FF (n = 145) or the Post-LH FF (n = 160) groups. After 72 h of IVM, the morphology of the cumulus–oocyte complexes (COC) in the post-LH group was different from that of the others: cumulus cells appeared more compact and darker. Analysis of the nuclear stages showed that the degeneration rate was significantly higher (P &lt; 0.05) in the post-LH group (58.7%) than in the pre-LH (40.9%) or in the control group (34.4%). No significant differences (P &gt; 0.05) were observed between the 3 groups in the rate of immature GVA-B oocytes (36.4, 28.5, and 25.3% in the control, Pre-LH, and Post-LH groups, respectively), in the rate of meiotic resumption (GV-C/D/E, MI, MII stages, 44.4, 51.9, and 38.7% in the control, Pre-LH, and Post-LH groups, respectively). Metaphase II rates were not significantly different (12.1, 8.6, and 4.8% in the control, Pre-LH, and Post-LH groups, respectively). In conclusion, canine COC may survive when exposed to IVM in pure follicular fluid, but the degeneration rate was higher in the post-LH group. The presence of follicular fluid did not inhibit meiosis resumption, but did not significantly improve IVM rates. To better mimic in vivo conditions, IVM in a sequence of media, such as IVM in follicular fluid followed by IVM in oviducal fluid remains to be tested.

207 EFFECT OF REPEAT HORMONE STIMULATION ON THE YIELD AND IN VITRO DEVELOPMENT OF JUVENILE CALF OOCYTES

Juvenile in vitro embryo transfer can markedly reduce animal generation intervals. The purpose of this study was to investigate the ovarian response of juvenile calves and in vitro oocyte developmental capacity after superstimulation. Experiments on calves were performed in accordance with the Animal Welfare Regulations. A total of 36 donor juvenile calves on standard nutrition and in a disease-free environment, were selected from the breeding farm of the Beijing Dairy Cattle Center. At 60 days of age, calves were randomly assigned into three groups of four calves each, replicated three times. On day 1, Group 1 received a progesterone vaginal insert (CIDR, 300 mg per device); Group 2 received a CIDR and 0.5 mg oestrogen benzoate (China); Group 3 received a CIDR, 0.5 mg oestrogen benzoate, and 50 mg progesterone (China). Then, calves were injected with FSH (Folltropin-V, Bioniche Animal Health, Belleville, ON, Canada) twice daily on days 5 (40 mg/40 mg) and 6 (30 mg/30 mg) at 12 h intervals. Cumulus–oocyte complexes (COCs) were recovered from the superstimulated calves 12 to 14 h after the final FSH treatment. COCs were considered usable unless they were damaged or had expanded cumulus layers. Usable COCs were matured in vitro for 24 h in maturation medium consisting of TCM199, 10% FBS, 10 μg mL–1 FSH, 1 μg mL–1 LH, 1 μg mL–1 E2–17β, 100 IU mL–1 penicillin, 100 μg mL–1 streptomycin, with (+Cys) or without (–Cys) 100 μM Cysteamine. Each calf oocyte was cultured in one well. The final concentration added to each fertilization drop was 5 × 106 sperm mL–1. Sperm and oocytes were co-cultured in IVF-100 medium (BO liquid+10 μg mL–1 heparin, Japan) at 38.5°C, 5% CO2 and a saturated humidity for 6 to 8 h. Blastocyst production rates were determined after 7 and 8 d of in vitro culture in CR1aa medium without the addition of cysteamine. Differences among treatments in each experiment were determined by one-way ANOVA and a multiple range test. Superstimulatory results indicated that more follicles were aspirated (63.2 per calf) and more usable oocytes were recovered (48.0 per calf) in Group 1 than in the other two groups (Group 2–45.2 and 31.8, respectively; Group 3–35.4 and 28.3, respectively; P &lt; 0.05). No difference was observed between Groups 2 and 3. Superstimulation of calves twice at 30 day intervals in Group 2 (n = 12) did not affect the number of follicles or usable oocytes (overall, 44.2 and 28.0 per calf). Maturation rates (86.5% v. 85.0%, respectively) and cleavage rates (84.4% v. 80.0%, respectively) did not differ whether cysteamine was not (–Cys; n = 318) or was (+Cys; n = 330) added to the maturation medium. However, the blastocyst rate differed significantly (12.9% v. 35.2%, respectively; P &lt; 0.01). This study established a protocol for the superstimulation of juvenile calves with an average of 48 oocytes obtained per calf. Superstimulation and surgical oocyte recovery twice at an interval of 30 days had no adverse effect on follicle development or oocyte recovery. The novelty of this research is that the blastocyst production rate of calf oocytes (35.2%) in maturation medium supplemented with cysteamine was similar to that reported in the cow.

Effect of potential oocyte transport protocols on blastocyst rates after intracytoplasmic sperm injection in the horse

Intracytoplasmic sperm injection (ICSI) is used to produce foals from otherwise infertile mares and from stallions with limited sperm stores, but requires expensive equipment and is technically demanding. Methods to transport oocytes to ICSI laboratories would allow collection of oocytes by the referring veterinarian and enable greater application of this technique. This study was conducted to evaluate protocols that could be used to transport immature and maturing oocytes for ICSI. In vitro experiment. Oocytes were recovered by transvaginal ultrasound-guided follicular aspiration either from dominant follicles 24 h after deslorelin administration (dominant stimulated follicle [DSF]), or from subordinate (immature) follicles at the same time. To mimic transport, DSF oocytes were incubated overnight under differing conditions before ICSI; immature oocytes were placed in varying conditions overnight before in vitro maturation, followed by ICSI. The rate of blastocyst production was compared among treatments. Blastocysts were produced in all groups. Dominant stimulated follicle oocytes held in sealed tubes in pre-equilibrated control maturation medium maintained at 37°C yielded blastocyst development equal to that obtained for control incubated oocytes (70%). Dominant stimulated follicle oocytes held similarly in a warm passive device yielded poor blastocyst development (10%). Immature oocytes held for one or 2 nights in modified M199 medium, or for one night in commercial embryo holding solution, in air at room temperature, yielded 35-37% blastocyst development per injected oocyte. A commercially available medium can be used for shipping immature oocytes at room temperature with good resulting blastocyst rates. Better blastocyst rates per oocyte are obtained from DSF oocytes; however, these require maintenance at 37°C and as they are already maturing at the time of collection, are more sensitive to delays. This new, practical information supporting transport of both immature and DSF oocytes for ICSI may allow wider use of this procedure.

Aromatase-null mice expressing enhanced green fluorescent protein in germ cells provide a model system to assess estrogen-dependent ovulatory responses

Enhanced green fluorescent protein (EGFP) has provided us with valuable approaches for tracking living cells. We established a novel line of transgenic mice, which express EGFP in the testis and ovary. Histological analysis demonstrated that spermatids in the testis and oocytes in ovarian follicles beyond preantral stages were positive for EGFP. By exploiting these features, we evaluated ovulatory responses of aromatase-gene (Cyp19a) knockout mouse expressing the EGFP transgene, which is totally anovulatory due to 17β-estradiol (E2) deficiency. Ovulation in the knockout mice was induced by sequential injections of E2 on days 1, 4 and 5, pregnant mare serum gonadotropin on day 4 and human chorionic gonadotropin on day 6. Fluorescent oocytes were readily detectable at 15 h after the last gonadotropin injection in the oviduct under a fluorescence stereomicroscope, even when only one oocyte was present. However, when E2 supplementation on day 4 or day 5 in the regimen was omitted, no ovulated oocytes were detected, indicating that exogenous E2 supplementation at the time of gonadotropin stimulation is necessary to induce ovulation in aromatase-gene knockout mice. Our results further demonstrated that the current mouse line can provide an alternative tool to study germ cell biology, including oogenesis, ovulation and senescence.

Roles of thyroid hormones in follicular development in the ovary of neonatal and immature rats

Thyroid hormones (TH) play a critical role in ovarian follicular development, maturation and the maintenance of various endocrine functions. However, whether TH can affect ovarian follicular development in neonatal and immature rats remains unclear. Therefore, the aim of the present study was to elucidate the effect of TH on ovarian follicular development in neonatal and immature rats. Thirty female post-lactation mothers of Sprague-Dawley rat pups were randomly divided into three groups: control, hyperthyroid (hyper), and hypothyroid (hypo). On postnatal days (PND) 10 and 21, body weights, serum hormones, ovarian histologic changes, and immunohistochemistry of thyroid hormone receptor alpha 1 (TRα1) and nitric oxide synthase types (NOS), and NOS activities, were determined. The data showed that body weights significantly decreased in both hyper and hypo groups compared with the control group (P < 0.05). In addition, the hyper group had increased serum concentrations of T3, T4, and E2; whereas the hypo group manifested reduced serum concentrations of T3, T4, and E2 on PND 10 and 21. The hyper and hypo groups showed significantly reduced total number of primordial, primary and secondary follicles on PND 10 and 21 compared with the control group (P < 0.05). Similarly, antral follicle numbers in the hyper and hypo groups were significantly decreased on PND 21 compared with the control group (P < 0.05). Immunostaining indicated that TRα1 and NOS were expressed in ovarian surface epithelium and oocytes of growing and antral follicles, with strong staining of the granulosa and theca cells of follicles. NOS activities were significantly augmented in the hyper, but diminished in the hypo groups on PND 10 and 21. In summary, our findings suggest that TH play important roles in ovarian functions and in the regulation of NOS activity. Our results also indicate that a relationship exists between the TH and NO signaling pathways during the process of ovarian follicular development in neonatal and immature rats.

Rapamycin‐sensitive mTORC1 signaling is involved in physiological primordial follicle activation in mouse ovary

In mammals, resting female oocytes reside in primordial ovarian follicles. An individual primordial follicle may stay quiescent for a protracted period of time before initiating follicular growth, which is also termed “activation.” Female reproductive capacity is sustained by the gradual, streamlined activation of the entire population of primordial follicles, but this process also results in reproductive senescence in older animals. Based on the recent findings that genetically triggered, excessive mammalian target of rapamycin complex 1 (mTORC1) activation in mouse oocytes leads to accelerated primordial follicle activation, we examined the necessity of mTORC1 signaling in physiological primordial follicle activation. We found that induction of oocyte mTORC1 activity is associated with early follicular growth in neonatal mouse ovaries. Pharmacological inhibition of mTORC1 activity in vivo by rapamycin treatment leads to a marked, but partial, suppression of primordial follicle activation. The suppressive effect of rapamycin on primordial follicle activation was reproduced in cultured ovaries. While rapamycin did not apparently affect several plausible cellular targets in neonatal mouse ovaries, such as mTORC2, AKT, or cyclin-dependent kinase (CDK) inhibitor p27-KIP1, its inhibitory effect on Cyclin A2 gene expression implies that mTORC1 signaling in oocytes may engage a Cyclin A/CDK regulatory network that promotes primordial follicle activation. The current work strengthens the concept that mTORC1-dependent events in the oocytes of primordial follicles may represent potential targets for intervention in humans to slow the depletion of the ovarian reserve.

Confirmation of ovarian homogeneity in post-vitellogenic cultured white sturgeon, Acipenser transmontanus

Assessing stage of oocyte maturity in female sturgeon by calculating oocyte polarization index (PI) is a necessary tool for both conservation propagation managers and caviar producers to know when to hormonally induce spawning. We tested the assumption that sampling ovarian follicles from one section of one ovary is sufficient for calculating an oocyte PI representative of oocyte maturity for an individual animal. Short-wavelength near-infrared spectroscopy (SW-NIR) scans were performed on three positions per ovary for five fish prior to caviar harvest. Samples of ovarian follicles were subsequently taken from the exact location of the SW-NIR scans for calculation of oocyte PI and follicle diameter. Oocyte PI was statistically different though not biologically relevant within an ovary and between ovaries in four of five fish. Follicle diameter was statistically different but not biologically relevant within an ovary in three of five fish. There were no differences in follicle diameter between ovaries. No statistical differences were observed between SW-NIR spectra collected at different locations within an ovary or between ovaries. These results emphasize the importance of utilizing both oocyte PI measurement and progesterone-induced oocyte maturation assays while deciding when to hormonally induce spawning in sturgeon females.

Viability analysis of oocyte–follicle complexes and gonadal fragments of zebrafish as baseline for toxicity testing

To achieve more information about growth and development of oocytes in teleost fish or concerning toxicity testing, it is necessary to develop adequate in vitro oocyte culture conditions. Herein, initial stages of zebrafish oocytes (I, primary; II, cortical; III, vitellogenic) were analyzed under serum-free medium conditions as gonadal fragments or as separated oocyte–follicle complexes. Two vital dye staining methods (MTT, trypan blue) were applied to assess mitochondrial activity and membrane integrity of the oocytes during 4 days, and compared to morphological alterations studied by transmission electron microscopy. Vital dye staining indicated reduced viability at day 4 for all stages in both in vitro culture methods. Additionally, the viability decreased significantly in gonadal fragments at day 2 for stages III (MTT, TB) and II (TB only). Signs of degradation at the ultrastructural level (vacuoles, disintegration of endoplasmic reticulum and detachment of follicular cell layers) appeared in gonadal fragments at day 4 for stages II and III, and in separated oocyte–follicle complexes both at day 4 for stages I–III, and at day 2 for stage III. In conclusion, zebrafish oocytes at stages I and II seemed viable for 2 days as separated oocyte–follicle complexes considering their mitochondrial activity, membrane integrity and ultrastructural morphology. Cultured as gonadal fragments, the majority of analyses indicated similar results for stages I and II oocytes. In contrast, stage III oocytes seemed viable for not longer than 24 h. Results should be taken into consideration for the experimental design of in vitro assays using teleost fish oocytes.

SAS1B protein [ovastacin] shows temporal and spatial restriction to oocytes in several eutherian orders and initiates translation at the primary to secondary follicle transition

Sperm Acrosomal SLLP1 Binding (SAS1B) protein (ovastacin) is an oolemmal binding partner for the intra-acrosomal sperm protein SLLP1. Immunohistochemical localization revealed that SAS1B translation is restricted among adult tissues to the ovary and oocytes, SAS1B appearing first in follicles at the primary-secondary transition. Quiescent oocytes within primordial follicles and primary follicles did not stain for SAS1B. Examination of neonatal rat ovaries revealed SAS1B expression first as faint signals in postnatal day 3 oocytes, with SAS1B protein staining intensifying with oocyte growth. Irrespective of animal age or estrus stage, SAS1B was seen only in oocytes of follicles that initiated a second granulosa cell layer. The precise temporal and spatial onset of SAS1B expression was conserved in adult ovaries in seven eutherian species, including nonhuman primates. Immunoelectron micrographs localized SAS1B within cortical granules in MII oocytes. A population of SAS1B localized on the oolemma predominantly in the microvillar region anti-podal to the nucleus in ovulated MII rat oocytes and on the oolemma in macaque GV oocytes. The restricted expression of SAS1B protein in growing oocytes, absence in the ovarian reserve, and localization on the oolemma suggest this zinc metalloprotease deserves consideration as a candidate target for reversible female contraceptive strategies.

Effect of flutamide on folliculogenesis in the fetal porcine ovary – Regulation by Kit ligand/c-Kit and IGF1/IGF1R systems

In pigs, primordial to primary follicle transition occur in the late pregnancy. The interactions between Kit ligand (KL) and its receptor (c-Kit), as well as insulin-like growth factor 1 (IGF1) and cognate receptor (IGF1R) are crucial for the primordial follicle activation. It is well established that hormonal disruption induces abnormalities in the developing reproductive system. Hence, this study investigated the influence of antiandrogen, flutamide, on genes involved in the primordial to primary follicle transition. Pregnant gilts were injected with flutamide (50mg/kg bw, seven times, every day) or corn oil (control groups) starting on gestation days 83 (GD90) or 101 (GD108). Fetal ovaries were excised on days 90 and 108 of gestation. The proportion of primordial and primary follicles was determined, and immunohistochemistry for c-Kit and IGF1R was conducted. To assess KL, c-Kit, IGF1 and IGF1R mRNA expression real-time PCR was performed. Ovaries from both GD90 and GD108 animals exhibited a greater proportion of primordial to primary follicles when compared to respective control groups. C-Kit and IGF1R were immunolocalized in the oocytes of primordial and primary follicles. Both c-Kit mRNA and protein levels and KL mRNA expression were diminished in GD90 group. IGF1R expression decreased at mRNA and protein levels, whereas IGF1 mRNA expression was increased in GD90 and GD108 groups. In summary, our findings may indicate that the interactions between KL and c-Kit as well as IGF1 and IGF1R are relevant to the initiation of follicular transition from primordial into primary follicles and can be affected by AR signaling.