Rictor/mTORC2 Pathway in Oocytes Regulates Folliculogenesis,and Its Inactivation Causes Premature Ovarian Failure

Molecular basis of ovarian folliculogenesis and etiopathogenesis of premature ovarian failure (POF), a common cause of infertility in women, are not fully understood. Mechanistic target of rapamycin complex 2 (mTORC2) is emerging as a central regulator of cell metabolism, proliferation, and survival. However, its role in folliculogenesis and POF has not been reported. Here, we showed that the signaling activity of mTORC2 is inhibited in a 4-vinylcyclohexene diepoxide (VCD)-induced POF mouse model. Notably, mice with oocyte-specific ablation of Rictor, a key component of mTORC2, demonstrated POF phenotypes, including massive follicular death, excessive loss of functional ovarian follicles, abnormal gonadal hormone secretion, and consequently, secondary subfertility in conditional knock-out (cKO) mice. Furthermore, reduced levels of Ser-473-phosphorylated Akt and Ser-253-phosphorylated Foxo3a and elevated pro-apoptotic proteins, Bad, Bax, and cleaved poly ADP-ribose polymerase (PARP), were observed in cKO mice, replicating the signaling alterations in 4-VCD-treated ovaries. These results indicate a critical role of the Rictor/mTORC2/Akt/Foxo3a pro-survival signaling axis in folliculogenesis. Interestingly, loss of maternal Rictor did not cause obvious developmental defects in embryos or placentas from cKO mice, suggesting that maternal Rictor is dispensable for preimplantation embryonic development. Our results collectively indicate key roles of Rictor/mTORC2 in folliculogenesis, follicle survival, and female fertility and support the utility of oocyte-specific Rictor knock-out mice as a novel model for POF.     

Development of an animal model for ovotoxicity using 4-vinylcyclohexene: a case study

BACKGROUND: The occupational chemical 4-vinylcyclohexene (VCH) has been shown to cause destruction of small pre-antral follicles in ovaries of mice. Further, its monoepoxide metabolites, 1,2-VCH epoxide, 7,8-VCH epoxide, and the diepoxide, VCD, have been shown to cause pre-antral follicle loss in rats as well as mice. Chemicals that destroy small pre-antral follicles are of concern to women because exposure can result in premature ovarian failure (early menopause). METHODS: Studies working with these chemicals over the past decade have determined a number of aspects of the mechanism(s) of small pre-antral destruction, and a variety of questions have been answered. RESULTS: Specifically, it has been determined that the diepoxide (VCD) is the bioactive form and it directly targets the ovary in mice and rats. Mice are more susceptible to VCH than rats because they are capable of its metabolic bioactivation. Follicle destruction by VCD is selective for primordial and primary follicles. Mechanistic studies in rats have determined that VCD causes ovotoxicity by accelerating the natural process of atresia (apoptosis) and this requires repeated exposures. Pro-apoptotic signaling events in the Bcl-2 and mitogen activated protein kinase families have been shown to be selectively activated in fractions of small pre-antral follicles (targets for VCD). Finally, a whole ovarian culture system using neonatal mouse and rat ovaries has been developed to expand the potential for more in depth investigations into ovotoxicity caused by VCD. CONCLUSIONS: This article provides an overview of the questions asked and the approaches taken in studying VCH and VCD to support these conclusions.     

Genomewide discovery and classification of candidate ovarian fertility genes in the mouse

Female infertility syndromes are among the most prevalent chronic health disorders in women, but their genetic basis remains unknown because of uncertainty regarding the number and identity of ovarian factors controlling the assembly, preservation, and maturation of ovarian follicles. To systematically discover ovarian fertility genes en masse, we employed a mouse model (Foxo3) in which follicles are assembled normally but then undergo synchronous activation. We developed a microarray-based approach for the systematic discovery of tissue-specific genes and, by applying it to Foxo3 ovaries and other samples, defined a surprisingly large set of ovarian factors (n = 348, approximately 1% of the mouse genome). This set included the vast majority of known ovarian factors, 44% of which when mutated produce female sterility phenotypes, but most were novel. Comparative profiling of other tissues, including microdissected oocytes and somatic cells, revealed distinct gene classes and provided new insights into oogenesis and ovarian function, demonstrating the utility of our approach for tissue-specific gene discovery. This study will thus facilitate comprehensive analyses of follicle development, ovarian function, and female infertility.     

The follicle-deplete mouse ovary produces androgen

The follicle-depleted postmenopausal ovary is enriched in interstitial cells that produce androgens. This study was designed to cause follicle depletion in mice using the industrial chemical, 4-vinylcyclohexene diepoxide (VCD), and characterize the steroidogenic capacity of cells in the residual ovarian tissue. From a dose-finding study, the optimal daily concentration of VCD was determined to be 160 mg/kg. Female B6C3F(1) immature mice were treated daily with vehicle control or VCD (160 mg kg(-1) day(-1), 15 days, i.p.). Ovaries were removed and processed for histological evaluation. On Day 15 following onset of treatment, primordial follicles were depleted and primary follicles were reduced to about 10% of controls. On Day 46, primary follicles were depleted and secondary and antral follicles were reduced to 0.7% and 2.6% of control, respectively. Seventy-five percent of treated mice displayed disruptions in estrous cyclicity. All treated mice were in persistent diestrus (acyclic) by Day 58. Plasma FSH levels were increased (P < 0.05) relative to controls on Day 37 and had plateaued by Day 100. Relative to age-matched cyclic controls, by Day 127, the significant differences in VCD-treated mice included reduced ovarian and uterine weights, elevated plasma LH and FSH, and reduced plasma progesterone and androstenedione. Furthermore, plasma 17beta-estradiol levels were nondetectable. Unlike controls, immunostaining for LH receptor, and the high density lipoprotein receptor (SR-BI), was diffuse in ovarian sections from VCD-treated animals. Ovaries from Day 120 control and VCD-treated animals were dissociated and dispersed cells were placed in culture. Cultured cells from ovaries of VCD-treated animals produced less LH-stimulated progesterone than control cells. Androstenedione production was nondetectable in cells from cyclic control animals. Conversely, cells from VCD-treated animals produced androstenedione that was doubled in the presence of insulin and LH (1 and 3 ng/ml). Collectively, these data demonstrate that VCD-mediated follicle depletion results in residual ovarian tissue that may be analogous to the follicle-deplete postmenopausal ovary. This may serve as a useful animal model to examine the dynamics of follicle loss in women as ovarian senescence ensues.     

p27kip1 (cyclin-dependent kinase inhibitor 1B) controls ovarian development by suppressing follicle endowment and activation and promoting follicle atresia in mice

In humans, the molecular mechanisms underlying ovarian follicle endowment and activation, which are closely related to the control of female reproduction, occurrence of menopause, and related diseases such as premature ovarian failure, are poorly understood. In the current study, we provide several lines of genetic evidence that the cyclin-dependent kinase (Cdk) inhibitor 1B (commonly known as p27(kip1) or p27) controls ovarian development in mice by suppressing follicle endowment and activation, and by promoting follicle death. In p27-deficient (p27(-/-)) mice, postnatal follicle assembly was accelerated, and the number of endowed follicles was doubled as compared with p27(+/+) mice. Moreover, in p27(-/-) ovaries the primordial follicle pool was prematurely activated once it was endowed, and at the same time the massive follicular death that occurs before sexual maturity was rescued by loss of p27. In early adulthood, however, the overactivated follicular pool in p27(-/-) ovaries was largely depleted, causing premature ovarian failure. Furthermore, we have extensively studied the molecular mechanisms underlying the above-mentioned phenotypes seen in p27(-/-) ovaries and have found that p27 controls follicular development by several distinct mechanisms at different stages of development of the ovary. For example, p27 controls oocyte growth by suppressing the functions of Cdk2/Cdc2-cyclin A/E1 in oocytes that are arrested at the diplotene stage of meiosis I. This function of p27 is distinct from its well-known role as a suppressor of cell cycle progression. In addition, we have found that p27 activates the caspase-9-caspase-3-caspase-7-poly (ADP-ribose) polymeraseapoptotic cascade by inhibiting Cdk2/Cdc2-cyclin A/B1 kinase activities in follicles, thereby inducing follicle atresia. Our results suggest that the p27 gene is important in determining mammalian ovarian development. This study therefore provides insight into ovary-borne genetic aberrations that cause defects in folliculogenesis and infertility in humans.     

Inhibition of PIK3 signaling pathway members by the ovotoxicant 4-vinylcyclohexene diepoxide in rats

4-Vinylcyclohexene diepoxide (VCD), an occupational chemical that specifically destroys primordial and small primary follicles in the ovaries of rats and mice, is thought to target an oocyte-expressed tyrosine kinase receptor, Kit. This study compared the temporal effect of VCD on protein distribution of KIT and its downstream PIK3-activated proteins, AKT and FOXO3. Postnatal Day 4 Fischer 344 rat ovaries were cultured in control media ± VCD (30 μM) for 2-8 days (d2-d8). KIT, AKT, phosphorylated AKT, FOXO3, and pFOXO3 protein levels were assessed by Western blotting and/or immunofluorescence staining with confocal microscopy. Phosphorylated AKT was decreased (P < 0.05) in oocyte nuclei in primordial (39% decrease) and small primary (37% decrease) follicles within 2 days of VCD exposure. After d4, VCD reduced (P < 0.05) oocyte staining for KIT (primordial, 44% decrease; small primary, 39% decrease) and FOXO3 (primordial, 40% decrease; small primary, 36% decrease) protein. Total AKT and pFOXO3 were not affected by VCD at any time. Akt1 mRNA, as measured by quantitative RT-PCR, was reduced (P < 0.05) by 23% on d4 of VCD exposure, but returned to control levels on d6 and d8. VCD exposure reduced Foxo3a mRNA by 26% on d6 (P < 0.05) and by 23% on d8 (P < 0.1). These results demonstrate that the earliest observed effect of VCD is an inhibition of phosphorylation and nuclear localization of AKT in the oocyte of primordial and small primary follicles. This event is followed by reductions in KIT and FOXO3 protein subcellular distribution prior to changes in mRNA. Thus, these findings further support that VCD induces ovotoxicity by directly targeting the oocyte through posttranslational inhibition of KIT-mediated signaling components.     

Involvement of the KIT/KITL signaling pathway in 4-vinylcyclohexene diepoxide-induced ovarian follicle loss in rats

Repeated daily dosing of rats with the occupational chemical 4-vinylcyclohexene diepoxide (VCD) depletes the ovary of primordial and primary follicles through an increase in the natural process of atresia. Additionally, in vitro exposure of Postnatal Day 4 (PND 4) rat ovaries to VCD causes similar follicular depletion. This study was designed to investigate survival signaling pathways that may be associated with VCD-induced ovotoxicity in small preantral follicles. Female Fischer 344 rats (PND 28) were dosed daily (80 mg/kg/day VCD i.p.; 12 days in vivo), and PND 4 ovaries were cultured (VCD 20 or 30 microM; 8 days in vitro). Microarray analysis identified a subset of 14 genes whose expression was increased or decreased by VCD in both experiments (i.e., via both exposure routes). Particularly, the analysis showed that relative to controls, VCD did not affect mRNA expression of growth and differentiation factor 9 (Gdf9), whereas there were decreases in mRNA encoding bone morphogenic protein receptor 1a (Bmpr1a) and Kit. To confirm findings from microarray, the genes Gdf9, Bmpr1a, and Kit were further examined. When growth factors associated with these pathways were added to ovarian cultures during VCD exposure, GDF9 and BMP4 had no effect on VCD-induced ovotoxicity; however, KITL attenuated this follicle loss. Additionally, there was a decrease in Kit and an increase in Kitl expression (mRNA and protein) following VCD exposure, relative to control. These results support that VCD compromises KIT/KITL signaling, which is critical for follicular survival in primordial and primary follicles.     

In utero exposure of mice to diethylstilbestrol alters neonatal ovarian follicle growth and development

The prenatal exposure of mice to diethylstilbestrol (DES, 10 micrograms/kg on day 15 of gestation) caused both quantitative and structural alterations in ovarian follicles within the neonatal ovary. At birth, control ovaries consisted of small type 1 and 2 ovarian follicles located in the ovarian cortex. By postnatal day 7, ovarian follicle development had advanced to the type 4 stage with larger follicles located within the ovarian medulla. In DES-exposed animals, ovarian follicle maturation was advanced with type 3b and 4 follicles appearing 24 h prior to their appearance in control animals. Also, type 5 ovarian follicles were present on postnatal day 6 in experimental animals but were never seen in control animals. In addition to an alteration in ovarian follicle dynamics, the diameter of individual ovarian follicles was transit time between the various stages of follicular development which results in a greater number of developmentally advanced ovarian follicles being present during neonatal ovarian development. The mechanism by which prenatal exposure to DES alters ovarian follicle dynamics during neonatal development is not known.     

New strategy for in vitro activation of primordial follicles with mTOR and PI3K stimulators

It had been known for decades that primordial follicles in mammalian ovaries are assembled with definite numbers and represent the ovarian reserve throughout the reproductive life. Intra-oocyte PI3K/mTOR pathways have been indicated to play a central role on the activation of primordial follicles. Genetic modified mouse models with chronic activation of PI3K/mTOR signals in primordial oocytes showed premature activation of all primordial follicles and eventually their exhaustion. On the other hand, this may suggest that, unlike chronic activation of PI3K/mTOR, its acute activation in infertility would activate primordial follicles, permitting fertility during the treatment. Previously, PI3K stimulators were reported as a temporary measure to accelerate primordial follicle activation and follicular development in both mouse and human, and were applied in the treatment of infertility in premature ovarian failure (POF) patients. To address whether mTOR stimulators could play similar role in the process, we transiently treated neonatal and aged mouse ovaries with mTOR stimulators-phosphatidic acid (PA) and propranolol. Our results demonstrated the stimulators increased activation of primordial follicles and the production of progeny. Human ovarian cortex cubes were also treated with mTOR or/and PI3K stimulators in vitro. When they were used separately, both of them showed similar promotive effects on primordial follicles. Surprisingly, after joint-treatment with the 2 kinds of stimulators together, synergistic effects on follicular development were observed. Based on increased efficiency of follicular activation in humans, here we propose in vitro transient treatment with mTOR and PI3K stimulators as an optimized protocol for the application in different clinical conditions with limited follicle reserve.     

Effects of impending ovarian failure induced by 4-vinylcyclohexene diepoxide on fertility in C57BL/6 female mice

Repeated daily dosing of mice with 4-vinylcyclohexene diepoxide (VCD) causes a gradual onset of ovarian failure, providing a model for perimenopause. Because increasing numbers of women are delaying starting a family, infertility in aging women is of concern. This study was designed to determine the effects of impending ovarian failure on fertility in VCD-treated mice. Female C57BL/6J mice were dosed daily (17 d) with vehicle control or VCD (160 mg/kg, intraperitoneally) to deplete primordial follicles and then were divided into 2 groups. Group 1 was mated soon after dosing; group 2 was mated on day 20 after dosing, during impending ovarian failure. Fertility was evaluated on gestational day 16. In group 1, cycle length, pregnancy rate, and number of live fetuses did not differ between VCD-treated animals and controls, but VCD-treated mice required more matings to become pregnant and had more resorptions. In group 2, VCD-treated mice demonstrated proestrus and copulatory plugs, but only 1 animal became pregnant, and she had no viable fetuses. Ovaries from pregnant and nonpregnant controls contained similar numbers of follicles and corpora lutea. Ovaries from VCD-treated animals contained no follicles, and corpora lutea were seen only in pregnant animals. In VCD-treated mice mated soon after dosing, conception was more difficult and more resorbed fetuses were seen, whereas in those mated closer to impending ovarian failure, no successful pregnancies were achieved. These results demonstrate that VCD-treated mice can be used to model infertility in perimenopausal women.     

HDAC6 regulates primordial follicle activation through mTOR signaling pathway

Primordial follicle pool established perinatally is a non-renewable resource which determines the female fecundity in mammals. While the majority of primordial follicles in the primordial follicle pool maintain dormant state, only a few of them are activated into growing follicles in adults in each cycle. Excessive activation of the primordial follicles accelerates follicle pool consumption and leads to premature ovarian failure. Although previous studies including ours have emphasized the importance of keeping the balance between primordial follicle activation and dormancy via molecules within the primordial follicles, such as TGF-β, E-Cadherin, mTOR, and AKT through different mechanisms, the homeostasis regulatory mechanisms of primordial follicle activation remain unclear. Here, we reported that HDAC6 acts as a key negative regulator of mTOR in dormant primordial follicles. In the cytoplasm of both oocytes and granulosa cells of primordial follicles, HDAC6 expressed strong, however in those activated primordial follicles, its expression level is relatively weaker. Inhibition or knockdown of HDAC6 significantly promoted the activation of limited primordial follicles while the size of follicle pool was not affected profoundly in vitro. Importantly, the expression level of mTOR in the follicle and the activity of PI3K in the oocyte of the follicle were simultaneously up-regulated after inhibiting of HDAC6. The up-regulated mTOR leads to not only the growth and differentiation of primordial follicles granulosa cells (pfGCs) into granulosa cells (GCs), but the increased secretion of KITL in these somatic cells. As a result, inhibition of HDAC6 awaked the dormant primordial follicles of mice in vitro. In conclusion, HDAC6 may play an indispensable role in balancing the maintenance and activation of primordial follicles through mTOR signaling in mice. These findings shed new lights on uncovering the epigenetic factors involved physiology of sustaining female reproduction.Subject terms: TOR signalling, Cell proliferation, Endocrine reproductive disorders     

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.     

Apoptosis induced in rats by 4-vinylcyclohexene diepoxide is associated with activation of the caspase cascades.

Previous studies have shown that ovotoxicity induced in rats by dosing with 4-vinylcyclohexene diepoxide (VCD) is likely via acceleration of the normal rate of atresia (apoptosis). The present study was designed to investigate the apoptosis-related caspase cascades as a component of this phenomenon in isolated ovarian small follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., on Day 1; a time when ovotoxicity has not been initiated), or dosed daily for 15 days (80 mg/kg, i.p., on Day 15; a time when significant ovotoxicity is underway). Ovaries were collected after the final dose. Small preantral follicles (25-100 microm in diameter) were isolated, cellular fractions were prepared, and cleavage activity or protein expression levels of caspases-3, -8, and -9 were measured. Cytosolic caspase-3 activity was increased in small follicles (P < 0.01) by VCD treatment (Day 1, 2.86 +/- 0.23; Day 15, 3.25 +/- 0.64, VCD/control, n = 3). This activation was not seen in large or antral follicles (not targeted by VCD). Procaspase-3 protein was increased(P < 0.05) by VCD treatment 212% over controls in small ovarian follicles in Day 15, but not Day 1-dosed rats. Immunofluorescence staining intensity was evaluated by confocal microscopy. Caspase-3 protein, located in the cytosolic compartment of oocytes and granulosa cells of preantral follicles in various stages of development, was selectively increased (P < 0.05) in primordial and small primary follicles from Day 15 VCD-dosed rats. Caspase-8 activity was increased in small follicles in Day 15, but not in Day 1-treated rats; whereas caspase-9 activity was increased by VCD on Day 1 in the mitochondrial fraction. Thus, these data provide evidence that accelerated atresia induced in small ovarian follicles in rats by VCD is associated with activation of a caspase-mediated cascade.     

Suppression of SEMA6C promotes preantral follicles atresia with decreased cell junctions in mice ovaries

Mammalian oocytes go through a long and complex developmental process, while acquiring the competencies that are required for fertilization and embryogenesis. Recent studies revealed that the communication between oocytes and granulosa cells (GCs) is a critical process for female follicle development. In the current study, we aimed to study whether and how semaphorin 6C (Sema6c) regulated the cell junctions between oocytes and GCs in mice preantral follicles. The attenuation of SEMA6C expression by siRNA decreased the cell–cell junctions and accelerated follicle atresia in vitro. PI3K-AKT pathway was activated when SEMA6C expression was downregulated. And the LY294002, a PI3K inhibitor, could reverse the effect of low SEMA6C expression on cell junctions in preantral follicles. Our findings revealed that Sema6c was involved in follicle development, and the suppression of SEMA6C led to cell junction defection by activating the PI3K/AKT pathway, which might also provide valuable information for understanding premature ovarian failure and ovarian aging.     

骨髓问充质干细胞移植对VCD所致卵巢损伤的修复研究

目的：探讨小鼠骨髓间充质干细胞（MSCs）移植对去氧乙烯基环己烯（VCD）所致卵巢早衰治疗的可行性。方法：采用VCD（160mgkg^-l,day^-1）连续腹腔注射来诱导小鼠卵巢早衰。每侧卵巢注射转染了绿色荧光基因小鼠骨髓来源的MSCs,于移植后14、28天及45天,取各组血液标本及卵巢组织,同时观察小鼠动情周期的变化;酶联免疫法检测血清FSH、LH水平,显微镜下观察MSC在卵巢的分布。结果：MSCs移植后各组均可见绿色荧光,并且主要分布于卵巢间质区,卵巢泡膜细胞区也可见绿色荧光细胞。MSCs组动情周期较实验对照组缩短,FSH与LH水平较实验对照组低,差异具有显著性。结论：骨髓间充质干细胞可改善卵巢早衰小鼠的卵巢内分泌功能,并且长时间存在于卵巢组织。骨髓间充质干细胞可能成为卵巢早衰治疗的新方法。     

Infertility caused by retardation of follicular development in mice with oocyte-specific expression of Foxo3a

In recent years, mammalian oocytes have been proposed to have important roles in the orchestration of ovarian follicular development and fertility. To determine whether intra-oocyte Foxo3a, a component of the phosphatidylinositol 3-kinase (PI3K) signaling pathway, influences follicular development and female fertility, a transgenic mouse model was generated with constitutively active Foxo3a expressed in oocytes. We found that the female transgenic mice were infertile, which was caused by retarded oocyte growth and follicular development, and anovulation. Further mechanistic studies revealed that the constitutively active Foxo3a in oocytes caused a dramatic reduction in the expression of bone morphogenic protein 15 (Bmp15), connexin 37 and connexin 43, which are important molecules for the establishment of paracrine and gap junction communications in follicles. Foxo3a was also found to facilitate the nuclear localization of p27(kip1) in oocytes, a cyclin-dependent kinase (Cdk) inhibitor that may serve to inhibit oocyte growth. The results from the current study indicate that Foxo3a is an important intra-oocyte signaling molecule that negatively regulates oocyte growth and follicular development. Our study may therefore give some insight into oocyte-borne genetic aberrations that cause defects in follicular development and anovulation in human diseases, such as premature ovarian failure.     

Immunization with recombinant murine cytomegalovirus expressing murine zona pellucida 3 causes permanent infertility in BALB/c mice due to follicle depletion and ovulation failure

Zona pellucida (ZP) glycoproteins are promising candidate antigens for use in immunocontraceptive vaccines because of their crucial role in mammalian fertilization. A single intraperitoneal immunization with recombinant murine cytomegalovirus engineered to express murine ZP3 (rMCMV-mZP3) induces permanent infertility with no evident systemic illness in female BALB/c mice. To investigate the mechanisms underpinning reproductive failure elicited by rMCMV-mZP3, ovarian parameters and reproductive function were evaluated at time points spanning 10 days to 5 wk after virus inoculation. Fertility was substantially impaired by 14 days after inoculation with rMCMV-mZP3 and was fully ablated by 21 days. Pregnancies established after inoculation but before complete infertility showed no adverse effects on fetal viability assessed at Day 17.5 post coitum (pc). Infertile mice retained estrous cycling activity and remained receptive to mating; however, at Day 3.5 pc there were fewer developing embryos and corpora lutea, plasma progesterone content was reduced, and there was no evidence of excess unfertilized oocytes. Consistent with this, profound ovarian pathology was evident from 10 days after rMCMV-mZP3 inoculation, with a decline first in mature ovarian follicles and then in immature ovarian follicles and with diminished expression of genes regulating follicle development, including Nobox, Gdf9, and Gja1 (connexin43). Follicle loss was associated with mild focal oophoritis and with recruitment of inflammatory leukocytes, predominantly CD4(+) and CD8(+) T cells evident from 10 days after virus inoculation. These data indicate that vaccination with rMCMV-mZP3 causes permanent infertility in BALB/c mice principally due to induction of ovarian autoimmune pathology leading to progressive oocyte depletion and eventual ovulation failure.     

The effect of icariin on autoimmune premature ovarian insufficiency via modulation of Nrf2/HO-1/Sirt1 pathway in mice

Primary ovarian insufficiency (POI) is a common gynecological disease. Autoimmunity is a common cause of POI. Icariin (ICA) plays a therapeutic role in many autoimmune diseases. This study aims to investigate the effect of ICA on autoimmune POI mice and its effect on immune regulation. Sixty-three female BALB/c mice were randomized into three groups (control, POI, POI + ICA). POI and POI + ICA group were hypodermically injected with zona pellucida three peptides (pZP3) to induce autoimmune POI. Then the POI + ICA group was gavaged with ICA. A vaginal smear was to observe estrous cycles, hematoxylin-eosin staining was to count follicles. Enzyme-linked immunosorbent analysis determined serum FSH, LH, AMH, and anti-zona pellucida antibody (AZPAb) levels. In addition, flow cytometry detected the expression of Th1 cells and Treg cells, and Western blot was used to detect the expression of Nuclear factor E2 related factor 2(Nrf2), heme oxygenase-1 (HO-1), and Sirtuin-1 (Sirt1) proteins. pZP3 treatment decreased serum AMH levels and increased FSH, LH, and AZPAb levels. Additionally, decreases in the number of healthy follicles at all stages and an increase in the number of atretic follicles. Abnormal ovarian structure and an arrested estrous cycle were also noted. However, ICA rescued POI through up-regulating Nrf2, HO-1, and Sirt1 expressions and up-regulating Treg expressions. ICA treatment improved the structure of the injured ovarian and its function in autoimmune POI mice. The mechanism is achieved by increasing the expression of Nrf2/HO-1/Sirt1 pathway in the ovary and increasing Treg cells' expression.