Primordial Follicle Development Research Articles

Identification and analysis of key circRNAs in the mouse embryonic ovary provides insight into primordial follicle development.

CircRNAs are a class of noncoding RNAs with tissue- and development-specific expression characteristics. In many mammals, primordial follicle development begins in the embryonic stage. However, the study of circRNAs in primordial follicle development in mice has not been reported. In this study, ovaries were collected from mouse foetuses at 15.5 days post coitus (dpc) and 17.5 dpc, which are two key stages of primordial follicle development. A total of 4785 circRNAs were obtained by using RNA-seq.Of these, 83 differentially expressed circRNAs were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that these differential circRNAs were mainly involved in the regulation of reproductive development. Through qRT-PCR, back-splice sequence detection and enzyme digestion protection experiments, we found that circ-009346, circ-014674, circ-017054 and circ-008296 were indeed circular. Furthermore, circ-009346, circ-014674 and circ-017054 were identified as three key circRNAs by analysing their expression in the ovaries of mice at different developmental stages. The circRNA-miRNA-mRNA interaction network was constructed and validated for target miRNA and mRNA using qRT-PCR. The interacting genes circ-009346, circ-014674, and circ-017054 were subjected to KEGG enrichment analysis. We found that circ-014674 may participate in the assembly and reserve of primordial follicles through oestrogen and the Janus kinase (JAK) signal transducer and activator of transcription (STAT) signalling pathway (JAK-SATA). Circ-009346 and circ-017054 may have similar functions and are involved in the activation and growth of primordial follicles through the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signalling pathways. Based on our findings, three circRNAs associated with primordial follicle development were identified, and their potential mechanisms of regulating primordial follicle development were revealed. These findings will help us better understand the molecular mechanism of circRNAs in primordial follicles and provide important references and targets for the development of primordial follicles.

In Vitro Maturation, In Vitro Oogenesis, and Ovarian Longevity.

This paper will review a remarkable new approach to in vitro maturation "IVM" of oocytes from ovarian tissue, based on our results with in vitro oogenesis from somatic cells. As an aside benefit we also have derived a better understanding of ovarian longevity from ovary transplant. We have found that primordial follicle recruitment is triggered by tissue pressure gradients. Increased pressure holds the follicle in meiotic arrest and prevents recruitment. Therefore recruitment occurs first in the least dense inner tissue of the cortico-medullary junction. Many oocytes can be obtained from human ovarian tissue and mature to metaphase 2 in vitro with no need for ovarian stimulation. Ovarian stimulation may only be necessary for removing the oocyte from the ovary, but this can also be accomplished by simple dissection at the time of ovary tissue cryopreservation. By using surgical dissection of the removed ovary, rather than a needle stick, we can obtain many oocytes from very small follicles not visible with ultrasound. A clearer understanding of ovarian function has come from in vitro oogenesis experiments, and that explains why IVM has now become so simple and robust. Tissue pressure (and just a few "core genes" in the mouse) direct primordial follicle recruitment and development to mature oocyte, and therefore also control ovarian longevity. There are three distinct phases to oocyte development both in vitro and in vivo: in vitro differentiation "IVD" which is not gonadotropin sensitive (the longest phase), in vitro gonadotropin sensitivity "IVG" which is the phase of gonadotropin stimulation to prepare for meiotic competence, and IVM to metaphase II. On any given day 35% of GVs in ovarian tissue have already undergone "IVD" and "IVG" in vivo, and therefore are ready for IVM.

Effect of Ningxin-Yishen Formula on D-galactose-induced Premature Ovarian Insufficiency Mice by Inhibiting p53

: The prevalence of premature ovarian insufficiency (POI) is gradually increasing, safer and more effective treatments are urgently needed. The aim of this study was to evaluate the effects of Ningxin-Yishen formula (NXYSF) on D -galactose-induced POI mice as well as to shed a light on its potential mechanisms. : Six to eight weeks old C57BL/6 female mice were used in this study and randomly divided into six groups: control group; model group; estradiol valerate (EV) treatment group and NXYSF treatment group with graded doses (9.5, 19, and 38 g∙kg −1 /day). Both EV and NXYSF treatments were initiated at the 15th day of modeling and lasted for 28 days. Afterwards, the ovarian function was evaluated in each group by analyzing the proportion of primordial follicles as well as the serum sex hormone levels. Furthermore, network pharmacology approach was performed to elucidate the potential targets of NXYSF, which were verified through western blotting experiments finally. : NXYSF could significantly reverse the inefficiency of weight gain caused by POI, and promote the development of primordial follicles. In addition, it could restore the abnormal serum anti-Müllerian hormone (AMH), estradiol (E 2 ), luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Moreover, some crucial key gene targets including TP53 were proposed to be related with the effect of NXYSF through network pharmacology analysis. Last but importantly, western blotting experiments confirmed that NXYSF could inhibit the expression of p53 protein in mouse ovary. : Our findings proposed that NXYSF might be an effective alternative treatment for POI.

Optimisation of hormonal treatment to improve follicular development in one-day-old mice ovaries cultured under in vitro condition.

Base medium containing knock-out serum replacement (KSR) has been found to support formation and maintenance of follicles in one-day-old mice ovaries, but has not been shown to properly support activation and growth of primordial follicles. The present study was conducted to tailor the hormonal content of base medium containing KSR to enhance development of primordial follicles in neonatal ovaries. One-day-old mice ovaries were initially cultured with base medium for four days, and then, different hormonal treatments were added to the culture media and the culture was proceeded for four additional days until day eight. Ovaries were collected for histological and molecular assessments on days four and eight. In experiment I, the main and interactive effects of FSH and testosterone were investigated and FSH promoted activation of primordial follicles and development of primary and preantral follicles, and upregulated genes of phosphoinositide 3-kinase (Pi3k ), KIT ligand (Kitl ), growth differentiation factor 9 (Gdf9 ) and follicle stimulating hormone receptor (Fshr ) (P Bmp15 ), Connexin-43 (Cx43 ) and luteinising hormone and choriogonadotropin receptor (Lhcgr ) (P P Lhcgr (P P >0.05). Supplementation of culture medium containing KSR with gonadotropins, particularly hMG, could improve follicular growth and expression of factors regulating follicular development. This study was a step forward in formulating an optimal medium for development of follicles in cultured one-day-old mice ovaries.

ROCK1 is a multifunctional factor maintaining the primordial follicle reserve and follicular development in mice.

The follicle is the basic structural and functional unit of the ovary in female mammals. The excessive depletion of follicles will lead to diminished ovarian reserve or even premature ovarian failure, resulting in diminished ovarian oogenesis and endocrine function. Excessive follicular depletion is mainly due to loss of primordial follicles. Our analysis of published human ovarian single-cell sequencing results by others revealed a significant increase in rho-associated protein kinase 1 (ROCK1) expression during primordial follicle development. However, the role of ROCK1 in primordial follicle development and maintenance is not clear. This study revealed a gradual increase in ROCK1 expression during primordial follicle activation. Inhibition of ROCK1 resulted in reduced primordial follicle activation, decreased follicular reserve, and delayed development of growing follicles. This effect may be achieved through the HIPPO pathway. The present study indicates that ROCK1 is a key molecule for primordial follicular reserve and follicular development.NEW & NOTEWORTHY ROCK1, one of the Rho GTPases, plays an important role in primordial follicle reserve and follicular development. ROCK1 was primarily expressed in the cytoplasm of oocytes and granulosa cell in mice. Inhibition of ROCK1 significantly reduced the primordial follicle reserve and delayed growing follicle development. ROCK1 regulates primordial follicular reserve and follicle development through the HIPPO signaling pathway. These findings shed new lights on the physiology of sustaining female reproduction.

Co-culture of human cryopreserved fragmented ovarian tissue with theca progenitor cells derived from theca stem cells.

Despite the significant advances in the in vitro development of human primordial follicles, it is still a challenging approach with great potential for improvements. Therefore, the present study aimed to investigate the effect of a feeder layer of human theca progenitor cells (hTPCs) on the development of primordial follicles embedded in human ovarian tissue. Fragments of frozen-thawed ovarian tissue were activated using the vanadate-derivative dipotassium bisperoxo (5-hydroxy-pyridine-2-carboxylic) oxovanadate (V) and kit ligand for 24h. Then, the specimens were divided into the co-culture and mono-culture groups and were cultured with and without a hTPC feeder layer for 6days, respectively. Afterward, the follicles were counted and classified, and the hormone levels and expression levels of apoptosis- and folliculogenesis-related genes were assessed. Both culture groups showed significant follicle growth (P < 0.05). However, the co-culture group had a significantly higher number of growing follicles compared to the other group (P < 0.05). Moreover, the expression levels of ZP1, ZP2, ZP3, BMP-7, AMH, and GDF9 were significantly higher in the co-culture group compared to the other group (P < 0.05), while the expression levels of P53 and CASP3 were significantly lower (P < 0.05). Also, the concentrations of estradiol, progesterone, testosterone, and androstenedione were significantly higher in the co-culture group compared to the other group (P < 0.05). The present study results provided novel evidence on the direct role of hTPCs in the growth and development of human primordial follicles. However, there is a need for future studies to illustrate the underlying mechanisms. Schematic summary of the results. According to our results, the expression of ZP1, ZP2, ZP3, and GDF9 in the oocytes, AMH in the granulosa cells, and BMP4 in the theca cells of the co-culture group were significantly higher than those of the mono-culture and non-culture groups, while the expression of apoptotic genes (BAX, CASP3, and P53) was significantly lower. Moreover, the co-culture group showed significantly increased levels of estradiol, progesterone, testosterone, and androstenedione in its culture media compared to the mono-culture groups.

Ovarian dysfunction in women with Turner syndrome.

Ovarian dysfunction is one of the most common features of women with Turner syndrome. In these women, oocyte apoptosis is markedly accelerated from the early stage of fetal life. Reduction in the number of germ cells disturbs primordial follicle development and thereby leads to the formation of streak gonads. There are three possible causes of accelerated germ cell loss in 45,X ovaries. First, chromosomal pairing failure due to X chromosomal aneuploidy is believed to induce meiotic arrest. Indeed, it has been suggested that the dosage of the X chromosome is more critical for the survival of the oocytes than for other cells in the ovary. Second, impaired coupling between oocytes and granulosa cells may also contribute to germ cell apoptosis. Previous studies have shown that 45,X ovaries may tend to lose tight junctions which are essential for intercellular interactions. Lastly, ovarian dysfunction in women with Turner syndrome is partly attributable to the reduced dosage of several genes on the X chromosome. Specifically, BMP15, PGRMC1, and some other genes on the X chromosome have been implicated in ovarian function. Further studies on the mechanisms of ovarian dysfunction are necessary to improve the reproductive outcomes of women with Turner syndrome.

Oocyte Arrested at Metaphase II Stage were Derived from Human Pluripotent Stem Cells in vitro

Initiation of meiosis is the most difficult aspect of inducing competent oocytes differentiation from human stem cells in vitro. Human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) were cultured with follicle fluid, cytokines and small molecule to induced oocyte-like cells (OLCs) formation through a three-step induction procedure. Expression of surface markers and differentiation potential of germ cells were analyzed in vitro by flow cytometry, gene expression, immunocytochemistry, western blotting and RNA Sequencing. To induce the differentiation of hiPSCs into OLCs, cells were firstly cultured with a primordial germ cell medium for 10 days. The cells exhibited similar morphological features to primordial germ cells (PGCs), high expressing of germ cell markers and primordial follicle development associated genes. The induced PGCs were then cultured with the primordial follicle-like cell medium for 5 days to form the induced follicle-like structures (iFLs), which retained both primordial oocytes-like cells and granulosa-like cells. In the third step, the detached iFLs were harvested and transferred to the OLC-medium for additional 10 days. The cultured cells developed cumulus-oocyte-complexes (COCs) structures and OLCs with different sizes (50–150 μm diameter) and a zona pellucida. The in vitro matured OLCs had polar bodies and were arrested at metaphase II (MII) stage. Some OLCs were self-activated and spontaneously developed into multiple-cell structures similar to preimplantation embryos, indicating that OLCs were parthenogenetically activated though in vitro fertilization potential of OLCs are yet to be proved. in vitro maturation of OLCs derived from hiPSCs provides a new means to study human germ cell formation and oogenesis.Graphical

Immunolocalization of melatonin receptor type 1 in the sheep ovary and involvement of the PI3K/Akt/FOXO3a signaling pathway in the effects of melatonin on survival and in vitro activation of primordial follicles.

This study characterized the expression of melatonin receptor type 1 (MT1 ) protein in sheep ovaries, evaluated melatonin effects on primordial follicle survival and development after in vitro culture of ovarian tissue and verified the possible involvement of the phosphatidylinositol-3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FOXO3a) pathway in the melatonin actions. Ovine ovarian fragments were cultured in α-modified minimum essential mediumalone (α-MEM+ ) or supplemented with 100, 500, or 1000 pg/ml melatonin for 7 days. PI3K inhibition was performed through pretreatment of ovarian fragments with LY294002. Thereafter, immunohistochemistry was performed to evaluate the expression of cleaved caspase-3, Akt, phosphorylated-Akt, and phosphorylated-FOXO3a(p-FOXO3a). The immunohistochemical localization of the MT1 receptor protein was documented in sheep preantral and antral follicles. After in vitro culture, 100 pg/ml melatonin showed higher follicular survival and activation than α-MEM+ and other melatonin concentrations. After PI3K inhibition, there was an increase in cleaved caspase-3-positive follicles, and a decrease in the primordial follicle activation, Akt phosphorylation, and nuclear exclusion of p-FOXO3a. In conclusion, MT1 receptor protein is present in the sheep ovary. Furthermore, 100 pg/ml melatonin maintains survival and stimulates activation of primordial follicles through the PI3K/Akt/FOXO3a signaling pathway after in vitro culture of sheep ovarian tissue.

The effect of mTOR activation and PTEN inhibition on human primordial follicle activation in ovarian tissue culture.

The effect of PTEN inhibitor (Bpv(HOpic); Bpv) and mTOR activators (phosphatidic acid (PA) and propranolol (PP)), were evaluated on the activation and subsequent development of human primordial follicles in ovarian tissue culture. Slow frozen-thawed human ovarian cortical strips were incubated for 24h in different groups: (1) Control (base medium), (2) Bpv (100µM), (3) PA (200µM), (4) PA + PP (50µm), and (5) Bpv + PA + PP. Afterward, the medium was exchanged, and all groups were cultured without stimulators for 6 additional days. The proportion of normal and degenerated follicles, estradiol secretion, and expression of RPS6, FOXO3a, and AKT proteins was evaluated and compared between groups. After 24h of culture, there was no significant difference between the proportion of primordial and growing follicles in either of the experimental groups. This non-significant change was also observed for the phosphorylated protein to total protein ratios of RPS6, FOXO3a, and AKT proteins. After 7days of culture, the proportion of the transitional follicles was significantly higher in comparison to the primordial follicles for the PA, PA + PP, and Bpv + PA + PP groups. The estradiol level was significantly higher on the last day compared to the first day, in PA, PA + PP, and Bpv + PA + PP groups. Hormonal secretion was significantly higher in the PA and PA + PP groups and lower in the Bpv and Bpv + PA + PP groups compared to the control on day 7 of culture. Temporary in vitro treatment of human ovarian tissue with mTOR activators enhances the initiation of primordial follicle development and positively influences steroidogenesis after short-term culture.

New insights into the reverse of chromium-induced reprotoxicity of pregnant mice by melatonin

Hexavalent chromium Cr(VI) is a well-known environmental toxic metal that causes reprotoxicity in pregnant females. There are currently no appropriate interventions or treatments for Cr(VI) exposure during pregnancy. Herein, the protective effect of melatonin (MLT) against Cr(VI)-induced reprotoxicity is investigated by administrating MLT to pregnant mice exposed to Cr(VI). The results indicate that MLT effectively alleviates Cr(VI)-induced adverse pregnancy outcomes, restoring the decreased fetal weight and increased fetal resorption and malformation caused by Cr(VI) exposure to normal levels. MLT reduces the negative effects of Cr(VI) on follicular atresia and the development of primordial follicle in the maternal ovarian, thereby mitigating the decline in the reserve of primordial follicles. MLT alleviates Cr(VI)-induced oxidative stress, hence reducing the excessive accumulation of malondialdehyde in the maternal ovary. MLT inhibits Cr(VI)-induced apoptosis of ovarian granulosa cells and the expression of cleaved caspase-3 in the ovary. MLT reduces the increase in serum follicle-stimulating hormone caused by Cr(VI) exposure, while elevating anti-Mullerian hormone levels. We demonstrate that MLT reverses Cr(VI)-induced reprotoxicity in pregnant mice, opening up a new avenue for treating reproductive defects caused by environmental stress.

Mechanical stress-induced Hippo signaling in respect to primordial follicle development and polycystic ovary syndrome pathogenesis

Abstract Polycystic ovary syndrome (PCOS) is a heterogeneous reproductive disease that can cause infertility. The Hippo signaling pathway, a network highly conserved throughout evolution, maintains the balance between follicle proliferation and dormancy. Dynamic changes in primordial follicles cannot occur without the participation of biological signals and mechanical force; however, little is known about the mechanism by which biomechanical signaling triggers PCOS, especially in the context of primordial follicle development. To investigate the contribution of mechanical stress and the Hippo signaling pathway to the onset of PCOS, we searched the literature via the PubMed database, and inclusion and exclusion criteria were established to ensure the rigor of this research. We eventually included 54 publications in which Hippo signaling and mechanical force were suggested to play a vital role in the development of primordial follicles as well as elucidate the pathogenesis of PCOS. The Hippo pathway modulating follicle growth can be perturbed via extracellular mechanical stress caused by the stiff ovarian cortical environment in PCOS. Clinical intervention targeting the Hippo pathway can alter the activity of core Hippo members, such as the Yes-associated protein/transcriptional co-activator PDZ-binding motif complex. In some patients with PCOS, follicle overactivation can be attributed to the dysfunction of Hippo signal transduction. PCOS, a condition with various patterns, cannot be accurately explained by a single, specific mechanism. The present review identifies potential targets and therapeutic strategies for PCOS.

In-vitro maturation and transplantation of cryopreserved ovary tissue: understanding ovarian longevity

Research questionIs it possible to use experience gained from 24 years of frozen ovarian transplantation, and from recent experience with in-vitro gametogenesis to accomplish simple and robust in-vitro maturation (IVM) of oocytes from human ovarian tissue? DesignA total of 119 female patients between age 2 and 35 years old underwent ovary cryopreservation (as well as in-vitro maturation of oocytes and IVM in the last 13 individuals) over a 24-year period. Up to 22 years later, 17 returned to have their ovary tissue thawed and transplanted back. ResultsEvery woman had a return of ovarian function 5 months after transplant, similar to previous observations. As observed before, anti-Müllerian hormone (AMH) concentration rose as FSH fell 4 months later. The grafts continued to work up to 8 years. Of the 17, 13 (76%) became pregnant with intercourse at least once, resulting in 19 healthy live births, including six live births from three women who had had leukaemia. Of the harvested germinal vesicle oocytes, 35% developed with simple culture media into mature metaphase II oocytes. ConclusionsThe authors concluded the following. First, ovary tissue cryopreservation is a robust method for preserving fertility even for women with leukaemia, without a need to delay cancer treatment. Second, many mature oocytes can often be obtained from ovary tissue with simple media and no need for ovarian stimulation. Third, ovarian stimulation only be necessary for removing the oocyte from the ovary, which can also be accomplished by simple dissection at the time of ovary freezing. Finally, pressure and just eight ‘core genes’ control primordial follicle recruitment and development.

Proliferating cell nuclear antigen presentation, as a marker of folliculogenesis, in the transplanted ovarian tissue.

One of the most important ways to understand the ovarian biology is studding the initiation of primordial follicle development and subsequent folliculogenesis control. In this study, proliferating cell nuclear antigen (PCNA) presentation was used as a marker of follicular development in the thawed ovarian tissue (OT) following transplantation onto chick embryo chorioallantoic membrane (CAM) using two methods of freezing of slow freezing and vitrification. Samples of OT from 10 patients were subjected to slow freezing and vitrification. After warming, CAM transplantation was done and PCNA proliferation index (PI; percent of PCNA-positive granulosa cells) was calculated for each follicle stage. Image J software was used to determine the mean staining intensity. PCNA was positive for granulosa cells and oocytes nuclei, but negative for ooplasm. There were no remarkable PCNA staining in the granulosa cells of primordial follicles, but increased significantly as follicle progression (p< 0.05). Proliferation rate was also insignificantly higher in the vitrified than slow freezing group, before and after transplantation (p< 0.05). Lower PCNA presentation index was observed after CAM transplantation (p< 0.05). The earliest stage of follicular recruitment took place in the transitional follicles, before squamous cells transform to cuboidal cells. PCNA showed that follicles had proliferation power after cryopreservation. Higher presentation after vitrification may indicate accelerated folliculogenesis in the thawed OT.

LIF and bFGF enhanced chicken primordial follicle activation by Wnt/β-catenin pathway

The cytokines leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) are closely related to the development of primordial follicles. In this study, the functions and correlation of LIF and bFGF in the development of chicken primordial follicles were examined, along with the signaling pathways including protein kinase B (AKT), extracellular regulated protein kinase (ERK) and Wnt/β-catenin signaling pathways. Ovarian tissues were collected from four-day-old chicks and incubated with LIF and bFGF alone or in combination for three days to observe the changes in follicular development. Results showed that there was a time-dependent correlation between the changes in expression of LIF/its receptor (LIFR) and the developmental process of primordial follicles. LIF and bFGF exerted a synergistic effect on the activation of primordial follicles. However, SC144 (an antagonist of LIFR) inhibited this stimulating action. The effect by LIF and bFGF were shown to operate at AKT and ERK signaling pathways to suppress cell apoptosis and promote proliferation (P < 0.05) via the Wnt/β-catenin signaling (P < 0.05). In conclusion, local cytokines LIF and bFGF functioned to enhance the activation of chicken primordial follicles by increasing cell proliferation and decreasing apoptosis in the ovary involving AKT, ERK and Wnt/β-catenin signaling.

Translational Physiology of Anti-Müllerian Hormone: Clinical Applications in Female Fertility Preservation and Cancer Treatment.

BackgroundWhilst the ability of AMH to induce the regression of the Müllerian ducts in the male fetus is well appreciated, AMH has additional biological actions in relation to steroid biosynthesis and ovarian follicle dynamics. An understanding of the physiology of AMH illuminates the potential therapeutic utility of AMH to protect the ovarian reserve during chemotherapy and in the treatment of female malignancies. The translation of the biological actions of AMH into clinical applications is an emerging focus of research, with promising preliminary results.Objective and RationaleStudies indicate AMH restrains primordial follicle development, thus administration of AMH during chemotherapy may protect the ovarian reserve by preventing the mass activation of primordial follicles. As AMH induces regression of tissues expressing the AMH receptor (AMHRII), administration of AMH may inhibit growth of malignancies expressing AMHR II. This review evaluates the biological actions of AMH in females and appraises human clinical applications.Search MethodsA comprehensive search of the Medline and EMBASE databases seeking articles related to the physiological functions and therapeutic applications of AMH was conducted in July 2021. The search was limited to studies published in English.OutcomesAMH regulates primordial follicle recruitment and moderates sex steroid production through the inhibition of transcription of enzymes in the steroid biosynthetic pathway, primarily aromatase and 17α-hydroxylase/17,20-lyase. Preliminary data indicates that administration of AMH to mice during chemotherapy conveys a degree of protection to the ovarian reserve. Administration of AMH at the time of ovarian tissue grafting has the potential to restrain uncontrolled primordial follicle growth during revascularization. Numerous studies demonstrate AMH induced regression of AMHR II expressing malignancies. As this action occurs via a different mechanism to traditional chemotherapeutic agents, AMH has the capacity to inhibit proliferation of chemo-resistant ovarian cancer cells and cancer stem cells.Wider ImplicationsTo date, AMH has not been administered to humans. Data identified in this review suggests administration of AMH would be safe and well tolerated. Administration of AMH during chemotherapy may provide a synchronistic benefit to women with an AMHR II expressing malignancy, protecting the ovarian reserve whilst the cancer is treated by dual mechanisms.

O-218 The effect of mTOR activation on human primordial follicle activation during in-situ culture

Abstract Study question What is the effect of mTOR pathway activation on human primordial follicles in-situ activation and subsequent development following tissue cryopreservation? Summary answer Temporary treatment of cryopreserved human ovarian tissue with mTOR activators cause the initiation of primordial follicle development and influence steroidogenesis. What is known already In-vitro activation of primordial follicles to produce mature oocytes provides an alternative technique for fertility preservation. The employment of different stimulators of PI3K pathway has been successfully used to activate resting follicles during culture or prior to grafting in patients with premature ovarian insufficiency. The addition of phosphatidic acid (PA) and propranolol (PP), as mTOR stimulators, in the culture medium has promoted primordial follicle activation morphologically in mouse and human ovaries. Molecular and functional evaluations of primordial follicle activation after treatment with the mentioned stimulators has not been conducted. Study design, size, duration Ovarian tissues which were donated from 6 transsexual patients (23-35 years old), were dissected and cryopreserved with slow-freezing technique. After thawing, they were cut into 1x1x1 mm fragments and incubated with different stimulators in three groups: 1) Control (without stimulators), 2) PA (200µM), and 3) PA+PP (200µM &amp; 50µM respectively). In groups two and three, ovarian fragments were cultured for 24 hours in presence of stimulators and then cultured for additional 6 days without stimulators. Participants/materials, setting, methods The cultured ovarian fragments were directly processed for Hematoxylin and Eosin staining and Western blot analysis. The proportion of morphologically normal and degenerated primordial and growing follicles and 17β-estradiol (E2) level in the culture medium were compared after 1 and 7 days of culture to assess follicular development and function. Western blot analysis for phosphorylated and non-phosphorylated status of FOXO3a and RSP6 proteins expression were compared after 24 hours of incubation. Main results and the role of chance The proportion of primordial and growing follicles were not significantly different in the experimental groups after 24 hours of incubation with either of the stimulators. Western blot analyses indicated a significant reduction of FOXO3a in the PA+PP group compared to the control group. The phosphorylation level of RPS6 protein did not significantly change in either of the groups. The proportion of transitional follicles were significantly higher in the PA group compared to other groups after 7 days of culture. The E2 secretion level was significantly higher at the last day of culture compared to day 1 for all groups. At the end of the culture period, E2 levels was significantly higher in both PA and PA+PP groups compared to the control group. Limitations, reasons for caution Due to ovarian fragmentation before culture, the HIPPO pathway downstream molecules should have also been evaluated by western blot, which was not contributed in this study. Wider implications of the findings The results demonstrate the beneficial effect of mTOR signaling to accelerate early primordial follicle recruitment in cryopreserved-thawed human ovarian fragments. Trial registration number not applicable

Rutin promotes activation and reduces apoptosis of primordial follicles by regulating Akt phosphorylation after in vitro culture of ovine ovarian tissue

The aims of this study were to analyze the effects of different concentrations of rutin on primordial follicle survival and development after in vitro culture of sheep ovarian tissue, and to verify the possible involvement of the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway in the rutin actions. Ovarian fragments were fixed for histological analysis (fresh control) or cultured in α-minimum essential medium alone (α-MEM+: control medium) or in α-MEM+supplemented with different concentrations of rutin (0.1; 1 or 10 μg/mL) for 7 days. Inhibition of the PI3K activity was performed in fragments cultured with 50 μM LY294002. Thereafter, immunohistochemistry was performed to evaluate the expression of cleaved caspase-3 (apoptosis) and Akt phosphorylation (p-Akt). The results showed that 1 μg/mL rutin has a greater percentage of normal follicles (P < 0.05) than those of α-MEM+ and other rutin treatments. In addition, 1 μg/mL rutin maintained the follicular apoptosis similar (P > 0.05) to that of the fresh control and lower than α-MEM+ and 10 μg/mL rutin. All rutin concentrations increased (P < 0.05) follicular activation compared to fresh control and α-MEM+. Furthermore, follicular and oocyte diameters increased (P < 0.05) only after culture with 1 μg/mL rutin. After PI3K inhibition, there was a reduction (P < 0.05) of rutin follicular effects. In conclusion, rutin at 1 μg/mL reduces apoptosis, promotes activation and growth of sheep primordial follicles through the modulation of the PI3K/Akt signaling pathway after in vitro culture of ovine ovarian tissue.

MiR-145 regulates steroidogenesis in mouse primary granulosa cells through targeting Crkl

AimsIt has been demonstrated that miR-145 is expressed in primordial follicles and modulates the initiation of primordial follicle development. We aimed to explore the function of miR-145 in mouse granulosa cells (mGCs). Materials and methodsThe proliferation and differentiation of GCs were examined via MTT, EDU assay, QRT-PCR, ELISA and electron microscope analysis. The target of miR-145 was determined by bioinformatics analysis and luciferase reporter assay and the molecular mechanisms were examined via western blot and quantitative Real-Time RT-PCR. Key findingsWe proved that down-regulation of miR-145 could inhibit GCs proliferation and differentiation. In addition, we provided evidence that Crkl was the target gene of miR-145. The miR-145 antagomir caused an increase in Crkl expression and activation of the JNK/p38 MAPK pathway. Overexpression of Crkl with pEGFP-N1-Crkl vector inhibited GCs differentiation and progesterone synthesis as well as activation of the JNK/p38 MAPK pathway. SignificanceOur study shows that miR-145 targets Crkl and through the JNK/p38 MAPK signaling pathway promotes the GCs proliferation, differentiation, and steroidogenesis. MiR-145 may play an important role in the ovarian physiology and pathology.

The inhibition of WIP1 phosphatase accelerates the depletion of primordial follicles

Research questionWhat role does wild-type p53-induced phosphatase 1 (WIP1) play in the regulation of primordial follicle development? DesignWIP1 expression was detected in the ovaries of mice of different ages by western blotting and immunohistochemical staining. Three-day-old neonatal mouse ovaries were cultured in vitro with or without the WIP1 inhibitor GSK2830371 (10 μM) for 4 days. Ovarian morphology, follicle growth and follicle classification were analysed and the PI3K–AKT–mTOR signal pathway and the WIP1–p53-related mitochondrial apoptosis pathway evaluated. ResultsWIP1 expression was downregulated with age. Primordial follicles were significantly decreased in the GSK2830371-treated group, without a significant increase in growing follicles. The ratio of growing follicles to primordial follicles was not significantly different between the control and GSK2830371 groups, and no significant variation was observed in the PI3K–AKT–mTOR signal pathway. The inhibition of WIP1 phosphatase accelerated primordial follicle atresia by activating the p53–BAX–caspase-3 pathway. ConclusionsThese findings reveal that WIP1 participates in regulating primordial follicle development and that inhibiting WIP1 phosphatase leads to massive primordial follicle loss via interaction with the p53–BAX–caspase-3 pathway. This might also provide valuable information for understanding decreased ovarian reserve during ovarian ageing.