Oocytes Of Primordial Follicles Research Articles

Primary oocytes with cellular senescence features are involved in ovarian aging in mice

In mammalian females, quiescent primordial follicles serve as the ovarian reserve and sustain normal ovarian function and egg production via folliculogenesis. The loss of primordial follicles causes ovarian aging. Cellular senescence, characterized by cell cycle arrest and production of the senescence-associated secretory phenotype (SASP), is associated with tissue aging. In the present study, we report that some quiescent primary oocytes in primordial follicles become senescent in adult mouse ovaries. The senescent primary oocytes share senescence markers characterized in senescent somatic cells. The senescent primary oocytes were observed in young adult mouse ovaries, remained at approximately 15% of the total primary oocytes during ovarian aging from 6 to 12 months, and accumulated in aged ovaries. Administration of a senolytic drug ABT263 to 3-month-old mice reduced the percentage of senescent primary oocytes and the transcription of the SASP factors in the ovary, in addition, led to increased numbers of primordial and total follicles and a higher rate of oocyte maturation. Our study provides experimental evidence that primary oocytes, a germline cell type that is arrested in meiosis, become senescent in adult mouse ovaries and that senescent cell clearance reduced primordial follicle loss and mitigated ovarian aging phenotypes.

A rat study on the PTEN expression in ovarian tissue in PCOS and folliculogenesis

The objective of this investigation was to examine alterations in PTEN expression within ovarian tissue in a rat model of polycystic ovary syndrome (PCOS). The analysis also encompassed the examination of PTEN alterations in the ovarian tissue throughout the process of folliculogenesis in rats with normal ovulatory cycles. The study involved 12 adult female Sprague‒Dawley rats randomly assigned to the letrozole-induced polycystic ovary syndrome (PCOS) group as part of an animal-based research endeavour. The sections derived from the ovaries were subjected to immunohistochemical staining for PTEN. The evaluation of PTEN staining levels in ovarian tissues was conducted using electron microscopy. Follicle counts, as well as hormonal and biochemical analyses (serum luteinising hormone (LH), follicle-stimulating hormone (FSH), anti-Müllerian hormone (AMH), testosterone, oestradiol levels and serum glucose, triglyceride, HDL and LDL-cholesterol levels), were conducted to provide evidence of the manifestation of polycystic ovary syndrome (PCOS) in rats. The number of primordial and Graafian follicles in the PCOS group decreased significantly, and the number of primary, secondary and antral follicles increased significantly. PTEN expression was found to be significantly higher in the PCOS group than in the control group in the primordial follicle oocyte cytoplasm, primordial follicle granulosa cells, primary follicle oocyte cytoplasm, primary follicle granulosa cells, antral follicle oocyte cytoplasm, antral follicle granulosa cells, and corpus luteum (p = 0.007, p = 0.001, p = 0.001, p = 0.001, p = 0.001, p = 0.002, and p = 0.018, respectively). In the non-PCOS group, a time-dependent comparison of the amount of oocyte cytoplasm and PTEN staining in granulosa cells of the oocytes at different stages of development was performed. While the follicles were developing from the primordial follicle to the primary and antral follicle, the amount of PTEN staining in the oocyte cytoplasm decreased, whereas the PTEN activity in the granulosa cells increased as the oocyte developed (p = 0.001 and p = 0.001, respectively). The current investigation demonstrated changes in PTEN expression in ovarian tissue throughout the course of normal folliculogenesis, as well as in instances of disrupted folliculogenesis, with a focus on rats with PCOS.

CHEK2 signaling is the key regulator of oocyte survival after chemotherapy.

Cancer treatments can damage the ovarian follicle reserve, leading to primary ovarian insufficiency and infertility among survivors. Checkpoint kinase 2 (CHEK2) deficiency prevents elimination of oocytes in primordial follicles in female mice exposed to radiation and preserves their ovarian function and fertility. Here, we demonstrate that CHEK2 also coordinates the elimination of oocytes after exposure to standard-of-care chemotherapy drugs. CHEK2 activates two downstream targets-TAp63 and p53-which direct oocyte elimination. CHEK2 knockout or pharmacological inhibition preserved ovarian follicle reserve after radiation and chemotherapy. However, the lack of specificity for CHEK2 among available inhibitors limits their potential for clinical development. These findings demonstrate that CHEK2 is a master regulator of the ovarian cellular response to damage caused by radiation and chemotherapy and warrant the development of selective inhibitors specific to CHEK2 as a potential avenue for ovario-protective treatments.

Xrcc5/KU80 is not required for the survival or activation of prophase-arrested oocytes in primordial follicles.

The non-growing, meiotically-arrested oocytes housed within primordial follicles are exquisitely sensitive to genotoxic insults from endogenous and exogenous sources. Even a single DNA double-strand break (DSB) can trigger oocyte apoptosis, which can lead to accelerated depletion of the ovarian reserve, early loss of fertility and menopause. Therefore, repair of DNA damage is important for preserving the quality of oocytes to sustain fertility across the reproductive lifespan. This study aimed to evaluate the role of KU80 (encoded by the XRCC5 gene) - an essential component of the non-homologous end joining (NHEJ) pathway - in the repair of oocyte DNA DSBs during reproductive ageing, and following insult caused by the DNA-damaging chemotherapies cyclophosphamide and cisplatin. To investigate the importance of KU80 following endogenous and exogenous DNA damage, ovaries from conditional oocyte-specific Xrcc5 knockout (Xrcc5 cKO) and wildtype (WT) mice that were aged or exposed to DNA damage-inducing chemotherapy were compared. Ovarian follicles and oocytes were quantified, morphologically assessed and analysed via immunohistochemistry for markers of DNA damage and apoptosis. In addition, chemotherapy exposed mice were superovulated, and the numbers and quality of mature metaphase- II (MII) oocytes were assessed. The number of healthy follicles, atretic (dying) follicles, and corpora lutea were similar in Xrcc5 cKO and WT mice at PN50, PN200 and PN300. Additionally, primordial follicle number and ovulation rates were similar in young adult Xrcc5 cKO and WT mice following treatment with cyclophosphamide (75mg/kg), cisplatin (4mg/kg), or vehicle control (saline). Furthermore, KU80 was not essential for the repair of exogenously induced DNA damage in primordial follicle oocytes. These data indicate that KU80 is not required for maintenance of the ovarian reserve, follicle development, or ovulation during maternal ageing. Similarly, this study also indicates that KU80 is not required for the repair of exogenously induced DSBs in the prophase-arrested oocytes of primordial follicles.

The dynamic expression of SOX17 in germ cells from human female foetus and adult ovaries after specification.

SOX17 has been identified as a critical factor in specification of human primordial germ cells, but whether SOX17 regulates development of germ cells after sex differentiation is poorly understood. We collected specimens of gonadal ridge from an embryo (n=1), and ovaries of foetuses (n=23) and adults (n=3). Germ cells were labelled with SOX17, VASA (classic germ cells marker), phosphohistone H3 (PHH3, mitosis marker) and synaptonemal complex protein 3 (SCP3, meiosis marker). SOX17 was detected in both cytoplasm and nucleus of oogonia and oocytes of primordial and primary follicles from 15 to 28 gestational weeks (GW). However, it was exclusively expressed in cytoplasm of oogonia at 7 GW, and in nucleus of oocytes in secondary follicles. Co-expression rates of SOX17 in VASA+ germ cells ranged from 81.29% to 97.81% in foetuses. Co-staining rates of SOX17 and PHH3 or SCP3 were 0%-34% and 0%-57%, respectively. Interestingly, we distinguished a subpopulation of SOX17+VASA- germ cells in fetal ovaries. These cells clustered in the cortex and could be co-stained with the mitosis marker PHH3 but not the meiosis marker SCP3. The dynamic expression of SOX17 was detected in human female germ cells. We discovered a population of SOX17+ VASA- germ cells clustering at the cortex of ovaries. We could not find a relationship between mitosis or meiosis and SOX17 or VASA staining in germ cells. Our findings provide insight into the potential role of SOX17 involving germ cells maturation after specification, although the mechanism is unclear and needs further investigation.

Fetal Ovarian Reserve: the Dynamic Changes in Ubiquitin C-Terminal Hydrolase L1.

The regulation of protein turnover by the unique deubiquitinating enzyme ubiquitin C-terminal hydrolase L1 (UCHL1) is only seen in oocytes, spermatogonia, and neurons. Our objective was to investigate variation in expression of UCHL1 across fetal maturation of oocytes that result in lifelong ovarian reserve. We performed a retrospective cohort study of 25 fetal autopsy specimens from 21 to 36weeks. This was an IRB-approved protocol with parental permission for use of tissues for research purposes. Tissues were stained for expression of the oocyte-specific protein UCHL1, and expression levels were evaluated using quantitative immunofluorescence across gestational ages after correction for the area and background absorbance. Corrected total cell fluorescence (CTCF) for expression of UCHL1 within human oocytes was compared across fetal gestational ages and oocyte size. Trends were analyzed using a locally weighted scatterplot smoothing algorithm. Local expression of UCHL1 increases in oocytes across ovarian development reaching a plateau at 27weeks with the maintenance of elevated levels through 36weeks gestational age. This maturation trend is also evidenced by the increase in protein expression as oocyte area increases (r = 0.5530, p ≤ 0.001) with the largest rise occurring as oocytes are enveloped into primordial follicles. The increase in expression as oocytes transition from oogonia into oocytes in primordial follicles and beyond may be part of the preparation of both oocytes and the surrounding somatic cells for the long-term maintenance of the ovarian reserve.

Oocyte aging in comparison to stem cells in mice.

To maintain homeostasis, many tissues contain stem cells that can self-renew and differentiate. Based on these functions, stem cells can reconstitute the tissue even after injury. In reproductive organs, testes have spermatogonial stem cells that generate sperm in men throughout their lifetime. However, in the ovary, oocytes enter meiosis at the embryonic stage and maintain sustainable oogenesis in the absence of stem cells. After birth, oocytes are maintained in a dormant state in the primordial follicle, which is the most premature follicle in the ovary, and some are activated to form mature oocytes. Thus, regulation of dormancy and activation of primordial follicles is critical for a sustainable ovulatory cycle and is directly related to the female reproductive cycle. However, oocyte storage is insufficient to maintain a lifelong ovulation cycle. Therefore, the ovary is one of the earliest organs to be involved in aging. Although stem cells are capable of proliferation, they typically exhibit slow cycling or dormancy. Therefore, there are some supposed similarities with oocytes in primordial follicles, not only in their steady state but also during aging. This review aims to summarise the sustainability of oogenesis and aging phenotypes compared to tissue stem cells. Finally, it focuses on the recent breakthroughs in vitro culture and discusses future prospects.

Adolescent exposure to low-dose Δ9-tetrahydrocannabinol depletes the ovarian reserve in female mice.

Cannabis use by adolescents is widespread, but its effects on the ovaries remain largely unknown. Δ9-tetrahydrocannabinol (THC) exerts its pharmacological effects by activating, and in some conditions hijacking, cannabinoid receptors (CBRs). We hypothesized that adolescent exposure to THC affects ovarian function in adulthood. Peripubertal female C57BL/6N mice were given THC (5 mg/kg) or its vehicle, once daily by intraperitoneal injection. Some mice received THC from postnatal day (PND) 30-33 and their ovaries were harvested PND34; other mice received THC from PND30-43, and their ovaries were harvested PND70. Adolescent treatment with THC depleted ovarian primordial follicle numbers by 50% at PND70, 4 weeks after the last dose. The treatment produced primordial follicle activation, which persisted until PND70. THC administration also caused DNA damage in primary follicles and increased PUMA protein expression in oocytes of primordial and primary follicles. Both CB1R and CB2R were expressed in oocytes and theca cells of ovarian follicles. Enzymes involved in the formation (N-acylphosphatidylethanolamine phospholipase D) or deactivation (fatty acid amide hydrolase) of the endocannabinoid anandamide were expressed in granulosa cells of ovarian follicles and interstitial cells. Levels of mRNA for CBR1 were significantly increased in ovaries after adolescent THC exposure, and upregulation persisted for at least 4 weeks. Our results support that adolescent exposure to THC may cause aberrant activation of the ovarian endocannabinoid system in female mice, resulting in substantial loss of ovarian reserve in adulthood. Relevance of these findings to women who frequently used cannabis during adolescence warrants investigation.

OR06-1 Cyclophosphamide Depletes Primordial Follicles via the p63 Apoptotic Pathway

Abstract Cancer therapies often cause serious side effects, affecting the quality of life for cancer survivors. The ovary can be affected by cancer therapies, causing premature ovarian insufficiency, leading to endocrine dysfunction and infertility. Thus, maintaining ovarian function against cancer treatment is an unmet need for female cancer patients. Cyclophosphamide (CPA), a common chemotherapeutic agent, is metabolized in vivo and forms DNA crosslinks, inducing apoptosis in rapidly proliferating tumor cells. However, the underlying mechanism of the CPA-induced oocyte death in ovarian reserve remains unclear. This study aims to investigate the mechanism of oocyte death in primordial follicles by generating oocyte-specific Abl1 and p63 knockout and Pik3ca* knockin mouse models using Gdf9-iCre+. Postnatal day 7 female mice were i.p. injected w/wo CPA. Notably, we found CPA drastically reduced the number of primordial follicles without increasing the number of growing follicles. The quantification of surviving follicles validated that 90% of the primordial follicles from oocyte-specific Abl1 knockout mice were lost following CPA treatment in vivo and in vitro. Concurrently, high expression of CHK2 was detected in the oocytes of the ovary cultured with CPA metabolite in vitro. This implied CHK2 rather than ABL is a possible tyrosine kinase for CPA-induced oocyte death. Most of all, p63 was a key player for CPA-induced oocyte death, supported by the result that p63 knockout oocytes were rescued after CPA treatment. To clarify whether the ovaries lose follicles via activation or apoptosis pathway, PIK3CA* mice were examined with CPA. As expected, primordial follicles and primordial follicles with activated oocytes in the ovary of PIK3CA* mice survived against CPA due to constitutive PI3K activity in them. This indicates activated follicles resist gonadotoxic reagents, although p63 is expressed inside their oocytes. Accordingly, the apoptosis markers, BAX and cleaved PARP were highly induced with CPA injection in a time-dependent manner in the ovaries from wild-type female mice but not from the p63 knockout. The double-strand break also occurred in the nucleus of oocytes post CPA administration as γH2AX was detected. Interestingly, OPA1, a protein required for mitochondrial fusion, was highly induced inside the oocyte cytoplasm of p63 knockout, while oocytes in wild-type mice time-dependently lost the OPA1 expression by CPA treatment. This indicates that oocytes without p63 in the nucleus survive and induce mitochondrial fusion to escape apoptosis by mitochondrial damages. In conclusion, CPA induces depletion of oocytes of primordial follicles through the CHK2-TAp63 apoptotic pathway. Presentation: Saturday, June 11, 2022 11:30 a.m. - 11:45 a.m.

OTSSP167 leads to follicular dysplasia and negatively affects oocyte quality in mice

OTSSP167 is an anti-tumor drug significantly inhibiting tumor growth in xenotransplantation studies using mouse breast, lung, prostate, and pancreatic cancer cell lines. Its phase I clinical trial has been completed, indicating its great potential for future treatment of solid tumors. However, its drug-related adverse effects on reproductive systems have not yet been reported. In this study, we evaluated the effects of OTSSP167 on reproduction of female mice by determining oocyte quality and follicular development. We selected four-week-old female ICR mice for a 21-day intraperitoneal injection of OTSSP167 at a dose of 5 mg/kg/d. We found that OTSSP167 could block the meiotic process of oocytes, leading to a decrease in oocyte maturation and ovulated oocyte numbers, as well as a decrease in the quality of oocytes. The results showed that OTSSP167 treatment caused disordered spindle assembly, decreased mitochondria membrane potential, and increased accumulation of reactive oxygen species in oocytes. Further investigation showed that OTSSP167 induced DNA double-strand breaks, as indicated by increased levels of γH2AX in oocytes of primordial follicles and granulosa cells of growing follicles, which induced follicular atresia and decreased the numbers of follicles at various growing stages. Our study suggests that OTSSP167 treatment may have serious effects on the ovary and consequences for female cancer patients, providing strong evidence for the necessity of protecting female fertility in clinical OTSSP167 trials.

Proliferating Cell Nuclear Antigen (PCNA) Localization in the Ovaries of Buffalo, Dog and Pig by Immunohistochemistry

Background: It was expected from the previous studies that the immune histochemical staining for PCNA within follicular cells altered depending on the stage of follicular growth. Therefore, we aimed at determining the expression of proliferating cell nuclear antigen protein (PCNA) in the buffalo, dog and pig ovaries. Methods: The localization of PCNA was demonstrated in formalin fixed, paraffin embedded tissue sections of buffalo, dog and pig ovaries using primary mouse monoclonal anti-PCNA antibody. Result: Staining for PCNA was not observed in pregranulosa cells but observed in oocytes of primordial follicles. In primary to early secondary follicles, in oocytes and in few granulosa cells the positive staining was detected. The late secondary preantral follicles and actively growing small to large antral follicles showed strong PCNA labelling in the layers of granulosa and theca cells. PCNA staining was expressed in nuclei of oocytes in preantral and small antral follicles. The follicles demonstrating advanced atresia showed mild to no PCNA labelled granulosa and theca cells. The results of our study demonstrated that follicular growth and development in ovaries may be effectively monitored by determining the rate of granulosa cell proliferation of PCNA.

Oocyte CTR1 is not essential for cisplatin-induced oocyte death of primordial follicle.

Accumulated evidence indicates that cisplatin, a platinum-based alkylating agent, causes preferential DNA damage to oocytes of primordial follicles (PFs) in the ovary, suggesting oocyte-favored accumulation of cisplatin. Copper transporter 1 (CTR1; Slc31a1 ) is implicated in facilitating cisplatin uptake in cells. Here we found that oocytes of PFs had constitutively higher expression of CTR1 than other cell types in mouse ovary. However, oocyte-specific Slc31a1 knockout was not sufficient to prevent cisplatin-induced depletion of PFs in vitro . Our data indicate that CTR1 would not be the only route for cisplatin to be transported inside the oocytes of PFs in the ovary.

BCAS2 is involved in alternative splicing and mouse oocyte development.

Alternative splicing (AS) is an important mechanism to regulate organogenesis and fertility. Breast carcinoma amplified sequence 2 (BCAS2) is one of the core components of the PRP19 complex, a multiple function complex including splicing, and it is involved in the initiation of meiosis through regulating AS in male mice. However, the role of BCAS2 in mouse oogenesis remains largely unknown. In this study, we found that BCAS2 was highly expressed in the oocytes of primordial follicles. Vasa-Cre-mediated deletion of Bcas2 caused poor oocyte quality, abnormal oogenesis and follicular development. The deletion of Bcas2 in mouse oocytes caused alteration in 991 AS events that corresponded to 706 genes, including Pabpc1l, Nobox, Zfp207, Mybl2, Prc1, and Spc25, which were associated with oogenesis and spindle assembly. Moreover, the disruption of BCAS2 led to degradation of PRP19 core proteins in mouse oocytes. These results suggested that BCAS2 was involved in the AS of functional genes through PRP19 complex during mouse oocyte development.

P–416 Radiotherapy inflicts long-term damage upon the uterus, causing uterine artery endothelial dysfunction and pregnancy loss in mice

Abstract Study question Does the uterus sustain direct and long-term damage following radiotherapy, independent of ovarian damage? Summary answer Radiotherapy causes direct and long-term uterine damage. Ovariectomised and hormonally supplemented mice experience uterine artery endothelial dysfunction and pregnancy loss following transfer of healthy blastocysts. What is known already The detrimental off-target impacts of cancer therapies on the ovary are well established, with some fertility preservation techniques available to ensure patients maintain their fertility following gonadotoxic treatment. Low doses of radiotherapy kill the majority of primordial follicle oocytes in the ovary, reducing the ovarian reserve and fertile lifespan. Patients who have received radiotherapy experience higher rates of pregnancy complications including preterm birth, low birth weight, and stillbirth. However, no studies have investigated if radiation inflicts direct, fertility compromising damage to the uterus. Study design, size, duration Adolescent female mice were untreated or exposed to whole body y-irradiation (7Gy), then ovariectomised. Immediate damage was assessed up to 24 hours post-irradiation (n = 4/group). Four weeks following treatment, mice were hormone primed to induce endometrial receptivity (n = 7/group), artificial decidualisation (n = 7–8/group), or receive embryo transfers from healthy, unexposed donor mice to assess embryo implantation (n = 11–13/group), and mid-gestation development (n = 8–10/group). Participants/materials, setting, methods Four week old C57BL6/CBA (F1) female mice were used for this study. Immunofluorescence and in situ hybridisation were utilised to localise markers of immediate DNA damage and cell death following irradiation, and markers of receptivity in hormone primed uteri. Measurements of uterine artery blood flow were recorded using Doppler ultrasonography, and indices of pulsatility and resistance calculated. Uterine artery wire myography was performed to assess competency of endothelial and smooth muscle compartments following irradiation. Main results and the role of chance Within 24 hours of irradiation, DNA damage (yH2AX) and apoptosis (Puma/TUNEL) were elevated in uteri, compared to control. Irradiated mice that received embryo transfers from untreated donors had similar numbers of implantation sites 3-days post-transfer versus controls, however uteri were pale and atrophic suggesting impaired vascularisation. By 10-days post-transfer, implantation sites in irradiated mice were resorbing (p < 0.001), although uterine artery Doppler ultrasound measurements demonstrated no change in pulsatility or resistance indices. When the brain was shielded from irradiation to protect the hypothalamic-pituitary-gonadal axis, resorption still occurred (p < 0.001), suggesting direct uterine damage is the likely cause of pregnancy loss. To investigate uterine damage in the absence of an embryo, endometrial receptivity was induced artificially. Uteri from irradiated animals were lighter compared to control (p < 0.05), however localisation of receptivity markers (E-cadherin, Mucin1, Ki67) was normal. When decidualisation was artificially induced irradiated uteri were also lighter (p < 0.01) indicating impaired decidualisation and reduced capacity to adapt to pregnancy. Wire myography performed on uterine arteries demonstrated endothelial dysfunction in irradiated mice (p < 0.0001). Limitations, reasons for caution Here, only a single age and dose of radiotherapy exposure are modelled. Patients of all ages can receive many doses of radiotherapy in combination with various chemotherapies. Our highly manipulable model enables any treatment variation to be modelled and the effect on the uterus and pregnancy examined. Wider implications of the findings: Reproductive aged cancer patients need to be appropriately counselled regarding the risks to their long-term fertility following treatment. Until now, potential permanent impacts to the uterus following cancer treatment have not been considered. It is clear radiotherapy can impose long-term damage to the uterus and influence pregnancy success and fertility. Trial registration number NA

Distinct Signaling Pathways Distinguish in vivo From in vitro Growth in Murine Ovarian Follicle Activation and Maturation.

Women with cancer and low ovarian reserves face serious challenges in infertility treatment. Ovarian tissue cryopreservation is currently used for such patients to preserve fertility. One major challenge is the activation of dormant ovarian follicles, which is hampered by our limited biological understanding of molecular determinants that activate dormant follicles and help maintain healthy follicles during growth. Here, we investigated the transcriptomes of oocytes isolated from dormant (primordial) and activated (primary) follicles under in vivo and in vitro conditions. We compared the biological relevance of the initial molecular markers of mature metaphase II (MII) oocytes developed in vivo or in vitro. The expression levels of genes involved in the cell cycle, signal transduction, and Wnt signaling were highly enriched in oocytes from primary follicles and MII oocytes. Interestingly, we detected strong downregulation of the expression of genes involved in mitochondrial and reactive oxygen species (ROS) production in oocytes from primordial follicles, in contrast to oocytes from primary follicles and MII oocytes. Our results showed a dynamic pattern in mitochondrial and ROS production-related genes, emphasizing their important role(s) in primordial follicle activation and oocyte maturation. The transcriptome of MII oocytes showed a major divergence from that of oocytes of primordial and primary follicles.

DNA repair in primordial follicle oocytes following cisplatin treatment.

Genotoxic chemotherapy and radiotherapy can cause DNA double stranded breaks (DSBs) in primordial follicle (PMF) oocytes, which then undergo apoptosis. The development of effective new fertility preservation agents has been hampered, in part, by a limited understanding of DNA repair in PMF oocytes. This study investigated the induction of classical DSB repair pathways in the follicles of wild type (WT) and apoptosis-deficient Puma-/- mice in response to DSBs caused by the chemotherapy agent cisplatin. Adult C57BL/6 WT and Puma-/- mice were injected i.p. with saline or cisplatin (5 mg/kg); ovaries were harvested at 8 or 24 h. Follicles were counted, and H2A histone family member (γH2AX) immunofluorescence used to demonstrate DSBs. DNA repair protein RAD51 homolog 1 (RAD51) and DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) immunofluorescence were used to identify DNA repair pathways utilised. Puma-/- mice retained 100% of follicles 24 h after cisplatin treatment. Eight hours post-treatment, γH2AX immunofluorescence showed DSBs across follicular stages in Puma-/- mice; staining returned to control levels in PMFs within 5 days, suggesting repair of PMF oocytes in this window. RAD51 immunofluorescence eight hours post-cisplatin was positive in damaged cell types in both WT and Puma-/- mice, demonstrating induction of the homologous recombination pathway. In contrast, DNA-PKcs staining were rarely observed in PMFs, indicating non-homologous end joining plays an insignificant role. PMF oocytes are able to conduct high-fidelity repair of DNA damage accumulated during chemotherapy. Therefore, apoptosis inhibition presents a viable strategy for fertility preservation in women undergoing treatment.

Ovaries of patients recently treated with alkylating agent chemotherapy indicate the presence of acute follicle activation, elucidating its role among other proposed mechanisms of follicle loss

To investigate mechanisms of primordial follicle (PMF) loss invivo in human ovaries shortly after alkylating agent (AA) chemotherapy. Cohort study. Tertiary university medical center. Ninety-six women aged 15-39 years who underwent ovarian tissue cryopreservation for fertility preservation. Fresh ovarian tissue samples were harvested from women treated with AA (n = 24) or non-AA (n = 24) chemotherapy <6 months after treatment and age-matched untreated women (n = 48). Differential follicle counts, time from chemotherapy exposure, immunostaining for apoptosis (cleaved caspase-3) and FOXO3A on tissue harvested within ultrashort time intervals (4-12 days), collagen (Sirius red) and neovascularization (CD34). AA-treated ovaries had significant loss of PMFs, and significant increase in absolute numbers of growing follicles compared with untreated control ovaries. The number of growing follicles was inversely correlated with time from chemotherapy. Representative staining for FOXO3A observed decreased nuclear localization in PMF oocytes in AA-treated ovaries removed within the ultrashort time interval compared with untreated ovaries. Neither significant loss of PMFs, increase in growing follicles, nor decrease in nuclear FOXO3A were observed in non-AA-treated ovaries. No increased expression of cleaved caspase-3 was seen in PMFs within the ultrashort time interval after AA or non-AA chemotherapy. Significant stromal fibrosis and neovascularization were observed in AA-treated ovaries only after follicle loss had already occurred (4-6 months). Follicle activation occurs invivo in ovaries of patients treated with AA, indicating a pathologic mechanism which may contribute to chemotherapy-induced follicle loss.

Individual-oocyte transcriptomic analysis shows that genotoxic chemotherapy depletes human primordial follicle reserve\xa0in vivo by triggering proapoptotic pathways without growth activation

Gonadotoxic chemotherapeutics, such as cyclophosphamide, can cause early menopause and infertility in women. Earlier histological studies showed ovarian reserve depletion via severe DNA damage and apoptosis, but others suggested activation of PI3K/PTEN/Akt pathway and follicle ‘burn-out’ as a cause. Using a human ovarian xenograft model, we performed single-cell RNA-sequencing on laser-captured individual primordial follicle oocytes 12 h after a single cyclophosphamide injection to determine the mechanisms of acute follicle loss after gonadotoxic chemotherapy. RNA-sequencing showed 190 differentially expressed genes between the cyclophosphamide- and vehicle-exposed oocytes. Ingenuity Pathway Analysis predicted a significant decrease in the expression of anti-apoptotic pro-Akt PECAM1 (p = 2.13E-09), IKBKE (p = 0.0001), and ANGPT1 (p = 0.003), and reduced activation of PI3K/PTEN/Akt after cyclophosphamide. The qRT-PCR and immunostaining confirmed that in primordial follicle oocytes, cyclophosphamide did not change the expressions of Akt (p = 0.9), rpS6 (p = 0.3), Foxo3a (p = 0.12) and anti-apoptotic Bcl2 (p = 0.17), nor affect their phosphorylation status. There was significantly increased DNA damage by γH2AX (p = 0.0002) and apoptosis by active-caspase-3 (p = 0.0001) staining in the primordial follicles and no change in the growing follicles 12 h after chemotherapy. These data support that the mechanism of acute follicle loss by cyclophosphamide is via apoptosis, rather than growth activation of primordial follicle oocytes in the human ovary.

The Effect of Isotretinoin on Oocyte Maturation in Adolescent Female Rats

Objective: Isotretinoin is used in acne vulgaris treatment for more than 20 years. Isotretinoin has serious side effects on many organs, but there are no comprehensive studies investigating its possible toxic effects on reproductive organs. Thus, we aimed to investigate the possible toxic effects of isotretinoin administration on oocyte maturation in female rat gonads in this study. Methods: Thirty-two adolescent female rats (Wistar Albino, 220 ± 35 g) were randomly divided into 4 groups with 8 subjects in each group: group 1, group 2, group 3, and group 4. Different doses of isotretinoin which was dissolved in sesame oil were given to rats by gavage: 7.5 mg/kg/day in group 3 and 15 mg/kg/day in group 4. The rats in group 2 received sesame oil by gavage. To create gavage stress, only gavage was administered to the rats in group 1. The gavages for each group continued once a day and at a certain time for 30 days. To determine the effect of isotretinoin on oocyte maturation, the periodic acid-Schiff reaction was performed for histochemical and histomorphometric evaluation of the zona pellucida, and staining of growth differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15) was performed for immunohistochemical analysis. Results: When the thickness of the zona pellucida was evaluated, a statistically significant difference was found between group 1 and experimental groups (group 3 and group 4). In the experimental groups, it was determined that the thickness of the zona pellucida was decreased depending on the increase in dose. GDF-9 and BMP-15 expressions in oocytes of primordial and primary follicles decreased significantly in the experimental groups compared to group 1 and group 2. However, the expression of GDF-9 and BMP-15 in oocytes of secondary follicles was not significantly different between group 1 and group 2 and the experimental groups. Conclusions: In our study, we showed toxic effect of isotretinoin on oocyte maturation in female rats.

Continuous treatment with cisplatin induces the oocyte death of primordial follicles without activation.

Chemotherapy directly or indirectly affects organs in a short-term or continuous manner. Endocrine organs are especially sensitive to cancer treatment, leading to concerns among patients regarding their quality of life afterward. Side effects to the ovary include damage to the ovarian reserve, resulting in follicle loss, endocrine hormone deficiency, and infertility. It has been previously demonstrated that continuous treatment with 2mg/kg cisplatin for 15days can activate primordial follicles, suggesting that the response in the oocytes of primordial follicles was dependent on cisplatin concentration and administration frequency. However, our results demonstrate that continuous treatment with 2mg/kg cisplatin for 15days leads to the same consequence as with the continuous treatment of 5mg/kg cisplatin: the death of oocytes in primordial follicles without indication of activation. Moreover, animals co-injected with melatonin and cisplatin did not display any significant differences from those treated with cisplatin only contrary to the known results. 6-hydroxymelatonin, a metabolite of melatonin, could not prevent follicle destruction, implying that melatonin does not confer the protection of ovarian follicles, either directly or indirectly. Altogether, our data support that fertoprotectants against cisplatin must target molecules that control cell death pathways in the oocytes of primordial follicles.