Oocyte Loss Research Articles

Changes in DNA repair compartments and cohesin loss promote DNA damage accumulation in aged oocytes.

Oocyte loss, a natural process that accelerates as women approach their mid-30s, poses a significant challenge to female reproduction. Recent studies have identified DNA damage as a primary contributor to oocyte loss, but the mechanisms underlying DNA damage accumulation remain unclear. Here, we show that aged oocytes have a lower DNA repair capacity and reduced mobility of DNA damage sites compared to young oocytes. Incomplete DNA repair in aged oocytes results in defective chromosome integrity and partitioning, thereby compromising oocyte quality. We found that DNA repair proteins are arranged in spatially distinct DNA repair compartments that form during the late stages of oocyte growth, accompanied by changes in the activity of DNA repair pathways. We demonstrate alterations in these compartments with age, including substantial changes in the levels of key DNA repair proteins and a shift toward error-prone DNA repair pathways. In addition, we show that reduced cohesin levels make aged oocytes more vulnerable to persistent DNA damage and cause changes in DNA repair compartments. Our study links DNA damage accumulation in aged oocytes, a leading cause of oocyte loss, to cohesin deterioration and changes in the organization, abundance, and response of DNA repair machinery.

Robotic Manipulation of Cumulus–Oocyte Complexes for Cumulus Cell Removal

The removal of cumulus cells from cumulus–oocyte complexes is a critical step in clinical in vitro fertilization. Since the oocyte is partially occluded by the surrounding cumulus cells and individual cumulus cells are small in size, it is difficult for embryologists to assess the oocyte's maturity before cumulus cell removal and to completely remove all the cumulus cells manually . Furthermore, it is easy for the oocyte to become lost inside the micropipette during aspiration due to the inaccuracy of manual control. To deal with these difficulties, a robotic system was developed to completely remove cumulus cells from mature oocytes. In this study, an EPSANet50 network was developed to accurately assess the maturity of oocytes, avoiding the removal of cumulus cells around the immature oocyte. An adaptive controller was designed to accurately position oocytes at the target position, reducing the loss of oocytes inside the micropipette. An improved Yolov5s network was proposed to quantify the number and size of cumulus cells and assess the completeness of cumulus cell removal. The experimental results on mouse cumulus–oocyte complexes showed that the robotic system had a higher success rate (98.0 ± 1.8% vs. 85.3 ± 2.4%) and lower discard rate (4.1 ± 2.7% vs. 19.6 ± 3.5%) than the manual operation. Moreover, a higher amplification rate and lower non-specific rate were also achieved by the robotic system in the subsequent genetic testing procedure, indicating reduced genetic contamination from the cumulus cells.

Sendai virus-mediated RNA delivery restores fertility to congenital and chemotherapy-induced infertile female mice.

Current infertility treatment strategies focus on mature gametes, leaving a significant proportion of cases with gamete progenitors that stopped complete differentiation. On the other hand, recent advancements in next-generation sequencing have identified many candidate genes that may promote maturation of germ cells. Although gene therapy has shown success in mice, concerns about the integration of DNA vectors into oocytes hinder clinical applications. Here, we present the restoration of fertility in female mice through Sendai virus (SeV)-mediated RNA delivery. Ovaries lacking Kitl expression exhibit only primordial follicles due to impaired signaling to oocytes expressing the KIT tyrosine kinase. Despite SeVs being immunogenic and larger than the blood-follicle barrier, the administration of Kitl-expressing SeVs reinitiated oogenesis in genetically infertile mice that have only primordial follicles, resulting in the birth of normal offspring through natural mating. This virus also effectively addressed iatrogenic infertility induced by busulfan, a widely used cancer chemotherapy agent. Offspring born through SeV administration and natural mating displayed normal genomic imprinting patterns and fertility. Since SeVs pose no genotoxicity risk, the successful restoration of fertility by SeVs represents a promising approach for treating congenital infertility with somatic cell defects and protecting fertility of cancer patients who may become infertile due to loss of oocytes during cancer therapy.

O-038 Impact of maternal aging on the DNA repair landscape in mouse oocytes

Abstract Study question How does maternal aging impact DNA damage accumulation and DNA repair capacity in mammalian oocytes? Summary answer Age-related changes in DNA repair compartments and chromatin organisation drive DNA damage accumulation in aged oocytes, ultimately contributing to the decline in DNA repair capacity. What is known already Oocyte loss occurs progressively from birth and accelerates as women approach their mid-30s, posing a significant challenge to female reproductive health. Recent studies have highlighted the role of the DNA repair response in driving ovarian ageing and oocyte loss. Aged oocytes accumulate elevated levels of DNA damage. Unless repaired, persistent DNA damage activates DNA repair checkpoints, causing the elimination of defective oocytes. Although we know DNA double-strand breaks (DSBs) in oocytes increase with maternal age, surprisingly, little is known about why this damage is not repaired efficiently. Study design, size, duration Oocytes were isolated from ovaries of 8 weeks or 68-86-week-old CD1 mice, which correspond to reproductively young and old females respectively. Over 600 and 1000 oocytes from aged any young females were used to analyse DNA damage levels, DNA repair effciency, DNA repair compartments and for other live assays. Participants/materials, setting, methods DNA damage was induced in CD1 oocytes from young and aged mice using neocarzinostatin. Levels of DNA damage and DNA repair proteins were quantified using immunofluoresnce confocal/STED microscopy. High-resolution light sheet microscopy was used to perform live imaging of dynamics of DNA damage foci and chromosome segregation during meiosis. Main results and the role of chance Our work demonstrates that aged oocytes have a lower DNA repair capacity and reduced mobility of DNA damage sites compared to young oocytes. We further show that incomplete DNA repair results in defective chromosome integrity and partitioning, thereby compromising oocyte quality. To investigate the causes of the age-related decline in DNA repair, we systematically studied the localization and abundance of several DNA repair proteins in oocytes from young and aged mice. We found that DNA repair proteins localized in spatially distinct DNA repair compartments in oocyte nuclei, that form during the late stages of oocyte growth, accompanied by changes in the activity of DNA repair pathways. Several DNA repair proteins, their corresponding compartments, and their response to DNA damage were altered in aged oocytes. Lastly, we demonstrate that age-related cohesin loss increases DNA damage levels and reduces DNA repair capacity. We hypothesize that cohesin dissociation with maternal ageing may make chromosomes prone to accumulate DNA damage without proper DNA repair. Thus, age-related changes in DNA repair compartments and chromatin organisation drive DNA damage accumulation in aged oocytes, ultimately contributing to the decline in reproductive potential. Limitations, reasons for caution Neocarzinostatin, a radiomimetic drug used to induce DNA DSBs in mouse oocytes in vitro, may not fully represent sources of DNA damage during aging. The CD1 mouse model and ages were selected to recapitulate equivalent advanced maternal age in women. Further investigation is needed to confirm these findings in humans. Wider implications of the findings Overall, we propose that altered DNA repair machinery and cohesin loss contribute to persistent DNA damage and reduced DNA repair capacity in aged oocytes, which can cause chromosome defects and fragmentation, and eventually trigger oocyte death, thereby accelerating the loss of female fertility. Trial registration number Not Applicable

P-217 Keep calm and carry on: assessing the effect of below-benchmark vitrified/warmed donor oocyte survival rates in subsequent laboratory and clinical key performance indicators

Abstract Study question When we experience below benchmark donor oocyte survival rates, are the laboratory and clinical outcomes of the recipient cycle affected? Summary answer Donor-oocyte cryo-survival rates below the benchmark (95-100%) result in slightly lower laboratory KPIs, equivalent live birth rates (LBR) and lower cumulative LBR (CLBR) per cycle. What is known already The use of vitrified/warmed donor oocytes allows overcoming many logistic challenges, but it is hindered by the loss of oocytes due to degeneration after warming. The 2012 Vienna Consensus established the benchmark rate of oocyte survival at 95-100%, competency level at > 85%. Results of < 85% cryo-survival are considered below competency, <70% can be considered poor, and <50% very poor. Below-benchmark rates of survival may not only limit the number of donor oocytes for the cycle, but they can also translate into lower per-oocyte cycle KPIs if the lower survival correlates with lower performance of the surviving oocytes. Study design, size, duration We analyzed 12690 vitrified-warmed donor oocyte recipient cycles, encompassing a total 144793 vitrified/warmed donor oocytes, carried out in a network of European private IVF units from 2018-2022. Cycles with PGT-A and spermatozoa from testicular biopsy were excluded. The primary endpoint of the study was the blastocyst utilization rate per 2PN zygote. Secondary endpoints were fertilization rate, live birth rate after the first single blastocyst transfer (SET-LBR) of each cycle, and cumulative LBR (CLBR). Participants/materials, setting, methods In all cases, fertilization was carried out by ICSI, and embryos were cultured to the blastocyst stage at low oxygen tension. Blastocysts with good or fair quality (A, B & C by ASEBIR Criteria) were considered usable (transferred + cryopreserved blastocyts / zygotes). Differences in proportions were analyzed using chi-square and the adjusted odds ratio (OR) was calculated using multivariable logistic regression. Main results and the role of chance Baseline donor and patient characteristics such as age, stimulation duration and dose, total donor oocytes per cycle, male factor, and fresh/frozen/donor spem were compared between groups and adjusted for when necessary. The mean number of warmed oocytes per cycle was 11.4±3.22 with a mean survival rate of 89.2%. More oocytes per cycle were consumed in the lower survival groups, yet the total oocytes available for ICSI was smaller in the below competency, poor and very poor survival groups. There were differences in the proportion of usable blastocysts per zygote amongst the survival rate groups (benchmark: 48.9%; competence: 47.0%, below-competence: 46.0%; poor: 43.6%; very-poor:43.6%, p < 0.001). Fertilization rates also differed between survival groups (benchmark: 76.9%; competence: 76.5%, below-competence: 75.6%; poor: 74.7%; very-poor:75.4%, p < 0.001) The adjusted odds ratio of live birth on the first SET of the cycle was not lower to the benchmark survival group in all the lower survival groups (Benchmark: 40.9%; Competence: OR:0.995; 95%CI:0.971-1.018; below-competence: OR:0.996; 95%CI:0.970-1.023; poor: OR:1.044; 95%CI:1.003-1.086, and very-poor: OR:1.069; 95%CI:0.976-1.170). The CLBR decreased in lower survival groups, with 80.8% for the benchmark survival group, 80.3% (p = 0.539) for competence, 76.2% (p < 0.001) for below-competence, 72.5% (p < 0.001) for poor, and 74.8% (p = 0.056) for very-poor. Limitations, reasons for caution These results are limited to a donor-oocyte population. Wider implications of the findings Lower oocyte-cryo-survival rates resulted in a lower total number of usable blastocysts obtained for the recipient, and consequently, lower CLBR. However, the competence of surviving oocytes is encouraging, with laboratory KPIs in very close range irrespectively of the cohort survival rates, and similar implantation potential of the obtained blastocysts. Trial registration number not applicable

Potential clinical application of anti-Müllerian hormone testing in radioiodine treatment of thyroid carcinoma

Differentiated thyroid cancer is the most common endocrinological malignancy. Radioiodine treatment has a clear benefit in locally aggressive and metastatic cancers. There are discussions about long-term and acute adverse events.Anti-Müllerian hormone is regarded as the best endocrine marker for evaluating the physiological loss of oocytes in healthy women with regard to age. The impact of radioiodine treatment on anti-Müllerian hormone levels has been more significantly reported in patients over 35 years of age. About reproductive dysfunction, calculations of individual absorbed doses of radioiodine in ovaries after thyroid cancer therapy have not been performed yet. The aim of our ongoing prospective study is to determine serum anti-Müllerian hormone to estimate ovarian reserve for premenopausal women treated with radioiodine and to compare anti-Müllerian hormone levels before and after radioiodine treatment. Predicting radioiodine side effects by evaluating a simple serum biomarker may help to select an appropriate treatment strategy for young women planning pregnancy, specifically in the assessment of ovarian reserve and premature ovarian failure with early onset of menopause.

Macrophage activation drives ovarian failure and masculinization in zebrafish.

BMP15 is a conserved regulator of ovarian development and maintenance in vertebrates. In humans, premature ovarian insufficiency is caused by autoimmunity and genetic factors, including mutation of BMP15. The cellular mechanisms underlying ovarian failure caused by BMP15 mutation and immune contributions are not understood. Using zebrafish, we established a causal link between macrophage activation and ovarian failure, which, in zebrafish, causes sex reversal. We define a germline-soma signaling axis that activates macrophages and drives ovarian failure and female-to-male sex reversal. Germline loss of zebrafish Bmp15 impairs oogenesis and initiates this cascade. Single-cell RNA sequencing and genetic analyses implicate ovarian somatic cells that express conserved macrophage-activating ligands as mediators of ovarian failure and sex reversal. Genetic ablation of macrophages or elimination of Csf1Rb ligands, Il34 or Csf1a, delays or blocks premature oocyte loss and sex reversal. The axis identified here provides insight into the cells and pathways governing oocyte and ovary maintenance and potential therapeutic targets to preserve female fertility.

Chronic stress induces meiotic arrest failure and ovarian reserve decline via the cAMP signaling pathway.

Chronic stress is suspected to be a causal factor of female subfertility; however, the underlying mechanisms remain unclear. Here, we found that chronic stress inhibited the cyclic adenosine 3',5'-monophosphate (cAMP) signaling pathway, leading to ovarian reserve decline in mice. A chronic stress model was constructed using restraint stress for 8 weeks. An elongated estrous cycle and a significant increase in the number of atretic follicles were observed in the stress group. We identified a significant increase in meiotic arrest failure (MAF) in oocytes in the stress group, characterized by condensed metaphase chromosomes, assembled spindles, or polar bodies in the oocytes. Whole-mount ovarian reserve estimation at the single-oocyte level using the CUBIC method (clear, unobstructed brain/body imaging cocktails and computational analysis) revealed a significant decrease in quiescent oocytes from 2,261/ovary in the control group to 1,373/ovary in the stress group. The number of growing oocytes also significantly decreased from 220/ovary in the control group to 150/ovary in the stress group. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of the meiotic arrest maintenance pathways revealed significant downregulation of Gpr3, Nppc, and Npr2 in the stress group. These results indicate that blocking cAMP production contributes to MAF and a decline in ovarian reserve. Overall, we present new insights into the mechanisms underlying chronic-stress-induced oocyte loss and potential targets for ovarian reserve preservation.

Maternal exposure to cetylpyridinium chloride impairs oogenesis by causing mitochondria disorder in neonates

Cetylpyridinium Chloride (CPC) is a common disinfectant with potential mitochondrial toxicity. However, the effects of CPC on female reproduction remains unclear. In the present study, pregnant mice were exposed to environmentally relevant doses of CPC for 3 days, the effects were evaluated in the female offspring. Maternal exposure to CPC caused loss of oocytes in neonatal ovaries. TEM analysis of neonatal ovaries showed CPC caused aberrant mitochondrial morphology including vacuolated and disorganized structure, reduced functional cristae. In addition, CPC decreased mitochondrial membrane potential in neonatal oocytes. Seahorse analysis showed that CPC hampered mitochondrial reserve, manifested as reduced spare respiratory capacity. Furthermore, CPC damaged mitochondrial function and impaired developmental competence of MII oocytes, suggesting a persisting impact into adulthood. In summary, this is the first known demonstration that maternal exposure to CPC caused mitochondrial disorders in neonatal ovaries and had long-term effects on fertility of the female offspring.

A filtration medium replacement method that increases the efficiency of immunofluorescence staining of oocytes

ABSTRACT Immunofluorescence staining is used to investigate proteins and protein interactions in oocytes. In typical protocols, the medium that suspends the oocytes requires replacement more than ten times during the staining procedure; this is time-consuming, technically challenging and not amenable to automation. We developed a filtration method using negative pressure to replace manual replacement of medium. We investigated oocyte loss, time required and staining results using our filtration method compared to traditional procedure. We found that our filtration method reduced oocyte loss by at least 60% and decreased the time required to obtain comparable staining outcomes. It provides an efficient and fast way to replace culture medium for oocytes.

P-187 Vitrified-warmed donor oocytes’ cryo-survival rate is not associated with subsequent embryological outcomes and clinical pregnancy rate per first transfer

Abstract Study question Is the survival rate of a cohort of donor oocytes used for each recipient associated with the outcomes of the cycle? Summary answer Oocyte cryo-survival rates (benchmark = 95-100%, competency = 85-95%, below-competency = 70-85%, and poor = 50-70%) are not associated with embryological outcomes and clinical pregnancy in the first transfer of the cycle. What is known already The use of vitrified-warmed donor oocytes allows overcoming many logistic challenges, but it is hindered by the loss of oocytes due to degeneration after warming. The 2012 Vienna Consensus established the benchmark rate of oocyte survival at 95-100%, competency level at > 85%. Results <85% cryo-survival are considered below competency, and <70% can be considered poor. Below-benchmark rates of survival may not only limit the number of oocytes, but also could translate into lower cycle KPIs. Study design, size, duration We analyzed 2190 vitrified-warmed donor oocyte recipient cycles, encompassing a total 20280 vitrified oocytes from 1036 donors. Cycles were carried out in 6 private IVF units from the same group. Cycles from 2018 to 2022 were included in the analysis. Cycles with PGT-A and spermatozoa from testicular biopsy were excluded. The outcome of the first transfer of each cycle was analyzed. Participants/materials, setting, methods Vitrification was performed using open carriers and two different commercial media (Irvine-Scientific and Kitazato). In all cases, fertilization was carried out by ICSI, and embryos were cultured to the blastocyst stage at low oxygen tension. Blastocysts with good or fair quality (A, B & C by ASEBIR Criteria) were considered usable, and the usable blastocyst rate was the sum of all blastocysts transferred and cryopreserved, divided by the number of zygotes on day1. Main results and the role of chance The mean number of warmed oocytes per cycle was 9.3, with a survival rate of 93.7%. A pairwise comparison of fertilization rates with Bonferroni correction revealed that the benchmark survival rate group had similar results to the 85-95%, 70-85% and the 50-70% groups (95-100%: 73.7%; 85-95: 72.1%, 70-85%: 70.9%, 50-70%: 73.1%, p = 1 in all cases). The usable blastocyst rate was similar between the benchmark survival rate and the 85-95% and 50-70% groups, but higher than the 70-85% group (95-100%: 49.0%; 85-95: 48.5%, p = 1.00; 70-85%: 43.5%, p = 0.012, 50-70%: 51.3%, p = 1.00). Of 1368 Day5, SET, first-attempt transfers, 53.5% had a clinical pregnancy. The mean age of recipients undergoing embryo transfer was 42.6 ± 4.5 years and they had undergone 1.1 ± 1.8 previous IVF cycles. Adjusted-OR of clinical pregnancy was only significantly lowered in comparison to the benchmark survival rate in the 70-85% survival group (OR:0.49; 95%CI:0.34−0.71), but not it in the 50-70% group (OR:1.50, 95%CI: 0.92-2.46) nor the 85-95% group (OR:1.00, 95%CI: 0.70-1.42). The use of a sperm donor was positively correlated with clinical pregnancy (OR:1.39; 95%CI:1.04-1.88). Recipient age, male factor, and number of previous IVF cycles were not correlated with clinical pregnancy. Limitations, reasons for caution Retrospective study. Only open carriers were used for vitrification. Cumulative outcomes were not assessed. Wider implications of the findings Sub-benchmark levels of oocyte cryo-survival were not directly associated with worse laboratory outcomes or clinical pregnancy rates. These results are encouraging for cases with lower cryo-survival and useful to counsel patients in this regard. Nevertheless, striving to meet benchmark cryo-survival rates is essential. Trial registration number Not Applicable

TP63 gain-of-function mutations cause premature ovarian insufficiency by inducing oocyte apoptosis.

The transcription factor p63 guards genome integrity in the female germline, and its mutations have been reported in patients with premature ovarian insufficiency (POI). However, the precise contribution of the TP63 gene to the pathogenesis of POI needs to be further determined. Here, in 1,030 Chinese patients with POI, we identified 6 heterozygous mutations of the TP63 gene that impaired the C-terminal transactivation-inhibitory domain (TID) of the TAp63α protein and resulted in tetramer formation and constitutive activation of the mutant proteins. The mutant proteins induced cell apoptosis by increasing the expression of apoptosis-inducing factors in vitro. We next introduced a premature stop codon and selectively deleted the TID of TAp63α in mice and observed rapid depletion of the p63+/ΔTID mouse oocytes through apoptosis after birth. Finally, to further verify the pathogenicity of the mutation p.R647C in the TID that was present in 3 patients, we generated p63+/R647C mice and also found accelerated oocyte loss, but to a lesser degree than in the p63+/ΔTID mice. Together, these findings show that TID-related variants causing constitutive activation of TAp63α lead to POI by inducing oocyte apoptosis, which will facilitate the genetic diagnosis of POI in patients and provide a potential therapeutic target for extending female fertility.

Ameliorative Effect of Citrus Lemon Peel Extract and Resveratrol on Premature Ovarian Failure Rat Model: Role of iNOS/Caspase-3 Pathway

Premature ovarian failure (POF) is described as a loss of oocytes and the absence of folliculogenesis and is considered an adverse effect of chemotherapeutic drugs, which leads to infertility. Subsequently, the existing inquiry was achieved by exploring the potential suspicious influences of lemon peel extract (LPE), and resveratrol (RES) on cyclophosphamide (CPA) induced-POF. The results showed that CPA-induced POF significantly decreased serum estradiol (E2) and progesterone levels, along with a considerable rise in serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels. Moreover, CPA administration to rats significantly increased the serum level of Malondialdehyde (MDA) and significantly lowered the levels of reduced glutathione (GSH) and superoxide dismutase (SOD); in addition, it increased nuclear factor kappa B (NF-κB) levels, tumor necrosis factor-α (TNF-α), as well as cyclooxygenase 2 (COX-2) with the spread expression of inducible nitric oxide synthase (iNOS) mRNA levels and caspase-3 (Casp3) levels in ovarian tissues versus the control rats. However, treatment with LPE and RES suppressed the triggering of NF- κB pathways, evidenced by a considerable reduction in Casp3 & iNOS mRNA expression level and significant ameliorative effects in all evaluated parameters, as confirmed by the histological and immunohistochemical investigation when comparing the model group. In overall findings, both lemon peel extract and resveratrol can mitigate the adverse effects of CPA-induced POF. Most crucially, its combination therapy is a promising pharmacological agent for this disease.

Oocyte Casein kinase 1α deletion causes defects in primordial follicle formation and oocyte loss by impairing oocyte meiosis and enhancing autophagy in developing mouse ovary

Casein kinase 1α is a member of CK1 family, which is ubiquitously expressed and plays multiple functions, including its potential roles in regulating cell division. But the functions of CK1α in mammalian oogenesis and folliculogenesis remain elusive. In this study, we assayed the cell type of CK1α expression in the developing mouse ovary and confirmed that CK1α was highly expressed in ovaries after birth. The oocyte-specific CK1α knockout (cKO) mouse model was then established by crossing Ddx4-Cre mice with Csnk1a1-floxp mice, and the effects of CK1α deletion on oogenesis and folliculogenesis were identified. The results showed that oocyte CK1α deletion impaired the progression of oocyte meiosis and primordial follicle formation during meiotic prophase I, which subsequently caused oocyte loss and mouse infertility. Further, the in vivo CK1α deletion and in vitro inhibition of CK1 activity resulted in the defects of DNA double-strand break (DSB) repair, whereas apoptosis and autophagy were enhanced in the developing ovary. These may contribute to oocyte loss and infertility in cKO mice. It is thus concluded that CK1α is essential for mouse oogenesis and folliculogenesis by involving in regulating the processes of oocyte meiosis and DNA DSB repair during meiotic prophase I of mouse oocytes. However, the related signaling pathway and molecular mechanisms need to be elucidated further.

ASH1L contributes to oocyte apoptosis by regulating DNA damage.

In female mammals, the size of the initially established primordial follicle pool within the ovaries determines the reproductive life span. Interestingly, the establishment of the primordial follicle pool is accompanied by a remarkable programmed oocyte loss for unclear reasons. Here, we identify a new role of ASH1-like histone lysine methyltransferase (ASH1L) in controlling the apoptosis of oocytes during meiotic prophase I in mice. Our results showed that overexpression of Ash1l led to a dramatic loss of fetal oocytes via apoptosis, which subsequently resulted in a reduced capacity of the primordial follicle pool. Overexpression of Ash1l also led to a deficiency in DNA double-strand break repair associated with premature upregulation of p63 and phosphorylated checkpoint kinase 2 (p-CHK2), the major genome guardian of the female germline, following Ash1l overexpression in fetal ovaries. In summary, ASH1L is one of the indispensable epigenetic molecules that acts as a guardian of the genome. It protects oocyte genome integrity and removes oocytes with serious DNA damage by regulating the expression of p63 and p-CHK2 during meiotic prophase I in mice. Our study provides a perspective on the physiological regulatory role of DNA damage checkpoint signaling in fetal oocyte guardianship and female fertility.

Dominant TP63 missense variants lead to constitutive activation and premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is a leading form of female infertility, characterised by menstrual disturbance and elevated follicle‐stimulating hormone before age 40. It is highly heterogeneous with variants in over 80 genes potentially causative, but the majority of cases having no known cause. One gene implicated in POI pathology is TP63. TP63 encodes multiple p63 isoforms, one of which has been shown to have a role in the surveillance of genetic quality in oocytes. TP63 C‐terminal truncation variants and N‐terminal duplication have been described in association with POI, however, functional validation has been lacking. Here we identify three novel TP63 missense variants in women with nonsyndromic POI, including one in the N‐terminal activation domain, one in the C‐terminal inhibition domain, and one affecting a unique and poorly understood p63 isoform, TA*p63. Via blue‐native page and luciferase reporter assays we demonstrate that two of these variants disrupt p63 dimerization, leading to constitutively active p63 tetramer that significantly increases the transcription of downstream targets. This is the first evidence that TP63 missense variants can cause isolated POI and provides mechanistic insight that TP63 variants cause POI due to constitutive p63 activation and accelerated oocyte loss in the absence of DNA damage.

Autophagy participates in germline cyst breakdown and follicular formation by modulating glycolysis switch via Akt signaling in newly-hatched chicken ovaries

In females, the establishment of the primordial follicle pool is accompanied by a remarkable programmed oocyte loss for unclear reasons. In this study, the role of autophagy was investigated to serve as a protective mechanism for oocyte survival during chicken folliculogenesis. Inhibition of autophagy by 3-methyladenine (3-MA) led to a remarkable delay in germ cell cyst breakdown that resulted in fewer primordial follicles and retarded sequent follicular development either in vivo or in the ovarian organ culture. Furthermore, the glycolysis level was downregulated in ovaries treated with 3-MA, while Recilisib (a specific activator of Akt) reversed this inhibiting effect of 3-MA on primordial folliculogenesis. Collectively, these data indicate that autophagy functions to maintain germ cell cyst breakdown and primordial follicle assembly by regulating ovarian glycolysis involving Akt signaling in the ovaries of newly-hatched chickens.

Maternal starvation primes progeny response to nutritional stress

Organisms adapt to environmental changes in order to survive. Mothers exposed to nutritional stresses can induce an adaptive response in their offspring. However, the molecular mechanisms behind such inheritable links are not clear. Here we report that in Drosophila, starvation of mothers primes the progeny against subsequent nutritional stress. We found that RpL10Ab represses TOR pathway activity by genetically interacting with TOR pathway components TSC2 and Rheb. In addition, starved mothers produce offspring with lower levels of RpL10Ab in the germline, which results in higher TOR pathway activity, conferring greater resistance to starvation-induced oocyte loss. The RpL10Ab locus encodes for the RpL10Ab mRNA and a stable intronic sequence RNA (sisR-8), which collectively repress RpL10Ab pre-mRNA splicing in a negative feedback mechanism. During starvation, an increase in maternally deposited RpL10Ab and sisR-8 transcripts leads to the reduction of RpL10Ab expression in the offspring. Our study suggests that the maternally deposited RpL10Ab and sisR-8 transcripts trigger a negative feedback loop that mediates intergenerational adaptation to nutritional stress as a starvation response.

Effects of Bacillus subtilis ZJ-2019-1 on Zearalenone Toxicosis in Female Gilts.

The purpose of this research was to investigate the toxicity of zearalenone (ZEN) on the growth performance, genital organs, serum hormones, biomarkers, and histopathological changes of female gilts and to evaluate the efficacy of Bacillus subtilis ZJ-2019-1 in alleviating ZEN toxicosis in gilts. Twenty-four female gilts were randomly allocated to four groups with six replicates per group and one gilt per replicate, fed on four feeds prepared previously, which were basic diet (control group, C group), ZEN diet (Z group), Zlb diet (Zlb group) containing B. subtilis ZJ-2019-1 in liquid form, and Zdb diet (Zdb group) containing B. subtilis ZJ-2019-1 in dehydrated form. The results showed that the vulva size and relative weight of reproductive organs had no significant difference in the control group, Zlb group, and Zdb group, but were significantly lower than in the Z group (p < 0.05); the relative weight of the liver was lower in the C group, Zlb group, and Zbd group than in the Z group (0.05 < p < 0.1). The concentration of serum glutamate dehydrogenase (GLDH) was lower, but follicle-stimulating hormone (FSH) was higher in the Z group, Zlb group, and Zdb group than in the Z group (0.05 < p < 0.1). Additionally, serum luteinizing hormone (LH) concentration had no significant difference in the C group, Zlb group, and Zdb group but was significantly lower than in the Z group (p < 0.05); estradiol (E2) was significantly lower in the Zlb group and Zdb group than that in C group, but significantly higher than that in Z group (p < 0.05); PRL was significantly higher in the Zlb group and Zdb group than in the C group, but was significantly lower than in Z group (p < 0.05). ZEN and its reduced metabolites were measured in biological samples after enzymatic hydrolysis of the conjugated forms. The concentration of serum ZEN and its metabolite, α-zeralenol (α-ZOL), had no significant difference in Zlb, Zdb, and control groups but was significantly lower than in the Z group (p < 0.05); urine ZEN and its metabolites, α-ZOL and β-zeralenol (β-ZOL), had no significant difference in Zlb, Zdb, and control groups but was significantly lower than in the Z group (p < 0.05). Cell damages were observed in the liver, uterus, and ovary of gilts in the Z group and alleviated in Zlb and Zdb groups, but the loss of oocytes was irreversible in the ovary. The ZEN-contaminated diet caused serious changes in female hormones and brought harm to the livers and reproductive organs, but B. subtilis ZJ-2019-1 could naturally remove the ZEN significantly, which ameliorated the reproductive impairment in gilts caused by ZEN. The addition of B. subtilis ZJ-2019-1 to ZEN-contaminated feeds could ameliorate the toxic effects effectively, regardless of liquid or dry culture. Therefore, the B. subtilis ZJ-2019-1 strain has great potential industrial applications.

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse.

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans. Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.