The long-term in vitro culture (LIVC) of cumulus-oocyte complexes (COCs) derived from early antral follicles (EAFs) has shown significant potential in producing meiotically competent oocytes in adult ewes. However, the effectiveness of this approach in prepubertal ewes remains unexplored. This study aimed to evaluate the efficiency of the LIVC system in prepubertal ewes, addressing the challenge of limited EAF availability in adults. Both adult and prepubertal ewe ovaries were used to isolate COCs from EAFs (350-450 µm), which were then cultured in TCM199 supplemented with 0.15 μg/mL Zn++, 10-4 IU/mL FSH, 10 ng/mL estradiol, 50 ng/mL testosterone, 50 ng/mL progesterone, and 5 μM cilostamide. After five days of LIVC, a group of COCs underwent in vitro maturation, while others were evaluated for LIVC effectiveness, including morphology, oocyte diameter, chromatin configuration, gap junction communication, meiotic competence, intracellular reactive oxygen species (ROS) levels, mitochondrial activity, and distribution. Results showed a significant increase in oocyte diameter (116.3 vs. 113.4 µm, p < 0.01), higher COC morphological integrity (61.8% vs. 42.6%), and active gap junction communication in adults compared to prepubertal ewes. Moreover, prepubertal ewes exhibited a higher mitochondrial activity, along with spontaneous meiosis resumption (26.7% vs. 0%) after LIVC and a lower rate of meiosis resumption (21.0% vs. 59.4%) following in vitro maturation. No significant differences in ROS levels or mitochondrial distribution were observed between groups. In conclusion, COCs isolated from EAFs of prepubertal ewes showed restricted development during LIVC, highlighting significant age-related differences in optimizing fertility preservation techniques for prepubertal animals. Fertility preservation in adult livestock can be achieved by culturing growing oocytes from early antral follicles, a stage characterized by a small fluid-filled cavity. Still, its effectiveness in prepubertal animals remains uncertain. This study compared the quality and growth of oocytes from adult and prepubertal sheep. After five days in culture, oocytes from adult sheep showed better growth, structure, and cell-to-cell communication. In contrast, those from prepubertal sheep grew less but showed higher energy activity and early signs of maturation. These findings reveal clear age-related differences and suggest that current methods may need adjustments for younger animals. This work supports the development of better fertility preservation strategies for immature livestock.

Polystyrene and polyethylene terephthalate nanoplastics differentially impact mouse ovarian follicle function.

17β-hydroxysteroid dehydrogenase 1 (HSD17B1) is the primary enzyme responsible for the activation of estrone (E1) to estradiol (E2) in ovaries and extra-gonadal tissues of both humans and rodents. In the present study, molecular modeling identified the substitution of His222 in the human HSD17B1 enzyme with glycine in the mouse as the key determinant for the different steroid specificity between the species. Furthermore, Ser143Ala mutation at the active site of mouse HSD17B1 resulted in a total loss of E1 to E2 conversion by HSD17B1. This resulted in elevated intraovarian and circulating E1 concentrations in adult HSD17B1 Ser143Ala knock-in (HSD17B1-KI) females, but no changes in E2 concentrations were observed compared to the wild-type mice. Androstenedione and dihydrotestosterone were also elevated in the HSD17B1-KI ovaries, associated with elevated circulating LH. However, the effect of HSD17B1 inactivation on female reproductive development and function was mild, primarily resulting in a slight decrease in ovarian weight in older HSD17B1-KI mice, without notable effects on fertility. Expression of genes related to steroid biosynthesis, mitochondrial metabolism, and known markers of polycystic ovary syndrome was found to be upregulated in adult HSD17B1-KI ovaries. However, no alterations in the structure or function of extra-gonadal tissues were observed, and the uterus and bone phenotypes in the HSD17B1-KI females were unaffected. Our results demonstrate that the blockade of HSD17B1-dependent E2 synthesis is successfully compensated for in mouse in vivo, resulting in only a mild ovarian estrogen and androgen imbalance but no significant adverse effects on reproductive or bone health.

Cell death shapes multicellular organism development and sustains tissue and organ homeostasis. Great progress has been made in understanding the breadth of physiological and biochemical diversity in cell death and clearance pathways, which play vital roles in organismal development and health. While apoptosis and necrosis have been studied extensively across many model systems and contexts, the discovery of non-apoptotic paradigms of cell death and their roles in disease has greatly expanded the field. Collectively called Regulated Cell Death, these death pathways operate in a tissue and context-dependent manner. Germ cells in many organisms develop in cysts of interconnected cells, and may die in response to environmental or developmental cues. Recent findings suggest that germ cell cysts may use a common mechanism of non-apoptotic cell death involving phagocytic and lysosomal activity of surrounding somatic cells. Cyst cell death has been analyzed most thoroughly in the Drosophila adult ovary and testis, with remarkable similarity to cyst cell death in mouse adult testis and fetal ovary. In this review, we highlight recent progress in deciphering germline cyst cell death.

Paracrine signaling is critical for ovarian development and maintenance of non-growing, gonadotropin-independent follicles. During puberty the human ovary develops defined compartments as gonadotropin signaling triggers follicle activation and maturation. Adjacent follicles utilize paracrine signaling but also benefit from nearby interstitial cell populations. Here, we characterized human ovarian interstitial cells and their effects on follicle growth through a cross-species culture assay. Both prepubertal and postpubertal primary ovarian interstitial populations improved follicle growth, and postpubertal cells conferred a significant increase in follicle estradiol production but not maturation rates. In one cohort, interstitial cell-conditioned media was not sufficient for the growth advantage observed with co-culture. To investigate how follicle-interstitial cell crosstalk changes during puberty, single-cell RNA-sequencing was performed on 11 ovarian tissue cryopreservation samples (participants 0.34–22.8 years). Results confirmed the presence of heterogenous interstitial cell populations previously identified in the adult ovary, including abundant theca/stromal cells expressing genes enriched for ECM-related processes. Finally, over 100 proteins were identified in co-culture media using bottom-up proteomics representing the human ovarian interstitial secretome. This study uniquely characterized human ovarian interstitial cells across the pubertal transition and advances our understanding of the human pediatric and adolescent ovarian follicular microenvironment.

Autophagy is a central pathway involved in maintaining cellular homeostasis during development, metabolism, and regeneration. The selective autophagy receptor p62/SQSTM1 is a multifunctional protein that plays a critical role in regulating autophagic activity by interacting with LC3/Atg8 proteins and ubiquitinated substrates. Aedes albopictus has rapidly spread worldwide and poses a serious threat to human health by transmitting dengue and other arboviral diseases. In the present study, we identified a putative p62/SQSTM1 homolog protein (AaSQSTM1) in Ae. albopictus, which contains conserved structural regions, including the Atg8 family interaction motif (AIM) and ubiquitin-associated (UBA) domain. The expression levels of AaSQSTM1 and AaAtg8 varied from the egg stage to the adult stage in Ae. albopictus. Quantitative analyses revealed that blood-feeding upregulated the expression levels of AaSQSTM1 and AaAtg8 in the ovaries of female adults. AaSQSTM1 was applied to monitor autophagic activity alone or with AaAtg8 by assaying endogenous protein levels and overexpressing fluorescent reporters in C6/36 cells. Our study revealed that AaSQSTM1 regulates the autophagic activity with AaAtg8, providing a basis for studying the autophagy process in mosquitoes.

Innexins are the main components of gap junctions in invertebrates. However, the evolution and function of these in insects remain elusive. Phylogenetic analysis indicated that insect innexin genes originated from one ancestral gene, and they were divided into 6 clusters. Totally, 8 innexin (inx) genes were identified from Tribolium castaneum. These genes were highly expressed in different stages, including inx1 and inx7 in late pupae, inx2, inx3, and inx7-like2 in early pupae, shaking-B and inx7-like1 at postembryonic stages, and inx4/5/6 at early embryos and late adults. Tissue expression analysis indicated they had high transcription in distinct tissues, containing inx1 and inx7-like1 in the wing and gut of late pupae, inx7 in the gut and central nervous system of late pupae, shaking-B in most tissues except wings of late pupae, inx2 and inx3 in all tissues of early pupae, inx7-like2 in the gut and fat body of early pupae, and inx4/5/6 in the ovary of late adults. RNA interference (RNAi) experiments revealed that inx2 and inx4/5/6 were required for metamorphosis, fertility, and ovarian development, inx3 functioned during eclosion, inx1, inx7, and inx7-like1 played important roles in embryonic development, and shaking-B was critical for adult survival. Furthermore, silencing inx2, inx4/5/6, inx7, or shaking-B downregulated the expression of vitellogenin (Vg) and Vg receptor in female adults. These results indicated that innexin genes had crucial roles in the metamorphosis, fertility, and embryonic development of insects and provided the theoretical basis for developing inx2, inx3, and inx4/5/6 as potential targets of RNAi to control pests.

Mammalian ovary development is essential for female fertility, involving the complex spatial patterning of diverse cell types to establish the finite reserve of ovarian follicles. While single-cell transcriptome analyses have provided important insights into the mechanisms driving specification and developmental trajectories of ovarian cells, they disrupt this crucial spatial context. To overcome this limitation, we used 10X Genomics Visium HD spatial transcriptomics to analyze the developing mouse ovary while maintaining its native cellular architecture. We captured all ovarian cell types at eight key fetal and postnatal timepoints, generating a near single cell resolution library of spatial gene expression across ovarian development. This comprehensive dataset allows analysis of dynamic transcriptional signatures associated with unique spatial patterning throughout development, including the establishment of cortex and medulla and assembly of ovarian follicles in each region. This dataset represents a fundamental resource for the investigation of regulatory mechanisms driving spatial patterning of the ovary and opens new avenues to explore the spatial determinants of female fertility and reproductive longevity.

ABSTRACTObjective:To compare the clinical and biochemical profiles of adolescent and adult patients with Polycystic Ovary Syndrome (PCOS) and to evaluate the impact of insulin resistance on metabolic parameters.Methodology:A cross-sectional study was conducted at a tertiary care hospital from June to December 2024. A total of 150 PCOS patients, diagnosed using the Rotterdam criteria, were included. Clinical features (BMI, menstrual irregularities, hirsutism and family history) and biochemical parameters (fasting glucose, insulin, HOMA-IR, lipid profile and hormone levels) were analyzed. Patients were stratified into insulin-resistant and non-insulin-resistant groups for comparison. Insulin resistance was defined using HOMA-IR cut off value of more than 2.5.Results:Adolescents had lower BMI (25.5 ± 3.8 vs. 29.1 ± 4.9 kg/m², p < 0.001) and more frequent menstrual irregularities (90.7% vs. 49.3%, p < 0.001) than adults. Insulin resistance was observed in 85 (56.7%) patients and was associated with significantly higher fasting insulin (18.2 ± 4.8 vs. 9.8 ± 2.9 μU/mL, p < 0.001), HOMA-IR (4.8 ± 1.2 vs. 2.3 ± 0.6, p < 0.001) and higher LDL with lower HDL levels.Conclusions:Significant age-related differences in clinical and biochemical profiles exist among PCOS patients, along with insulin resistance status. Early identification of metabolic risks is crucial for timely intervention.

Ovarian aging profoundly impacts reproductive health and accelerates the overall aging process, yet the development of effective therapeutic strategies remains a formidable challenge. In this study, we report the rejuvenating effects of HEP14, a natural activator of protein kinase C (PKC) pathway, on aged ovarian function by inducing mitophagy and effectively clearing reactive oxygen species. To ensure controlled and sustained delivery of HEP14 in vivo, we develop HEP14-loaded PLGA microspheres. Transcriptomic analysis reveals a significant overlap between the transcriptional profiles of HEP14-treated aged ovaries and those of adult ovaries, suggesting molecular rejuvenation process closely associated to HEP14-induced mitophagy. Histopathological evaluations further substantiate these findings, showing that HEP14 enhances mitophagy, exhibits antioxidative properties and promotes follicular regeneration. Consequently, ovarian endocrine function in aged mice is substantially restored. Using transmission electron microscopy, confocal microscopy, and western blot analysis alongside pharmocological inhibitors and PKC-specific siRNA, in vitro studies further demonstrate the restorative effect of HEP14 on mitophagy, leading to improved mitochondrial function and subsequent alleviation of oxidative stress in senescent ovarian granulosa cells. This effect is mediated through the activation of the PKC-ERK1/2 pathway, which plays an pivotal role in the action mechanism in HEP14. These discoveries offer new therapeutic hope for ovarian aging.

Abstract The negative slope of human ovulatory fidelity begins with a high follicular fund balance with gradual withdrawals over time. This depletion of reserve is a sentinel event foreshadowing severe reproductive loss and, eventually, systemic aging. Conversely, the youthful ovarian phenotype requires coordination among endothelial, granulosa, immune, perivascular, stromal, and perhaps germline stem cells. This diverse tissue matrix theoretically can be modified by platelet (PLT)-derived moieties, but this awaits experimental confirmation in adult ovaries. Indeed, while the cellular entropy states of menopause and low reserve could follow a final common pathway, cell kinetics are not always irreversible within this set. We propose an experimental design to track nuclear factor κB (NF-κB) oscillations, tumor necrosis factor-alpha (TNF-α), selected gene expression, apoptosis, and key immune/inflammatory actions as entropy drivers antagonizing reproductive fitness. Since NF-κB and TNF-α are discharged in activated PLT releasate (or react to its cargo proteins), our investigation audits response markers pre- vs post-injection of processed platelet-rich plasma (PRP), connecting discrete signals to transcriptional output, cell function, and ovarian cytoarchitecture. This may reveal intraovarian PRP operating as a local entropy rectifier, with organ field function shifting to support oocyte competence incidentally, where “menopause reversal” is merely a beneficial secondary effect.

Maternal ovo represses the expression of transposable elements in adult ovaries.

Exposure to micro- and nanoplastics is unavoidable. Foods and beverages contain plastic particles from environmental contamination and processing and packaging materials, which are frequently made of polyethylene terephthalate (PET). Micro- and nanoplastics have been detected in human tissues such as the brain, liver, and placenta, as well as in ovarian follicular fluid, but little is known about the effects nanoplastics have on the female reproductive system. In addition, few studies on the health impacts of nanoplastics have been performed using environmentally relevant plastic types and concentrations. Thus, this research tested the hypothesis that nanoplastics made of spherical polystyrene (PS), a common model nanoplastic, would have different effects on cultured mouse ovarian follicles compared to secondary PET nanoplastics at environmentally relevant doses. The ovary is a highly sensitive reproductive organ responsible for the development of follicles, which contain the oocyte, and production of steroid hormones. Follicles were harvested from adult mouse ovaries and cultured for 96 h with vehicle, spherical commercially available 200 nm PS nanoplastics (1–100 μg/mL), or lab-generated 240 nm PET nanoplastics (0.1–10 μg/mL). PS and PET nanoplastic exposure inhibited follicle growth and altered expression of genes related to steroid synthesis, cell cycle, and oxidative stress. PET nanoplastics increased levels of pregnenolone and decreased expression of Cyp17a1. Overall, both plastic types altered ovarian function, but they impacted different genes in similar pathways. These findings suggest that nanoplastic exposure at environmentally relevant concentrations may pose a risk to female reproductive health by disrupting hormonal and molecular pathways. In addition, environmentally relevant plastic types and doses are necessary for studying health impacts of nanoplastics.

Norfloxacin inhibited the growth, development and reproduction of Grapholita molesta by reducing the abundance of Enterobacteriaceae bacteria in the guts of larva and ovaries of female moth.

Mealybugs are highly aggressive pests that infest various plants and cause substantial economic losses. Histone lysine methyltransferases (KMT) are evolutionarily conserved and proposed to be essential in early embryo development in animals. However, few KMTs have been reported in mealybugs. Here, we identified a novel KMT gene, PsASH2, in the cotton mealybug, Phenacoccus solenopsis Tinsley. This gene was highly expressed in the ovary of female adults. Through RNA interference (RNAi) of PsASH2 by dsRNA microinjection, we found a reduction in the number of male embryos and total embryos in the ovaries of pregnant females. Continuous downregulation of PsASH2 in mated females until their death resulted in few changes in sex ratio but significant decreases in the number of both male and female offspring. Therefore, we believe that PsASH2 plays essential roles in embryo survival for both sexes of the cotton mealybug which may provide a potential target gene for the management of cotton mealybug by disrupting embryo development.

Insulin receptor (InR) mediates the highly conserved insulin/insulin-like growth factor signaling pathway that regulates a broad range of life-history traits. Most insects encode 2 InR paralogs, but their functional divergence and redundancy remain to be explored. Here, we pursued the functionality of 2 InRs, LmInR1, and LmInR2, in the migratory locust Locusta migratoria. LmInR1 is clustered into the clade of ancestral InR, and LmInR2 belongs to the clade of InR gene duplication. While LmInR1 expression was at the highest levels in nymphal stage and adult ovary, LmInR2 was predominantly expressed in adult fat body. Loss of LmInR1 function led to defective nymph-adult transition and reproduction. LmInR2 depletion had little effect on metamorphosis, but caused severe defects in female reproduction, including remarkable reduction of Vitellogenin (Vg) expression and arrested egg development. Interestingly, LmInR1 expression responded to lower levels of glucose, whereas LmInR2 was expressed in response to increasing concentrations of glucose. Moreover, the expression of LmInR2 but not LmInR1 was responsive to juvenile hormone (JH) and its receptor. The results suggest that LmInR2 has evolved neofunctionalization for massive Vg synthesis required for synchronous maturation of dozens of oocytes in a high-fecundity insect. This study reveals a novel aspect of differential functions of InR paralogs and provides new insights into understanding of the insulin signaling cascade in insects.

The orphan nuclear receptor Nr2f2, also known as COUP-TFII, plays important roles in the development and function of multiple organs, including the reproductive system. NR2F2 is expressed in the interstitial cells of both embryonic and adult testes, ovaries, and reproductive tracts. Taking advantage of such unique expression pattern, we have developed a tamoxifen inducible Cre mouse model, Nr2f2-iCreERT2, which specifically and efficiently targets interstitial cells in both male and female reproductive organs across embryonic and adult stages. This model offers a powerful tool for gene knockout studies specifically in the interstitial compartment, without affecting the supporting or germ cells. Additionally, it enables interstitial cell lineage tracing, facilitating the assessment of the interstitial's contribution to non-interstitial cell types during development and differentiation.

This study investigated the effects of dietary Antarctic krill meal (AKM) on the physiological metabolism and flavor quality of adult Eriocheir sinensis ovaries during the postharvest temporary rearing. The AKM concentrations tested were 0% (including negative control group and positive control group), 2%, 4%, 6%, and 8%. The results indicate that the E. sinensis ovaries in 8% AKM group produced the highest levels of aroma compounds after thermal processing, including hexanal, heptanal, phenylacetaldehyde, 3-octanone, and 2-methylbutanoic acid ethyl ester. The 8% AKM and negative control group were analyzed by UPLC-MS/MS combined with the nontargeted and widely targeted metabolomics technique. The AKM altered the composition of aroma precursors by adjusting the metabolism of glycerophospholipid, linoleic acid, α-linolenic acid, and amino acid in ovaries. Moreover, lipids composed of polyunsaturated fatty acids (PUFAs) were significantly upregulated (p &lt; 0.05). Dietary supplementation with 8% AKM had the best effect on improving the ovarian flavor quality of E. sinensis. During the postharvest temporary rearing, more aromatic precursors were produced by regulating physiological metabolism. The ovarian flavor was enhanced by lipid oxidation, Maillard reaction, and Strecker degradation during thermal processing.

The adult ovary at single cell resolution: an expert review.

The rete ovarii (RO) is an appendage of the ovary that has been given little attention. Although the RO appears in drawings of the ovary in early versions of Gray's Anatomy, it disappeared from recent textbooks, and is often dismissed as a functionless vestige in the adult ovary. Using PAX8 immunostaining and confocal microscopy, we characterized the fetal development of the RO in the context of the mouse ovary. The RO consists of three distinct regions that persist in adult life, the intraovarian rete (IOR), the extraovarian rete (EOR), and the connecting rete (CR). While the cells of the IOR appear to form solid cords within the ovary, the EOR rapidly develops into a convoluted tubular epithelium ending in a distal dilated tip. Cells of the EOR are ciliated and exhibit cellular trafficking capabilities. The CR, connecting the EOR to the IOR, gradually acquires tubular epithelial characteristics by birth. Using microinjections into the distal dilated tip of the EOR, we found that luminal contents flow toward the ovary. Mass spectrometry revealed that the EOR lumen contains secreted proteins potentially important for ovarian function. We show that the cells of the EOR are closely associated with vasculature and macrophages, and are contacted by neuronal projections, consistent with a role as a sensory appendage of the ovary. The direct proximity of the RO to the ovary and its integration with the extraovarian landscape suggest that it plays an important role in ovary development and homeostasis.