Search Results (73)

Ferroptosis is a novel kind of regulated cell death that occurs when redox equilibrium is disrupted, leading to iron-dependent lipid peroxidation. Ferroptosis is defined by the buildup of deleterious lipid hydroperoxides, the inactivation of glutathione peroxidase 4 (GPX4), and mitochondrial shrinkage, setting it apart from apoptosis and necrosis. The relevance of this route to human reproduction remains unknown, despite its thorough investigation in neurodegeneration and cancer. Recent studies demonstrate that the ovarian follicular milieu is especially susceptible to ferroptosis owing to its high content of polyunsaturated fatty acids, iron-dependent metabolism, and the generation of reactive oxygen species. Dysregulation of ferroptosis may result in infertility by affecting granulosa cell survival, oocyte maturation, and embryonic competence. Ferroptotic activity correlates with oxidative stress indicators identified in clinical diseases including polycystic ovary syndrome, reduced ovarian reserve, and insufficient responsiveness to ovarian stimulation. Potential indicators include GPX4 expression, decreased glutathione levels, and the accumulation of lipid reactive oxygen species in granulosa cells and follicular fluid. Melatonin, which boosts antioxidant defences, and ferrostatin-1, a prototype inhibitor of ferroptosis that lowers lipid peroxidation, are two early candidates for treatment. For future evaluations, these agents should be used with standardised FF biomarker panels. Significantly, vitamin E, coenzyme Q10, and small-molecule ferroptosis inhibitors have shown efficacy in halting ferroptosis in experimental settings. These approaches have shown protective benefits in alternative systems and may signify viable treatment options for assisted reproduction. This narrative review encapsulates ferroptosis inside the ovarian follicle, its influence on oocyte quality, and the implications for in vitro fertilization results. Full article

Hypoxic pulmonary vasoconstriction (HPV) optimizes gas exchange but, when impaired, can result in life-threatening hypoxemia. Moreover, under conditions of generalized alveolar hypoxia, HPV can result in pulmonary hypertension. Voltage-gated K⁺ channels (K_v channels) are key to HPV: a change in the intracellular hydrogen peroxide (H₂O₂) levels during acute hypoxia is assumed to modulate these channels’ activity to trigger HPV. However, there are longstanding conflicting findings on whether H₂O₂ inhibits or activates K_v channels. Therefore, we hypothesized that H₂O₂ affects K_v channels depending on the experimental conditions, i.e., the H₂O₂ concentration, the channel’s subunit configuration or the experimental clamping potential in electrophysiological recordings. Therefore, cRNAs encoding the K_v1.5 channel and the auxiliary K_vβ subunits (K_vβ1.1, K_vβ1.4) were generated via in vitro transcription before being injected into Xenopus laevis oocytes for heterologous expression. The K⁺ currents of homomeric (Kv1.5) or heteromeric (K_v1.5/K_vβ1.1 or K_v1.5/K_vβ1.4) channels were assessed by two-electrode voltage clamp. The response of the K_v channels to H₂O₂ was markedly dependent on (a) the clamping potential, (b) the H₂O₂ concentration, and (c) the K_v channel’s subunit composition. In conclusion, our data highlight the importance of the choice of experimental conditions when assessing the H₂O₂ sensitivity of K_v channels in the context of HPV, thus providing an explanation for the long-lasting controversial findings reported in the literature. Full article

In addition to the male genome, the fertilizing spermatozoon delivers to the oocyte several factors whose deficiency can cause embryo dysfunction. Sperm oocyte-activating factor, identified as phoshoplipase C zeta (PLCζ), drives oocyte exit from meiotic arrest through a signaling pathway initiated by periodic rises of free cytosolic Ca²⁺ concentration (calcium oscillations). Sperm centrioles, together with oocyte proteins, form centrosomes that are responsible for aster formation, pronuclear migration, and DNA polarization before nuclear syngamy and subsequent mitotic divisions. Sperm DNA fragmentation can be at the origin of aneuploidies, while epigenetic issues, mainly abnormal methylation of DNA-associated histones, cause asynchronies of zygotic gene activation among embryonic cells. Sperm long and short non-coding RNAs are important epigenetic regulators affecting critical developmental processes. Dysfunction of sperm PLCζ, centrioles, DNA, and RNA mostly converge to aneuploidy, developmental arrest, implantation failure, miscarriage, abortion, or offspring disease. With the exception of DNA fragmentation, the other sperm issues are more difficult to diagnose. Specific tests, including heterologous human intracytoplasmic sperm injection (ICSI) into animal oocytes, genetic testing for mutations in PLCZ1 (the gene coding for PLCζ in humans) and associated genes, and next-generation sequencing of sperm transcriptome, are currently available. Oral antioxidant treatment and in vitro selection of healthy spermatozoa can be used in cases of sperm DNA fragmentation, while ICSI with assisted oocyte activation is useful to overcome oocyte-activation defects. No clinically confirmed therapy is yet available for sperm RNA issues. Full article

Donkeys (Equus asinus) hold significant agricultural value in China, particularly for their hides and meat, which possess notable medicinal and dietary importance. However, their reproductive efficiency remains suboptimal compared with other livestock. Ovarian function is a key determinant of fertility, yet the molecular mechanisms underlying donkey ovarian biology remain largely unexplored. To address this gap, we performed single-cell RNA sequencing of donkey ovaries, generating a high-resolution transcriptomic atlas comprising 17,423 cells. Cross-species comparative analysis revealed a high degree of evolutionary conservation in core ovarian cell types, including endothelial, epithelial, immune, and smooth muscle cells, among vertebrates. In contrast, granulosa and theca cells exhibited distinct transcriptional profiles across species, reflecting lineage-specific adaptations. Notably, we identified key genes with donkey-specific expression patterns, including NR3C1 in endothelial cells, LIPE in granulosa cells, and DHRS9 in theca interna cells. Furthermore, an in vitro cumulus–oocyte complex model demonstrated the critical role of GATM in mammalian oocyte maturation. Collectively, these findings provide a comprehensive characterization of ovarian cell-type conservation and species-specific adaptations, offering key molecular insights into the mechanisms underlying cross-species differences in reproductive efficiency. Full article

Background: This paper briefly reviews the most important endocrine features of primary ovarian insufficiency (POI) and shows their relevance for the diagnosis and treatment of this condition. Introduction: Endocrine disturbances in POI cause problems for both the fertility and general health status of the affected women. Both subfertility and infertility result from the depletion of growing ovarian follicles which, in its turn, is the causative factor of hypoestrogenism; this is responsible for most of the general health problems affecting women. Method: Search of literature. Results and conclusion: A combination of high-serum follicle-stimulating hormone (FSH) and low 17β-estradiol (E2) concentrations is a key feature characterizing POI and is the decisive element for POI diagnosis. However, an in-depth search for possible genetic and non-genetic causes is important for adequate counseling regarding prevention and early intervention. The treatment of general health problems, based on correcting hypoestrogenism through hormone replacement therapy (HRT), is relatively easy. On the other hand, resolving infertility is a much more difficult task, and oocyte donation is the only really efficient instrument. Fertility preservation is a suitable alternative in patients with early POI diagnosis, in whom some viable follicles are still present in the ovaries. In patients who refuse oocyte donation, intraovarian injection of autologous platelet-rich plasma and in vitro activation of dormant follicles may be considered. Other innovative treatments, such as stem cell therapies or nuclear transfer, are currently under investigation. Full article

In vitro embryo production has accelerated in the cattle industry in recent years. Because women are similar to cows, this represents an opportunity to improve women’s reproductive protocols. This review focuses on crosstalk communication during folliculogenesis for an in-depth understanding of the events involved in developing the oocyte competence necessary to generate an embryo after fertilization. This knowledge can be used to improve oocytes in in vitro maturation cultures, which would allow us to obtain oocytes of high quality and competence, resulting in successful pregnancies in both women and cows. The first part of this review covers the concepts of cellular crosstalk before puberty in the primordial, primary, and secondary follicles. The next part involves cellular crosstalk after puberty, when gonadotropin hormones act on the ovary, promoting oocyte maturation. The final part comprises a perspective on using cow models to study human ovary physiology. Full article

The CRISPR/Cas9 system has become a powerful tool for molecular design breeding in livestock such as sheep. However, the efficiency of the Cas9 system combined with zygote microinjection remains suboptimal. In this study, mature sheep oocytes were used for microinjection to assess the impact of various factors on Cas9 editing efficiency. We found that the in vitro maturation efficiency of oocytes is related to environmental factors such as air temperature, pressure, and humidity. Our results indicate that high-efficiency gene editing can be achieved when targeting the SOCS2, DYA, and TBXT, using a microinjection mixture with a concentration of 10 ng/μL Cas9 and sgRNA. By optimizing the injection capillary, we significantly reduced the oocyte invalidation rate post-microinjection to 3.1–5.3%. Furthermore, we observed that using either Cas9 protein or mRNA in the microinjection process resulted in different genotypes in the edited oocytes. Importantly, parthenogenetic activation did not appear to affect the editing efficiency. Using this high-efficiency system, we successfully generated SOCS2 or DYA gene-edited sheep, with all lambs confirmed to be genetically modified. This study presents a highly efficient method for producing gene-edited sheep, potentially enabling more precise and effective strategies for livestock breeding. Full article

Background/Objectives: Recent studies, typically using patient cerebrospinal fluid (CSF), have suggested that different autoantibodies (Aabs) acting on their respective receptors, may underlie neuropsychiatric disorders. The GluN1 (NR1) subunit of the N-methyl-D-aspartate receptor (NMDAR) has been identified as a target of anti-NMDAR Aabs in a number of central nervous system (CNS) diseases, including encephalitis and autoimmune epilepsy. However, the role or the nature of Aabs responsible for effects on neuronal excitability and synaptic plasticity is yet to be established fully. Methods: Peptide immunisation was used to generate Aabs against selected specific GluN1 extracellular sequences based on patient-derived anti-NMDAR Aabs that have been shown to bind to specific regions within the GluN1 subunit. ‘Protein A’ purification was used to obtain the total IgG, and further peptide purification was used to obtain a greater percentage of NMDAR-target specific IgG Aabs. The binding and specificity of these anti-NMDAR Aabs were determined using a range of methodologies including enzyme-linked immunosorbent assays, immunocytochemistry and immunoblotting. Functional effects were determined using different in vitro electrophysiology techniques: two-electrode voltage-clamps in Xenopus oocytes and measures of long-term potentiation (LTP) in ex vivo hippocampal brain slices using multi-electrode arrays (MEAs). Results: We show that anti-NMDAR Aabs generated from peptide immunisation had specificity for GluN1 immunisation peptides as well as target-specific binding to the native protein. Anti-NMDAR Aabs had no clear effect on isolated NMDARs in an oocyte expression system. However, peptide-purified anti-NMDAR Aabs prevented the induction of LTP at Schaffer collateral-CA1 synapses in ex vivo brain slices, consistent with causing synaptic NMDAR hypofunction at a network level. Conclusions: This work provides a solid basis to address outstanding questions regarding anti-NMDAR Aab mechanisms of action and, potentially, the development of therapies against CNS diseases. Full article

Advances in genome editing technology have made it possible to create genome-edited (GE) animals, which are useful for identifying isolated genes and producing models of human diseases within a short period of time. The production of GE animals mainly relies on the gene manipulation of pre-implantation embryos, such as fertilized eggs and two-cell embryos, which can usually be achieved by the microinjection of nucleic acids, electroporation in the presence of nucleic acids, or infection with viral vectors, such as adeno-associated viruses. In contrast, GE animals can theoretically be generated by fertilizing ovulated oocytes with GE sperm. However, there are only a few reports showing the successful production of GE animals using GE sperm. Artificial insemination (AI) is an assisted reproduction technology based on the introduction of isolated sperm into the female reproductive tract, such as the uterine horn or oviductal lumen, for the in vivo fertilization of ovulated oocytes. This approach is simpler than the in vitro fertilization-based production of offspring, as the latter always requires an egg transfer to recipient females, which is labor-intensive and time-consuming. In this review, we summarize the various methods for AI reported so far, the history of sperm-mediated gene transfer, a technology to produce genetically engineered animals through in vivo fertilization with sperm carrying exogenous DNA, and finally describe the possibility of AI-mediated creation of GE animals using GE sperm. Full article

Estrogen is a steroid hormone that plays a key role in regulating many physiological processes, such as follicle activation and development and oocyte maturation in mammals. Ca²⁺ is crucial in oogenesis, oocyte maturation, ovulation, and fertilization. However, the mechanism by which estrogen regulates Ca²⁺ during oocyte maturation in mice has not been reported. This study revealed that Ca²⁺ levels in oocytes significantly increase during the 4–12 h period in vitro. Oocytes treated with 0.1 µM estrogen and 1 µM G1, a G-protein-coupled estrogen receptor (GPER) agonist, showed significantly increased Ca²⁺ levels, while treatment with 1 µM G15, an antagonist of GPER, significantly decreased Ca²⁺ levels. Notably, estrogen regulates Ca²⁺ in oocytes through the GPER pathway and promotes the expression of the Ca²⁺-producing protein EPAC1. In addition, estrogen alleviates the inhibitory effect of the Ca²⁺ chelator BAPTA-AM during oocyte maturation by promoting Ca²⁺ production. Furthermore, estrogen can promote the expression of the mitochondrial generation-associated protein SIRT1 through the GPER pathway, alleviate mitochondrial oxidative damage caused by BAPTA-AM, and restore the mitochondrial membrane potential level. Collectively, this study demonstrates that estrogen can regulate Ca²⁺ through the GPER-EPAC1 pathway and promote the expression of SIRT1, which promotes oocyte mitochondrial function during oocyte maturation. Full article

Ex vivo follicle growth is an essential tool, enabling interrogation of folliculogenesis, ovulation, and luteinization. Though significant advancements have been made, existing follicle culture strategies can be technically challenging and laborious. In this study, we advanced the field through development of a custom agarose micromold, which enables scaffold-free follicle culture. We established an accessible and economical manufacturing method using 3D printing and silicone molding that generates biocompatible hydrogel molds without the risk of cytotoxicity from leachates. Each mold supports simultaneous culture of multiple multilayer secondary follicles in a single focal plane, allowing for constant timelapse monitoring and automated analysis. Mouse follicles cultured using this novel system exhibit significantly improved growth and ovulation outcomes with comparable survival, oocyte maturation, and hormone production profiles as established three-dimensional encapsulated in vitro follicle growth (eIVFG) systems. Additionally, follicles recapitulated aspects of in vivo ovulation physiology with respect to their architecture and spatial polarization, which has not been observed in eIVFG systems. This system offers simplicity, scalability, integration with morphokinetic analyses of follicle growth and ovulation, and compatibility with existing microphysiological platforms. This culture strategy has implications for fundamental follicle biology, fertility preservation strategies, reproductive toxicology, and contraceptive drug discovery. Full article

Ovarian follicular fluid (FF) has a direct impact on oocyte quality, playing key roles in fertilization, implantation, and early embryo development. In our recent study, we found FF thromboxane (TX) to be a novel factor inversely correlated with oocyte maturation and identified thrombin, transforming growth factor β (TGFβ), TNF-α, and follicular granulosa cells (GCs) as possible contributors to FF TX production. Therefore, this study sought to investigate the role of TGFβ3 in regulating TX generation in human ovarian follicular GCs. TGFβ3 was differentially and significantly present in the FF of large and small follicles obtained from IVF patients with average concentrations of 68.58 ± 12.38 and 112.55 ± 14.82 pg/mL, respectively, and its levels were correlated with oocyte maturity. In an in vitro study, TGFβ3 induced TX generation/secretion and the converting enzyme-COX-2 protein/mRNA expression both in human HO23 and primary cultured ovarian follicular GCs. While TGFβRI and Smad2/3 signaling was mainly required for COX-2 induction, ERK1/2 appeared to regulate TX secretion. The participation of Smad2/3 and COX-2 in TGFβ3-induced TX generation/secretion could be further supported by the observations that Smad2/3 phosphorylation and nuclear translocation and siRNA knockdown of COX-2 expression compromised TX secretion in GCs challenged with TGFβ3. Taken together, the results presented here first demonstrated that FF TGFβ3 levels differ significantly in IVF patients’ large preovulatory and small mid-antral follicles and are positively associated with oocyte maturation. TGFβ3 can provoke TX generation by induction of COX-2 mRNA/protein via a TGFβR-related canonical Smad2/3 signaling pathway, and TX secretion possibly by ERK1/2. These imply that TGFβ3 is one of the inducers for yielding FF TX in vivo, which may play a role in folliculogenesis and oocyte maturation. Full article

BRCA1 and BRCA2 mutations are responsible for a higher incidence of breast and ovarian cancer (from 55% up to 70% vs. 12% in the general population). If their functions have been widely investigated in the onset of these malignancies, still little is known about their role in fertility impairment. Cancer patients treated with antineoplastic drugs can be susceptible to their gonadotoxicity and, in women, some of them can induce apoptotic program in premature ovarian follicles, progressive depletion of ovarian reserve and, consequently, cancer treatment-related infertility (CTRI). BRCA variants seem to be associated with early infertility, thus accelerating treatment impairment of ovaries and making women face the concrete possibility of an early pregnancy. In this regard, fertility preservation (FP) procedures should be discussed in oncofertility counseling—from the first line of prevention with risk-reducing salpingo-oophorectomy (RRSO) to the new experimental ovarian stem cells (OSCs) model as a new way to obtain in vitro-differentiated oocytes, several techniques may represent a valid option to BRCA-mutated patients. In this review, we revisit knowledge about BRCA involvement in lower fertility, pregnancy feasibility, and the fertility preservation (FP) options available. Full article

Infertility is considered a global health issue as it currently affects one in every six couples, with female factors reckoned to contribute to partly or solely 50% of all infertility cases. Over a thousand genes are predicted to be highly expressed in the female reproductive system and around 150 genes in the ovary. However, some of their functions in fertility remain to be elucidated. In this study, 13 ovary and/or oocyte-enriched genes (Ccdc58, D930020B18Rik, Elobl, Fbxw15, Oas1h, Nlrp2, Pramel34, Pramel47, Pkd1l2, Sting1, Tspan4, Tubal3, Zar1l) were individually knocked out by the CRISPR/Cas9 system. Mating tests showed that these 13 mutant mouse lines were capable of producing offspring. In addition, we observed the histology section of ovaries and performed in vitro fertilization in five mutant mouse lines. We found no significant anomalies in terms of ovarian development and fertilization ability. In this study, 13 different mutant mouse lines generated by CRISPR/Cas9 genome editing technology revealed that these 13 genes are individually not essential for female fertility in mice. Full article

Information on long-term effects of postovulatory oocyte aging (POA) on offspring is limited. Whether POA affects offspring by causing oxidative stress (OS) and mitochondrial damage is unknown. Here, in vivo-aged (IVA) mouse oocytes were collected 9 h after ovulation, while in vitro-aged (ITA) oocytes were obtained by culturing freshly ovulated oocytes for 9 h in media with low, moderate, or high antioxidant potential. Oocytes were fertilized in vitro and blastocysts transferred to produce F1 offspring. F1 mice were mated with naturally bred mice to generate F2 offspring. Both IVA and the ITA groups in low antioxidant medium showed significantly increased anxiety-like behavior and impaired spatial and fear learning/memory and hippocampal expression of anxiolytic and learning/memory-beneficial genes in both male and female F1 offspring. Furthermore, the aging in both groups increased OS and impaired mitochondrial function in oocytes, blastocysts, and hippocampus of F1 offspring; however, it did not affect the behavior of F2 offspring. It is concluded that POA caused OS and damaged mitochondria in aged oocytes, leading to defects in anxiety-like behavior and learning/memory of F1 offspring. Thus, POA is a crucial factor that causes psychological problems in offspring, and antioxidant measures may be taken to ameliorate the detrimental effects of POA on offspring. Full article