Purpose:Ovulation is a highly regulated inflammatory process that involves the initiation and timely resolution of the inflammation for efficient oocyte maturation. During this process, glucocorticoids accumulate in the pre-ovulatory follicular environment as an anti-inflammatory mediator. However, their temporal regulation, endogenous synthesis, and functional significance on oocyte maturation and subsequent developmental competence remain unclear. Methods:Corticosterone levels in reproductive tract fluid and serum, alongside the expression and localization of related markers, including glucocorticoid receptors (NR3C1), were analyzed in granulosa cells (GC) and cumulus cells (CC) during gonadotropin stimulation. The functional relevance of corticosterone was evaluated by inhibiting endogenous corticosterone synthesis using metyrapone (MET) and by supplementing exogenously during in vitro maturation (IVM) conditions. Results:Human chorionic gonadotropin (hCG) increased follicular corticosterone at 8 h and upregulated steroidogenic markers (Cyp11a1, Cyp11b1, and Cyp21a1) while downregulating the inactivating enzyme Hsd11b2. MET significantly inhibited FSH-mediated endogenous corticosterone synthesis, resulting in poor IVM outcomes, and supplementation of exogenous corticosterone enhanced the IVM outcome, mitigated the MET effect, and maintained inflammatory balance. Conclusions:Gonadotropin induces a transient surge of ovarian corticosterone that supports in vivo maturation. Supplementing physiological levels of corticosterone during in vitro maturation (IVM) promotes oocyte maturation probably through cumulus cells.

Seminal plasma, traditionally regarded as a passive vehicle for sperm transport, is now recognized as a biologically active fluid that is important for normal sperm function and optimal male fertility. This review summarizes how androgen-regulated secretions from the seminal vesicles, prostate, and epididymis influence sperm competence at ejaculation. Key components include seminal vesicle-derived lipids (e.g., oleic acid), prostatic citrate, carbohydrates, ions, and antioxidants that rapidly remodel membranes, activate motility, and preserve genome integrity. Androgen signaling via testosterone and dihydrotestosterone coordinates metabolic programs such as oleic-acid synthesis in the seminal vesicles and citrate secretion in the prostate. Seminal-vesicle fatty acids fuel mitochondrial respiration and support linear motility, whereas prostatic citrate primarily buffers pH and chelates divalent cations that stabilize membranes and acrosomal status. In addition, seminal plasma supplies precursors for cysteine and glutathione biosynthesis, supporting redox homeostasis in sperm. Beyond physiology, timed supplementation with whole seminal plasma or defined components improves motility and fertilization outcomes in livestock and humans. By focusing on how androgen-regulated seminal plasma synthesis influences sperm function, this review outlines mechanisms that are relevant to both male fertility biology and reproductive technologies.

Various cells are localized to appropriate positions in tissues and induced a unique gene expression and functions. In this study, we show for the first time that age-related changes in ovarian hardness and elasticity are linked to abnormal signal transduction affects in secondary follicles. Immature ovaries had lower hardness and elasticity indices, whereas aged ovaries had lower elasticity and higher hardness indices than mature ovaries. Importantly, an optimal hardness and elasticity is important for normal gene expression profile of secondary follicles. We reproduced the ovarian hardness and elasticity using alginate beads with different concentration and viscosities, for three-dimensional culture of secondary follicles. In the aged-ovarian hardness group, follicular diameter decreased significantly and the expression of inflammation-related markers increased significantly. In the immature-ovarian hardness group, follicular diameter increased significantly and the expression of differentiation and proliferation markers increased significantly. The mechanism transmitted the hardness and elasticity to the change of gene expression was that YAP, transmit the physical conditions to nucleus via actin network, was significantly increased in both condition of immature or aged ovary. The abnormal signaling associated with the age-related changes in ovarian hardness and elasticity may aid in the development of treatments for abnormal follicular development in infertility.

BACKGROUND:Mammalian sperm need to undergo hyperactivation, capacitation, and acrosome reaction to achieve fertilization competence. However, the female reproductive tract presents contrasting metabolic environments. The uterus is glucose-rich, and the oviduct is lactate-rich and glucose-limited. Furthermore, our previous study reported that lactate plays a regulatory role in glucose metabolism through suppressing glucose uptake and glycolysis. How bull sperm maintain glycolysis-associated functions when exogenous glucose is limited remains unclear. OBJECTIVES:To characterize lactate-responsive changes in sperm glycogen content and determine whether CP91149-sensitive processes are associated with glycolysis-related sperm functions and cleavage after in vitro fertilization (IVF) under glucose-limited conditions. MATERIALS AND METHODS:Fresh bull ejaculates were incubated in substrate-free HTF medium to deplete the stored energy. The energy-depleted sperm were treated with lactate, followed by lactate withdrawal and subsequent incubation with glycogen phosphorylase inhibitor (CP91149) in the presence or absence of glucose. Sperm motility, kinematics, acrosome integrity, metabolic flux, glycogen level, and cleavage were evaluated in vitro. RESULTS:Lactate pretreatment prolonged sperm motility under substrate depletion. Lactate withdrawal increased a hyperactivated-like motility subpopulation. Flow cytometry showed that the lactate withdrawal increased membrane permeability of sperm and elevated the percentage of acrosome-reacting sperm. Glycolytic activity of sperm was increased after withdrawal, but the induction was suppressed by the glycogen phosphorylase inhibitor CP91149. Lactate increased sperm glycogen content, which declined after withdrawal. Immunoblotting and immunofluorescence detected proteins associated with gluconeogenesis and glycogen metabolism in bull sperm. CP91149 reduced motility after lactate withdrawal in a dose-dependent manner, and glucose supplementation restored motility. Under glucose-free IVF conditions, CP91149 reduced the post-IVF functional outcome, which was restored by glucose supplementation. DISCUSSION AND CONCLUSION:These findings show lactate-responsive changes in sperm glycogen content and a CP91149-sensitive phenotype affecting glycolytic activity, motility, and the post-IVF functional outcome. The data are consistent with a contribution of endogenous glycogen-derived substrates to sperm function under glucose-limited conditions.

Background:Toll-like receptors (TLRs) are critical components of the innate immune system and are expressed in various cells, including the reproductive system. Although their roles in female reproductive tissues such as the ovaries and uterus, including their involvement in fertilization and implantation, have been extensively reviewed, their expression and function in male germ cells, particularly in sperm, remain underexplored. Methods:This review provides a comprehensive summary of research on TLRs expressed in sperm, including findings from experimental models in mice, humans, and industrial livestock. Results:The activation of TLR2 and TLR4, which detect Gram-positive and Gram-negative bacteria, has been shown to reduce sperm motility and viability, thereby impairing fertilization. Conversely, low levels of TLR2 activation have been reported to promote the fertilization of bull sperm, suggesting that TLR2/4 may act as regulators of fertilization. TLR7 and TLR8, which are exclusively expressed in X chromosome-bearing sperm (X-sperm), have attracted increasing research interest. These receptors modulate sperm metabolism, selectively reduce the motility of X sperm, and enable the separation of X and Y sperm. Conclusion:TLRs in the sperm serve as immune receptors that detect bacterial and viral infections, thereby reducing sperm functionality, preventing miscarriage, protecting maternal health, and sex selection.

BackgroundEnergy metabolism and substrate balance are critical determinants of sperm motility and fertility. Linear motility is necessary for sperm forward movement, whereas hyperactivated motility is a prerequisite for fertilization. The preference of metabolic pathways depends on substrate availability which controls sperm motility. However, there are differences in substrate composition in seminal plasma, vagina, uterus and oviducts.ObjectivesThis study aims to clarify how the spermatozoa maintains its metabolic homeostasis and functions in the presence of glucose and lactate either alone or in combinations.Materials and MethodsFresh bull spermatozoa was incubated with modified human tubal fluid (mHTF) medium containing either no-energy, glucose, lactate, or a combination of glucose and lactate. Sperm motility, kinematics, adenosine triphosphate (ATP) production, mitochondrial membrane potential, glucose incorporation, glycolysis, oxidative phosphorylation (OXPHOS), and acrosome reaction were systematically assessed.ResultsGlucose resulted in zigzag motility and lactate induced linear motility. Glucose-derived zigzag motility was suppressed by lactate with increasing lactate concentration in a dose-dependent manner. The addition of lactate with glucose showed higher mitochondrial membrane potential, higher oxygen consumption rate (OCR), and lower extracellular acidification rate (ECAR). Lactate suppressed glucose incorporation in midpiece and tail regions, reduced glycolysis, and shifted sperm metabolism toward OXPHOS which resulted in linear motility and maintained acrosome integrity.DiscussionLactate played a metabolic and regulatory role in bull sperm metabolism. As a metabolic role, it oxidized through OXPHOS and maintained linear motility. The metabolic changes by lactate suppressed glucose-induced acrosome reactions and maintained linear motility, which might be beneficial for sperm transportation toward the fertilization site of the female reproductive tract and results in successful fertilization.ConclusionThis is a novel finding that explores the regulatory role of lactate over glucose metabolism in bull sperm functionality. Optimum balancing of glucose and lactate maintains the motility and functionality of bull spermatozoa. These outcomes might have substantial implications for the enhancement of sperm preservation techniques, sperm handling, and fertility outcomes.

Male factors account for almost half of the causes of infertility. In rodents and humans, most of the components of semen are supplied by the seminal vesicles, and they support male reproductive ability, but there are many unknown details. This study focused on metabolic changes in seminal vesicle epithelial cells and investigated how testosterone affects seminal plasma composition. A factor improving the linear motility of sperm was secreted from the seminal vesicles, and it was produced in an androgen-dependent manner. Bioassays, gene expression, and flux analysis studies demonstrated that testosterone promotes glucose uptake in seminal vesicle epithelial cells via GLUT4, resulting in fatty acid synthesis. ACLY was a critical factor in this metabolic change, which produced fatty acids, especially oleic acid. In conclusion, the critical role of testosterone-induced metabolic changes in the seminal vesicles is to ensure the synthesis of fatty acids. These findings suggest that testosterone-dependent lipid remodeling may contribute to sperm straight-line motility, and further functional verification is required.

Purpose:We demonstrated that briefly treating ovaries with collagenase before exposure to a hyperosmotic solution preserved secondary follicles. However, preserving antral follicles remained difficult. We investigated whether the absence of lipids in warming (thawing) solution is a limiting factor. Methods:Frozen ovary was thawed in a lipid-containing solution and investigated for the effect on the structure, function, and viability of granulosa cells, which are important for antral follicle function. Results:Lipids incorporated into the granulosa cell membrane protected the distribution of actin structures lining the cell membrane and maintained the expression of paxillin, actin-related adapter proteins, and YAP, which induces genes critical for granulosa cell function, to support the survivability of granulosa cells in antral follicles. It also preserved the function of frozen ovaries for 3 months after transplantation into the ovarian bursa of recipient hosts, significantly increasing the total number of viable offspring. Conclusions:Lipids-key components of serum-protected the granulosa cell membrane during warming by preventing the efflux of fatty acids for maintenance of proper localization of the actin cytoskeleton and membrane proteins, supporting normal antral follicle function. The novel method combining collagenase pretreatment with a lipid-containing warming solution offers a promising approach to maintaining the function of cryopreserved ovaries.

Sperm capacitation, a prerequisite for fertilization, is regulated not only by intrinsic signaling but also by paracrine factors within the female tract. Analysis of previously published RNA-seq datasets identified the ectodysplasin-A2 receptor (EDA2R), an X-linked member of the TNF-receptor superfamily, as a candidate regulator of this process. This study was conducted to test the hypothesis that the EDA-A2/EDA2R axis is a regulator that directly regulates sperm capacitation during fertilization process. Western blotting and immunofluorescence showed that EDA2R was localized in late spermatogenic cells and in the midpiece of epididymal sperm. Incubation of mouse sperm in HTF medium containing the corresponding ligand EDA-A2 (0–1 µg/mL) resulted in a dose-dependent improvement in the amplitude of lateral head displacement and curvilinear velocities. Ligand exposure promoted the appearance of capacitation hallmarks: tyrosine phosphorylation level was elevated within 30 min and the proportion of FITC-PNA positive, acrosome-reacted cells increased at 30 and 60 min (p < 0.05). The EDA-A2 treated sperm yielded a higher cleavage rate (78.5

Improving the quality and developmental competence of in vitro-produced (IVP) embryos is crucial for assisted reproductive technologies. Recently, we demonstrated that TLR2, an innate immune receptor, is expressed in bovine sperm and their activation improves fertilization and subsequent embryo development. However, its role in early embryo development is poorly understood. This study investigated the impact of activation of early embryonic TLR2 on IVP bovine embryo development. The immunofluorescence and western blot analyses confirmed robust TLR2 expression in early embryonic stages. Embryos were treated at 36 h post-insemination (hpi), with 100 ng/ml of TLR2 agonist and cultured in vitro and showed increased blastocyst rates and faster growth speed. Additionally, TLR2 activation slightly increased basal calcium levels and induced autophagy while suppressing cathepsin B activity and DNA damage, leading to reduced apoptosis in embryos. Together, these findings indicate that embryo TLR2 is involved in embryo development competence via a slight increase in the basal cytosolic calcium and subsequent embryo metabolic activities, autophagy induction, and reduction of apoptosis levels. These results provide a promising approach for producing highly competent good-quality embryos for improving the efficiency of IVP bovine embryos.

Purpose:Glucose plays a critical role in early embryonic development, influencing metabolic dynamics and developmental competence in a sex-specific manner. This study investigates the complex interplay between glucose availability, developmental competence, and sex-specific outcomes in preimplantation mouse embryos. Methods:Mouse embryos were cultured in a modified KSOM medium with varying glucose concentrations (0-20 mM), monitored via time-lapse microscopy, and analyzed for developmental competence, sex determination by PCR, and X-linked metabolic gene expression. Stage-specific glucose addition/removal experiments and PDHA1 immunofluorescence staining were performed to assess temporal glucose dependency and sex-specific metabolic patterns. Results:Glucose is essential during the morula-to-blastocyst transition. Analysis of developmental dynamics showed that glucose concentration affected the variability in developmental rates, particularly at the four-cell and eight-cell stages. Interestingly, sex ratio skewing was observed, with male embryos dominating the early developmental groups regardless of glucose levels. Expression analysis of X-linked metabolic genes revealed stage-specific patterns, with PDHA1 exhibiting the highest activity at the eight-cell stage. Conclusions:Glucose availability accelerated embryonic development and created sex-specific patterns of developmental timing, with male embryos exhibiting faster progression rates, which might be associated with differential X-linked PDHA1 metabolic gene expression during early mouse embryogenesis.

Background:Platelet derivatives improve the uterine immune environment and increase pregnancy success in humans and animals. Platelet-conditioned media (PCM) contain all molecules derived from platelets in vitro (platelet secretions). The present study aimed to investigate the immunological impacts of platelet secretions on polymorphonuclear neutrophils (PMNs) and bovine endometrial epithelial cells (BEECs), in vitro. Methods:Platelets (10×107 platelets/mL) from Holstein dairy cows were incubated for 0.5 h or lysed to obtain the PCM and platelet lysate (Lysate), respectively. PMNs were stimulated with PCM for 3h. While BEECs were exposed to PCM or Lysate for 24 h. Real-time PCR was performed to detect the expression of targeted genes (cytokines), including TNFA, IL1B and PGES1. Lipoxin A4 (LXA4; anti-inflammatory mediator) and PGE2 concentrations in the supernatants of PMNs cultured with PCM were measured via ELISA. Cell proliferation in BEECs was assessed using the Cell Counting Kit-8 (CCK-8). Additionally, uterine explants were prepared and processed for immunofluorescence to determine the expression of the LXA4 receptor. Results:In PMNs, platelet secretions downregulated the mRNA expression of pro-inflammatory cytokines (TNFA and IL1B) and increased LXA4 production. In both PMNs and BEECs, platelet secretions upregulated PGES1 expression and PGE2 production. In BEECs, platelet secretions and Lysate upregulated TGFB1. While Lysate suppressed IL1B mRNA expression. Further, platelet secretions showed an anti-proliferative effect in BEECs and increased the LXA4 receptor protein expression in the endometrial epithelia. Conclusions:Our findings reveal for the first time that platelet secretions directly act on PMNs and BEECs in vitro, thereby assisting the uterine immune network to shift anti-inflammatory environment toward pregnancy. The present study can explain, in part, the successful applications of platelet derivatives in reproductive medicine.

Succinylation is a recently identified post-translational modification (PTM) that modulates enzyme activity and metabolism. However, how it regulates metabolic reprogramming in sperm remains unclear. In this study, we show that succinylation of pyruvate kinase M2 (PKM2) promotes oxidative phosphorylation (OXPHOS) and linear motility in boar sperm. Under low glucose (LG) extender, sperm linear motility was significantly enhanced, mitochondrial activity increased, glycolysis was inhibited, and the pentose phosphate pathway (PPP) was promoted. PKM2 interacted with voltage-dependent anion channel 3 (VDAC3), increasing mitochondrial permeability. Supplementing high glucose (HG) extender with succinic acid (SA) enhanced these effects. In contrast, the addition of resveratrol (RES) to LG extender, significantly reduced progressive motility (PM), decreased mitochondrial activity, and promoted the glycolysis pathway. Additionally, LG extender facilitated PKM2 succinylation and mitochondrial translocation. These findings demonstrate that PKM2 succinylation reprograms glucose metabolism from glycolysis to PPP, enhancing ATP production and supporting sustained linear motility, providing insights into optimizing sperm preservation.

Purpose: LH induces the expression of EGF-like factors and their shedding enzyme (ADAM17) in granulosa cells (GCs), which is essential for ovulation via activation of the ErbB-ERK1/2 pathway in cumulus cells (CCs). Neurotensin (NTS) is reported as a novel regulator of ovulation, whereas the NTS-induced maturation mechanism in oocytes remains unclear. In this study, we focused on the role of NTS in the expression of EGF-like factors and ErbBs, and ADAM17 activity, during oocyte maturation and ovulation in mice. Methods: The expression and localization in GC and CC were examined. Next, hCG and NTS receptor 1 antagonist (SR) were injected into eCG-primed mice, and the effects of SR on ERK1/2 phosphorylation were investigated. Finally, we explored the effects of SR on the expression of EGF-like factors and ErbBs, and ADAM17 activity in GC and CC. Results: NTS was significantly upregulated in GC and CC following hCG injection. SR injection suppressed oocyte maturation and ERK1/2 phosphorylation. SR also downregulated part of the expression of EGF-like factors and their receptors, and ADAM17 activity. Conclusions: NTS induces oocyte maturation through the sustainable activation of the ERK1/2 signaling pathway by upregulating part of the EGF-like factor-induced pathway during oocyte maturation in mice.

LH induces the expression of EGF-like factors and their shedding enzyme (ADAM17) in granulosa cells (GCs), which is essential for ovulation via activation of the ErbB–ERK1/2 pathway in cumulus cells (CCs). Neurotensin (NTS) is reported as a novel regulator of ovulation, whereas the NTS-induced maturation mechanism in oocytes remains unclear. In this study, we focused on the role of NTS in the expression of EGF-like factors and ErbBs, and ADAM17 activity, during oocyte maturation and ovulation in mice. The expression and localization in GC and CC were examined. Next, hCG and NTS receptor 1 antagonist (SR) were injected into eCG-primed mice, and the effects of SR on ERK1/2 phosphorylation were investigated. Finally, we explored the effects of SR on the expression of EGF-like factors and ErbBs, and ADAM17 activity in GC and CC. NTS was significantly upregulated in GC and CC following hCG injection. SR injection suppressed oocyte maturation and ERK1/2 phosphorylation. SR also downregulated part of the expression of EGF-like factors and their receptors, and ADAM17 activity. NTS induces oocyte maturation through the sustainable activation of the ERK1/2 signaling pathway by upregulating part of the EGF-like factor-induced pathway during oocyte maturation in mice.

Sperm cells are highly susceptible to oxidative stress, which decreases their motility and fertility. However, glutathione (GSH) plays a critical role in protecting sperm cells from oxidative damage, a common byproduct of mitochondrial oxidative phosphorylation. On the other hand, GSH biosynthesis in sperm is limited by the availability of cysteine (Cys), which is inherently unstable and found at low concentrations in boar seminal plasma. In somatic cells, Cys can be produced through the transsulfuration pathway, catalyzed by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). In this study, we report that a group of enzymes involved in GSH synthesis is present in boar sperm. Notably, CBS and CTH protein levels increase during incubation, suggesting active regulation of their synthesis. This increase is inhibited by cycloheximide (CHX), indicating that ongoing protein synthesis is necessary for maintaining these levels. Our study also identified the presence of translation factors, such as eukaryotic initiation factor 4E (eIF4E), and their activation through phosphorylation of the ERK1/2-RSK-eIF4E pathway during incubation. Additionally, we found that CBS mRNA transcripts with short poly(A) tails are present in boar sperm, and polyadenylation of these short-tailed mRNAs occurs during incubation to enhance their translation. The use of cordycepin, a polyadenylation inhibitor, significantly reduced the translation of CBS, leading to decreased GSH synthesis and impaired sperm motility. However, the addition of cysteine counteracted the inhibitory effects of cordycepin, underscoring the essential role of cysteine in maintaining GSH levels. These findings provide new insights into the post-transcriptional regulation of GSH synthesis in sperm and suggest potential strategies for enhancing sperm preservation and fertility by targeting polyadenylation and translation mechanisms.

Oxidative stress, caused by both endogenous and exogenous factors, affects sperm function by damaging morphology and reducing metabolic activity, leading to reduced fertilization ability. The purpose of this study was to investigate the effects of oxidative stress on bull sperm and to evaluate the efficacy of targeted antioxidants in mitigating these detrimental effects. Fresh bull semen samples were subjected to hydrogen peroxide (H2O2) and antimycin treatments to induce oxidative stress, and the antioxidants PQQ, ergothioneine, and vitamin C were applied to counteract the induced stress. Sperm motility, viability, and reactive oxygen species (ROS) levels in the cytoplasm and mitochondria of sperm were assessed using computer-assisted sperm analysis (CASA) and flow cytometry. The treatment with H2O2 rapidly decreased sperm viability, and antimycin-induced mitochondrial ROS mainly decreased sperm motility; PQQ and vitamin C effectively reduced mitochondrial ROS, while ergothioneine and vitamin C reduced cytosolic ROS. In frozen-thawed sperm, oxidative stress was elevated in both cytoplasm and mitochondria, and all three antioxidants improved sperm motility by inhibiting ROS production. Furthermore, the localization of oxidized lipids (4-hydroxynonenal) in sperm was detected using immunofluorescence, indicating that oxidative stress affects the head and midpiece of sperm. These findings highlight the potential of targeted antioxidants to mitigate the detrimental effects of oxidative stress on bull sperm and provide valuable insights to improve semen quality and optimize the use of antioxidants in artificial insemination.

Ovarian fibrosis contributes to age-related ovarian dysfunction. In our previous study, we observed ovarian fibrosis in both obese and aging mice with intracellular lipid droplets in the fibrotic ovaries. Although the importance of mitochondria in ovarian fibrosis has been recognized in pharmacological studies, their role in lipid metabolism remains unclear. Globin peptide (GP), derived from hemoglobin, enhances lipid metabolism in obese mice. This study aimed to elucidate the importance of lipid metabolism in ovarian fibrosis by using GP. Treatment of ovarian stromal cells with GP increased mitochondrial oxygen consumption during β-oxidation. Lipid accumulation was also observed in the ovaries of granulosa cell-specific Nrg1 knockout mice (gcNrg1KO), and the administration of GP to gcNrg1KO mice for two months reduced ovarian lipid accumulation and fibrosis in addition to restoring the estrous cycle. GP holds promise for mitigating lipid-related ovarian issues and provides a novel approach to safeguarding ovarian health by regulating fibrosis via lipid pathways.