Male infertility has been increasing globally, raising concerns for reproductive health. Ornidazole (ORN) emerges as a novel environmental pollutant and compromises male fertility. However, the protective role and underlying mechanisms of docosahexaenoic acid (DHA) against ORN-induced testicular damage remain unexplored. Our clinical data showed that elevated serum ORN levels were negatively correlated with sperm quality. In vivo, ORN exposure led to impaired spermatogenesis, including meiotic disorders. Specifically, ORN impaired redox balance and reduced the expression of mitochondrial respiratory chain proteins (Ndufs1 and SdhB) in spermatocytes. Supplementation with docosahexaenoic acid significantly restored the quantity of DDX4-positive germ cells and SYCP3-positive spermatocytes and facilitated the progression from zygotene to pachytene stage. Mechanistically, DHA restored mitochondrial function and ROS levels by stimulating peroxisome proliferator-activated receptor gamma (PPARγ) signaling. Moreover, DHA reduced the expression of mitochondria-associated endoplasmic reticulum membranes (MAMs)-tethered voltage-dependent anion channel 1 (VDAC1), restoring MAMs balance and mitochondrial calcium homeostasis in a PPARγ-dependent manner. The DHA/PPARγ/VDAC1 axis in spermatocytes functions as a critical metabolic switch for regulating MAMs and ensuring mitochondrial homeostasis during meiosis. DHA is a promising therapeutic metabolite for oligoasthenozoospermia induced by environmental pollution.

Female reproductive aging is the most critical determinant of decreased fertility because the quality of oocytes gradually deteriorates with age, leading to poor embryo development, increased miscarriage rates, and reduced live births. Oocyte growth and maturation depend on bidirectional communication with surrounding granulosa cells through gap junctions, which are composed mainly of connexin 43 (CX43). Although intercellular communication mediated by CX43 is essential for folliculogenesis, how it becomes altered in the context of ovarian aging and drives oocyte deterioration remains largely unclear. This study aimed to clarify age-related alterations in gap junction communication and investigate whether augmenting CX43-mediated coupling via the peptide ZP123 might restore oocyte competence and enhance fertility outcomes in older females. Our data demonstrated that CX43 expression in mouse ovaries and granulosa cells markedly decreased in an age-dependent manner, and this reduction was evident at both the protein and mRNA levels. The level of phosphorylated CX43 at Ser373 decreased markedly, indicating that total CX43 loss was the primary contributor to impaired gap junction communication. In vitro, our study revealed that ZP123 treatment significantly enhanced dye transfer between KGN cells but did not increase CX43 expression, suggesting an improvement in gap junction intercellular communication without increasing CX43 protein abundance. In vivo, chronic ZP123 administration to aged mice restored the normal gap junction between granulosa cells, improved follicle development. Functionally, ZP123 enhanced oocyte maturation (GVBD and MII rates), corrected spindle abnormalities, improved mitochondrial distribution, and increased ATP production while reducing ROS accumulation. Moreover, the mitochondrial membrane potential and Ca²⁺ homeostasis were restored, and excessive opening of the mitochondrial permeability transition pore was prevented. Consistent with these cellular improvements, ZP123-treated aged mice presented increased ovulation numbers and enhanced preimplantation embryo development, including increased blastocyst formation rates. This study revealed that disrupted CX43-mediated communication is a key mechanism underlying the decrease in oocyte quality during ovarian aging. Pharmacological enhancement of gap junctions with ZP123 effectively restored ovarian function, oocyte competence, and developmental potential, highlighting gap junction modulation as a novel and promising therapeutic avenue to mitigate age-related fertility decline in women of advanced maternal age.

Granulosa cells play a pivotal role in follicle initiation and development. Metabolic disorders can inflict damage on granulosa cells, ultimately leading to abnormal ovarian function. It is well established that a high dietary intake of fructose can induce a range of metabolic diseases, such as insulin resistance and non-alcoholic fatty liver disease. However, the underlying mechanism by which fructose affects ovarian function remains unclear. In this study, we subjected wild-type mice to a 30% fructose solution in their drinking water for 16 weeks to establish high-fructose animal models. The results obtained showed that in mice exposed to high fructose, hormone levels and estrous cycles were abnormal, and the number of atretic follicles increased. Simultaneously, senescence and apoptosis of granulosa cells were also observed in high-fructose mice, and the level of cellular protein O-GlcNAcylation significantly increased. Treatment of granulosa cells with the O-GlcNAcylation activator Thiamet G and inhibitor OSMI-1 demonstrated that elevated O-GlcNAcylation induces granulosa cell senescence and promotes apoptosis. We utilized immunoprecipitation-mass spectrometry to investigate O-GlcNAcylated proteins in the ovaries of high-fructose mice and the results indicated that numerous proteins had significantly elevated O-GlcNAcylation levels, mainly centered on the chromatin "remodeling" and "nucleocytoplasmic transport" pathways. Among them, increases in the O-GlcNAcylation levels of nucleoporin 54 and glucose-regulated protein 78 were confirmed by co-immunopreciptitation. These results uncover a new mechanism of fructose-induced ovarian function impairment, providing potential targets for the treatment of diet-related fertility disorders.

Obesity has become an important factor affecting male fertility and is often accompanied by abnormal levels of fatty acids, especially saturated fatty acids (SFAs). Palmitic acid (PA; C16:0) is the most frequent SFA in high-fat diet (HFD) and impairs blood-testis barrier (BTB) integrity and decreases sperm quality. RNA N6-methyladenosine (m6A) is involved in lipid metabolism and affects BTB integrity and spermatogenesis. However, it is unclear whether RNA m6A modification plays a role in the BTB damage caused by PA. Herein, we unraveled that PA induced the upregulation of ALKBH5 in TM4 and primary Sertoli cells and this upregulation of ALKBH5 was involved in PA-induced BTB disruption. Conversely, suppressing Alkbh5 expression could alleviate the BTB disruption induced by PA. Moreover, we found Srgn might be a potential downstream target of Alkbh5-mediated BTB integrity suppression. Mechanistically, the Alkbh5 upregulation caused by PA disrupted BTB integrity via enhancing Srgn stability in a RNA m6A dependent manner, and Srgn impaired the integrity of the BTB by activating the NF-κB and MAPK pathways and thereby elevating the expression of Ccl2 and TNF-α in TM4 and primary Sertoli cells. Our study revealed an important role of the regulatory network of ALKBH5-m6A-Srgn in the PA-induced BTB disruption and provided potential therapeutic strategies for improving fertility potential in obese patients.

Although dietary factors are increasingly implicated as crucial determinants of male fertility, specific dietary risk factors and their metabolic mechanisms remain poorly understood. In this study, patients with non-obstructive azoospermia (NOA) or severe oligospermia (EO) demonstrated significantly elevated erucic acid (EA) levels, with a nearly 3-fold increase in serum (P < 0.0001) and a 27 % increase in semen (P = 0.0147) compared to controls, suggesting a potential dose-dependent negative impact of EA exposure on spermatogenesis. Mice fed a high-EA diet exhibited a significant 52.5 % reduction in sperm concentration compared with the control group, indicating impaired reproductive function (P < 0.0001). Mechanistic experiments in testicular organoid (TO) models have demonstrated that high-dose erucic acid (EA) directly interferes with meiotic progression. Transcriptomic analysis revealed significant disruptions in retinol metabolism pathways, which are essential for regulating spermatogenesis. The key gene involved in retinoic acid (RA) biosynthesis, retinol dehydrogenase (Rdh10), showed an approximate 38.5 % decrease in expression (P = 0.0467). Liquid chromatography-mass spectrometry (LC-MS) further confirmed that elevated EA levels significantly suppressed RA synthesis in Sertoli cells (P = 0.0078). The underlying molecular mechanism involves the binding of EA to the GPR120 receptor, which subsequently downregulates the expression of Rdh10. Co-culture experiments with Sertoli cells and spermatogonial stem cells complemented by TO assays demonstrated that RA supplementation may be a promising therapeutic strategy to reverse EA-induced meiotic and spermatogenic impairment. These findings provide novel insights into how prolonged dietary EA exposure adversely affects male reproductive health by disrupting retinol metabolism and RA signalling pathways and offer evidence for defining dietary EA safety thresholds and informing policies to safeguard male fertility.

Oocyte aging is closely related to a decline in female fertility, accompanied by increased reactive oxygen species levels and changes in protein posttranslational modifications. However, the role of protein palmitoylation in oocyte aging has not been investigated. In the present study, a new association between redox and palmitoylation in aging oocytes was found. We found that the protein level of palmitoyl-protein thioesterase 1 (PPT1), a depalmitoylation enzyme, was increased in maternally aged mice oocytes and follicular fluid of aged (age >35 years) patients with decreased ovarian reserve (DOR). Elevated PPT1 led to decreased S-palmitoylation levels in oocytes, which impaired oocyte maturation and spindle formation. Tubulin was identified as a critical palmitoylated protein regulated by PPT1, whose palmitoylation was also decreased by advanced age, accompanied by abnormalities in membrane localization and microtubule polymerization. Melatonin was found to down-regulate excessive PPT1 and rescue PPT1-induced damage in mouse oocytes, not only by regulating oxidative stress, but also by binding with PPT1 to regulate its lysosomal degradation. In summary, our data demonstrate that PPT1 participates in oocyte aging by regulating tubulin palmitoylation, providing evidence that oxidative stress regulates protein palmitoylation and revealing a novel mechanism of oocyte aging.

PurposeWe aimed to demonstrate the genetic factors of primary gonadal dysgenesis in a consanguineous family characterized by underdeveloped testes and non-obstructive azoospermia (NOA) in a male and primary amenorrhoea and primary ovarian insufficiency (POI) in a female.MethodsDNA was extracted from the male proband with infertility from the consanguineous family for whole-exome sequencing and bioinformatics analysis to screen for potential pathogenic genes and mutations. Sanger sequencing was used for further validation of his family pedigree. The effects of the identified novel mutation were evaluated in the male testes tissue by immunohistochemistry and in HEK293T cells by Western blot.ResultsA homozygous frameshift mutation c.998delG (p. Gly333Glufs*50) in MCM8 was identified in the two siblings. The testes tissue sections in the male showed a Sertoli cell-only syndrome (SCOS). Functional analysis in vitro suggested that the mutation results in a truncated protein of MCM8 in HEK293T cells, and immunohistochemistry in vivo showed decreased expression of MCM8 protein.ConclusionWe identified a novel homozygous frameshift mutation of MCM8 in two siblings diagnosed with primary gonadal dysgenesis from a consanguineous family. Functional analysis confirmed the pathogenicity of this mutation. Our study not only further reveals the essential role of MCM8 in human gonadal development, but also expands the mutational spectrum of MCM8 involved in male NOA and female POI and provides a new molecular marker for genetic counseling of infertility.

Dietary components or patterns have been shown to affect male fertility. The increasing intake of processed foods rich in advanced glycation end products (AGEs) may threaten spermatogenesis. However, the key cell type affected by AGEs in spermatogenic microenvironment remains unspecified. Furthermore, given that subcellular organelle interactions, particularly communications between mitochondria and endoplasmic reticulum (ER), are of paramount importance in male fertility, it is worthwhile to investigate dynamic changes of mitochondria-ER contacts (MERCs) in AGE-driven spermatogenesis dysfunction. In this study, we found that serum AGEs levels increased in patients with oligoasthenozoospermia (OAZ), which was accompanied by decreased inhibin B levels, leading us to explore the effect of AGEs on Sertoli cells. In vivo experiments revealed that AGEs-rich diet disrupted spermatogenesis and induced Sertoli cell senescence and dysfunction in mice. We further confirmed that AGEs elicited an increase in MERCs, as well as ER stress and mitochondrial dysfunction in Sertoli cells. Omega-3 polyunsaturated fatty acids (omega-3), which are a category of dietary supplements with the potential to improve male fertility, were employed in the rescue experiment. We demonstrated that omega-3 mitigate dietary AGE-induced Sertoli cell senescence and OAZ via the remodeling of MERCs, highlighting the AGE-RAGE axis as a potential target for treating male infertility.

Male subfertility is a global health concern, with spermatogenic dysfunction being a critical cause. Abnormally high level of palmitic acid (PA), a main component of dietary saturated fatty acid, has been reported to be implicated in the spermatogenic dysfunction, accompanied with a decrease of inhibin B (INHB). However, the mechanism underlying PA-induced downregulation of INHB, and the specific function of INHB in the spermatogenesis microenvironment, remain unclear. Since PA is the main substrate of palmitoylation, a common post-translational lipid modification, we investigated the role of palmitoylation in INHB synthetic defects and subsequent dyszoospermia induced by PA in this study. Mice were treated with PA for 30 days to establish a high PA model, and a palmitoylation inhibitor 2-bromopalmitate (2BP) was used for spermatogenesis rescuing. Concentrations and motilities of sperms in the cauda epididymides were analyzed, and pathological examinations were performed to assess spermatogenic function. Hormone levels were detected using ELISA. Primary mouse Sertoli cells and TM4 Sertoli cell line were used for in vitro exploration of mechanisms. Acyl biotin exchange assay was used to explore protein palmitoylation. Co-culture of TM4 and GC1 cells was used to explore the effects of Sertoli cell-secreted INHB on spermatogonia in a paracrine manner. In this study, we found that excessive PA downregulates testicular INHB levels by suppressing expression of its βB subunit (InhβB) in Sertoli cells, with hyper-palmitoylation of the transcription factor SRY-box containing gene 9 (Sox9) serving as a key regulatory node in this process. We further identified the palmitoyl transferase ZDHHC16 as the primary enzyme responsible for PA-induced Sox9 hyper-palmitoylation. Furthermore, INHB was shown to promote spermatogonial proliferation and differentiation in a paracrine manner within the spermatogenic microenvironment, thereby mediating the modulation of spermatogenesis by palmitoylation in Sertoli cells. Overall, this study demonstrated that INHB synthesis can be suppressed by PA-induced hyper-palmitoylation of Sox9, and decreased secretion of INHB by Sertoli cells directly leads to spermatogenic dysfunction in the testis microenvironment. These findings highlight Sox9 palmitoylation as a candidate target for treatment of dyszoospermia accompanied with dyslipidemia, and underscore the critical role of INHB in regulating spermatogenesis within the testicular microenvironment.

In brief:O-GlcNAc plays an important role in many age-related diseases. This study shows that O-GlcNAc participates in oocyte aging and that reducing O-GlcNAc levels in aging oocytes improves oocyte quality. Abstract:With an increase in the mean age at parturition worldwide, female reproductive aging has become a key health problem. Advanced maternal age is reflected by decreased oocyte quality; however, the molecular mechanisms of oocyte aging are uncharacterized. O-linked N-acetylglucosamine (O-GlcNAc), a dynamic posttranslational modification, plays a critical role in the development of many age-related diseases; yet, it remains unclear whether and how O-GlcNAc participates in oocyte aging. Here, we found that global O-GlcNAc was elevated in normal biological aging mice oocytes (9 months), which were characterized by meiotic maturation failure and impaired mitochondrial function. Specifically, O-GlcNAc targeted the mitochondrial fission protein dynamic-related protein 1 to mediate mitochondrial distribution in the process of aging. Using the O-GlcNAcase (OGA) pharmacological inhibitor Thiamet-G and Oga knockdown (Oga-KD) to mimic the age-related high O-GlcNAc in young oocytes from 6-8 week-old mice mimicked the phenotype of oocyte aging. Moreover, reducing O-GlcNAc levels in aging oocytes restored spindle organization to improve oocyte quality. Our results demonstrate that O-GlcNAc is a key regulator of meiotic maturation that participates in the progression of oocyte aging.

Age-related declines in oocyte quality and ovarian function are pivotal contributors to female subfertility in clinical settings. Yet, the mechanisms driving ovarian aging and oocyte senescence remain inadequately understood. The present study evaluated the alterations in N-glycoproteins associated with ovarian aging and noted a pronounced elevation in N221 glycopeptides of cathepsin L (Ctsl) in the ovaries of reproductive-aged mice (8-9 months and 11-12 months) compared to younger counterparts (6-8 weeks). Subsequent analysis examined the involvement of Ctsl in oocyte aging and demonstrated a significant elevation in Ctsl levels in aged oocytes. Further, it was revealed that the overexpression of Ctsl in young oocytes substantially diminished their quality, while oocytes expressing an N221-glycosylation mutant of Ctsl did not suffer similar quality degradation. This finding implies that the N221 glycosylation of Ctsl is pivotal in modulating its effect on oocyte health. The introduction of a Ctsl inhibitor into the culture medium restored oocyte quality in aged oocytes by enhancing mitochondrial function, reducing accumulated reactive oxygen species (ROS), lowering apoptosis, and recovering lysosome capacity. Furthermore, the targeted downregulation of Ctsl using siRNA microinjection in aged oocytes enhanced fertilization capability and blastocyst formation, affirming the role of Ctsl knockdown in fostering oocyte quality and embryonic developmental potential. In conclusion, these findings underscore the detrimental effects of high expression of N-glycosylated Ctsl on oocyte quality and its contribution to oocyte senescence, highlighting it as a potential therapeutic target to delay ovarian aging and enhance oocyte viability.

Abstract Background The management of male infertility continues to encounter an array of challenges and constraints, necessitating an in-depth exploration of novel therapeutic targets to enhance its efficacy. As an eight-carbon medium-chain fatty acid, octanoic acid (OCA) shows promise for improving health, yet its impact on spermatogenesis remains inadequately researched. Methods Mass spectrometry was performed to determine the fatty acid content and screen for a pivotal lipid component in the serum of patients with severe spermatogenesis disorders. The sperm quality was examined, and histopathological analysis and biotin tracer tests were performed to assess spermatogenesis function and the integrity of the blood-testis barrier (BTB) in vivo. Cell-based in vitro experiments were carried out to investigate the effects of OCA administration on Sertoli cell dysfunction. This research aimed to elucidate the mechanism by which OCA may influence the function of Sertoli cells. Results A pronounced reduction in OCA content was observed in the serum of patients with severe spermatogenesis disorders, indicating that OCA deficiency is related to spermatogenic disorders. The protective effect of OCA on reproduction was tested in a mouse model of spermatogenic disorder induced by busulfan at a dose 30 mg/kg body weight (BW). The mice in the study were separated into distinct groups and administered varying amounts of OCA, specifically at doses of 32, 64, 128, and 256 mg/kg BW. After evaluating sperm parameters, the most effective dose was determined to be 32 mg/kg BW. In vivo experiments showed that treatment with OCA significantly improved sperm quality, testicular histopathology and BTB integrity, which were damaged by busulfan. Moreover, OCA intervention reduced busulfan-induced oxidative stress and autophagy in mouse testes. In vitro, OCA pretreatment (100 µM) significantly ameliorated Sertoli cell dysfunction by alleviating busulfan (800 µM)-induced oxidative stress and autophagy. Moreover, rapamycin (5 µM)-induced autophagy led to Sertoli cell barrier dysfunction, while OCA administration exerted a protective effect by alleviating autophagy. Conclusions This study demonstrated that OCA administration suppressed oxidative stress and autophagy to alleviate busulfan-induced BTB damage. These findings provide a deeper understanding of the toxicology of busulfan and a promising avenue for the development of novel OCA-based therapies for male infertility.

Female infertility due to declining oocyte quality with age remains a significant challenge for patients and physicians, despite extensive research efforts. Recent studies suggest that microRNAs (miRNAs), which respond to various stressors in the aging process, may provide a promising solution. With the approval of small RNA drugs for clinical use, miRNA-based treatment of oocyte aging appears to be a viable option. Through high-throughput sequencing, miR-425-5p was identified as the only miRNA elevated under natural aging and oxidative stress. Microinjection of inhibitors to inhibit miR-425-5p effectively improved compromised phenotypes of old oocytes in vitro. Further investigation revealed that Crebzf acts as a mediator of miR-425-5p 's age-related functions in old oocytes. In vivo treatment with miR-425-5p antagomirs significantly improved impaired oocyte development in reproductively old females by targeting Crebzf . Single-cell RNA sequencing revealed that Crebzf plays a vital role in regulating mRNAs targeting histone H3, trimethylated lysine 4 (H3K4me3), a crucial marker for transcriptional silencing. Overexpression of miR-425-5p could hinder oocyte maturation by downregulating Crebzf expression and disrupting transcriptional regulation. Our findings provide new insights into the potential of miR-425-5p antagomirs as a treatment for female infertility and highlight an elegant mechanism by which miR-425-5p inhibition of Crebzf inhibits a developmental switch in GV oocytes by regulating a group of histone methyltransferase mRNAs.

In recent years, the postponement of childbearing has become a critical social issue. Male fertility is negatively associated with age because of testis aging. Spermatogenesis is impaired with age, but the molecular mechanism remains unknown. The dynamic posttranslational modification O-linked N-acetylglucosamine (O-GlcNAc), which is a type of monosaccharide modification, has been shown to drive the process of aging in various systems, but it has not yet been investigated in the testis and male reproductive aging. Thus, this study aims to investigate the alteration of O-GlcNAc with aging and explore the role of O-GlcNAc in spermatogenesis. Here, we demonstrate that the decline in spermatogenesis in aged mice is associated with elevation of O-GlcNAc. O-GlcNAc is specifically localized in differentiating spermatogonia and spermatocytes, indicating its crucial role in meiotic initiation and progression. Mimicking the age-related elevation of O-GlcNAc in young mice by disabling O-GlcNAcase (OGA) using the chemical inhibitor Thiamet-G can recapitulate the impairment of spermatogenesis in aged mice. Mechanistically, the elevation of O-GlcNAc in the testis leads to meiotic pachytene arrest due to defects in synapsis and recombination. Furthermore, decreasing O-GlcNAc in aged testes using an O-GlcNAc transferase (OGT) inhibitor can partially rescue the age-related impairment of spermatogenesis. Our results highlight that O-GlcNAc, as a novel posttranslational modification, participates in meiotic progression and drives the impairment of spermatogenesis during aging.

RESEARCH QUESTION:Is early embryo development in mice influenced by RNA binding protein with multiple splicing 2 (RBPMS2), a maternal factor that accumulates and is stored in the cytoplasm of mature oocytes? DESIGN:The expression patterns of RBPMS2 in mouse were analysed using quantitative real-time PCR (qRT PCR) and immunofluorescence staining. The effect of knockdown of RBPMS2 on embryo development was evaluated through a microinjection of specific morpholino or small interfering RNA. RNA sequencing was performed for mechanistic analysis. The interaction between RBPMS2 and the bone morphogenetic protein (BMP) pathway was studied using BMP inhibitor and activator. The effect on the localization of E-cadherin was determined by immunofluorescence staining. RESULTS:Maternal protein RBPMS2 is highly expressed in mouse oocytes, and knockdown of RBPMS2 inhibits embryo development from the morula to the blastocyst stage. Mechanistically, RNA sequencing showed that the differentially expressed genes were enriched in the transforming growth factor-β (TGF-β) signalling pathway. BMPs are members of the TGF-β superfamily of growth factors. It was found that the addition of BMP inhibitor to the culture medium led to a morula-stage arrest, similar to that seen in RBPMS2 knockdown embryos. This morula-stage arrest defect caused by RBPMS2 knockdown was partially rescued by BMP activator. Furthermore, the localization of E-cadherin to the membrane was impaired in response to a knockdown of RBPMS2 or inhibition of the BMP pathway. CONCLUSION:This study suggests that RBPMS2 activates the BMP pathway and thus influences the localization of E-cadherin, which is important for early mouse embryo development during blastocyst formation.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder affecting women of reproductive age and is a significant cause of female subfertility. Our previous research demonstrated that the abnormal palmitoylation of heat shock protein-90α (HSP90α) plays a role in the development of PCOS. However, the palmitoyl acyltransferases in HSP90α palmitoylation remain poorly understood. Herein, we identified ZDHHC17 as a major palmitoyl acyltransferase for HSP90α palmitoylation in granulosa cells. ZDHHC17 protein expression was diminished under excess androgen conditions in vitro and in vivo. Consistently, ovarian ZDHHC17 expression was found to be attenuated in patients with PCOS. ZDHHC17 depletion decreased HSP90α palmitoylation levels and hampered the conversion of androgen to estrogen via CYP19A1. Furthermore, ZDHHC17-mediated regulation of CYP19A1 expression was dependent on HSP90α palmitoylation. Our findings reveal that the regulatory role of HSP90α palmitoylation by ZDHHC17 is critical in PCOS pathophysiology and provide insights into the role of ZDHHC17 in reproductive endocrinology.

Busulfan is an antineoplastic, which is always accompanied with the abnormal of spermatogonia self-renewal and differentiation. It has been demonstrated that the omega-3 polyunsaturated fatty acids (PUFAs) benefits mature spermatozoa. However, whether omega-3 can protect endogenous spermatogonia and the detailed mechanisms are still unclear. Evaluate of spermatogenesis function (in vivo) were examined by histopathological analysis, immunofluorescence staining, and western blotting. The levels of lipid metabolites in testicular tissue were determined via liquid chromatography. We investigated the effect of lipid metabolites on Sertoli cells provided paracrine factors to regulate spermatogonia proliferation and differentiation using co-culture system. In our study, we showed that omega-3 PUFAs significantly improved the process of sperm production and elevated the quantity of both undifferentiated Lin28+ spermatogonia and differentiated c-kit+ spermatogonia in a mouse model where spermatogenic function was disrupted by busulfan. Mass spectrometry revealed an increase in the levels of several omega-3 metabolites in the testes of mice fed with omega-3 PUFAs. The eicosapentaenoic acid metabolite 12-hydroxyeicosapentaenoic acid (12-HEPE) up-regulated bone morphogenic protein 4 (BMP4) expression through GPR120-ERK1/2 pathway activation in Sertoli cells and restored spermatogonia proliferation and differentiation. Our study provides evidence that omega-3 PUFAs metabolite 12-HEPE effectively protects spermatogonia and reveals that GPR120 might be a tractable pharmacological target for fertility in men received chemotherapy or severe spermatogenesis dysfunction.

Background Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disorder that frequently exhibits low-grade inflammation, pro-oxidant activity, and gut dysbiosis. PCOS has become one of the leading causes of female infertility worldwide. Recently, omega-3 polyunsaturated fatty acids (PUFAs) have been proven to benefit metabolic disorders in PCOS patients. However, its roles in the regulation of metabolic and endocrinal balances in PCOS pathophysiology are not clear. In the present study, we aimed to explore how omega-3 PUFAs alleviate ovarian dysfunction and insulin resistance in mice with dehydroepiandrosterone (DHEA)-induced PCOS by modulating the gut microbiota. Methods We induced PCOS in female mice by injecting them with DHEA and then treated them with omega-3 PUFAs. 16S ribosomal DNA (rDNA) amplicon sequencing, fecal microbiota transplantation (FMT) and antibiotic treatment were used to evaluate the role of microbiota in the regulation of ovarian functions and insulin resistance (IR) by omega-3 PUFAs. To further investigate the mechanism of gut microbiota on omega-3-mediated ovarian and metabolic protective effects, inflammatory and oxidative stress markers in ovaries and thermogenic markers in subcutaneous and brown adipose tissues were investigated. Results We found that oral supplementation with omega-3 PUFAs ameliorates the PCOS phenotype. 16S rDNA analysis revealed that omega-3 PUFA treatment increased the abundance of beneficial bacteria in the gut, thereby alleviating DHEA-induced gut dysbiosis. Antibiotic treatment and FMT experiments further demonstrated that the mechanisms underlying omega-3 benefits likely involve direct effects on the ovary to inhibit inflammatory cytokines such as IL-1β, TNF-α and IL-18. In addition, the gut microbiota played a key role in the improvement of adipose tissue morphology and function by decreasing multilocular cells and thermogenic markers such as Ucp1, Pgc1a, Cited and Cox8b within the subcutaneous adipose tissues. Conclusion These findings indicate that omega-3 PUFAs ameliorate androgen-induced gut microbiota dysbiosis. The gut microbiota plays a key role in the regulation of omega-3-mediated IR protective effects in polycystic ovary syndrome mice. Moreover, omega-3 PUFA-regulated improvements in the ovarian dysfunction associated with PCOS likely involve direct effects on the ovary to inhibit inflammation. Our findings suggest that omega-3 supplementation may be a promising therapeutic approach for the treatment of PCOS by modulating gut microbiota and alleviating ovarian dysfunction and insulin resistance.

Ovarian granulosa cells (GCs) in the follicle are the important mediator of steroidogenesis and foster oocyte maturation. Evidences suggested that the function of GCs could be regulated by S-palmitoylation. However, the role of S-palmitoylation of GCs in ovarian hyperandrogenism remains elusive. Here, we demonstrated that the protein from GCs in ovarian hyperandrogenism phenotype mouse group exhibits lower palmitoylation level compared with that in the control group. Using S-palmitoylation-enriched quantitative proteomics, we identified heat shock protein isoform α (HSP90α) with lower S-palmitoylation levels in ovarian hyperandrogenism phenotype group. Mechanistically, S-palmitoylation of HSP90α modulates the conversion of androgen to estrogens via the androgen receptor (AR) signalling pathway, and its level is regulated by PPT1. Targeting AR signaling by using dipyridamole attenuated ovarian hyperandrogenism symptoms. Our data help elucidate ovarian hyperandrogenism from perspective of protein modification and provide new evidence showing that HSP90α S-palmitoylation modification might be a potential pharmacological target for ovarian hyperandrogenism treatment.

n-3 PUFAs are classic antioxidant that can be used to treat follicular dysplasia and hyperinsulinemia caused by excessive oxidative stress in PCOS women. To investigate the effect of n-3 PUFA supplementation on the oocyte quality of polycystic ovary syndrome (PCOS) mice during in vitro maturation, a PCOS mouse model was established by dehydroepiandrosterone (DHEA). The GV oocytes of the control and PCOS groups were collected and cultured in vitro with or without n-3 PUFAs. After 14 h, the oocytes were collected. Our data demonstrated that the oocyte maturation rate of PCOS mice significantly increased after the addition of 50 µM n-3 PUFAs. The results of immunofluorescence showed that the abnormal rates of spindles and chromosomes in the PCOS + n-3 PUFA group were lower than those in the PCOS group. The mRNA expression of an antioxidant-related gene (Sirt1) and DNA damage repair genes (Brca1/Msh2) was found to be significantly rescued after n-3 treatment. Additionally, the results of living cell staining showed that the addition of n-3 PUFAs could reduce the levels of reactive oxygen species and mitochondrial superoxide in PCOS oocytes. In conclusion, the addition of 50 µM n-3 PUFAs during the in vitro maturation of PCOS mouse oocytes can improve the maturation rate by reducing the level of oxidative stress and the rate of spindle/chromosome abnormalities, providing valuable support during the IVM process.