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Calcium ions (Ca2+) play crucial roles in sperm motility and fertilization. The copine (CPNE) family comprises several Ca2+-dependent phospholipid-binding proteins. Of these, CPNE1 is extensively expressed in mammalian tissues; however, its precise role in testicular development and spermatogenesis is yet to be fully characterized. In this study, we used proteomics to analyze testicular biopsies and found that levels of CPNE1 were significantly reduced in patients with non-obstructive azoospermia (defective spermatogenesis) compared to those in patients with obstructive azoospermia (physiological spermatogenesis). In mice, CPNE1 is expressed at various stages of germ cell development and is associated with the Golgi apparatus. Ultimately, CPNE1 is expressed in the flagella of mature sperms. To further examine the role of CPNE1, we developed a Cpne1 knockout mouse model. Analysis showed that the loss of Cpne1 did not impair testicular development, spermatogenesis, or sperm morphology and motility in physiological conditions. When treated with gadolinium (III) chloride or 2-aminoethoxydiphenyl borate, known inhibitors of store-operated Ca2+ entry, Ca2+ signals and sperm motility were significantly compromised in wild-type mice; however, both mechanisms were conserved in KO mice. These results suggested that CPNE1 is dispensable for testicular development, spermatogenesis or sperm motility in physiological conditions. In addition, CPNE1 may represent a target of Ca2+ channel inhibitors and may therefore be implicated in the regulation of Ca2+ signaling and sperm motility.

BackgroundPolycystic ovary syndrome (PCOS) is a common heterogeneous disorder linked with endocrine and metabolic disturbances. The underlying mechanism of PCOS, especially its effect on oocyte competence, remains unclear. The study aimed to identify abnormal follicular metabolic changes using a multi-omics approach in follicular fluid from PCOS patients and to determine their effects on oocyte competence.MethodsA total of 36 women with PCOS and 35 women without PCOS who underwent in vitro fertilization and embryo transfer were included in the study. Cumulus cells and follicular fluid samples were collected. Follicular fluid samples underwent metabolomic analysis, while cumulus cell clusters from the same patients were assessed using transcriptomic analysis. Clinical information of patients and assisted reproductive technology (ART) results were recorded. Transcriptomics and metabolomics were integrated to identify disrupted pathways, and receiver operation characteristics (ROC) analysis was conducted to identify potential diagnostic biomarkers for PCOS. Pearson correlation analysis was conducted to assess the relationship between metabolites in follicular fluid and oocyte competence (fertilization and early embryo development potential).ResultsThrough multi-omics analysis, we identified aberrantly expressed pathways at both transcriptional and metabolic levels, such as the citrate cycle (TCA cycle), oxidative phosphorylation, the cAMP signaling pathway, the mTOR signaling pathway, and steroid hormone biosynthesis. Ten candidate metabolites were identified based on metabolic profiling data from these altered pathways. Phytic acid, succinic acid, 2’-deoxyinosine triphosphate, and 4-trimethylammoniobutanoic acid in the follicular fluid exhibited high specificity and sensitivity in distinguishing PCOS. Among these metabolites, L-arginine showed a negative correlation with the 2PN fertilization rate and cleavage rate, while estrone sulfate showed a negative correlation with the high-quality embryo rate in the in-vitro fertilization (IVF) cycle.ConclusionsWe have conducted a preliminary study of a novel metabolic signature in women with PCOS using a multi-omics approach. The alterations in key metabolic pathways may enhance our understanding of the pathogenesis of PCOS.

IntroductionMales with acute spinal cord injury (SCI) frequently exhibit testosterone deficiency and reproductive dysfunction. While such incidence rates are high in chronic patients, the underlying mechanisms remain elusive.Methods and resultsHerein, we generated a rat SCI model, which recapitulated complications in human males, including low testosterone levels and spermatogenic disorders. Proteomics analyses showed that the differentially expressed proteins were mostly enriched in lipid metabolism and steroid metabolism and biosynthesis. In SCI rats, we observed that testicular nitric oxide (NO) levels were elevated and lipid droplet-autophagosome co-localization in testicular interstitial cells was decreased. We hypothesized that NO impaired lipophagy in Leydig cells (LCs) to disrupt testosterone biosynthesis and spermatogenesis. As postulated, exogenous NO donor (S-nitroso-N-acetylpenicillamine (SNAP)) treatment markedly raised NO levels and disturbed lipophagy via the AMPK/mTOR/ULK1 pathway, and ultimately impaired testosterone production in mouse LCs. However, such alterations were not fully observed when cells were treated with an endogenous NO donor (L-arginine), suggesting that mouse LCs were devoid of an endogenous NO-production system. Alternatively, activated (M1) macrophages were predominant NO sources, as inducible NO synthase inhibition attenuated lipophagic defects and testosterone insufficiency in LCs in a macrophage-LC co-culture system. In scavenging NO (2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO)) we effectively restored lipophagy and testosterone levels both in vitro and in vivo, and importantly, spermatogenesis in vivo. Autophagy activation by LYN-1604 also promoted lipid degradation and testosterone synthesis.DiscussionIn summary, we showed that NO-disrupted-lipophagy caused testosterone deficiency following SCI, and NO clearance or autophagy activation could be effective in preventing reproductive dysfunction in males with SCI.

Oligoasthenoteratozoospermia is a human infertility syndrome caused by defects in spermatogenesis, spermiogenesis, and sperm maturation, and its etiology remains unclear. Kelch-like 10 (KLHL10) is a component of ubiquitin ligase E3 10 (KLHL10) and plays an important role in male fertility. Deletion or mutation of the Klhl10 gene in Drosophila or mice results in defects in spermatogenesis or sperm maturation. However, the molecular mechanisms by which KLHL10 functions remain elusive. In this study, we identified a missense mutation (c.1528A→G, p.I510V) in exon 5 of KLHL10, which is associated with oligoasthenoteratozoospermia in humans. To investigate the effects of this mutation on KLHL10 function and spermatogenesis and/or spermiogenesis, we generated mutant mice duplicating the amino acid conversion using the clustered regularly interspaced palindromic repeat/caspase 9 (CRISPR/Cas9) system and designated them Klhl10I510V mice. However, the Klhl10I510V mice did not exhibit any defects in testis development, spermatogenesis, or sperm motility at ten-weeks-of-age, suggesting that this mutation does not disrupt the KLHL10 function, and may not be the cause of male infertility in the affected individual with oligoasthenoteratozoospermia.