High concentrations of GW9508 enhance sperm velocity in mice via peroxisome proliferator-activated receptor γ.

Diquat (DQ) is a widely used herbicide, whose residues have been detected in food products, raising concerns about potential health risks. Nevertheless, the mechanisms underlying male reproductive damage after chronic DQ exposure remain unclear. In this study, we observed that chronic DQ exposure reduces Sertoli cell numbers and triggers mitochondrial quality control (MQC) impairment, oxidative stress, apoptosis, and blood-testis barrier (BTB) disruption in testicular tissue. Mechanistically, DQ downregulates NBR1 in Sertoli cells, leading to impaired mitophagy flux, increased mitochondrial fission, and elevated oxidative stress, which ultimately promote Sertoli cell apoptosis and compromise cellular barrier function. Metabolomic analysis indicated that chronic DQ exposure lowers spermidine (SPD) levels in the mouse testes. Exogenous SPD supplementation attenuated DQ-induced MQC dysfunction, oxidative stress, apoptosis, and BTB damage. Together, these results establish NBR1 as a critical regulator of DQ-triggered Sertoli cell injury and propose SPD as a potential therapeutic candidate for alleviating DQ-induced male reproductive impairment.

CTLA-2 alpha, a cysteine protease inhibitor, shares structural homology with pro-region of cathepsin L, to which it exhibits specific and potent inhibitory activity. Sertoli cells secrete cathepsin L which participates in spermatozoa production and maturation in testis and epididymis. Sertoli cells also regulate activity of regulatory T cells, helper T cells and cytotoxic T cells to prevent autoimmune attack on developing spermatogenic cells. Mast cells and activated T cells were first cells identified to express CTLA-2 alpha. CTLA-2 alpha mRNA is expressed by undifferentiated spermatogonia but its protein has never been demonstrated in testis, not even in other male reproductive organs. This study therefore aimed at investigating by immunohistochemistry CTLA-2 alpha localization in testis, rete testis, efferent duct, epididymis, deferens duct, seminal vesicles and prostate. Results showed localization of CTLA-2 alpha in Leydig cells, Sertoli cells, spermatogonia, spermatocytes, spermatids and spermatozoa, as well as in epithelial cells lining rete testis, efferent ducts, epididymis including spermatozoa, deferens duct and prostatic urethra. Basal cells and principal cells of initial segment of epididymis showed weak staining. In accessory sex glands, staining was detected in apical portion of cytoplasm of epithelial cells lining seminal vesicles. In prostate, staining was diffusely distributed in cytoplasm of luminal epithelial cells. Extension of prostate cell apical cytoplasm into lumen was frequently encountered in anterior and dorsal prostate, representing apocrine release of secretory products. Localization of CTLA-2 alpha in male reproductive system is suggestive of its potential roles in regulation of spermatogenesis in testicular tissues, maturation of spermatozoa in tubular structures and secretion of proteins and peptides by seminal vesicles and prostate essential for male fertility.

In the mouse testis, spermatogonial stem cells (SSCs) are sparsely distributed and migrate along the basement membrane of seminiferous tubules. Although the basement membrane is generally thought to be formed by surrounding somatic cells, whether SSCs also produce basement membrane proteins and, if so, whether SSC-produced laminin affects SSC behavior remains unknown. In this study, we found that mouse GFRα1+ spermatogonia, which include SSCs, expressed several laminin subunit genes, including Lamc1, whose expression declined upon differentiation. To test whether GFRα1+ spermatogonia-derived laminin regulates their behavior, we used two conditional knockout mouse models. In the Vasa-Cre model, which induces recombination in all germ cells, heterozygous deletion of Lamc1 increased both cell death and proliferation of GFRα1+ spermatogonia, while maintaining an apparent steady state of GFRα1+ cell density and spermatogenesis. In the tamoxifen-inducible GFRα1-CreER model carrying Lamc1flox/flox, tamoxifen-induced Lamc1 deletion in GFRα1+ spermatogonia caused a rapid reduction in their cell density within a few days, followed by increased proliferation and an imbalance between proliferation and differentiation of GFRα1+ spermatogonia that led to the restoration of GFRα1+ spermatogonial density. Collectively, these genetic findings suggest that GFRα1+ spermatogonia modulate their survival and behavior through laminin expression, likely by influencing the basement membrane around GFRα1+ spermatogonia. Such cell-autonomous regulation allows GFRα1+ spermatogonia, including SSCs, to form an appropriate local microenvironment wherever they reside within the testicular open niche, supporting stable behavior of spermatogonia during spermatogenesis.

Sodium pentachlorophenol induces inflammatory damage via the ATP2B4/Ca2+/ROS signaling axis in mouse testes.

Polylactic acid microplastic exposure induced male reproductive toxicity and decreased testosterone levels by accelerating Leydig cell senescence.

Polyglutamine-binding protein 1 protects mouse testes from heat stress.

Messenger RNA delivery into Sertoli cells restores fertility to congenitally infertile male mice.

Retinoic acid plays a critical role in spermatogenesis during both spermatogonial differentiation and spermiation. Mice lacking enzymes that synthesize retinoic acid, Aldh1a1/1a2, in the testes are infertile. WIN 18,446 reversibly inhibits spermatogenesis by inhibiting ALDH1A1/1A2 and is a promising approach to male contraception. Previously, we demonstrated the kinetics, efficacy, and reversibility of continuous treatment with WIN18,446 as a male contraceptive. We sought to investigate whether cyclic administration of WIN 18,446 could be as effective at inhibiting spermatogenesis as continuous treatment with WIN 18,446 while reducing the risk of potential testicular damage from continuous treatment. We studied testicular histology and sperm counts in response to continuous vs. cyclic treatment of WIN 18,446 and assessed recovery from the two treatment regimens. Male mice were fed a diet containing WIN 18,446 for 15 weeks, followed by a diet without the drug for up to 16 weeks. For cyclic treatment, male mice were fed the WIN 18,446-containing diet for 4 weeks, followed by three cycles of 2-weeks of control diet, followed by 2-weeks of WIN 18,446 diet for 12 additional weeks. After the last cyclic treatment, drug treatment was discontinued, and animals were allowed to recover for 8 weeks. Mice were euthanized at predetermined time points to assess recovery of spermatogenesis by testicular histology and sperm counts. Testis weights and sperm counts were similar between cyclically treated mice and mice treated with WIN 18,446 continuously for 15 weeks. Testes of mice under cyclic treatment contained spermatocytes/spermatids, but not mature, normal spermatozoa, while those from the mice under continuous treatment had only spermatogonia. Importantly, spermatogenesis recovered more quickly when mice were treated cyclically compared to those receiving continuous treatment based on testes weight and histology. Cyclic treatment of mice with the ALDH1A inhibitor WIN 18,446 is as efficacious as continuous treatment in suppressing spermatogenesis and may allow for faster recovery of spermatogenesis after the cessation of drug treatment, and reduce the chance of injury to the testes from continuous treatment.

Organ development and function are orchestrated by intricate transcriptional circuits. Here, we present a comprehensive atlas profiling 1,904 transcription regulators in the brain, cerebellum, heart, kidney, liver, ovary, and testis of fetal, neonatal, and adult mice. Using this dataset, we uncover Thymocyte Selection-Associated High Mobility Group Box Family Member 3 (TOX3) as a potential coactivator of Atoh1 in cerebellar granule neuron progenitors (GNPs). Tox3-deficient mice display severe ataxia and cerebellar hypoplasia, driven by depletion of GNPs, diminished Atoh1 expression, and impaired primary cilia. Single-nucleus RNA-sequencing analyses reveals compromised maintenance of the progenitor pool. TOX3 is also highly expressed in subsets of medulloblastoma, and its deletion reduces cerebellar neoplasia and prolongs survival in a mouse model. Mechanistically, how lineage-defining factors such as Atoh1 drive robust gene expression despite weak intrinsic transactivation activity remains unclear. We show that Tox3 physically associates with Atoh1 and co-occupies shared regulatory elements, converting an otherwise weak single-copy Atoh1-responsive E-box into a highly active enhancer that drives transcriptional activation by up to 120-fold, including at an ultraconserved E-box downstream of Atoh1 itself. Cross-species single-cell comparisons further show an association between Tox3 expression and cerebellum expansion during vertebrate evolution. Together, this work supports Tox3 as a critical Atoh1 coactivator in cerebellar development, tumorigenesis, and evolution, while providing an atlas and screening strategy as a valuable resource for exploring novel transcriptional regulators in organogenesis and tissue physiology.

ObjectiveTo investigate the genetic and molecular role of CAPZA1 in asthenozoospermia and its impact on sperm motility and flagellar integrity.MethodsWhole-exome sequencing (WES) was first performed in an infertile family with asthenozoospermia to identify candidate variants. The CAPZA1 variant was further screened by Sanger sequencing in 20 infertile men with asthenozoospermia and 20 age-matched fertile controls. CAPZA1 expression and sperm motility parameters were assessed by Western blot and computer-assisted semen analysis, respectively. Structural abnormalities were examined using transmission electron microscopy (TEM). In vitro CAPZA1 knockout (KO-CAPZA1) was achieved in isolated mouse round spermatids using CRISPR-Cas9, followed by RT-qPCR, Western blot, ELISA for cystine levels, and thiol quantification to assess downstream effects. Protein localization of DNAH9 and FSCN1 was analyzed by immunofluorescence. In vivo CAPZA1 deletion was induced via adeno-associated virus (AAV)-mediated CRISPR-Cas9 delivery into mouse testes, and subsequent sperm motility, protein expression, and ultrastructure were evaluated.ResultsA rare homozygous missense mutation in CAPZA1 (c.11T>C, p.Phe4Ser) was first identified by WES in the proband of an infertile family and was subsequently detected by Sanger sequencing in 3 of 20 asthenozoospermic patients. CAPZA1 protein expression was significantly reduced in mutant sperm, with a strong positive correlation to progressive motility (r = 0.849, p < 0.001). TEM revealed disorganized flagellar ultrastructure, including asymmetric fibrous sheath and partial dynein arm loss. In KO-CAPZA1 mouse spermatids, p300/CBP, SLC7A11, H3K27ac expression were decreased. Reduced cystine content and increased DTNB-reactive thiol groups after TCEP reduction indicated disrupted thiol/disulfide homeostasis. DNAH9 and FSCN1 expression and localization were disrupted in KO-CAPZA1 cells. KO-CAPZA1 in mice resulted in significantly decreased sperm progressive motility (p < 0.001) and abnormal axonemal structure, without affecting testicular morphology or sperm count.ConclusionCAPZA1 deficiency impairs sperm motility and flagellar architecture through disrupted cytoskeletal protein regulation and redox imbalance, and represents a novel genetic contributor to asthenozoospermia.

STOML2 is not essential for spermatogenesis and male fertility in mice.

Perfluorooctane sulfonic acid impairs spermatogenesis via the liver-gut microbiota-testis axis: a central role of chenodeoxycholic acid metabolism.

LSM14A, an LSM family protein, is dispensable for spermatogenesis and male fertility in mice.

Mechanism of PP2A affecting ubiquitination pathway in spermatogenesis.

Spmip8, also known as Tepp, is a protein-coding gene which highly conserved in the mammals. Although SPMIP8 has been reported to be highly expressed in the testis, the function of SPMIP8 in spermatogenesis and male fertility remain unknown. In this study, we used CRISPR/cas9-mediated genome editing system to generate Spmip8-deficient mice. The phenotype of Spmip8 knockout (KO) male mice was performed by fertility tests, histology, and immunofluorescence. SPMIP8 is localization to the flagella of elongating spermatids in testis. Spmip8 KO male mice exhibited normal fertility. No significant differences were found in sperm count, motility, morphology and kinematic parameters between WT and Spmip8 KO mice. Furthermore, no detectable defects in spermatogenesis were found in KO mice. The transcription level of several Spmip genes (Spmip1, Spmip2, Spmip3, Spmip7 and Spmip11) was elevated in the testes of Spmip8 knockout mice, suggesting that Spmip8 gene in male fertility could be compensated by other Spmip family members. Overall, the findings of this study suggest that Spmip8 is not an essential gene for male fertility in mice. Our study helps researchers avoid duplication and repetitive work and explore genes that are integral to spermatogenesis and male fertility.

Accurate testicular tissue characterization is critical for diagnosing reproductive disorders and malignancies. We present a rapid, label-free 3D imaging technique that leverages endogenous autofluorescence and a fast, non-toxic optical clearing protocol to reveal the full 3D architecture of thick mouse testis samples, without sectioning. This approach clearly distinguishes structures like epithelial tubules, interstitial tissue, and key cell types (germ, Sertoli, Leydig) with high precision. Autofluorescence serves as a powerful structural counterstain, enhancing multiplexed analysis alongside immunofluorescent or transgenic markers. Compatible with high-resolution techniques like image-scanning confocal microscopy (AiryScan), ourapproach captures fine intracellular details while avoiding the artefacts of traditional histology. Accessible, affordable, and equally usable on standard confocal microscopes, this method democratizes advanced testis imaging and accelerates progress in reproductive research.

Leydig cells are testosterone synthesis cells in testes, a process tightly regulated by luteinizing hormone (LH) through the activation of steroidogenic enzymes such as steroidogenic acute regulatory protein (StAR) and 3-beta-hydroxy-Delta5-steroid dehydrogenase (3β-HSD). While calcium/calmodulin-dependent protein kinase 2 (CAMK2) is known to modulate diverse cellular processes, including hormone signaling, its role in testosterone production remains unclear. In this study, we investigated the expression and functions of CAMK2 in mouse testes, focusing on its potential involvement in testosterone synthesis. Our findings demonstrate that CAMK2 expression progressively increases from postnatal day 1 (PND 1) to adulthood. Pharmacological inhibition of CAMK2 with KN-62 markedly reduced serum testosterone levels and downregulated the expression of key steroidogenic enzymes, including StAR and 3β-HSD, at both mRNA and protein levels. In vitro experiments using primary Leydig cells further confirmed that CAMK2 inhibition suppressed testosterone production and steroidogenic enzyme expression, particularly after prolonged (12-24 h) KN-62 treatment. Additionally, CAMK2 expression was upregulated in response to LH stimulation, suggesting its involvement in LH-mediated signaling pathways, potentially through modulation of the epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase 1/2 (ERK1/2) cascade. These findings demonstrate that CAMK2 positively regulates testosterone synthesis in Leydig cells, likely via the EGFR/ERK1/2 cascade. The results of this study enhance our understanding of the regulation of testosterone synthesis and identifies CAMK2 as a potential therapeutic target for male reproductive endocrine disorders.

Arsenic exposure reduces testosterone synthesis partially by evoking Leydig cell ferroptosis in mouse testes.

The gene regulatory mechanism of individual genes during spermatogenesis is well understood in model animals, but not in non-model animals such as human. The mouse Prss/Tessp locus encompassing six testis-specific protease genes is a model of gene function and regulation in the testis. Here we investigated the expression and regulation of PRSS locus in primates, especially human. Unlike high expression of all six genes in mouse testis, one or two different PRSS genes were predominantly expressed in six primate species. In human testis, PRSS50 was a predominant gene, and the other five PRSS genes were expressed at low levels. Interestingly, promoters of four lowly expressed PRSS genes were marked with both histone H3K4 trimethylation and histone H3K27 trimethylation in human spermatocytes and spermatids, while their orthologous mouse genes were marked only with histone H3K4 trimethylation. Additionally, an ATAC-seq peak in human testis exhibited silencer activity for PRSS45 which is a lowly expressed gene in human but a predominant gene in chimpanzee. These results suggest that bivalent chromatins and an active silencer cooperatively function to achieve appropriate levels of expression for individual PRSS genes during human spermatogenesis. These findings provide mechanistic insights into gene regulation in human spermatogenesis.