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.

Glycogen and lactate metabolism in mouse fetal Sertoli cells sustain the germ line.

Epimedium polysaccharide alleviates adenine-induced oligoasthenozoospermia in mice by inhibiting ferroptosis and inflammation through TLR4/NF-κB and SLC7A11/GPX4 axis.

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.

Er-Chen decoction alleviates spermatogenic dysfunction in obese mice by tuning the SIRT1/p53 axis.

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.

Synergistic assault of DEHP and MPs: Unmasking the ER stress-triggered autophagic injury male fertility.

Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate (HFPO-DA; CAS#: 62037-80-3) was tested for potential toxicity and carcinogenicity in CD-1 mice administered 0, 0.05, 0.1, 0.5, or 5mg/kg-day HFPO-DA via oral gavage for 9 or 18 months. Histopathological examinations were conducted at each time point along with clinical chemistry measurements. Reduced survival was observed in male mice exposed to 5mg/kg-day for 18 months but not 9 months. Hepatocellular hypertrophy was the most sensitive histopathological response to HFPO-DA and was significantly increased in both sexes at 9 and 18 months of exposure. At 18 months, hepatocellular hypertrophy was not observed below 0.1mg/kg-day. Liver tumors were significantly increased at 5mg/kg-day in males at both timepoints and in females at 18 months. No other treatment related tumors were observed. Consistent with previously published studies in mice, transcriptomic responses in the liver of both sexes showed enrichment of peroxisome proliferator-activated receptor alpha (PPARα) signaling pathways. These changes demonstrate that the tumor response in the liver is consistent with a PPARα mode of action. Other noncancer histopathological effects were limited to the adrenal gland (5mg/kg-day at ≥9 months) and testes (≥0.5mg/kg-day at 18 months) of male mice.

Immune cells play a crucial role in maintaining tissue homeostasis during aging. However, the dynamics and functions of immune cells in testicular aging have not been well elucidated. In this study, we utilized single-cell RNA sequencing (scRNA-seq) to analyze CD45-enriched immune cells isolated from young and old mice testis. This approach yielded a comprehensive dataset comprising 6622 immune cells, encompassing macrophages, monocytes, and T cells. Our analysis revealed a significant decline in FOLR2 + resident macrophages, accompanied by a corresponding increase in pro-inflammatory CD74 + macrophages, CCR2 + monocytes, and CD8 + T cells in old mice testis. These findings were further validated by multiplex immunofluorescence staining. Notably, during testicular aging, FOLR2 + macrophages underwent a phenotypic transition towards a pro-inflammatory state. This transition subsequently facilitated the recruitment of monocytes and CD8 + T cells via the CCL8-CCR2/CCR5 axis. Furthermore, we discovered that mitochondrial metabolic dysfunction was a key driver of FOLR2 + macrophage activation. Specifically, inhibition of IDH2, a key catalytic enzyme in the TCA cycle, significantly induced this activation. Collectively, our findings provide a detailed immune atlas of testicular aging and suggest a potential role for FOLR2 + macrophages in maintaining testicular immune homeostasis.

Age-related male hypogonadism is often associated with obesity-related metabolic disorders and impaired regulation of the testicular anti-inflammatory microenvironment. However, how adipose-mediated signals intersect with local testicular immunity remains unclear. In this study, single-cell RNA sequencing (scRNA-seq) of human and mouse testes identified a conserved CD206LoMHCIIHi macrophage subset, which undergoes CMKLR1-mediated metabolic reprogramming toward glycolysis and pro-inflammatory state during middle age. This immunometabolic shift is further found to impair spermatogenesis. CMKLR1 is identified as a viable target in vivo for restoring immunometabolic balance in aging testes. Systemic administration of a newly developed CMKLR1 antagonist peptide (P12C5) or non-pharmacological intervention such as high-intensity interval training (HIIT) rescued spermatogenesis in middle-aged humans and mice, and reversed the pro-inflammatory immunometabolic phenotype in testicular macrophages. Together, these findings validate CMKLR1 as a key modulator of testicular immunometabolism and a therapeutic target for mitigating age-related immunometabolic dysfunction.

Aim: To study the effect of Musli Pak supplementation on male reproductive physiology in Adult Parkes strain mice Study design: Twenty-four adult Parkes strain male mice of twelve to fourteen weeks age were placed in 4 different groups (6 mice /group). Standard optimum conditions were maintained; mice were treated with the respective dose (50 mg/kg BW,100 mg/kg BW, and 200 mg/kg BW) of Musli Pak along with control group for 35 days. Place and Duration of Study: Ethical clearance was approved by the IAEC, Department of Zoology, Banaras Hindu University with a validity of two years. Methodology: Following autopsy, reproductive organ indices, sperm quality, testicular daily sperm production (TDSP), steroidogenic enzyme activities(3β-HSD,17β-HSD), serum hormones, epididymal sialic acid, seminal vesicle fructose, and histoarchitecture of testis, seminal vesicle and epididymis of adult Parkes strain mice were evaluated. Results: In Musli Pak treated mice testis weight increased at all doses however relative testis and epididymis weight increased in mice treated with doses 100 & 200 mg/kg, body weight. Seminal vesicle weight was significantly higher in all treated groups. Musli Pak treatment appeared to enhance sperm count, motility, viability, TDSP and protected against abnormal morphology. Serum testosterone, estradiol,3β-HSD and 17β-HSD expression were elevated in mice particularly at 100 & 200 mg/kg dose of Musli Pak. PCNA expression was found highest at 50 and 200 mg/kg. Histology of testis, epididymis and seminal vesicle showed normal architecture with germinal epithelium thickening and sperm-rich lumina without degenerative changes. Biochemical analysis showed increased sialic acid and fructose concentration indicated improved epididymal and seminal vesicle function. Conclusion: Musli Pak supplementation enhances spermatogenesis, steroidogenesis, and sperm maturation in a dose-dependent manner, with 200 mg/kg showing maximal benefits and 50 mg/kg triggering strong proliferative activity. The findings support its traditional fertility-boosting use of Musli Pak without histological or biochemical toxicity..

Testosterone (T) produced by Leydig cells (LCs) is essential for male reproduction; yet, the regulatory mechanisms underlying steroidogenesis remain incompletely understood. Here, we investigated the role of cyclin-dependent kinase 5 regulatory subunit-associated protein 3 (CDK5RAP3) in Leydig cell development and steroidogenesis, based on its identification by immunoprecipitation-mass spectrometry (IP-MS) as a protein associated with steroidogenesis and cholesterol metabolism in mouse testicular tissue. Using human samples, we found that CDK5RAP3 expression was significantly reduced in Leydig cells from patients with spermatogenic failure (T < 10.4 nmol/L). Notably, CDK5RAP3 expression increased during mouse postnatal Leydig cell maturation and regeneration in an ethane dimethanesulfonate (EDS)-induced rat model. Functional analyses in primary LCs and MLTC-1 cells showed that hCG stimulation triggered CDK5RAP3 nuclear translocation without altering its overall expression, while CDK5RAP3 knockdown markedly impaired hCG-induced testosterone production and reduced the expression of the steroidogenic regulator steroidogenic acute regulatory (STAR) protein, as well as key steroidgenic enzymes, including cytochrome P450 family 11 subfamily A member 1 (CYP11A1), 17a-hydroxylase (CYP17A1), and 3β-hydroxysteroid dehydrogenase (HSD3B). Conversely, CDK5RAP3 overexpression enhanced testosterone production in the absence of hCG. In vivo, AAV2/9-mediated CDK5RAP3 silencing in adult mouse testes resulted in a significant reduction in serum testosterone levels compared with controls (3.60 ± 0.38 ng/mL vs. 1.83 ± 0.37 ng/mL). Mechanistically, CDK5RAP3 interacted with SMAD4 and CEBPB, and BMP pathway inhibition by Noggin rescued the testosterone deficit caused by CDK5RAP3 loss. Together, these findings identify CDK5RAP3 as an essential regulator of Leydig cell steroidogenesis and provide insight into its potential relevance to male infertility associated with low testosterone.

PIWI-interacting RNAs (piRNAs) guide PIWI endoribonucleases to destroy transposon transcripts, ensuring animal fertility. Here, we report the cryo-electron microscopy structure of the MIWI-pachytene piRNA complex isolated from mouse testes. The piRNA is held via non-specific charge-based interactions with the RNA backbone and by specific recognition of the first nucleotide uridine by residues within the MID and PIWI domains. The first six nucleotides of the guide RNA take up the A-form conformation to facilitate pairing with the target. The RNA channel is wider than that observed in insect PIWI proteins, explaining the tolerance for piRNA seed:target mismatches. The PIWI endonuclease domain is in an inactive "un-plugged" state, with the loop containing a catalytic residue (E671) requiring structural re-orientation for activity. Furthermore, the PIWI domain reveals a conserved pre-formed pocket that may serve to accommodate a conserved tryptophan from the interacting factor GTSF1 to promote small RNA-guided endoribonuclease activity.