ABSTRACT In male mammals, spermatogonial stem cells (SSCs) are essential for sustaining lifelong spermatogenesis within the testicular open niche, a unique environment that allows SSC migration over an extended niche area. As SSCs undergo continuous mitotic division, mutations accumulate and are transmitted to the descendant SSC clones. Therefore, SSC clonal fate behaviors, in terms of their efficiencies in completing spermatogenesis and undergoing expansion within the niche, influence sperm genomic diversity. We aimed to elucidate the effects of physiological aging on SSC clonal fate behavior within the testicular open niche. We used single‐cell RNA sequencing, lineage tracing, and intravital live imaging to investigate SSC behavior in aged mouse testes, where spermatogenesis, although reduced, persists. We found that undifferentiated spermatogonia maintained gene expression heterogeneity during aging. Among these, GFRα1⁺ cells, which exhibited state heterogeneity, showed accelerated proliferation and persistent motility, continuing to function as SSCs in older mice. In contrast, a subset of SSCs characterized by low Egr4 and Cops5 expression did not contribute to spermatid formation. These non–sperm‐forming SSC clones increased in proportion among the total SSC clones and expanded spatially within the testicular open niche in old mice, a phenomenon not observed in young mice. The expansion of non–sperm‐forming SSC clones in aged testes suggests that they occupy a niche space, limiting the availability of functional SSCs and potentially reducing sperm production and genetic diversity. These findings highlight age‐specific clonal characteristics as hallmarks of stem cell aging within the testicular open niche and provide novel insights into the mechanisms governing reproductive aging.

A prolyl isomerase Pin1 deficient (Pin1-/-) male mice had severe testicular atrophy. We investigated the function of Pin1 in spermatogenesis by analyzing the Pin1-/- mice at reproductive age. Pin1-/- mice had lessαPLZF positive spermatogonia (undifferentiated spermatogonia) than wild type (WT). Nevertheless, the Pin1-/- testis contained approximately the same number of GFRα1 positive spermatogonia (SSCs in steady state) as the WT testis. Furthermore, degeneration of the spermatogenia appeared in seminiferous tubules of 10 months old Pin1-/- mouse testis, and abnormal shape GFRα1 positive spermatogonia were observed. In Pin1-/- spermatogonia, the ratio of the phospho-histone H3 positive cells (mitotic cells) in GFRα1-positive spermatogonia was higher than that of WT. These results suggest that Pin1 promotes the progression of the mitotic cell cycle of SSC in steady-state, which is required for the sperm production from SSCs.

BACKGROUND: While screening for an inhibitor of the peptidyl prolyl cis/trans isomerase, Pin1, we came across a brown algae polyphenol that blocks the differentiation of fibroblasts into adipocytes. However, its effectiveness on the accumulation of fat in the body has never been studied. METHODOLOGY/PRINCIPAL FINDINGS: Oral administration of brown algae polyphenol to mice fed with a high fat diet, suppressed the increase in fat volume to a level observed in mice fed with a normal diet. We speculate that Pin1 might be required for the differentiation of stem cell to adipocytes. We established wild type (WT) and Pin1-/- (Pin1-KO) adipose-derived mesenchymal stem cell (ASC) lines and found that WT ASCs differentiate to adipocytes but Pin1-KO ASCs do not. CONCLUSION AND SIGNIFICANCE: Oral administration of brown algae polyphenol, a Pin1 inhibitor, reduced fat buildup in mice. We showed that Pin1 is required for the differentiation of stem cells into adipocytes. We propose that oral intake of brown algae polyphenol is useful for the treatment of obesity.

OBJECTIVE: The uptake carrier organic anion-transporting polypeptide 1B3 (OATP1B3, gene SLCO1B3) is involved in the hepatic clearance of xenobiotics including statins, taxanes, and mycophenolic acid. We thought to assess the SLCO1B3 coding region for yet unidentified polymorphisms and to analyze their functional relevance. METHODS: We used DNA of ethnically diverse individuals for polymerase chain reaction, and determined polymorphisms by sequencing or temperature-dependent capillary electrophoresis. We then created variant OATP1B3 expression plasmids by site-directed mutagenesis, which were transiently expressed and functionally characterized in human cervical carcinoma (HeLa) cells using radiolabeled substrates. RESULTS: We identified six nonsynonymous polymorphisms including novel variants such as 439A>G (Thr147Ala), 767G>C (Gly256Ala), 1559A>C (His520Pro), and 1679T>C (Val560Ala). Allelic frequencies occurred to be ethnicity-dependent, with the latter observed only in African-Americans (3.6%). After expression in HeLa cells, His520Pro, Val560Ala, and Met233Ile or Met233Ile_Ser112Ala haplotype variants showed decreased uptake activity compared with wild type for cholecystokinin-8 and rosuvastatin, but not for atorvastatin. Kinetic cholecystokinin-8 analysis showed reduced Vmax without altering Km. His520Pro and Val560Ala exhibited decreased total and plasma membrane protein expressions. Val560 mapped onto a structural model of OATP1B3 showed that this is a key region for substrate-transporter interaction. His520 resides in a predicted extracellular region thought to be critical to the pH-dependent component of OATP1B3 activity. Loss of activity at pH 7.4 and 8.0 relative to pH 6.5 was significantly greater for the Pro520 variant. CONCLUSION: OATP1B3 polymorphisms that result in altered expression, substrate specificity, and pH-dependent activity may be of potential relevance to hepatic clearance of substrate drugs in vivo.