The d-galactose (d-gal)-injected animal model, which is typically established by administering consecutive subcutaneous d-gal injections to animals for approximately six or eight weeks, has been frequently used for aging research. In addition, this animal model has been demonstrated to accelerate aging in the brain, kidneys, liver and blood cells. However, studies on aging in male reproductive organs that have used this animal model remain few. Therefore, the current study aimed to optimize a model of male reproductive aging by administering d-gal injections to male mice and to determine the possible mechanism expediting senescence processes during spermatogenesis. In this study, C57Bl/6 mice were randomized into five groups (each containing 8–10 mice according to the daily intraperitoneal injection of vehicle control or 100 or 200 mg/kg dosages of d-gal for a period of six or eight weeks). First, mice subjected to d-gal injections for six or eight weeks demonstrated considerably decreased superoxide dismutase activity in the serum and testis lysates compared to those in the control group. The lipid peroxidation in testis also increased in the d-gal-injected groups. Furthermore, the d-gal-injected groups exhibited a decreased ratio of testis weight/body weight and sperm count compared to the control group. The percentages of both immotile sperm and abnormal sperm increased considerably in the d-gal-injected groups compared to those of the control group. To determine the genes influenced by the d-gal injection during murine spermatogenesis, a c-DNA microarray was conducted to compare testicular RNA samples between the treated groups and the control group. The d-gal-injected groups exhibited RNA transcripts of nine spermatogenesis-related genes (Cycl2, Hk1, Pltp, Utp3, Cabyr, Zpbp2, Speer2, Csnka2ip and Katnb1) that were up- or down-regulated by at least two-fold compared to the control group. Several of these genes are critical for forming sperm-head morphologies or maintaining nuclear integration (e.g., cylicin, basic protein of sperm head cytoskeleton 2 (Cylc2), casein kinase 2, alpha prime interacting protein (Csnka2ip) and katanin p80 (WD40-containing) subunit B1 (Katnb1)). These results indicate that d-gal-injected mice are suitable for investigating male reproductive aging.

According to recent estimates, 2%–15% of couples are sterile, and approximately half of the infertility cases are attributed to male reproductive factors. However, the reasons remain undefined in approximately 25% of male infertility cases, and most infertility cases exhibit spermatogenic defects. Numerous genes involved in spermatogenesis still remain unknown. We previously identified Male Germ Cells Rab GTPase-Activating Proteins (MGCRABGAPs) through cDNA microarray analysis of human testicular tissues with spermatogenic defects. MGCRABGAP contains a conserved RABGAP catalytic domain, TBC (Tre2/Bub2/Cdc16). RABGAP family proteins regulate cellular function (e.g., cytoskeletal remodeling, vesicular trafficking, and cell migration) by inactivating RAB proteins. MGCRABGAP is a male germ cell-specific protein expressed in elongating and elongated spermatids during mammalian spermiogenesis. The purpose of this study was to identify proteins that interact with MGCRABGAP during mammalian spermiogenesis using a proteomic approach. We found that MGCRABGAP exhibited GTPase-activating bioability, and several MGCRABGAP interactors, possible substrates (e.g., RAB10, RAB5C, and RAP1), were identified using co-immunoprecipitation (co-IP) and nano liquid chromatography-mass spectrometry/mass spectrometry (nano LC-MS/MS). We confirmed the binding ability between RAB10 and MGCRABGAP via co-IP. Additionally, MGCRABGAP–RAB10 complexes were specifically colocalized in the manchette structure, a critical structure for the formation of spermatid heads, and were slightly expressed at the midpiece of mature spermatozoa. Based on these results, we propose that MGCRABGAP is involved in mammalian spermiogenesis by modulating RAB10.

Solute carrier family 9 isoform 3 (SLC9A3), a Na⁺/H⁺ exchanger, regulates the transepithelial absorption of Na⁺ and water and is primarily expressed on the apical membranes of the intestinal epithelium, renal proximal tubule, epididymis, and vas deferens. Loss of the Slc9a3 allele in mice enhances intestinal fluid and causes diarrhoea as a consequence of diminished Na⁺ and HCO₃⁻ absorption. Hence, the loss also causes male infertility and reveals the abnormal dilated lumen of the rete testis and calcification in efferent ductules. However, whether loss of Slc9a3 alleles also disrupts mammalian spermatogenesis remains unknown. First, through immunoblotting, we determined that SLC9A3 is highly expressed in the murine testis compared with the small intestine, epididymis, and vas deferens. During murine spermatogenesis, SLC9A3 is specifically expressed in the acrosome region of round, elongating, and elongated spermatids through immunostaining. Furthermore, SLC9A3 signals are enriched in the acrosome of mature sperm isolated from the vas deferens. In Slc9a3 knockout (KO) mice, compared with the same-aged controls, the number of spermatids on the testicular section of the mice progressively worsened in mice aged 20, 35, and 60 days. Sperm isolated from the epididymis of Slc9a3 KO mice revealed severe acrosomal defects. Our data indicated that SLC9A3 has a vital role in acrosomal formation during spermiogenesis.

The main objective of this study was to evaluate the potential genetic effects of SEPT14 on male infertility through sequencing the SEPT14 coding region. To address this research gap, 254 men with sperm abnormalities and 116 normozoospermic men were recruited, and the whole-coding regions of SEPT14 were sequenced. Two heterozygous mutations, p.Ala123Thr (3/254 vs. 0/116) and p.Ile333Thr (3/254 vs. 0/116), were identified in these cases. A high percentage of defective sperm heads was found in sperm with mutated SEPT14. Both mutations are highly evolutionarily conserved among vertebrates. The results of a fine morphological and chromatin structural analysis indicated severely malformed sperm heads with abnormal chromatin packaging through transmission electron microscopy and Toluidine blue staining. Compared with controls, high DNA fragmentation was demonstrated in sperm from cases carrying SEPT14 mutations using the comet assay. In addition, these two mutations in SEPT14 affected its polymerization ability in vitro. These data revels that the two SEPT14 missense mutations impaired sperm head morphology and induced DNA damage. Our study suggests that genetic variant of SEPT14 is one of the effects for human sperm formation and male fertility.

Male infertility is observed in approximately 50% of all couples with infertility. Intracytoplasmic sperm injection (ICSI), a conventional artificial reproductive technique for treating male infertility, may fail because of a severe low sperm count, immotile sperm, immature sperm, and sperm with structural defects and DNA damage. Our previous studies have revealed that mutations in the septin (SEPT)-coding gene SEPT12 cause teratozoospermia and severe oligozoospermia. These spermatozoa exhibit morphological defects in the head and tail, premature chromosomal condensation, and nuclear damage. Sperm from Sept12 knockout mice also cause the developmental arrest of preimplantation embryos generated through in vitro fertilization and ICSI. Furthermore, we found that SEPT12 interacts with SPAG4, a spermatid nuclear membrane protein that is also named SUN4. Loss of the Spag4 allele in mice also disrupts the integration nuclear envelope and reveals sperm head defects. However, whether SEPT12 affects SPAG4 during mammalian spermiogenesis remains unclear. We thus conducted this study to explore this question. First, we found that SPAG4 and SEPT12 exhibited similar localizations in the postacrosomal region of elongating spermatids and at the neck of mature sperm through isolated murine male germ cells. Second, SEPT12 expression altered the nuclear membrane localization of SPAG4, as observed through confocal microscopy, in a human testicular cancer cell line. Third, SEPT12 expression also altered the localizations of nuclear membrane proteins: LAMINA/C in the cells. This effect was specifically due to the expression of SEPT12 and not that of SEPT1, SEPT6, SEPT7, or SEPT11. Based on these results, we suggest that SEPT12 is among the moderators of SPAG4/LAMIN complexes and is involved in the morphological formation of sperm during mammalian spermiogenesis.

Septin (SEPT) genes encode well-preserved polymerizing GTP-binding cytoskeletal proteins. The cellular functions of SEPTs consist of mitosis, cytoskeletal remodeling, cell polarity, and vesicle trafficking through interactions with various types of cytoskeletons. We discovered that mutated SEPTIN12 in different codons resulted in teratozoospermia or oligozoospermia. In mouse models with a defective Septin12 allele, sperm morphology was abnormal, sperm count decreased, and sperms were immotile. However, the regulators of SEPT12 are completely unknown. Some studies have indicated that CDC42 negatively regulates the polymerization of SEPT2/6/7 complexes in mammalian cell lines. In this study, we investigated whether CDC42 modulates SEPT12 polymerization and is involved in the terminal differentiation of male germ cells. First, through scanning electron microscopy analysis, we determined that the loss of Septin12 caused defective sperm heads. This indicated that Septin12 is critical for the formation of sperm heads. Second, CDC42 and SEPT12 were similarly localized in the perinuclear regions of the manchette at the head of elongating spermatids, neck region of elongated spermatids, and midpiece of mature spermatozoa. Third, wild-type CDC42 and CDC42Q61L (a constitutive-acting-mutant) substantially repressed SEPT12 polymerization, but CDC42T17N (a dominant-negative-acting mutant) did not, as evident through ectopic expression analysis. We concluded that CDC42 negatively regulates SEPT12 polymerization and is involved in terminal structure formation of sperm heads.