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The acrosome plays a critical role in sperm-oocyte interactions during fertilization. SP-10 is an acrosomal matrix protein, which is evolutionarily conserved among mammals. The SP-10 antibody has been shown to be useful for staging the seminiferous cycle in the mouse and human. A canonical acrosomal marker; however, has never been used for staging in the horse. The objectives of the present study were to investigate the presence of SP-10 within the horse acrosome using an anti-mouse SP-10 antibody, to classify spermatids based on the shape of the acrosome, and then to use that information to assign stages of the cycle of the seminiferous epithelium. Testes from mature stallions with history of normospermic ejaculates were used for immunohistochemistry. We found that the mouse SP-10 antibody stained the horse acrosome vividly in testis cross-sections, indicating evolutionary conservation. Previous methods based on morphology alone without the aid of an antibody marker showed 8 stages in the horse seminiferous epithelium. Morphological detail of the acrosome afforded by the SP-10 marker in this study identified 16 steps of spermatids. This, in turn, led to the identification of 12 distinct stages in the cycle of the seminiferous epithelium of the horse wherein stage I shows recently formed round spermatids and stage XII includes meiotic divisions; a classification that is consistent with other animal models. The SP-10 antibody marks the acrosome in a way that enables researchers in the field to identify stages of spermatogenesis in the horse easily. In conclusion, we demonstrated that immunolabeling for SP-10 can be an objective approach to stage the cycle of the seminiferous epithelium in normospermic stallions; future studies will determine if SP-10 could be used to assess testicular dysfunction.

Loss-of-function mutations in multiple morphological abnormalities of the sperm flagella (MMAF)-associated genes lead to decreased sperm motility and impaired male fertility. As an MMAF gene, the function of fibrous sheath-interacting protein 2 (FSIP2) remains largely unknown. In this work, we identified a homozygous truncating mutation of FSIP2 in an infertile patient. Accordingly, we constructed a knock-in (KI) mouse model with this mutation. In parallel, we established an Fsip2 overexpression (OE) mouse model. Remarkably, KI mice presented with the typical MMAF phenotype, whereas OE mice showed no gross anomaly except for sperm tails with increased length. Single-cell RNA sequencing of the testes uncovered altered expression of genes related to sperm flagellum, acrosomal vesicle and spermatid development. We confirmed the expression of Fsip2 at the acrosome and the physical interaction of this gene with Acrv1, an acrosomal marker. Proteomic analysis of the testes revealed changes in proteins sited at the fibrous sheath, mitochondrial sheath and acrosomal vesicle. We also pinpointed the crucial motifs of Fsip2 that are evolutionarily conserved in species with internal fertilization. Thus, this work reveals the dosage-dependent roles of Fsip2 in sperm tail and acrosome formation.

Enhancers are cis-elements that activate transcription and play critical roles in tissue- and cell type-specific gene expression. During spermatogenesis, genes coding for specialized sperm structures are expressed in a developmental stage- and cell type-specific manner, but the enhancers responsible for their expression have not been identified. Using the mouse acrosomal vesicle protein (Acrv1) gene that codes for the acrosomal protein SP-10 as a model, our previous studies have shown that Acrv1 proximal promoter activates transcription in spermatids; and the goal of the present study was to separate the enhancer responsible. Transgenic mice showed that three copies of the −186/−135 fragment (50 bp enhancer) placed upstream of the Acrv1 core promoter (−91/+28) activated reporter expression in testis but not somatic tissues (n = 4). Immunohistochemistry showed that enhancer activity was restricted to the round spermatids. The Acrv1 enhancer failed to activate transcription in the context of a heterologous core promoter (n = 4), indicating a likely requirement for enhancer-core promoter compatibility. Chromatin accessibility assays showed that the Acrv1 enhancer assumes a nucleosome-free state in male germ cells (but not liver), indicating occupancy by transcription factors. Southwestern assays (SWA) identified specific binding of the enhancer to a testis nuclear protein of 47 kDa (TNP47). TNP47 was predominantly nuclear and becomes abundant during the haploid phase of spermatogenesis. Two-dimensional SWA revealed the isoelectric point of TNP47 to be 5.2. Taken together, this study delineated a 50-bp enhancer of the Acrv1 gene for round spermatid-specific transcription and identified a putative cognate factor. The 50-bp enhancer could become useful for delivery of proteins into spermatids.

The study of spermatogenesis in the mouse requires accurate identification of the cycle stage of seminiferous epithelium. A stage refers to the unique association of germ-cell types at a particular phase of development, as seen in a testicular cross section. The process of staging, however, is a daunting task. There are 12 stages represented in the mouse seminiferous tubule. Stages are typically identified on the basis of the morphology of the developing acrosome of spermatids. Although the characteristic features of the acrosome are well-documented in ultrastructure images, a reagent that can highlight the subtle differences in acrosome shape under the light microscope is lacking. Here we demonstrate that a polyclonal antibody raised against the mouse acrosomal protein SP-10 is extremely useful for stage identification. Immunohistochemistry showed that the anti-SP-10 antibody is highly specific for the acrosome of spermatids, as no other cell type in the epithelium showed immunoreactivity. At lower magnification, the gross shape of the acrosome and the increasing intensity of immunostaining served as a guide for the identification of stages 1–12. At higher magnification, characteristic morphological features –such as whether the part of the acrosome that contacts the nuclear surface is round (stage 3) or flat (stage 4) or curved (stage 6)– could be identified unambiguously. Overall, we present evidence that SP-10 is a useful marker for staging the cycle of a seminiferous epithelium. The anti-SP-10 antibody works well in different fixatives, on paraffin-embedded as well as cryosections; it has also been shown to be useful for characterizing spermatogenic defects in mutant mice.

To identify the developmental expression of the ACRV1 gene in humans and mice, testes cDNA samples were collected at different post-natal days (days 4, 9, 18, 35, 54, and 6 months) from Balb/c mice and were hybridised to the mouse whole genome 430 2.0 Array (Affymetrix Inc.) chip. The characteristics of ACRV1 were analysed using various cellular and molecular biotechnologies. The results showed that the expression of mouse ACRV1 was not detected in mouse testes on days 4, 9, and 18 but was present on days 35, 54, and 6 months. Using RT-PCR analysis of mouse ACRV1, we determined that mouse ACRV1 was expressed specifically in the mouse testis, and its expression began at days 35. Western blot analysis demonstrated that human ACRV1 was primarily expressed in human testes, and immunofluorescent and immunohistochemistry staining showed that human ACRV1 protein was predominantly located in round and elongated spermatids in human testes, indicating that ACRV1 may play an important role in mammalian spermatogenesis and may be a target of a contraceptive vaccine.