SPATC1L maintains the integrity of the sperm head‐tail junction

Spermatogenesis is a tightly regulated process involving germ cell‐specific and germ cell‐predominant genes. Here we investigate a novel germ cell‐specific gene, Spatc1l (spermatogenesis and centriole associated 1 like). Expression analyses show that SPATC1L is expressed in mouse and human testes. We find that mouse SPATC1L localizes to the neck region in testicular sperm. Moreover, SPATC1L associates with the regulatory subunit of protein kinase A (PKA). Using CRISPR/Cas9‐mediated genome engineering, we generate mice lacking SPATC1L. Disruption of Spatc1l in mice leads to male sterility owing to separation of sperm heads from tails. The lack of SPATC1L is associated with a reduction in PKA activity in testicular sperm, and we identify capping protein muscle Z‐line beta as a candidate target of phosphorylation by PKA in testis. Taken together, our results implicate the SPATC1L‐PKA complex in maintaining the stability of the sperm head‐tail junction, thereby revealing a new molecular basis for sperm head‐tail integrity.     

Characterization of baboon testicular antigens using monoclonal anti-sperm antibodies

Sperm antigens that appear during spermatogenesis in the baboon were identified by using three monoclonal antibodies generated in culture from mice immunized with baboon caudal epididymal spermatozoa. Antibodies BSA1 and BSA2 recognize trypsin-sensitive 84,000 and 45,000 dalton determinants that are restricted to the tail and anterior acrosomal regions of the sperm, respectively, as determined by Western blot and immunofluorescence techniques. The tail antigen absent in 2- and 3-yr-old baboon testes first appears in spermatid cells at about 4 yr of age. In contrast, the acrosomal antigen recognized by BSA2 is present in 3-yr-old primitive testicular germ cells. In the mature testis, the 45,000 molecular weight determinant is predominantly localized in the nucleus of late pachytene spermatocytes and round spermatid cells as observed via the avidinbiotin immunoperoxidase method. Antibody BSA3 reacted only with sailidase-treated sections of adult testis. This trypsin-resistant determinant, not expressed on testicular sperm, is recognized by antibody BSA3 only on epididymal sperm, thus indicating a post-testicular sperm modification.     

Dynamic distribution of Spatial during mouse spermatogenesis and its interaction with the kinesin KIF17b

The Spatial gene is expressed in highly polarized cell types, such as epithelial cells in the thymus, neurons in the brain and germ cells in the testis. In this study, we report the characterization and distribution of Spatial proteins during mouse spermatogenesis. Besides Spatial-epsilon and -delta, we show that the newly described short isoform Spatial-beta is expressed specifically in round spermatids. Using indirect immunofluorescence, we detected Spatial in the cytosol of the early round spermatid. By the end stages of round spermatids, Spatial is concentrated at the opposite face of the acrosome near the nascent flagellum and in the manchette during the elongation process. Finally in mature sperm, Spatial persists in the principal piece of the tail. Moreover, we found that Spatial colocalizes with KIF17b, a testis-specific isoform of the brain kinesin-2 motor KIF17. This colocalization is restricted to the manchette and the principal piece of the sperm tail. Further, coimmunoprecipitation experiments of native proteins from testis lysates confirmed Spatial-KIF17b association through the long Spatial-epsilon isoform. Together, these findings imply a function of Spatial in spermatid differentiation as a new cargo of kinesin KIF17b, in a microtubule-dependent mechanism specific to the manchette and the principal piece of the sperm tail.     

Functional role of GKAP1 in the regulation of male germ cell spontaneous apoptosis and sperm number

G kinase‐anchoring protein 1 (GKAP1) is a G kinase‐associated protein that is conserved in many eutherians and is mainly expressed in the testis, especially in spermatocytes and round spermatids. The function of GKAP1 in the testis is largely unknown. Here, we revealed that deletion of GKAP1 led to an increase in sperm production with swollen epididymis, and germ cell apoptosis was found to decrease in GKAP1 knock‐out mice. Further investigations showed that a deficiency of GKAP1 could partly change the cellular location of cGK‐Iα and increase the amount of active cAMP response element‐binding protein (CREB) in the nucleus. Therefore, the expression of a particular inhibitor of apoptosis proteins (IAPs) was upregulated because of the activation of CREB, and this increase in IAPs was associated with a decrease in the level of activated caspase‐3. These results suggest that a deficiency of GKAP1 in mouse testis could increase sperm production through a reduction of the spontaneous apoptosis of germ cells in the testis, possibly because of a change in the activity of the cGK‐Iα pathway.     

Germ cell transplantation and testis tissue xenografting in domestic animals

Transplantation of germ cells from fertile donor mice to the testes of infertile recipient mice results in donor-derived spermatogenesis and transmission of the donor's genetic material to the offspring of recipient animals. Germ cell transplantation provides a bioassay to study the biology of male germ line stem cells, develop systems to isolate and culture spermatogonial stem cells, examine defects in spermatogenesis and treat male infertility. Although most widely studied in rodents, germ cell transplantation has been applied to larger mammals. In domestic animals including pigs, goats and cattle, as well as in primates, germ cells can be transplanted to a recipient testis by ultrasonographic-guided cannulation of the rete testis. Germ cell transplantation was successful between unrelated, immuno-competent pigs and goats, whereas transplantation in rodents requires syngeneic or immuno-compromised recipients. Genetic manipulation of isolated germ line stem cells and subsequent transplantation will result in the production of transgenic sperm. Transgenesis through the male germ line has tremendous potential in domestic animal species where embryonic stem cell technology is not available and current options to generate transgenic animals are inefficient. As an alternative to transplantation of isolated germ cells to a recipient testis, ectopic grafting of testis tissue from diverse mammalian donor species, including horses and primates, into a mouse host represents a novel possibility to study spermatogenesis, to investigate the effects of drugs with the potential to enhance or suppress male fertility, and to produce fertile sperm from immature donors. Therefore, transplantation of germ cells or xenografting of testis tissue are uniquely valuable approaches for the study, preservation and manipulation of male fertility in domestic animals.     

Novel alternatively spliced mRNA (1c) of the protein kinase A RIα subunit is implicated in haploid germ cell specific expression

By using 5' RACE on rat testis cDNA we identified three alternatively spliced mRNAs of the RIalpha subunit of cAMP-dependent protein kinase that differed in their 5' untranslated regions. Two of these 5'-regions showed similarity with the human RIalpha exons 1a and 1b, while the third (1c) constituted a novel mRNA splice variant. Northern blot analysis showed that the 1c mRNA was specifically expressed in testis and only in postmeiotic germ cells. In contrast, the RIalpha 1b and RIalpha 1a mRNAs were present both in premeiotic germ cells and somatic cells of the testis, and the expression of both RIalpha 1a and 1b mRNAs were stimulated by cAMP in Sertoli cells. In sperm, the RIalpha protein was expressed after meiosis, and targeted to various subcellular structures via anchoring proteins. The RIalpha 1c haploid-specific mRNA, therefore, may be important for the regulation of RIalpha expression in sperm.     

Olfactory receptors are displayed on dog mature sperm cells

Olfactory receptors constitute a huge family of structurally related G protein-coupled receptors, with up to a thousand members expected. We have shown previously that genes belonging to this family were expressed in the male germ line from both dog and human. The functional significance of this unexpected site of expression was further investigated in the present study. We demonstrate that a few dog genes representative of various subfamilies of olfactory receptors are expressed essentially in testis, with little or no expression in olfactory mucosa. Other randomly selected members of the family show the expected site of expression, restricted to the olfactory system. Antibodies were generated against the deduced amino acid sequence of the most abundantly expressed olfactory receptor gene in dog testis. The purified serum was able to detect the gene product (DTMT receptor) in late round and elongated spermatids, as well as in the cytoplasmic droplet that characterizes the maturation of dog sperm cells, and on the tail midpiece of mature spermatozoa. Western blotting further confirmed the presence of a 40-kD immunoreactive protein in the membrane of mature sperm cells. Altogether , these results demonstrate that the main expression site of a subset of the large olfactory receptor gene family is not olfactory mucosa but testis. This expression correlates with the presence of the corresponding protein during sperm cell maturation, and on mature sperm cells. The pattern of expression is consistent with a role as sensor for unidentified chemicals possibly involved in the control of mammalian sperm maturation, migration, and/or fertilization.     

Impaired spermatogenesis and fertility in mice carrying a mutation in the Spink2 gene expressed predominantly in testes

Spermatogenesis is a complex process involving an intrinsic genetic program composed of germ cell-specific and -predominant genes. In this study, we investigated the mouse Spink2 (serine protease inhibitor Kazal-type 2) gene, which belongs to the SPINK family of proteins characterized by the presence of a Kazal-type serine protease inhibitor-pancreatic secretory trypsin inhibitor domain. We showed that recombinant mouse SPINK2 has trypsin-inhibitory activity. Distribution analyses revealed that Spink2 is transcribed strongly in the testis and weakly in the epididymis, but is not detected in other mouse tissues. Expression of Spink2 is specific to germ cells in the testis and is first evident at the pachytene spermatocyte stage. Immunoblot analyses demonstrated that SPINK2 protein is present in male germ cells at all developmental stages, including in testicular spermatogenic cells, testicular sperm, and mature sperm. To elucidate the functional role of SPINK2 in vivo, we generated mutant mice with diminished levels of SPINK2 using a gene trap mutagenesis approach. Mutant male mice exhibit significantly impaired fertility; further phenotypic analyses revealed that testicular integrity is disrupted, resulting in a reduction in sperm number. Moreover, we found that testes from mutant mice exhibit abnormal spermatogenesis and germ cell apoptosis accompanied by elevated serine protease activity. Our studies thus provide the first demonstration that SPINK2 is required for maintaining normal spermatogenesis and potentially regulates serine protease-mediated apoptosis in male germ cells.     

The testicular form of hormone-sensitive lipase HSLtes confers rescue of male infertility in HSL-deficient mice

Inactivation of the hormone-sensitive lipase gene (HSL) confers male sterility with a major defect in spermatogenesis. Several forms of HSL are expressed in testis. HSLtes mRNA and protein are found in early and elongated spermatids, respectively. The other forms are expressed in diploid germ cells and interstitial cells of the testis. To determine whether the absence of the testis-specific form of HSL, HSLtes, was responsible for the infertility in HSL-null mice, we generated transgenic mice expressing HSLtes under the control of its own promoter. The transgenic animals were crossed with HSL-null mice to produce mice deficient in HSL in nongonadal tissues but expressing HSLtes in haploid germ cells. Cholesteryl ester hydrolase activity was almost completely blunted in HSL-deficient testis. Mice with one allele of the transgene showed an increase in enzymatic activity and a small elevation in the production of spermatozoa. The few fertile hemizygous male mice produced litters of very small to small size. The presence of the two alleles led to a doubling in cholesteryl ester hydrolase activity, which represented 25% of the wild type values associated with a qualitatively normal spermatogenesis and a partial restoration of sperm reserves. The fertility of these mice was totally restored with normal litter sizes. In line with the importance of the esterase activity, HSLtes transgene expression reversed the cholesteryl ester accumulation observed in HSL-null mice. Therefore, expression of HSLtes and cognate cholesteryl ester hydrolase activity leads to a rescue of the infertility observed in HSL-deficient male mice.     

Increased levels of comet-detected spermatozoa DNA damage following in vivo isotopic- or X-irradiation of spermatogonia.

To investigate whether DNA damage arising in spermatogenic germ cells can be detected in resultant sperm, we have irradiated murine testis and collected spermatozoa from the vas deferens 45 days later. These cells were derived from spermatogonia present at the time of irradiation. Two forms of irradiation were used, external X-rays (4Gy) and internal auger electrons from contamination of the male mouse with the isotope Indium-114m (1.85MBq), which was localised in the testis. Both forms of irradiation produced a profound fall in vas deferens sperm count and testis weight, Indium-114m being more effective. Using the neutral Comet assay for double strand break detection, significant increases in sperm comet tail length and moment were observed. The levels of damage were similar for both treatments. Care had to be taken during the assay to distinguish between sperm and somatic cells as the proportion of the latter increased after irradiation. We conclude that the comet assay can detect DNA damage in spermatozoa after the in vivo exposure of male germ cells to a known testicular genotoxic agent. The assay may be useful for the assessment of sperm DNA damage (double stranded) associated with male infertility and post-fertilization developmental abnormalities in the offspring.     

Expression and function of the testis-predominant protein LYAR in mice

Mammalian spermatogenesis is a complex process involving an intrinsic genetic program of germ cell-specific and -predominant genes. In the present study, we analyzed the Ly-1 reactive clone (Lyar) gene in the mouse. Lyar, which is known to be expressed abundantly in the testis, encodes a nucleolar protein that contains a LYAR-type C2HC zinc finger motif and three nuclear localization signals. We herein confirmed that Lyar is expressed predominantly in the testis, and further showed that this expression is specific to germ cells. Protein analyses with an anti-LYAR antibody demonstrated that the LYAR protein is present in spermatocytes and spermatids, but not in sperm. To assess the functional role of LYAR in vivo, we used a genetrap mutagenesis approach to establish a LYAR-null mouse model. Lyar mutant mice were born live and developed normally. Male mutant mice lacking LYAR were fully fertile and showed intact spermatogenesis. Taken together, our results demonstrate that LYAR is strongly preferred in male germ cells, but has a dispensable role in spermatogenesis and fertility.     

三角帆蚌精子的发生

报道了光镜和透射电镜下三角帆蚌精子的发生过程及其一系列重要的形态变化。包括核延长,染色质浓缩,线粒体逐渐融合并后移;胞质减少及鞭毛形成,精原细胞是精巢中体积最大的细胞,细胞膜界限不明显,内质网发达,精母细胞开始出现中心粒,精细胞分化可分为3个阶段。成熟精子属原生型,由头部、中段和尾部三部分组成。     

Possible function of the ADAM1a/ADAM2 Fertilin complex in the appearance of ADAM3 on the sperm surface

In mouse, two different isoforms of ADAM1 (fertilin alpha), ADAM1a and ADAM1b, are produced in the testis. ADAM1a is localized within the endoplasmic reticulum of testicular germ cells, whereas epididymal sperm contain only ADAM1b on the plasma membrane. In this study, we show that the loss of ADAM1a results in the male infertility because of the severely impaired ability of sperm to migrate from the uterus into the oviduct through the uterotubal junction. However, epididymal sperm of ADAM1a-deficient mice were capable of fertilizing cumulus-intact, zona pellucida-intact eggs in vitro despite the delayed dispersal of cumulus cells and the reduced adhesion/binding to the zona pellucida. Among testis (sperm)-specific proteins examined, only the level of ADAM3 (cyritestin) was strongly reduced in ADAM1a-deficient mouse sperm. Moreover, the appearance of ADAM3 on the sperm surface was dependent on the formation of a fertilin protein complex between ADAM1a and ADAM2 (fertilin beta) in testicular germ cells, although no direct interaction between the fertilin complex and ADAM3 was found. These results suggest that ADAM1a/ADAM2 fertilin may be implicated in the selective transport of specific sperm proteins including ADAM3 from the endoplasmic reticulum of testicular germ cells onto the cell surface. These proteins then can participate in sperm migration into the oviduct, the dispersal of cumulus cells, and sperm binding to the zona pellucida.