Mouse DCUN1D1 (SCCRO) is required for spermatogenetic individualization.

Squamous cell carcinoma-related oncogene (SCCRO, also known as DCUN1D1) is a component of the E3 for neddylation. As such, DCUN1D1 regulates the neddylation of cullin family members. Targeted inactivation of DCUN1D1 in mice results in male-specific infertility. Infertility in DCUN1D1-/- mice is secondary to primary defects in spermatogenesis. Time-dam experiments mapped the onset of the defect in spermatogenesis to 5.5 to 6 weeks of age, which temporally corresponds to defects in spermiogenesis. Although the first round of spermatogenesis progressed normally, the number of spermatozoa released into the seminiferous lumen and epididymis of DCUN1D1-/- mice was significantly reduced. Spermatozoa in DCUN1D1-/- mice had multiple abnormalities, including globozoospermia, macrocephaly, and multiple flagella. Many of the malformed spermatozoa in DCUN1D1-/- mice were multinucleated, with supernumerary and malpositioned centrioles, suggesting a defect in the resolution of intercellular bridges. The onset of the defect in spermatogenesis in DCUN1D1-/- mice corresponds to an increase in DCUN1D1 expression observed during normal spermatogenesis. Moreover, consistent with its known function as a component of the E3 in neddylation, the pattern of DCUN1D1 expression temporally correlates with an increase in the neddylated cullin fraction and stage-specific increases in the total ubiquitinated protein pool in wild-type mice. Levels of neddylated Cul3 were decreased in DCUN1D1-/- mice, and ubiquitinated proteins did not accumulate during the stages in which DCUN1D1 expression peaks during spermatogenesis in wild-type mice. Combined, these findings suggest that DCUN1D1-/- mice fail to release mature spermatozoa into the seminiferous lumen, possibly due to unresolved intercellular bridges. Furthermore, the effects of DCUN1D1 on spermatogenesis likely involve its regulation of cullin-RING-ligase (CRL)-type ubiquitin E3 activity during spermiogenesis through its role in promoting Cul3 neddylation. The specific CRLs required for spermiogenesis and their protein targets require identification.

Septins at the annulus of mammalian sperm.

The annulus is an electron-dense ring structure connecting the midpiece and the principal piece of the mammalian sperm flagellum. Proteins from the septin family have been shown to localize to the annulus. A septin complex is assembled early in spermiogenesis with the cochaperone DNAJB13 and, in mature sperm, associates with Testis Anion Transporter 1; SLC26A8 (Tat1), a transmembrane protein of the SLC26 family. Studies in mice have shown that the annulus acts as a barrier to protein diffusion and controls correct organization of the midpiece. Consistent with these findings, absence of the annulus is associated with flagellum differentiation defects and asthenozoospermia in humans.

The annulus of the mouse sperm tail is required to establish a membrane diffusion barrier that is engaged during the late steps of spermiogenesis.

The annulus is a higher order septin cytoskeletal structure located between the midpiece and principal piece regions of the sperm tail. The annulus has been hypothesized to generate the diffusion barrier that exists between these two membrane domains. We tested this premise directly on septin 4 knockout mice, whose sperm are viable but lack an annulus, by following the diffusing membrane protein basigin. Basigin is normally confined to the principal piece domain on testicular and caput sperm, but undergoes relocation into the midpiece during sperm epididymal transit. On Sept4(-/-) sperm, domain confinement was lost, and basigin localized over the entire plasma membrane. Both immunofluorescence and immunoblotting further revealed reduced levels of basigin expression on sperm from the knockout. Testicular immunohistochemistry showed similar basigin expression and tail targeting in wild-type (WT) and Sept4(-/-) tubules until step 15 of spermatid development, at which point basigin was redistributed throughout the plasma membrane of Sept4(-/-) spermatids. The basigin outside of the tail was subsequently lost around the time of sperm release into the lumen. The redistribution in the knockout coincides with the time in WT sperm when the annulus completes its migration from the neck down to the midpiece-principal piece junction. We posit that basigin may not diffuse freely until after the annulus arrives at the midpiece-principal piece junction to restrict lateral movement. These results are the strongest evidence to date of a mammalian septin structure establishing a membrane diffusion barrier.

Cyclic 3',5'-AMP causes ADAM1/ADAM2 to rapidly diffuse within the plasma membrane of guinea pig sperm.

Because sperm cannot synthesize new proteins as they journey to the egg, they use multiple mechanisms to modify the activity of existing proteins, including changes in the diffusion coefficient of some membrane proteins. Previously, we showed that during capacitation the guinea pig heterodimeric membrane protein ADAM1/ADAM2 (fertilin) transforms from a stationary state to one of rapid diffusion within the lipid bilayer. The cause for this biophysical change, however, was unknown. In this study we examined whether an increase in cAMP, such as occurs during capacitation, could trigger this change. We incubated guinea pig cauda sperm with the membrane-permeable cAMP analog dibutyryl cAMP (db-cAMP) and the phosphodiesterase inhibitor papaverine and first tested for indications of capacitation. We observed hypermotility and acrosome-reaction competence. We then used fluorescence redistribution after photobleaching (FRAP) to measure the lateral mobility of ADAM1/ADAM2 after the db-cAMP treatment. We observed that db-cAMP caused roughly a 12-fold increase in lateral mobility of ADAM1/ADAM2, yielding diffusion similar to that observed for sperm capacitated in vitro. When we repeated the FRAP on testicular sperm incubated in db-cAMP, we found only a modest increase in lateral mobility of ADAM1/ADAM2, which underwent little redistribution. Interestingly, testicular sperm also cannot be induced to undergo capacitation. Together, the data suggest that the release of ADAM1/ADAM2 from its diffusion constraints results from a cAMP-induced signaling pathway that, like others of capacitation, is established during epididymal sperm maturation.

GM1 dynamics as a marker for membrane changes associated with the process of capacitation in murine and bovine spermatozoa.

We previously showed that in live murine and bovine sperm heads, the ganglioside G(M1) localizes to the sterol-rich plasma membrane overlying the acrosome (APM). Labeling G(M1) using the pentameric cholera toxin subunit B (CTB) induced a dramatic redistribution of signal from the APM to the sterol-poor postacrosomal plasma membrane (PAPM) upon sperm death. We now show a similar phenomenon in the flagellum where CTB induces G(M1) redistribution to sterol-poor membrane subdomains of the annulus and flagellar zipper. Because sterol efflux from the plasma membrane is required for capacitation, we examined whether G(M1) localization might be useful to detect membrane changes associated with capacitation and/or acrosomal exocytosis. First, incubation of murine and bovine sperm with their respective stimuli for capacitation did not change G(M1) distribution in live cells. However, incubation of sperm of both species with specific stimuli for capacitation, followed by the use of specific fixation conditions, induced reproducible, stimulus-specific patterns of G(M1) distribution. By assessing changes in G(M1) distribution in response to progesterone-induced AE, we show that these patterns reflect the response of murine sperm populations to capacitating stimuli. These data suggest that G(M1) localization can be used as a diagnostic tool for evaluating sperm response to stimuli for capacitation and/or AE. Such information could be useful when deciding between technologies of assisted reproduction or when screening for male fertility. Furthermore, stimulus-specific changes in G(M1) distribution showed that sperm could respond to NaHCO(3) or mediators of sterol efflux independently, thereby refining existing models of capacitation.

Visualization of GM1 with cholera toxin B in live epididymal versus ejaculated bull, mouse, and human spermatozoa.

The organization of membrane subdomains in mammalian sperm has recently generated controversy, with several reports describing widely differing localization patterns for the ganglioside GM1. Using the pentameric B subunit of cholera toxin (CTB), we found GM1 to be restricted to the plasma membrane overlying the acrosome in the heads of live murine sperm. Interestingly, CTB had minimal binding to live bovine and human sperm. To investigate whether this difference in GM1 localization was because of species differences or differences between collection from the epididymis (mouse) or an ejaculate (bull, human), we examined epididymal bovine and human sperm. We found that GM1 localized to the plasma membrane overlying the acrosome in sperm from these species. To determine whether some component of seminal plasma was interfering with the ability of CTB to access GM1, we incubated epididymal mouse sperm with fluid from murine seminal vesicles and epididymal bull sperm with bovine seminal plasma. This treatment largely abolished the ability of the CTB to bind to GM1, producing a fluorescence pattern similar to that reported for the human. The most abundant seminal plasma protein, PDC-109, was not responsible for this loss. As demonstration that the seminal plasma was not removing GM1, sperm exposed to seminal plasma were fixed before CTB addition, and again displayed fluorescence over the acrosome. These observations reconcile inconsistencies reported for the localization of GM1 in sperm of different species, and provide evidence for the segregation of GM1 to a stable subdomain in the plasma membrane overlying the acrosome.

Immune complex deposition in adult male Sprague-Dawley rats chronically immunized with GnRH.

PROBLEM: This study was undertaken to evaluate whether the anti-GnRH antibodies and immune complexes (IC) generated by immunization with GnRH-TT cause cellular damage within the animal. METHOD OF STUDY: Chronic immunization of rats with GnRH-TT injected i.m. was followed by tissue/organ analysis for immune complex deposition by immunofluorescence microscopy. Two groups were studied: (1) those immunized throughout the experiment until their ultimate demise, and (2) those given a chance to recover from the effects of chronic immunization before final analysis. RESULTS: GnRH-TT was effective in stopping spermatogenesis, which resumed after withdrawal of the immunogen. Most tissues from chronically immunized animals were not significantly different than controls, however the kidneys of treated animals exhibited a higher accumulation of IC. Despite increased IC deposition, pathologic effects were not detected at the cellular level. CONCLUSIONS: GnRH-TT is an effective immunocontraceptive although the accumulation of glomerular IC represents a potential deleterious side effect.

The Sept4 septin locus is required for sperm terminal differentiation in mice.

The murine septin4 gene (Sept4) has been implicated in diverse cellular functions, including cytokinesis, apoptosis, and tumor suppression. Here, we investigated the function of Sept4 proteins during mouse development by creating a targeted deletion of the Sept4 genomic locus. Sept4 mutant mice are viable but male sterile due to immotile and structurally defective sperm. During spermatogenesis, Sept4 proteins were essential for proper mitochondrial architecture and establishment of the annulus, a ring-like structure in the tail region of sperm. In addition, Sept4 mutant sperm showed defects in the elimination of residual cytoplasm during sperm maturation and had increased staining for the caspase inhibitor XIAP. This is consistent with a role of the proapoptotic Sept4 protein ARTS in promoting caspase-mediated removal of cytoplasm via inhibition of XIAP. Our results indicate that Sept4 proteins play distinct but evolutionarily conserved functions in different cellular compartments.