Cloning and partial characterization of the cDNA encoding the fox sperm protein FSA-Acr.1 with similarities to the SP-10 antigen

We have isolated and characterized a cDNA, cFSA-Acr.1, encoding a testis-specific fox sperm antigen. The antigen is located on the inner acrosomal compartment, and is expressed during spermatogenesis on the developing acrosome of round and elongating spermatids. Database searches with the deduced amino acid sequence of cFSA-Acr.1 revealed that the clone has high homology to both human and baboon sperm protein SP-10, and the mouse sperm protein, MSA-63. The region of highest homology is within the carboxyl terminus. In the middle of the open reading frame, the fox sequence shows unique sequences absent from both the human, baboon SP-10, and mouse MSA-63 sequences. In addition to cFSA-Acr.1, two other clones were also isolated from the same fox testis cDNA library, and sequence analysis shows that they may represent alternatively spliced mRNAs coding for other FSA-Acr proteins. © 1995 Wiley-Liss, Inc.     

A testis-specific gene encoding a nuclear high-mobility-group box protein located in elongating spermatids.

A cDNA encoding a DNA-binding protein has been isolated by screening a mouse testicular expression cDNA library with a concatemer of a 12-bp putative protein-binding element present in the promoter of the testis-specific gene PGK-2. Sequence analysis of the isolated cDNA indicated the presence of an open reading frame that encodes a protein with two conserved DNA-binding motifs known as the high-mobility-group (HMG) boxes. Northern (RNA) blot analysis demonstrated that expression of the gene is restricted to the postpuberal testis. The DNA-binding activity and sequence specificity of the recombinant HMG protein were confirmed by DNA mobility shift assay using the initial concatemer of the PGK-2 promoter element as a probe as well as the wild-type or mutated versions of the 12-bp element within its natural sequence context. Immunocytochemical staining of adult testis sections with polyclonal antisera recognizing this recombinant HMG protein demonstrated that it is located predominantly in the nuclei of elongated spermatids at steps 9 and 10. These results suggest that this novel HMG box protein gene may be involved in the regulation of gene expression of the haploid male genome. The gene from which the cDNA was derived has been termed testis-specific HMG (tsHMG).     

Equatorial segment protein defines a discrete acrosomal subcompartment persisting throughout acrosomal biogenesis

The equatorial segment of the acrosome underlies the domain of the sperm that fuses with the egg membrane during fertilization. Equatorial segment protein (ESP), a novel 349-amino acid concanavalin-A-binding protein encoded by a two-exon gene (SP-ESP) located on chromosome 15 at q22, has been localized to the equatorial segment of ejaculated human sperm. Light microscopic immunofluorescent observations revealed that during acrosome biogenesis ESP first appears in the nascent acrosomal vesicle in early round spermatids and subsequently segregates to the periphery of the expanding acrosomal vesicle, thereby defining a peripheral equatorial segment compartment within flattened acrosomal vesicles and in the acrosomes of early and late cap phase, elongating, and mature spermatids. Electron microscopic examination revealed that ESP segregates to an electron-lucent subdomain of the condensing acrosomal matrix in Golgi phase round spermatids and persists in a similar electron-lucent subdomain within cap phase spermatids. Subsequently, ESP was localized to electron-dense regions of the equatorial segment and the expanded equatorial bulb in elongating spermatids and mature sperm. ESP is the earliest known protein to be recognized as a marker for the specification of the equatorial segment, and it allows this region to be traced through all phases of acrosomal biogenesis. Based on these observations, we propose a new model of acrosome biogenesis in which the equatorial segment is defined as a discrete domain within the acrosomal vesicle as early as the Golgi phase of acrosome biogenesis.     

Integration of the mouse sperm fertilization-related protein equatorin into the acrosome during spermatogenesis as revealed by super-resolution and immunoelectron microscopy

Spermatids must precisely integrate specific molecules into structurally supported domains that develop during spermatogenesis. Once established, the architecture of the acrosome contributes to the acrosome reaction, which occurs prior to gamete interaction in mammals. The present study aims to clarify the morphology associated with the integration of the mouse fertilization-related acrosomal protein equatorin (mEQT) into the developing acrosome. EQT mRNA was first detected by in situ hybridization in round spermatids but disappeared in early elongating spermatids. The molecular size of mEQT was approximately 65 kDa in the testis. Developmentally, EQT protein was first detected on the nascent acrosomal membrane in round spermatids at approximately step 3, was actively integrated into the acrosomal membranes of round spermatids in the following step and then participated in acrosome remodeling in elongating spermatids. This process was clearly visualized by high-resolution fluorescence microscopy and super-resolution stimulated emission depletion nanoscopy by using newly generated C-terminally green-fluorescent-protein-tagged mEQT transgenic mice. Immunogold electron microscopy revealed that mEQT was anchored to the acrosomal membrane, with the epitope region observed as lying 5–70 nm away from the membrane and was associated with the electron-dense acrosomal matrix. This new information about the process of mEQT integration into the acrosome during spermatogenesis should provide a better understanding of the mechanisms underlying not only acrosome biogenesis but also fertilization and male infertility.