Fumie Suzuki-Toyota

Immunohistochemical localization of a water channel, aquaporin 7 (AQP7), in the rat testis

Abstract Cell volume reduction is one of the most distinct morphological changes during spermiogenesis and may be largely attributable to water efflux from the cell. A strong candidate for a water efflux route, aquaporin 7 (AQP7), which is a water channel, was studied immunohistochemically in the rat testis. Immunoreactivity was restricted within the elongated spermatids, testicular spermatozoa, and residual bodies remaining in the seminiferous epithelium. Weak but distinct immunoreactivity was first observed in the cytoplasmic mass of the spermatid at step 8 of spermiogenesis. The Golgi-like apparatus became steadily immunoreactive at step 10. The plasma membrane covering the cytoplasmic mass showed strong immunoreactivity after step 16. At this step, the middle piece of the tail also showed immunoreactivity at the portion protruding into the lumen. The whole head and distal tail, where the elongated spermatid had only a limited amount of cytoplasm, showed no immunoreactivity throughout spermiogenesis. After spermiation, the immunoreactivity of AQP7 remained at the middle piece and in the cytoplasmic droplet in the testicular spermatozoon. The present observations suggest that AQP7 contributes to the volume reduction of spermatids, since this water channel protein is localized on the plasma membrane covering the condensing cytoplasmic mass of the elongated spermatid, and since the seminiferous tubule fluid is hypertonic.

Factors Maintaining Normal Sperm Tail Structure During Epididymal Maturation Studied in Gopc−/− Mice

Abstract Gopc (Golgi-associated PDZ- and coiled-coil motif-containing protein)−/− mice are infertile, showing globozoospermia, coiled tails, and a stratified mitochondrial sheath. Transmission electron microscope (TEM) images of the spermatozoa were studied quantitatively to analyze disorganization processes during epididymal passage. Factors maintaining straight tail and normal mitochondrial sheath were also studied by TEM and immunofluorescent microscopy. Sperm tails retained a normal appearance in the proximal caput epididymidis. Tail disorganization started between the proximal and the middle caput epididymidis, and the latter is the major site for it. The tail moved up through the defective posterior ring and coiled around the nucleus to various degrees. Tail coiling occurred in the caput epididymidis suggesting it was triggered by cytoplasmic droplet migration. SPATA19/spergen-1, a candidate mitochondrial adhesion protein, remained on the stratified mitochondria, while GPX4/PHGPx, a major element of the mitochondrial capsule, was unevenly distributed on them. From these findings, we speculate GPX4 is necessary to maintain normal sheath structure, and SPATA19 prevents dispersal of mitochondria, resulting in a stratified mitochondrial sheath formation in Gopc−/− spermatozoa. The epididymal epithelium was normal in structure and LRP8/apoER2 expression suggesting that tail abnormality is due to intrinsic sperm factors. Three cell structures are discussed as requisite factors for maintaining a straight tail during epididymal maturation: 1) a complete posterior ring to prevent invasion of the tail into the head compartment, 2) stable attachment of the connecting piece to the implantation fossa, and 3) a normal mitochondrial sheath supported by SPATA19 and supplied with sufficient and normally distributed GPX4.

The Equatorial Subsegment in Mammalian Spermatozoa Is Enriched in Tyrosine Phosphorylated Proteins

Abstract The equatorial subsegment (EqSS) was originally identified by atomic force microscopy as a discrete region within the equatorial segment of Artiodactyl spermatozoa. In this investigation, we show that the EqSS is enriched in tyrosine phosphorylated proteins and present preliminary evidence for its presence in mouse and rat spermatozoa. The anti-phosphotyrosine monoclonal antibody (McAb) 4G10 bound strongly and discretely to the EqSS of permeabilized boar, ram, and bull spermatozoa. It also bound to a small patch on the posterior acrosomal region of permeabilized mouse and rat spermatozoa, suggesting that the EqSS is not restricted to the order Artiodactyla. An anti-HSPA1A (formerly Hsp70) antibody recognized the EqSS in boar spermatozoa. Immunogold labeling with McAb 4G10 localized the tyrosine phosphorylated proteins to the outer acrosomal membrane. This was verified by freeze-fracture electron microscopy, which identified the EqSS in three overlying membranes, the plasma membrane, outer acrosomal membrane, and inner acrosomal membrane. In all five species, tyrosine phosphorylated proteins became restricted to the EqSS during sperm maturation in the epididymis. The major tyrosine phosphorylated proteins in the EqSS of boar and ram spermatozoa were identified by mass spectrometry as orthologs of human SPACA1 (formerly SAMP32). Immunofluorescence with a specific polyclonal antibody localized SPACA1 to the equatorial segment in boar spermatozoa. We speculate that the EqSS is an organizing center for assembly of multimolecular complexes that initiate fusion competence in this area of the plasma membrane following the acrosome reaction.

A model of the acrosome reaction progression via the acrosomal membrane-anchored protein equatorin

It is important to establish a reliable and progressive model of the acrosome reaction. Here, we present a progression model of the acrosome reaction centering around the acrosomal membrane-anchored protein equatorin (MN9), comparing the staining pattern traced by MN9 antibody immunofluorescence with that traced by Arachis hypogaea agglutinin (PNA)-FITC. Prior to the acrosome reaction, equatorin was present in both the anterior acrosome and the equatorial segment. Since sperm on zona pellucida showed various staining patterns, MN9-immunostaining patterns were classified into four stages: initial, early, advanced, and final. As the acrosome reaction progressed from the initial to the early stage, equatorin spread from the peripheral region of the anterior acrosome toward the center of the equatorial segment, gradually over the entire region of the equatorial segment during the advanced stage, and finally uniformly at the equatorial segment at the final stage. In contrast, the PNA-FITC signals spread more quickly from the peripheral region of the acrosome toward the entire equatorial segment, while decreasing in staining intensity, and finally became weak at the final stage. MN9-immunogold electron microscopy showed equatorin on the hybrid vesicles surrounded by amorphous substances at advanced stage of acrosome reaction. Equatorin decreased in molecular mass from 40-60 to 35 kDa, and the signal intensity of 35 kDa equatorin increased as the acrosome reaction progressed. Thus, the established equatorin-based progression model will be useful for analyzing not only the behavior of equatorin but also of other molecules of interest involved in the acrosome reaction.

Oocyte activation ability correlates with head flatness and presence of perinuclear theca substance in human and mouse sperm

BACKGROUNDnRecent studies indicate that round-headed sperm cannot activate oocytes and lack the postacrosomal sheath (PAS) or perinuclear theca (PT), although normal flat-headed sperm can activate oocytes and do have PAS (PT). In this study, we investigated how oocyte activation ability correlates with sperm head morphology (round and flat) and the presence of PT, by studying MN13, a representative molecule of the PT.nnnMETHODSnWe analyzed sperm with flat and round heads from infertile patients with globozoospermia (n = 1) and teratozoospermia (n = 1), and also from GOPC(-/-) mice, an animal model of human globozoospermia. Differential interference contrast image analysis, immunocytochemistry with MN13 antibody, transmission electron microscopy and an oocyte activation assay (assessing pronucleus formation) with ICSI were used.nnnRESULTSnFlat-headed (control) sperm from both a healthy fertile volunteer man and wild-type mice had MN13 and PAS (PT). Flat-headed sperm (<5% of the population) from GOPC(-/-) mice also had both MN13 and PAS (PT), and they showed high oocyte activation ability. In contrast, round-headed sperm from a globozoospermia patient (100%) and GOPC(-/-) mice (>95% of the population) had neither MN13, nor PAS (PT), nor oocyte activation ability. Oocyte activation was higher in flat- versus round-headed sperm from GOPC(-/-) mice (P < 0.05).nnnCONCLUSIONSnOocyte activation ability may be related to sperm head flatness and presence of MN13 and PAS (PT) in human and mouse sperm. This information is a first step towards the possibility of selecting good-quality sperm with high oocyte activation ability for ICSI.

Reduction of intramembranous particles in the periacrosomal plasma membrane of boar spermatozoa during in vitro capacitation: A statistical study

Membrane remodeling in the periacrosomal plasma membrane (PAPM) of boar spermatozoa during incubation in capacitation medium was examined by the freeze‐fracture technique. In the preservation medium (PM) group, the major small (about 8 nm) intramembranous particles (IMP) and the minor large (> 10 nm) IMP were distributed evenly in the PAPM. The IMP‐free area increased during capacitation. To correct the IMP‐free area, arithmetically redistributed (ARD)‐IMP density was used for statistical analysis. In the PM group, the mean density ± SD of large IMP was 379 ± 64 and 266 ± 58/μm2, and that of small IMP was 1450 ± 155 and 672 ± 252/μm2 in protoplasmic (P) and external (E) faces, respectively. During capacitation, the significant (P < 0.01) reduction of large IMP density was encountered only in the E face of a few incubation groups, while that of the small IMP density occurred in the P face by 2 h. Consequently, reduction of the total IMP density of both faces was not significant in the large IMP, but it was significant (P < 0.01) in the small IMP. One‐fifth of the total small IMP density reduced by 2 h. Filipin‐sterol complexes (FSC) were numerous in the PAPM, and FSC‐free areas also increased during capacitation. The mechanism of IMP‐free area formation and the behavior of the small IMP in the PAPM during capacitation were discussed in relation to membrane stability.

Localisation of RA175 (Cadm1), a cell adhesion molecule of the immunoglobulin superfamily, in the mouse testis, and analysis of male infertility in the RA175-deficient mouse.

RA175, a member of the immunoglobulin superfamily, plays an important role in cell adhesion, and RA175 gene‐deficient mice (RA175−/−) show oligoastheno‐teratozoospermia. To understand the function of RA175, location in the testis and the morphological features of its spermatogenic cells in RA175−/− mice were investigated. Immunohistochemical studies revealed that RA175 immunoreactivity was observed on the cell surface of the spermatogenic cells at specific stages. A strong reaction was detected from type A spermatogonia to pachytene spermatocytes at stage IV and from step 6 to step 16 spermatids during spermatogenesis. From pachytene spermatocytes at stage VI to step 4 spermatids, the reaction was not detected by the enzyme‐labelled antibody method and was faintly detected by the indirect immunofluorescence method. Abnormal vacuoles in the seminiferous epithelium, showing exfoliation of germ cells, and ultrastructural abnormality of the elongate spermatids were revealed in the RA175−/− testes. Other members of the immunoglobulin superfamily such as basigin, nectin‐2 and nectin‐3, which have an important role in spermatogenesis, were immunohistochemically detected in the RA175−/− testis. These observations indicate a unique expression pattern of RA175 in the testis and provide clues regarding the mechanism of male infertility in the testis.

Adhesion between plasma membrane and mitochondria with linking filaments in relation to migration of cytoplasmic droplet during epididymal maturation in guinea pig spermatozoa

High-resolution microscopy has been used to investigate the mechanism of the migration of cytoplasmic droplets during epididymal maturation of guinea pig spermatozoa. On testicular spermatozoa, droplets are located at the neck and, after passage through the middle cauda epididymidis, migrate only as far as the center of the midpiece. Initially, the space between the plasma membrane and outer mitochondrial membranes outside the droplet is 30.8±11.0xa0nm, whereas on mature spermatozoa, it significantly (P<0.01) narrows to a more consistent 15.9±1.3xa0nm. This is accompanied by the appearance of thin filaments cross-linking the two membranes above and below the droplet. Changes also occur in the arrangement of intramembranous particles (IMPs) in the plasma membrane overlying the midpiece. At the spermatid stage, linear arrays of IMPs are absent but appear on immature spermatozoa, where they are short with an irregular orientation, in the epididymis. On mature spermatozoa, numerous parallel linear arrays are present at the region where the plasma membrane adheres to the mitochondria. The membrane adhesion process can thus be observed two-dimensionally. The initial migration of the droplet from the neck is probably attributable to diffusion, with the formation of cross-linking filaments between the two membranes in the proximal midpiece preventing any backward flow and squeezing the droplet distally until it is arrested at the central midpiece by the filaments formed in the distal midpiece. The filaments might also stabilize the flagellum against hypo-osmotic stress encountered during ejaculation and within the female tract.

The involvement of immunoglobulin superfamily proteins in spermatogenesis and sperm-egg interaction

The immunoglobulin superfamily (IgSF) proteins are expressed on the plasma membrane between Sertoli cells and germ cells in the testis. IgSF proteins are specifically present at the apical Sertoli-germ cell junction, that is, ectoplasmic specialization and are involved in germ cell differentiation. Some IgSF proteins are present on the surface of germ cells and undergo further biochemical modifications during sperm maturation. These IgSF proteins undergo final modifications during capacitation and/or the acrosome reaction. The function and expression of IgSF proteins in the testis and spermatozoa, as they relate to spermatogenesis and sperm-egg interaction, are discussed.

The involvement of immunoglobulin superfamily proteins in spermatogenesis and sperm-egg interaction: Immunoglobulin superfamily proteins

The immunoglobulin superfamily (IgSF) proteins are expressed on the plasma membrane between Sertoli cells and germ cells in the testis. IgSF proteins are specifically present at the apical Sertoli‐germ cell junction, that is, ectoplasmic specialization and are involved in germ cell differentiation. Some IgSF proteins are present on the surface of germ cells and undergo further biochemical modifications during sperm maturation. These IgSF proteins undergo final modifications during capacitation and/or the acrosome reaction. The function and expression of IgSF proteins in the testis and spermatozoa, as they relate to spermatogenesis and sperm–egg interaction, are discussed. (Reprod Med Biol 2006; 5: 87–93)