血管内皮生长因子蛋白在大鼠睾丸和附睾的表达和定位研究

为探讨血管内皮生长因子(VEGF)在雄性生殖系精子发生发育和成熟过程中的调控作用,应用免疫组化、Periodic acid-Schiff(PAS)染色及蛋白质免疫印迹技术,检测VEGF蛋白在成年大鼠睾丸和附睾的表达和定位情况。Western-blots显示,在大鼠睾丸和附睾内均有VEGF蛋白(约45kD)的表达;免疫组化显示,睾丸内VEGF见于圆形和长形精子细胞、Sertoli细胞和Leydig细胞,免疫阳性产物位于细胞质内。精子细胞的VEGF表达伴随精子细胞项体发育的全过程,精子残余体呈强阳性。附睾内VEGF表达于附睾管上皮,且有区域和细胞特异性。附睾起始段的所有上皮主细胞内都有VEGF阳性颗粒;头、体、尾各段的VEGF阳性细胞多数与含PAS阳性颗粒的细胞重合,证明为亮细胞;近端附睾的管腔内可见精子头部呈VEGF阳性染色。睾丸、附睾间质血管内皮为VEGF阴性。上述结果表明,VEGF蛋白可由生殖细胞和附睾管上皮细胞直接产生,它可能以自分泌和／或旁分泌的形式共同作用于睾丸和附睾的生殖细胞和血管内皮,直接或间接影响精子的发生、发育和成熟过程,特别是精子顶体的形成过程,并可能与精子在附睾内的成熟有关。     

Different expression time of the 105-kDa protein and 90-kDa heat-shock protein in rat testis

To understand the physiological functions of the 105-kDa protein which is testis-specific and HSP90 (90-kDa heat-shock protein) related protein, the appearance of it in the testis has been followed during the development of rat. On immunoblotting analysis, the 105-kDa protein did not appear until after the age of five weeks, while HSP90 could be detected at three weeks. In the spermatozoa, the 105-kDa protein was much abundant but not in the LC-540 cells (a cell line from Leydig cell tumor in rat testis) cytosol. This finding has attracted much attention to the relationship between this protein and sperm functions.     

Distribution of sodium-potassium ATPase in the rat testis and epididymis.

Sodium-potassium ATPase (Na+K(+)-ATPase) is a ubiquitous plasma membrane enzyme which uses the hydrolysis of ATP to regulate cellular Na+ and K+ levels and fluid volume. This ion pumping action is also thought to be involved in fluid movement across certain epithelia. There are several different genes for this enzyme, some of which are tissue specific. Using an antibody specific for the catalytic subunit of canine kidney Na+K(+)-ATPase, we have localized immunoreactivity in the seminiferous and epididymal epithelium of rats of various ages. There was no specific staining of 10-day-old rat testis. Faint staining was detected at 13 days and appeared to be associated with the borders of Sertoli cells. At 16 days prominent apical and lateral staining but no basal staining of Sertoli cell membranes was observed. This type of distribution continued until spermatids were present in the epithelium. In the adult rat testis, specific staining was detected in Sertoli cell crypts associated with elongating spermatids, and on the apical and lateral Sertoli cell membrane. In some instances immunoreactivity was concentrated at presumed sites of junctional specializations. In the excurrent ducts of immature and mature rats, Na+K(+)-ATPase staining was heavy in the efferent ducts and somewhat lighter in the epididymis. In all regions, the staining was basolateral although there were variations in intensity among the different parts of the epididymis. These results show 1) that rat testis and epididymal Na+K(+)-ATPase share some immunological determinants with the canine enzyme; 2) that the epididymal enzyme is located in the conventional basolateral position; and 3) that the distribution of Sertoli cell Na+K(+)-ATPase is probably apical and lateral rather than basal.     

Localization and expression of ADAM2 in the dromedary camel testis,epididymis and sperm during rutting season

ADAM2 (fertilin β) is a sperm surface protein reported in several mammalian species. However, the presence of ADAM2 in the male reproductive system and sperm of the camel is not well known. The present study was to clarify the localization and expression of ADAM2 in the dromedary camel testis, epididymis and spermatozoa during rutting season using immunohistochemistry (IHC) and the quantitative real-time polymerase chain reaction (qPCR). Tissue samples were obtained from the testis (proximal and distal) and epididymis (caput, corpus, and cauda) from eight mature male camels. Epididymal and ejaculated sperms were collected from four other fertile camels. IHC analysis clearly showed the localization of ADAM2 protein in the spermatocytes and the round and elongated spermatids of the testis, in the epithelial cells along the epididymis tract, on the posterior head of the sperm within the cauda epididymis, and on the acrosomal cap of both the epididymal and ejaculated sperm. The expression of camel ADAM2 mRNA was significantly higher (P < 0.05) in the testis when compared with the epididymis. These findings may suggest an important role of ADAM2 in the fertility of male dromedary camels.     

Rat sperm AS-A: subcellular localization in testis and epididymis and surface distribution in epididymal sperm

In this study, we investigated the subcellular compartmentalization of arylsulfatase-A (AS-A) in the testis and epididymis as well as the surface distribution in rat epididymal sperm. Testicular AS-A was compartmentalized specifically to the area underneath the outer acrosomal membrane of the acrosomal granule and to the dorsal aspect of the sperm acrosome. Epididymal AS-A was synthesized in the endoplasmic reticular (ER) network of principal cells and secreted into epididymal lumen as evident by its reactivity in the apical cytoplasm and vesicles therein underneath stereocilia. In clear cells, AS-A reactivity was found throughout the cytoplasmic machineries involved in endocytosis. Surface distribution of AS-A was initially detectable at the concave ridge as early as in sperm of the initial segment (IS). AS-A was additionally localized to the post-acrosomal region in caput (CP), corpus (CO) and cauda (CD) epididymal sperm. The expression levels of surface AS-A gradually increased during sperm transit from IS to CD epididymidis. These results favored the adsorption of AS-A from epididymal fluid onto the sperm surface, rather than shunting from the acrosome as a consequence of capacitation-associated membrane priming.This work was supported by Research Initiate Grant funded by Faculty of Science, Mahidol University to W.W.     

Postnatal development of lectin-binding pattern in the rat testis and epididymis

Seven rhodamine-conjugated lectins were utilized to study the distribution of glycoproteins in the developing rat testis and epididymis. In the testis a clear developmental pattern was found in Leydig cells and the cell boundaries between Sertoli and spermatogenic cells, as well as during acrosome formation. Some of the first degenerating meiotic cells and the apical extensions of the Sertoli cells at the time of spermiation also displayed a characteristic lectin binding. The epididymal differentiation was characterized by an increasing lectin binding of the subapical Golgi zone and apical surface, and intratubular secretion prior to the arrival of sperm. After the accumulation of tubular secretion and sperm some epithelial cells were transformed into narrow (initial segment) and light cells (distal caput, cauda) with a strong affinity for some lectins. These cells appeared to be responsible for the absorption and digestion of tubular material derived from the testis and of surplus secretion and/or sperm structures.     

Recent advances in sperm maturation in the human epididymis

As spermatozoa move through the human epididymis they encounter a varied environment with respect to the proteins with which they come into contact. In the proximal epididymis sperm are subjected to the action of enzymes and exposure to proteins involved in membrane modification. In the middle region another set of proteins and enzymes predominates; those associated with sterol transport could modify the sperm membrane to permit the uptake of GPI-anchored zona binding proteins P34H and CD52. More distally sperm encounter increasing activities of lytic enzymes, proteins involved in both zona binding and oocyte fusion, the major maturation antigen CD52, antimicrobial activity and decapacitation factors, that help them to survive before ejaculation. Adherence of the proteins to different domains (e.g. anterior acrosome or equatorial acrosomal segment) may depend on the nature of the protein, the lipid composition of the particular membrane and the ionic environment in the epididymal lumen. The eventual location on a capacitated sperm (acrosomal membrane) or acrosome-reacted sperm (equatorial region) may dictate their role in, for example, zona-binding (P34H) or oocyte-binding (gp20). Both proteins and membranes may be modified during epididymal transit by the enzymes which may add to or remove carbo-hydrates and peptides from the sperm surface.     

Peroxisomes and ether lipid biosynthesis in rat testis and epididymis

Plasmalogens are a main component of the spermatozoon membrane, playing a crucial role in their maturation. The initial steps in plasmalogen biosynthesis are catalyzed by two peroxisomal enzymes, dihydroxyacetonephosphate acyltransferase and alkyl-dihydroxyacetonephosphate synthase. The localization of both enzymes in the membrane of peroxisomes implies that plasmalogen-producing cells should contain this organelle. To unravel the putative source of spermatozoan plasmalogens we investigated which cell types in the testis and epididymis are endowed with peroxisomes. To this extent, testicular and epididymal tissue was analyzed at the protein and RNA levels by means of light and electron microscopical immunocytochemistry as well as by Western and Northern blotting. Proteins and mRNAs of peroxisomal enzymes, especially those of dihydroxyacetonephosphate acyltransferase and alkyl-dihydroxyacetonephosphate synthase, were detected in the testis and epididymis. In the testis, peroxisomes were localized exclusively in Leydig cells and not in cells of the seminiferous tubules, implying that the latter do not contribute to the biosynthesis of plasmalogens of the sperm membrane. In contrast, peroxisomes could be clearly visualized in the epithelial cells of the epididymis. The results suggest that peroxisomes in epithelial cells of the rat epididymis play a pivotal role in the biosynthesis of plasmalogens destined for delivery to the sperm plasma membrane.     

Immunocytochemical localization of lipocalin-type prostaglandin D synthase in the bull testis and epididymis and on ejaculated sperm

Previously, we identified a 26-kDa fertility-associated protein in bull seminal plasma as lipocalin-type prostaglandin D synthase. The objective of the present study was to immunohistochemically localize this enzyme to the various cell types within the bull testis and seven subsegments of the epididymis, and on ejaculated sperm in order to gain further insight into its potential function in male reproduction. In the testis, immunoperoxidase staining was localized within the elongating spermatids and Sertoli cells of the seminiferous tubules, varying with the stage of the spermatogenic cycle. The highest level of staining occurred during stages III-VII. The cuboidal epithelial cells of the rete testis and efferent ducts were also immunoreactive. Expression of lipocalin-type prostaglandin D synthase was not uniform in the seven epididymal subsegments, suggesting a possible role in sperm maturation. In all epididymal regions, expression was limited to the epithelial principal cells; no immunoreactivity was apparent in other cell types. Lipocalin-type prostaglandin D synthase was strikingly localized in the caput epididymidis, while moderate to weak staining was observed in the remainder of the epididymis. Droplets of reaction product observed within the lumen increased progressively from the caput to cauda. Using fluorescence microscopy, we also localized lipocalin-type prostaglandin D synthase to the apical ridge of the acrosome on ejaculated sperm.     

A rat sperm flagellar surface antigen that originates in the testis and is expressed on the flagellar surface during epididymal transit.

We identified a rat sperm flagellar surface antigen using an IgG1 monoclonal antibody (MC31) against rat epididymal sperm. Avidin-biotin-peroxidase immunohistochemistry demonstrated that the antigen was first expressed in the cytoplasm of early primary spermatocytes, then gradually became restricted to the principal piece of the sperm flagellum during spermatogenesis. However, when the sperm reached the corpus epididymidis, the antigen was expressed on the surface of both the principal piece and the midpiece of the flagellum. The epithelial cells of the epididymis were not stained with MC31. Immunogold electron microscopy showed that the antigen was present on the surface of the sperm flagellar plasma membrane. Immunoblotting of Triton X-100 extracts of epididymal sperm after one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions demonstrated that MC31 detected a major antigen of 26,000-28,000 daltons (26-28K). Two-dimensional isoelectric focusing and SDS-PAGE indicated that the 26-28K antigen had an isoelectric focusing point (pl) of 5.8-5.3; minor antigens were also detected from 26K (pl 5.8) to 35K (pl 5.0). These results indicate that the antigen recognized by MC31 is an acidic 26-35K protein that originates in the testis, is integrated into the sperm flagellar plasma membrane of the principal piece during spermatogenesis, and then is expressed on the entire flagellar surface during epididymal transit.     

Expression and localization of PMCA4 in rat testis and epididymis

It has recently been shown in mice that the plasma membrane Ca²⁺-ATPase isoform 4 (PMCA4) is essential for sperm fertilization capacity. We analyzed whether sperm PMCA4 is formed in the rat during spermatogenesis or is synthesized in the epididymis and transferred onto sperm during sperm maturation. We could show that PMCA4 is conserved in sperm from testis to epididymis. In testis, PMCA4 mRNA was restricted to spermatogonia and early spermatocytes, while the PMCA4 protein was detected in spermatogonia, late spermatocytes, spermatids and in epididymal sperm. In epididymis PMCA4 mRNA was localized in basolateral plasma membranes of epithelial cells of the caput, corpus and cauda epididymidis. In contrast, the protein was only detectable in the epithelial cells of the caput, indicating that PMCA4 mRNA is only translated into protein in caput epithelium. In the epididymal corpus and cauda, PMCA4 mRNA and protein, respectively, was localized and in peritubular cells. Furthermore, we detected an identical distribution of PMCA4a and b splice variants in rat testis, epididymal corpus and cauda. In the caput epididymidis, where PMCA4 is located in the epithelium splice variant 4b was more prominent. Further experiments have to clarify the functional importance of the differences in the PMCA4 distribution.     

Immunohistochemical studies on the localization of cellular retinol-binding protein in rat testis and epididymis

The immunohistochemical localization of cellular retinol-binding protein (CRBP) was studied in rat testis and epididymis. Parallel studies were also carried out on the localization of plasma retinol-binding protein (RBP) and transthyretin (TTR) in testis. The studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of each antigen by the unlabeled peroxidase-antiperoxidase method. For RBP and TTR, specific immune staining was found in the interstitial spaces between the seminiferous tubules, and not in the tubules themselves. In contrast, strong specific immune staining for CRBP was found in the seminiferous tubules, with a striking localization within Sertoli cells. Moreover, a distinct cyclic variation of specific staining for CRBP within Sertoli cells was observed during the spermatogenic cycle. This cyclic variation was seen with regard to both the intensity of staining and to the anatomic distribution of CRBP within the Sertoli cells. Within the epididymis CRBP was selectively localized to the proximal portion of the caput epididymidis, with variations in intensity of the staining of the epithelium of the ducts in different histological zones. Specific immune staining for CRBP was very weak or absent in the other portions of the epididymis. These results were confirmed by radioimmunoassay. Vitamin A-deficient rats showed markedly reduced specific immune staining for CRBP in both testes and epididymides, and greatly reduced levels of CRBP in these tissues on radioimmunoassay. These studies on the localization of CRBP provide information concerning the specific cells and anatomic loci within the testis and epididymis where retinol may be playing an important role in sperm formation and maturation.     

Glycoprotein changes in the rat sperm plasma membrane during maturation in the epididymis.

To investigate surface glycoprotein changes during post-testicular maturation, plasma membranes were isolated from proximal caput, distal caput, and cauda epididymal rat spermatozoa. Membrane glycoproteins were identified on Western blots of SDS-PAGE fractionated samples using biotinylated lectins and Vecta-stain reagents; these were compared to glycoproteins present in cauda epididymal luminal fluid. Lens culinaris agglutinin, Pisum sativum agglutinin, peanut agglutinin, wheat germ agglutinin, Ricinus communis agglutinin, Ulaex europaeus agglutinin, and Dolichol biflorus agglutinin each bound a specific subset of the polypeptides present. Several types of glycoprotein changes were noted including their appearance, loss, alteration of staining intensity, and alteration of electrophoretic mobility. Some maturation-dependent sperm surface glycoproteins co-migrated with glycoproteins present in epididymal fluid. This approach of direct analysis of the glycoproteins in purified plasma membranes identifies a broader spectrum of maturation-related surface changes occurring within the epididymis than are noted with surface labeling procedures.