Acrosome-phase Spermatids Research Articles

Distribution of Lectin‐Bindings in the Testis of the Lesser Mouse Deer, Tragulus javanicus

The distribution of lectin bindings in the testis of the smallest ruminant, lesser mouse deer (Tragulus javanicus), was studied using 12 biotinylated lectins specific for d-galactose (peanut agglutinin PNA, Ricinus communis agglutinin RCA I), N-acetyl-d-galactosamine (Dolichos biflorus agglutinin DBA, Vicia villosa agglutinin VVA, Soybean agglutinin SBA), N-acetyl-D-glucosamine and sialic acid (wheat germ agglutinin WGA, s-WGA), D-mannose and d-glucose (Lens culinaris agglutinin LCA, Pisum sativum agglutinin PSA, Concanavalin A Con A), L-fucose (Ulex europaeus agglutinin UEA I), and oligosaccharide (Phaseolus vulgaris agglutinin PHA-E) sugar residues. In Golgi-, cap-, and acrosome-phase spermatids, lectin-bindings were found in the acrosome (PNA, RCA I, VVA, SBA, WGA and s-WGA), and in the cytoplasm (PNA, RCA I, VVA, SBA, WGA, LCA, PSA, Con A and PHA-E). s-WGA binding was confined to the spermatid acrosome, but other lectins were also observed in spermatocytes. In spermatogonia, VVA, WGA, Con A, and PHA-E bindings were observed. Sertoli cells were intensely stained with DBA and Con A, and weakly with PHA-E. In interstitial Leydig cells, RCA I, DBA, VVA, Con A, PSA, LCA, WGA and PHA-E were positive. UEA I was negative in all cell types including spermatogenic cells. Unusual distribution of lectin-bindings noted in the testis of lesser mouse deer included the limited distribution of s-WGA only in the spermatid acrosome, the distribution of DBA in Sertoli cells, Leydig cells and lamina propria, and the absence of UEA I in all type cells. The present results were discussed in comparison with those of other animals and their possible functional implications.

A Lectin Histochemical Study on the Testis of the Babirusa, Babyroussa babyrussa (Suidae)

The distribution of lectin bindings in the testis of babirusa, Babyrousa babyrussa (Suidae) was studied histochemically using 10 biotinylated lectins, Peanut agglutinin (PNA), Ricinus communis agglutinin (RCA I), Dolichos biflorus agglutinin (DBA), Vicia villosa agglutinin (VVA), Soybean agglutinin (SBA), Wheat germ agglutinin (WGA), Lens culinaris agglutinin (LCA), Pisum sativum agglutinin (PSA), Concanavalin A(Con A) and Ulex europaeus agglutinin (UEA I). Nine of 10 lectins showed a variety of staining patterns in the seminiferous epithelium and interstitial cells. The acrosome of Golgi-, cap- and acrosome-phase spermatids displayed various PNA, RCA I, VVA, SBA and WGA bindings, indicating the presence of glycoconjugates with D-galactose, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine sugar residues respectively. No affinity was detected in the acrosome of late spermatids. LCA, PSA and Con A which have affinity for D-mannose and D-glucose sugar residues were positive in the cytoplasm of spermatids and spermatocytes. DBA was positive only in spermatogonia. In addition to DBA, positive binding in spermatogonia was found for VVA, WGA and Con A, suggesting the distribution of glycoconjugates with N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, D-mannose and D-glucose sugar residues. Sertoli cells were stained intensely with RCA I, WGA and Con A. In Leydig cells, RCA I and Con A were strongly positive, while WGA, LCA and PSA reactions were weak to moderate. The present findings showed that the distribution pattern of lectin binding in the testis of babirusa is somewhat different from that of pig or other mammals reported previously.

Distribution of Lectin Bindings in the Testes of the Greater Japanese Shrew Mole, Urotrichus talpoides.

The binding patterns of 20 lectins in the testes of the greater Japanese shrew mole (Urotrichus talpoides) were investigated by light microscopy. Eleven lectins (WGA, sWGA, LEL, STL, Jacalin, PSA, LCA, BPA, Con-A, WGA and PHA-E) exhibited a positive reaction in the cytoplasm and acrosomal region of spermatids and in the cytoplasm of Sertoli cells. LEL, BSL-I, DSA, sWGA, BSL-I and PHA-L were intensely positive in the cytoplasm of Leydig cells. The binding patterns in the acrosomal region of spermatids were divided into three types. For LEL, ECL and STL, the reaction was detected in the acrosomal regions from Golgi to cap phase spermatids and disappeared in acrosome and maturation phase spermatids. For SBA, VVL, ECL and DSL, the reaction was detected in the acrosomal region from Golgi to acrosome phase spermatids. No reaction was recognized in the acrosome of maturation phase spermatids. In DBA and Jacalin, the cytoplasm of maturation phase spermatids was positive although no reaction was detected in the acrosomal region of all spermatids. sWGA, BPA and LEL exhibited a strong reaction in the plasma membrane of zygotene spermatocytes at stages X and XI. BSL-II revealed a strong reaction in the cytoplasm of spermatogonia.

Transport and rearrangement of the intra-acrosomal protein acrin1 (MN7) during spermiogenesis in the guinea pig testis.

We have recently shown that a 90-kDa glycoprotein, acrin1 (MN7), is exclusively localized in the dorsal region of the acrosomal apical segment of mature guinea pig sperm, and that its location changes during epididymal maturation. The present study examined the process of transport and organization of this protein in the acrosome during spermatogenesis in the guinea pig testis. Immunoperoxidase electron microscopy showed stage-specific localization of acrin1 within the developing acrosome, as follows: acrin1 first appeared in the proacrosomic vesicles of the early Golgi phase spermatids, and it was then localized in the electron-lucent matrix region of the acrosomic vesicles of the late Golgi phase spermatids. During the cap phase, acrin1 was abundant in the electron-lucent matrix of the acrosomal apical segment and in the head-cap region (principal segment). acrin1 became more restricted to the peripheral region of the electron-lucent matrix of acrosome phase spermatids and it was localized in the electron-lucent dorsal matrix region of maturation phase spermatids. In the final step of spermiogenesis, acrin1 disappeared from the equatorial and principal segments, and it was finally confined to the dorsal matrix region of the acrosomal apical segment. In addition, Western blot analysis showed that acrin1 of testes and epididymal sperm was of the identical size, indicating that acrin1 is not proteolytically modified during epididymal sperm maturation. These results indicate that acrosome morphogenesis is closely associated with the rearrangement of acrosomal proteins.

A Lectin-Histochemical Study on the Seminiferous Epithelium of the Northern Smooth-Tailed Tree Shrew (Dendrogale murina)and the Java Tree Shrew (Tupaia javanica)

Lectin-binding patterns in the testes of the northern smooth-tailed tree shrew, Dendrogale murina and Java tree shrew, Tupaia javanica were studied by light microscopy and compared the data with those of the common tree shrew. Four lectins (PNA, SBA, BPA and GS-II) were used in this study. Peanut (Arachis hypogaea) agglutinin (PNA), soybean (Glycine max) agglutinin (SBA) and Bauhinia purpurea agglutinin (BPA) showed a strong reaction in the acrosomal region from Golgi to acrosome-phase spermatids in three species of tree shrews. These lectins also showed a granular positive reaction in the cytoplasm from acrosome to maturation-phase spermatids in three species, except that BPA revealed no granular reaction (though it was positive) in the spermatid cytoplasm of the northern smooth-tailed tree shrew and that PNA revealed no reaction in the spermatid cytoplasm of the common tree shrew. While, Griffonia simplicifolia-II agglutinin (GS-II) showed a positive reaction in the acrosomal region of Golgi-phase spermatids in three species of tree shrews. Although GS-II was positive in the spermatocyte cytoplasm of three species, it showed granular in the northern smooth-tailed tree shrew and common tree shrew but not granular in the Java tree shrew. Thus, the lectin-binding patterns in testes were similar among three species belonging to the Order Scandentia. However, slight differences were also detected even among these phylogenetically-close species.

Lectin-Binding Patterns in the Testes of the Java Fruit Bat (Pteropus vampyrus) and the Japanese Lesser Horseshoe Bat (Rhinolophus cornutus).

Lectin binding patterns in the testes of the Java fruit bat, Pteropus vampyrus, and the Japanese lesser horseshoe bat, Rhinolophus cornutus, were investigated by light microscopy. Binding patterns were similar in both species, except for some slight differences. UEA-I, SBA, DBA and BSL-I revealed no reaction in the testes of either species. Con A and WGA gave a diffuse reaction all over the seminiferous epithelium in both species. In addition to this binding pattern, PHA-E exhibited a granular reaction within the cytoplasm of pachytene spermatocytes. PNA and RCA-I gave an intense reaction in the acrosomal region from Golgi to acrosome-phase spermatids in both species, but, in addition to this binding pattern, RCA-I reacted in the cytoplasm of spermatocytes and spermatids in the Java fruit bats. PSA revealed a granular reaction in the cytoplasm of spermatids in the Japanese lesser horseshoe bats. These binding patterns were similar to those of mammals studied before, except that a few specific bindings were detected in the bats.

Targeting of the domain-specific integral membrane protein PM52 to the periacrosomal plasma membrane during guinea pig spermiogenesis.

The sperm plasma membrane is segregated into functionally, biochemically, and structurally distinct domains yet the protein sorting pathways and assembly mechanisms that assemble these domains during spermiogenesis are incompletely understood. We previously characterized two structurally related size-variant, integral membrane proteins of 52 kDa (PM52) and 35 kDa localized to the periacrosomal plasma membrane of guinea pig cauda epididymal spermatozoa (Westbrook-Case et al., 1994). In this study we used light and electron microscopic immunocytochemistry to define the expression pattern and sorting pathway that establishes the domain-specific distribution of PM52 during spermiogenesis. The PM52 is first expressed in acrosome-phase spermatids and it localizes exclusively to the cytoplasmic lobe. Immunoelectron microscopy revealed that both cytoplasmic vesicles and the plasma membrane of the cytoplasmic lobe labeled with anti-PM52. During early stages of expression, PM52 appeared to be absent from the head region, but significant PM52 accumulation over the spermatid head was noted in late acrosomal phase spermatids. Throughout spermiogenesis PM52 extended posteriorly to the annulus, which represents a barrier preventing PM52 diffusion into the posterior tail. Following the migration of the annulus to the midpiece-principal piece junction, PM52 began to disappear from the flagellar region and at the completion of spermiogenesis most of the PM52 was restricted to the acrosomal segment. Spermatids and epididymal sperm PM52 exhibited identical sizes by SDS-PAGE and immunoblotting, indicating that they are not proteolytically modified during epididymal maturation. The PM52 antibodies were also used to screen a guinea pig testis cDNA library, and sequence determination of full-length PM52 clones demonstrated identity of a sperm membrane protein recently termed "sperad" (Quill and Garbers, 1996). Membrane barriers and potential mechanisms establishing the domain-specific residence of PM52 are discussed.

Sorting of the Domain-Specific Acrosomal Matrix Protein AM50 during Spermiogenesis in the Guinea Pig

We previously identified an insoluble 50-kDa acrosomal matrix protein (AM50) localized to the ventral region of the guinea pig sperm apical segment. AM50 is converted to a 42-kDa polypeptide and released during matrix dispersion in acrosome-reacting sperm. This study examines the sorting pathways and assembly processes which generate the domain-specific distribution of AM50 in the acrosome. AM50 was expressed during early acrosome development and localized to the matrix of proacrosomal granules and the acrosomal vesicle. Initial sorting of AM50 occurred in Golgi phase spermatids, where it became concentrated in the matrix surrounding the acrosomal granule. AM50 remained restricted to the apical segment of acrosome phase spermatids and was finally sorted to the ventral matrix of the apical segment in maturation phase spermatids. By reducing SDS-PAGE testicular AM50 exhibited a slightly higher M r of 52 kDa than the 50-kDa form of cauda epididymal spermatozoa. Nonreducing SDS-PAGE demonstrated that testicular and epididymal AM50 were assembled into homomeric complexes of 480 and 450 kDa respectively. Cauda epididymal sperm apical segments contained a second disulfide-cross-linked homomeric complex of 520 kDa composed of a 68-kDa subunit. These studies indicate that AM50 is first assembled into a disulfide-cross-linked complex and subsequently processed into mature AM50. These data also suggest that disulfide-linked complexes of different structural proteins may assemble into distinct acrosomal matrix compartments.

Ethanol-phosphotungstic acid and bismuth staining of spermatid nucleoli in mouse spermiogenesis

The nucleoli of developing mouse spermatids were examined with ethanol-phosphotungstic acid (E-PTA) staining, and also with bismuth staining following formaldehyde fixation (FA-Bi staining) and glutaraldehyde fixation (GA-Bi staining). Only the cortical zone of the nucleolar dense fibrillar component (DFC) in the round spermatids was stained with E-PTA, while the inner area remained either faintly (early Golgi-phase spermatids) or completely unstained (cap-phase spermatids). Incubation of the fixed testis with dithiothreitol before E-PTA staining resulted in homogeneously intense staining of the DFC. The facts suggest that numerous E-PTA-positive basic proteins were present in the DFC, but disulfide crosslinks formed in the DFC proteins prevent penetration of PTA into the DFC interior. The DFC was stained with bismuth after FA-Bi and GA-Bi staining until the disappearance of the nucleoli occurring in acrosomephase spermatids. The fibrillar center, homogeneously stained using E-PTA, FA-Bi, and GA-Bi methods was present in the nucleoli of Golgi-phase and early cap-phase spermatids, but disappeared in the nucleoli of late capphase spermatids. These results are discussed based on the previous studies dealing with the ribosomal RNA synthesis in mouse spermiogenesis.

Effects of cefamandole on spermatogenic development of young CD rats

The testicular toxicity of cefamandole, a β-lactam antibiotic with an N-methylthiotetrazole side chain, was evaluated in neonatal rats. Cefamandole caused delayed maturity of the germinal epithelium of neonatal rats when given on Postpartum Days 6 through 36. In rats given daily subcutaneous injections of 1000 mg/kg during this period, the most mature germinal cells were acrosome phase spermatids, whereas control rats had spermatids in the maturation phase. In studies of specific developmental phases, the effect of 1000 mg/kg daily cefamandole was primarily on the initial waves of spermatogonia during the period of rapid development (Postpartum Days 4 through 13). In animals treated from birth to Postpartum Day 8 and evaluated sequentially on Postpartum Days 5 through 9, there were no morphologically discernible effects on the transformation of gonocytes to immature spermatogonia, but there were slight degenerative changes in the first waves of developing spermatogonia. Cefamandole, 1000 mg/kg daily, given Postpartum Days 14 through 18 during the initial phase of spermatocyte development, also caused a slight degenerative change of the initial waves of pachytene spermatocytes. The significance of the findings in neonatal rats is unknown because differences in spermatogenic development between rat and human preclude direct extrapolation of the effects of cefamandole in neonatal rats to effects in humans.

Effects of Cefamandole on Spermatogenic Development of Young CD Rats

The testicular toxicity of cefamandole, a β-lactam antibiotic with an N-methylthio-tetrazole side chain, was evaluated in neonatal rats. Cefamandole caused delayed maturity of the germinal epithelium of neonatal rats when given on Postpartum Days 6 through 36. In rats given daily subcutaneous injections of 1000 mg/kg during this period, the most mature germinal cells were acrosome phase spermatids, whereas control rats had spermatids in the maturation phase. In studies of specific developmental phases, the effect of 1000 mg/kg daily cefamandole was primarily on the initial waves of spermatogonia during the period of rapid development (Postpartum Days 4 through 13). In animals treated from birth to Postpartum Day 8 and evaluated sequentially on Postpartum Days 5 through 9, there were no morphologically discernible effects on the transformation of gonocytes to immature spermatogonia, but there were slight degenerative changes in the first waves of developing spermatogonia. Cefamandole, 1000 mg/ kg daily, given Postpartum Days 14 through 18 during the initial phase of spermatocyte development, also caused a slight degenerative change of the initial waves of pachytene spermatocytes. The significance of the findings in neonatal rats is unknown because differences in spermatogenic development between rat and human preclude direct extrapolation of the effects of cefamandole in neonatal rats to effects in humans.

Centromere pattern in different mouse seminiferous tubule cells.

Centromere arrangement in different mouse seminiferous tubule cells was analyzed using an anticentromere antiserum from a patient with the CREST syndrome of scleroderma. A peptide of 18 kd was recognized by this serum on immunoblotting of mouse nuclear proteins from seminiferous tubule cells. In the cells studied by immunofluorescence, different patterns of centromere arrangement were observed. A speckled arrangement of centromeres was found in spermatogonia, double spots corresponding to meiotic bivalents were found in pachytene cells, and clusters of a haploid numer of centromeres were found in early and acrosome phase spermatids. In Sertoli cells, only three centromeric spots were detected, corresponding to the nucleolar organizer chromosome pairs. A relationship between the functional stage of the cell and the arrangement of and conformational changes in the centromeres is considered.

Relative volume of sertoli cells in monkey seminiferous epithelium: A stereological analysis

Techniques of quantitative stereology have been utilized to determine the relative volume occupied by the Sertoli cells and germ cells in two particular stages (I and VII) of the cycle of the seminiferous epithelium. Sertoli cell volume ranged from 24% in stage I of the cycle to 32% in stage VII. Early germ cells occupied 3.4% in stage I (spermatogonia) and 8.7% in stage VII (spermatogonia and preleptotene spermatocytes). Pachytene spermatocytes occupied 15% (Stage I) and 24% (stage VII) of the total volume of the seminiferous epithelium. In stage I the two generations of spermatids comprised 58% of the total epithelium by volume, whereas in stage VII, after spermiation, the acrosome phase spermatids occupied 35% of the total seminiferous epithelial volume.

Testicular development and testosterone concentrations in the testis and plasma of young male shrews ( Suncus murinus)

Testicular development and testosterone concentrations in the peripheral plasma and testes of young, adult, and castrated male musk shrews ( Suncus murinus) were examined. Fifty immature, 24 adult, and eight castrated males were used. Immature males ranged from 5–45 days of age. Plasma and testis homogenates were assayed for testosterone by radioimmunoassay. Testes were examined histologically for spermatogenic development. Plasma testosterone concentrations increased gradually from Day 5 to Day 24, then rapidly to Day 30. There was a sharp decrease between Day 30 and 33, then a gradual increase to Day 45. Testis testosterone concentrations followed the same pattern, except that on Day 5, the concentration was significantly higher than on Day 10. Adult males had higher testis testosterone and lower plasma testosterone concentrations than Day 45 males. Castrated males had barely detectable plasma testosterone levels. Testis weights increased linearly with age, except for a slight decrease between Day 30 and 33. Spermatogenic development was already initiated by Day 5. Cap-phase spermatids were present on Day 23, acrosome-phase spermatids on Day 30, and mature spermatozoa on Day 31. Epididymal sperm were present by Day 45.

Studies on the structure and function of the mammalian testis. II. Cytological and histochemical observations on the testis of the rat after a single exposure to heat applied for different lengths of time.

The scrotal regions of five groups of rats were exposed to a temperature of 43 °C for progressively longer periods, ranging from 10 to 30 min in steps of 5 min. The animals were then killed at 3 days, 7 days, 3 weeks and 6 weeks after heat-treatment, and their testes examined by various cytological and histochemical procedures. Appropriate control animals were killed alongside the experimental groups. There was essentially no change seen in the 10 min group. In the 15 min group two critical periods were established where further development of the germ cells failed; the first occurred as cells in the early transitional condition attempted to develop into late transitional forms, while the second operated from late pachytene to the end of the maturation division. This led to what has been termed the pattern of mild heat damage. As the exposure time was increased the pattern of mild heat damage was extended, and after 20 min the two critical periods merged. A third also began to appear, such that while cells in the Golgi phase were able to form cap-phase cells they could not develop into acrosome- phase spermatids. Twenty-five minutes exposure produced a similar result but the third critical period was then firmly established, since neither the Golgi or cap-phase spermatids developed into acrosome-phase spermatids. After 30 min all the previous critical periods were extended and merged and their extreme boundaries marked two new critical periods, one operating at the spermatogonial level and the other at the acrosome-phase. In animals treated for 15, 20 and 25 min there was a variable degree of recovery which occurred between 7 and 21 days after heating, during which time the critical periods disappeared. The damage to the germinal epithelium was accompanied by an increase in the lipid content of the Sertoli cells which progressively gave a red colour with Nile blue (non-acidic) and a positive reaction to Schultz’s test for unsaturated sterols. This effect is interpreted as an indirect one resulting from the damage to the germinal epithelium and the cessation of sperm atogenic activity. The high lipid/sterol content of the Sertoli cells was reduced to normal levels with the recovery of the germinal epithelium, notably during the maturation division of the primary and secondary spermatocytes corresponding to stages IX-XIV of the cycle of the seminiferous epithelium. Injections of follicle-stimulating hormone (FSH) into rats exposed to the heat for 30 min did not reduce the lipid/sterol content of the Sertoli cells to normal levels as found previously when the same gonadotrophin was administered to oestrogen-treated rats. This is attributed to the extensive damage caused to the germ cells in the 30 min group. The work on the heat-treated animals provides further evidence in support of the view that the lipids/sterols of the Sertoli cells represent material normally utilized by the germ cells in the course of their development; it emphasizes that the cells which primarily use this material are the spermatocytes and finally suggests that the last-mentioned cells govern or regulate sterol metabolism by the Sertoli cells. The Leydig cells in all groups appeared to be unaffected by the heat-treatment.

Reinitiation and Restoration of Spermatogenesis with Testosterone Propionate and Other Hormones After a Long-Term Post-hypophysectomy Regression Period

The ability of testosterone propionate (TP) to restore spermatogenesis was investigated in 67- to 70-day hypophysectomized rats administered TP either separately or in combination with somatotrophin (STH) and/or thyroxine (T4). Following TP treatment for 35 days, the testicular weights had increased to twice those of the hypophysectomized controls, and a few of the seminiferous tubules contained germinal cells as mature as acrosome phase spermatids. With more prolonged therapy (90–110 days), spermatogenesis was restored qualitatively but not quantitatively in 33% of the animals, while the remainder of the animals showed varying degrees of response. The testes of animals given TP+STH or TP+STH+T4 for 35 days were heavier than those of animals receiving TP alone, and a few of the seminiferous tubules contained maturation phase spermatids. Treatment for 90–110 days completely restored spermatogenesis in ½ of these animals. Consequently, although testosterone alone can restore spermatogenesis to a limited extent, the addition of STH appears to augment this effect significantly. Following total post-hypophysectomy regression of the testes, some seminiferous tubules apparently lose the ability to respond to hormonal stimulation. In contrast, complete morphologic and functional restoration of the male reproductive system is possible in some animals if therapy is continued for sufficient periods of time.

Role of the parathyroids in the production of osteitis fibrosa and soft tissue calcification by lead acetate.

The ability of testosterone propionate (TP) to restore spermatogenesis was investigated in 67- to 70-day hypophysectomized rats administered TP either separately or in combination with somatotrophin (STH) and/or thyroxine (T4). Following TP treatment for 35 days, the testicular weights had increased to twice those of the hypophysectomized controls, and a few of the seminiferous tubules contained germinal cells as mature as acrosome phase spermatids. With more prolonged therapy (90–110 days), spermatogenesis was restored qualitatively but not quantitatively in 33% of the animals, while the remainder of the animals showed varying degrees of response. The testes of animals given TP+STH or TP+STH+T4 for 35 days were heavier than those of animals receiving TP alone, and a few of the seminiferous tubules contained maturation phase spermatids. Treatment for 90–110 days completely restored spermatogenesis in ½ of these animals. Consequently, although testosterone alone can restore spermatogenesis to a limited ext...