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.     

金黄地鼠精子发生及附睾成熟过程中Ca~（2＋）的分布规律

应用焦锑酸钾原位沉淀法对金黄地鼠精子发生及附睾成熟过程中Ｃａ２＋的分布变化规律进行了系统的研究。在睾丸的曲细精管中，支持细胞和生精细胞的细胞核和细胞质有钙沉淀颗粒分布。在支持细胞、精原细胞、精母细胞、高尔基体期和顶体期精子细胞的胞质中钙沉淀主要分布于线粒体和内质网。支持细胞核仁的无定形部分、核仁相随染色质和核质中有大量的钙沉淀颗粒。在精原细胞、精母细胞、高尔基体期的精子细胞核中钙沉淀主要分布于浓缩的染色质周围及其内部，而分散的染色质中则少见钙沉淀。在顶体期的精子细胞核内钙沉淀主要分布于核膜上，核质中偶见Ｃａ２＋沉淀。成熟期的精子细胞钙沉淀颗粒分布于顶体外膜和顶体内膜的内侧，顶体内膜上有钙沉淀集中分布。在附睾中钙沉淀分布于精子顶体区的质膜内外两侧和顶体外膜外侧，精子尾部线粒体外膜和基质中也有钙沉淀分布。     

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. Anat Rec 259:131–140, 2000. © 2000 Wiley‐Liss, Inc.     

Germ cell degeneration in normal and microwave-irradiated rats: potential sperm production rates at different developmental steps in spermatogenesis

Germ cell degeneration in 14 normal and 14 microwave-irradiated, adult (400-500 gm), Sprague-Dawley rats was compared by evaluating potential sperm production rates at different developmental steps in spermatogenesis. Following 9 days of irradiation at 1.3 GHz (6 hours/day at 6.3 mW/gm using 1-mu sec pulsewidth at 600 pulses/second) or sham treatment, rats were killed at 6.5, 13.0, 26.0, or 52.0 days following treatment. Testes were perfused with 2% glutaraldehyde, embedded in Epon, and sectioned at 0.5 micron for morphometric analyses. Plasma LH and FSH concentrations were determined by radioimmunoassay from blood collected on the day of death. Considering nuclear size, percentage of nuclei in the parenchyma, and life span of different cells, potential daily sperm production was determined for type B spermatogonia, preleptotene or pachytene primary spermatocytes, or spermatids with round nuclei. No differences (P greater than .05) in parameters tested were found among time periods following irradiation. With the possible exception of sperm production per testis (P less than .05) based on pachytene spermatocytes, microwave irradiation had no effect on the parameters evaluated. No degeneration was detected in spermatogenesis when potential sperm production rates were determined either from type B spermatogonia to spermatids or from type B spermatogonia to a posttesticular approximation of sperm production rate. Thus, it appears that regulation of sperm production rates must take place during spermatogonial mitoses, since once the number of type B spermatogonia is determined, there is essentially no subsequent alteration in sperm production potential in normal or irradiated adult rats.     

Testicular development in cynomolgus monkeys

The testes from 136 male cynomolgus monkeys were examined histopathologically in order to investigate the relationship between the development of spermatogenesis and testis weight, age, and body weight. At Grade 1 (immature), Sertoli cells and spermatogonia were the only cell classes in the testis. At Grade 2 (pre-puberty), no elongated spermatids were observed in the testis, although a few round spermatids and small lumen formation were observed. At Grade 3 (onset of puberty), all classes of germ cells were observed in the testis, although seminiferous tubule diameters and numbers of germ cells were small. Slight debris in the epididymis was observed in almost all animals. At Grade 4 (puberty), almost complete spermatogenesis was observed in the seminiferous tubules and it was possible to ascertain the spermatogenesis stage as described by Clermont, although tubule diameters and numbers of germ cells were small. There was less debris in the epididymis than at Grade 3. At Grade 5 (early adult), complete spermatogenesis was observed in the seminiferous tubules. At Grade 6 (adult), complete spermatogenesis in the seminiferous tubules and a moderate or large number of sperm in the epididymis were observed. Moreover, sperm analysis using ejaculated sperm was possible. Logistic regression analysis showed that testis weight is a good indicator of testicular maturity.     

Cloning of cDNAs and the differential expression of A-type cyclins and Dmc1 during spermatogenesis in the Japanese eel, a teleost fish.

We cloned A-type cyclins (cyclins A1 and A2) and Dmc1 cDNAs from the eel testis. Cyclin A1 mRNA was predominantly expressed in the livers, ovaries, and testes of the eels. In contrast to cyclin A1 mRNA, a very high expression of cyclin A2 mRNA was observed in the brains, livers, kidneys, spleens, ovaries, and testes of the eels. Dmc1 mRNA was predominantly expressed in the testes and ovaries; expression in the brain was also detected. In the eel testis, a few type-A spermatogonia incorporating 5-bromo-2'-deoxyuridine (BrdU) were seen before the initiation of spermatogenesis by hormonal induction. On day 1 after hormonal induction, the number of BrdU-labeled spermatogonia increased remarkably, and after 3 and 6 days, many labeled type-B spermatogonia were also observed. The expression of cyclin A2 increased 1 day after the induction of spermatogenesis and reached a plateau after 6 days, when many type-B spermatogonia with high proliferative activity were found. In contrast, the expression of cyclin A1 mRNA was detected after 9 days, coincident with the first appearance of spermatocytes. Cyclin A1 mRNA was localized in germ cells of all stages, from primary spermatocytes to round spermatids, whereas cyclin A2 mRNA was specifically localized in spermatogonia, secondary spermatocytes, round spermatids, and testicular somatic cells, including Sertoli cells. Dmc1 was localized only in the earlier stages of primary spermatocytes; before this stage, cyclin A1 mRNA was not detectable. Overall, cyclin A2, Dmc1, and cyclin A1 are expressed in spermatogenic cells sequentially before and during meiosis in the eel testis.     

The expression pattern of PARP-1 and PARP-2 in the developing and adult mouse testis

Although the importance of the PARP family members in the adult testis has already been acknowledged, their expression in the developing testis has not been addressed. We performed immunohistochemistry by using PARP-1 and PARP-2 antibodies on the developing mouse testis at embryonic day (E) 15.5, E17.5, postnatal day (PN) 0, PN3, PN9, PN20 and adult. Our results showed that at embryonic and early postnatal days, the expression of PARP-1 was in the nuclei of gonocytes and spermatogonia. PARP-1 was positive in interstitial cells with nuclear localization at all studied ages. At embryonic and early postnatal days, the expression of PARP-2 was in the cytoplasm of gonocytes and spermatogonia. During the progress of spermatogenesis, PARP-2 was localized in the cytoplasm of pre-leptotene spermatocytes on PN9, in the cytoplasm of pachytene spermatocytes on PN15 and in the cytoplasm of round spermatids on PN20. In the adult, PARP-2 staining can still be observed in the cytoplasm of spermatogonia, but to a much lesser degree than in the round and elongating spermatids. For all the studied ages, PARP-2 was positive in Sertoli cells and interstitial cells with cytoplasmic localization. Our results indicate that PARP proteins are present in germ and somatic cells during testis development in mice.     

Chronic chromium exposure-induced changes in testicular histoarchitecture are associated with oxidative stress: study in a non-human primate (Macaca radiata Geoffroy)

BACKGROUND: Reproductive toxicity of chromium is in dispute despite positive findings in rodents. Recently we reported epididymal toxicity of hexavalent chromium (CrVI) in bonnet monkeys and in this paper we report its testicular toxicity. METHODS: Adult monkeys (Macaca radiata) were given drinking water containing CrVI (100, 200, 400 p.p.m.) for 6 months and testes were removed for ultrastructural and biochemical analyses. RESULTS: CrVI treatment disrupted spermatogenesis, leading to accumulation of prematurely released spermatocytes, spermatids and uni- and multinucleate giant cells in the lumen of seminiferous tubules. Transmission electron microscopy revealed granulation of chromatin and vacuolation between acrosomal cap and manchette microtubules of elongated spermatids and in the Golgi area of round spermatids. Pachytene spermatocytes had fragmented chromatin and swollen mitochondria with collapsed cristae. Spermatocytes and spermatogonia in the basal compartment were unaffected. Macrophages containing phagocytosed sperm and dense inclusions in Sertoli cells were seen. Specific activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase and concentrations of the non-enzymatic antioxidants glutathione, vitamins A, C and E decreased, while concentrations of H(2)O(2) and hydroxyl radicals increased in the testis of chromium-treated monkeys. Withdrawal of chromium treatment for 6 months normalized spermatogenesis and the status of pro- and antioxidants in the testis. CONCLUSIONS: CrVI disrupts spermatogenesis by inducing free radical toxicity, and supplementation of antioxidant vitamins may be beneficial to the affected subjects.     

中心体蛋白centrin在大鼠精子发生过程中的表达

目的：研究中心体蛋白centrin在大鼠生精细胞中的表达情况,以深入了解centrin在精子发生过程中的作用。方法：通过重力沉降法分离大鼠不同发育阶段的生精细胞,用免疫荧光和蛋白印迹实验检测各级生精细胞中centrin蛋白的表达,用定量RT－PCR检测centrin同源基因centrin1和centrin2mRNA的表达水平。结果：间接免疫荧光和蛋白印迹显示精母细胞、圆形、长形精子细胞均有centrin蛋白存在,位于中心粒上,而在附睾的成熟精子中centrin则消失。RT－PCR研究发现,centrin在睾丸组织中特异性表达,centrin2在多种组织中均有表达。在睾丸中,centrin1仅在生精细胞进入减数分裂后转录,其mRNA水平在圆形精子细胞中最高,而centrin2在精原细胞中即有表达,减数分裂后其mRNA难以检测到。结论centrin蛋白在大鼠雄性配子的发育过程中最终丢失;该基因家族中同源基因centrin1和centrin2表达呈现组织特异性和发育阶段特性,在精子发生过程中发挥不同功能,centrin1蛋白可能与减数分裂及鞭毛生成相关,centrin2则参与细胞有丝分裂过程。     

Seminiferous tubule stages in the prairie dog (Cynomys ludovicianus) during the annual breeding cycle

The male prairie dog (Cynomys ludovicianus) is an annual breeder with complete testicular regression between breeding periods. Knowledge of the seminiferous tubule cycle stages at all phases of the annual cycle is essential for evaluation of testicular effects of endogenous and exogenous hormones. Testis tubule diameter is directly correlated with testicular weight during the annual cycle. Seminiferous tubule stages found during testicular activity start with sperm release and round spermatids in the Golgi stage (I). Then they progress through the cap and acrosome stages (stages II to VI) until elongate spermatids are formed. During these stages preleptotene, leptotene and zygotene cells develop into pachytene cells which mature with the long spermatids (stage VII). Two distinct tubule associations (stages VIII, IX) follow during which the first and second meiotic metaphases occur. These stages are correlated with the middle and late phases of residual lobe retraction and condensation. The last stage (X) has final sperm development and is present with round spermatids that have no Golgi development. During regression changes are initially associated with the seminiferous tubule stages of active testes and end with relocation of Sertoli cell nuclei to a position above the basal layer of spermatogonia. Out of season testes are characterized by few spermatogonial mitoses and absence of viable spermatocytes. In recrudescent testes, Sertoli cell nuclei again become basal, spermatogonia resume mitoses and spermatocytes and spermatids progressively develop. After each cycle of proliferation of germ cells there is sloughing of the most differentiated spermatocytes and spermatids until the final tubule associations of the active testis are present. Anat. Rec. 247:355–367, 1997. © 1997 Wiley-Liss, Inc.     

Compensatory upregulation of myelin protein zero-like 2 expression in spermatogenic cells in cell adhesion molecule-1-deficient mice

The cell adhesion molecule-1 (Cadm1) is a member of the immunoglobulin superfamily. In the mouse testis, Cadm1 is expressed in the earlier spermatogenic cells up to early pachytene spermatocytes and also in elongated spermatids, but not in Sertoli cells. Cadm1-deficient mice have male infertility due to defective spermatogenesis, in which detachment of spermatids is prominent while spermatocytes appear intact. To elucidate the molecular mechanisms of the impaired spermatogenesis caused by Cadm1 deficiency, we performed DNA microarray analysis of global gene expression in the testis compared between Cadm1-deficient and wild-type mice. Out of the 25 genes upregulated in Cadm1-deficient mice, we took a special interest in myelin protein zero-like 2 (Mpzl2), another cell adhesion molecule of the immunoglobulin superfamily. The levels of Mpzl2 mRNA increased by 20-fold and those of Mpzl2 protein increased by 2-fold in the testis of Cadm1-deficient mice, as analyzed with quantitative PCR and western blotting, respectively. In situ hybridization and immunohistochemistry demonstrated that Mpzl2 mRNA and protein are localized in the earlier spermatogenic cells but not in elongated spermatids or Sertoli cells, in both wild-type and Cadm1-deficient mice. These results suggested that Mpzl2 can compensate for the deficiency of Cadm1 in the earlier spermatogenic cells.     

Molecular chaperone calmegin localization to the endoplasmic reticulum of meiotic and post-meiotic germ cells in the mouse testis.

Calmegin is a testis-specific Ca(2+)-binding protein that is homologous to calnexin. Recently, sperm from transgenic mice lacking calmegin have been shown to be infertile. To further characterize calmegin, we analyzed the precise stage of expression and the intracellular localization of this protein in germ cells during mouse spermatogenesis by an immunoperoxidase technique using the anti-calmegin monoclonal antibody TRA369. Light microscopic immunocytochemistry showed that calmegin appeared in early pachytene spermatocytes, with the highest expression in round and elongating spermatids, and disappeared in the maturation phase of spematids at step 15. Immunoelectron microscopy showed that selective localization was found at the endoplasmic reticulum membrane and the nuclear envelope of spermatogenic cells. During the maturation phase, a dramatic reduction in calmegin occurred in the endoplasmic reticulum of the spermatids, suggesting that the major function of calmegin has been completed by the time spermatids reach step 14. In addition, although the immunoreactivity was completely absent in the calmegin-deficient mutant mouse testis, ultrastructural analysis showed that mature sperm from the knockout mice were normal. This suggests that calmegin is not required for the morphogenesis of male germ cells. Thus, our results suggest that calmegin has a major role in mouse spermatogenesis, and also indicate that this protein would be useful as a maker molecule to study the functional role of the endoplasmic reticulum in the process of spermatid differentiation.     

Testis structure in the sys (symplastic spermatids) mouse

Testes of mice with the recessive insertional mutation termed symplastic spermatids (sys) were assessed for structural and developmental abnormalities. Homozygous (sys/sys) males are infertile due to an abnormality in spermatogenesis leading to azoospermia. The major interruption to spermatogenesis occurs when the intercellular bridges that connect round spermatids open prematurely resulting in the formation of symplasts. Symplasts contain as many as 285 nuclei. Development of spermatids within symplasts is arrested just before, or just after, elongation of the spermatid nuclei begins. Symplasts degenerate and appear to be phagocytized by Sertoli cells and by intratubular macrophages. In addition, degeneration of young round spermatids and also spermatocytes occasionally is observed. Spermatocyte degeneration is substantial in some tubules and leaves them depleted of cells other than basal compartment cells. Sertoli cell abnormalities are prominent and include intracellular vacuolation, absence of apical processes surrounding round spermatids, degeneration, and occasional sloughing. Although reduplication and infolding of the basal lamina is also seen, this does not appear as a common phenomenon. The sys phenotype is first manifest in animals between 19 days and 22 days of age. Considerable variability is seen in testis histology of prepubertal animals; some display degenerating pachytene spermatocytes and virtually no Sertoli cell vacuoles, while others display vacuoles without apparent elevated numbers of degenerating spermatocytes. Although this study has not revealed the primary cell type(s) affected by the insertional inactivation event, it is possible that the abnormalities in the Sertoli cells are responsible for germ cell degeneration as it is generally recognized that deficits in the Sertoli cell can result in major germ cell abnormalities but not vice versa.     

Quantitative (stereological) study of the effects of vasectomy on spermatogenesis in rabbits

Using stereological methods, especially the optical disector for unbiased estimation of nuclear number, our recent study demonstrated that long-term (6 or 12 months) vasectomy in the rhesus monkey had no significant effects on spermatogenesis (Peng et al. Reproduction 2002, 124, 847-856). This study aimed to determine the scenario in the rabbit using the same morphometric methodology. Three groups of normal male Japanese white rabbits (aged 4-5 months) were subjected to unilateral vasectomy; 10 days, 6 months and 12 months later both testes and epididymides were removed. Testicular and epididymal methacrylate-embedded sections were obtained for stereology. Vasectomy-induced damage to spermatogenesis was observed, primarily sloughing of spermatogenic cells with a greater reduction in the number of advanced (adluminal) cells. The damage was most severe at 10 days, occurring in all the testes on the vasectomized side and involving sloughing of even type A spermatogonia, the number of which returned to normal at 6 and 12 months. Damage was less severe at 6 and 12 months, being found in half of the testes of the vasectomy side, in which the total numbers of later germ cell types were 24.0-59.1% (spermatocytes) and 0.3-11.6% (spermatids) of control at 6 months, and 20.1-22.1% (spermatocytes) and 0.4-12.0% (spermatids) of control at 12 months. By contrast, Sertoli cell number per testis was unchanged following vasectomy in any group. Epididymis on the vasectomy side, especially at 10 days and 6 months, appeared larger than on the contralateral side, but this difference was not statistically significant, and no sperm granuloma was seen in the epididymis.     

Regulated expression of the ubiquitin protein ligase, E3(Histone)/LASU1/Mule/ARF-BP1/HUWE1, during spermatogenesis.

A ubiquitin protein ligase (E3), E3(Histone)/LASU1 (Mule/ARF-BP1/HUWE1), was recently identified that mediates ubiquitination of core histones, the Mcl-1 anti-apoptotic protein, and the p53 tumor suppressor protein. However, the expression of E3(Histone)/LASU1 remains poorly studied. Because we identified E3(Histone)/LASU1 from the testis, we explored its regulation during spermatogenesis. In the first wave of rat spermatogenesis, E3(Histone)/LASU1 mRNA and protein had peak expression at days 10 and 20, respectively, and decreased with age. Consistent with these findings, immunohistochemistry revealed that E3(Histone)/LASU1 was highly expressed in nuclei from spermatogonia to mid-pachytene spermatocytes. There was no obvious staining in spermatids, when histones are ubiquitinated and degraded. E3(Histone)/LASU1 was also expressed in other tissues. However, except in neuronal cells of the brain, expression was cytoplasmic. Thus, E3(Histone)/LASU1 may play a role in chromatin modification in early germ cells of the testis, but also has functions in other tissues.     

DOG1 immunohistochemical staining of testicular biopsies is a reliable tool for objective assessment of infertility

Testicular biopsy may be a component of the work-up of male infertility. However, no reliable diagnostic tools are available for objective quantitative assessment of spermatogenic cells. It is well known that MAGE-A4 is selectively expressed in spermatogonia and our group has previously demonstrated that DOG1 differentially stains germ cells. Therefore, we performed DOG1 and a double stain cocktail (DOG1 and 57b murine monoclonal anti-MAGE-A4) immunohistochemical stains on 40 testicular infertility biopsies (10 each with active spermatogenesis, Sertoli cell-only, hypospermatogenesis, and maturation arrest), 25 benign seminiferous tubules from radical orchiectomies, and 5 spermatocytic tumors (ST). In biopsies/resections with active spermatogenesis, DOG1 stained spermatocytes and spermatids and was absent in spermatogonia, while MAGE-A4 stained spermatogonia and primary spermatocytes (weak). In hypospermatogenesis, DOG1 highlighted decreased spermatocytes/spermatids and MAGE-A4 highlighted decreased spermatogonia. DOG1 staining confirmed decreased to absent spermatocytes in maturation arrest and MAGE-A4 staining established the presence of preserved spermatogonia in all cases. All STs were negative for DOG1 and positive for MAGE-A4, while all Sertoli cell-only cases were negative for DOG1 and the double stain cocktail. In conclusion, we confirmed that DOG1 is expressed in spermatocytes and spermatids and MAGE-A4 highlights primarily spermatogonia. Usage of these stains facilitates confirmation of maturation arrest, assessment of the percentage of testis involvement in hypospermatogenesis and identification of mixed patterns. Finally, this study supports that the differentiation of STs is more closely related to spermatogonia than the more mature spermatocytes.     

Chromosome aberrations and spermatogenic disorders in mice with Robertsonian translocation (11; 13)

Objective: To determine the diagnostic features of Robertsonian (Rob) translocation (11; 13) in mice and the mechanisms underlying the effect on spermatogenesis and reproductive decline. Methods: A Rob translocation (11; 13) mouse model was established by cross-breeding, and confirmed by chromosome analysis. Chromosome aberrations and translocation patterns were identified in mice with Rob translocation (11; 13) by fluorescence in situ hybridization (FISH). Spermatogenic disorders were investigated at different stages of spermatogenesis. Immunofluorescent analysis was performed on sections of testis and epididymis specimens during spermatogenic meiosis. The weight of the testes and reproductive decline were recorded. Results: The crossed Rob translocation (11; 13) mouse has 39 chromosomes, including a fusion chromosome (included chromosomes 11 and 13) using dual color FISH. There was no difference in the distribution pattern of SYCP3 and γH2AX in spermatocytes between Rob translocation and wild-type mice; however, round haploid spermatids presented characteristic morphologic changes of apoptosis and the number of haploid spermatids was decreased. Furthermore, the immature germ cells were released into the epididymis and the number of mature sperm was reduced. Conclusions: Chromosome aberrations and spermatogenic disorders may result from apoptosis of round haploid spermatids and a reduced number of mature sperm in Rob translocation (11; 13) mice. Abnormal sperm and reduced number of sperm may be one of the main reasons for reproductive decline and male infertility in Rob translocation (11; 13) mice.