Overexpression of ubiquitin carboxyl-terminal hydrolase L1 arrests spermatogenesis in transgenic mice

Ubiquitin carboxyl-terminal hydrolase 1 (UCH-L1) can be detected in mouse testicular germ cells, mainly spermatogonia and somatic Sertoli cells, but its physiological role is unknown. We show that transgenic (Tg) mice overexpressing EF1alpha promoter-driven UCH-L1 in the testis are sterile due to a block during spermatogenesis at an early stage (pachytene) of meiosis. Interestingly, almost all spermatogonia and Sertoli cells expressing excess UCH-L1, but little PCNA (proliferating cell nuclear antigen), showed no morphological signs of apoptosis or TUNEL-positive staining. Rather, germ cell apoptosis was mainly detected in primary spermatocytes having weak or negative UCH-L1 expression but strong PCNA expression. These data suggest that overexpression of UCH-L1 affects spermatogenesis during meiosis and, in particular, induces apoptosis in primary spermatocytes. In addition to results of caspases-3 upregulation and Bcl-2 downregulation, excess UCH-L1 influenced the distribution of PCNA, suggesting a specific role for UCH-L1 in the processes of mitotic proliferation and differentiation of spermatogonial stem cells during spermatogenesis.     

Involvement of the D-type cyclins in germ cell proliferation and differentiation in the mouse

Using immunohistochemistry, the expression of the D-type cyclin proteins was studied in the developing and adult mouse testis. Both during testicular development and in adult testis, cyclin D(1) is expressed only in proliferating gonocytes and spermatogonia, indicating a role for cyclin D(1) in spermatogonial proliferation, in particular during the G(1)/S phase transition. Cyclin D(2) is first expressed at the start of spermatogenesis when gonocytes produce A(1) spermatogonia. In the adult testis, cyclin D(2) is expressed in spermatogonia around stage VIII of the seminiferous epithelium when A(al) spermatogonia differentiate into A(1) spermatogonia and also in spermatocytes and spermatids. To further elucidate the role of cyclin D(2) during spermatogenesis, cyclin D(2) expression was studied in vitamin A-deficient testis. Cyclin D(2) was not expressed in the undifferentiated A spermatogonia in vitamin A-deficient testis but was strongly induced in these cells after the induction of differentiation of most of these cells into A(1) spermatogonia by administration of retinoic acid. Overall, cyclin D(2) seems to play a role at the crucial differentiation step of undifferentiated spermatogonia into A(1) spermatogonia. Cyclin D(3) is expressed in both proliferating and quiescent gonocytes during testis development. Cyclin D(3) expression was found in terminally differentiated Sertoli cells, in Leydig cells, and in spermatogonia in adult testis. Hence, although cyclin D(3) may control G(1)/S transition in spermatogonia, it probably has a different role in Sertoli and Leydig cells. In conclusion, the three D-type cyclins are differentially expressed during spermatogenesis. In spermatogonia, cyclins D(1) and D(3) seem to be involved in cell cycle regulation, whereas cyclin D(2) likely has a role in spermatogonial differentiation.     

The new function of two ubiquitin C-terminal hydrolase isozymes as reciprocal modulators of germ cell apoptosis.

Ubiquitination is required throughout all developmental stages of mammalian spermatogenesis. The two ubiquitin C-terminal hydrolase (UCH) enzymes, UCH-L1 and UCH-L3, deubiquitinate ubiquitin-protein conjugates and control the cellular balance of ubiquitin. These two UCH isozymes have 52% amino acid identity and share significant structural similarity. A new function of these two closely related UCH enzymes during spermatogenesis which is associated with germ cell apoptosis has been analyzed. Apoptosis, in general, is thought to be partly regulated by the ubiquitin-proteasome system. During spermatogenesis, apoptosis controls germ cell numbers and eliminates defective germ cells to facilitate testicular homeostasis. In this paper, I review the distinct function of the two UCH isozymes in the testis of gad and Uchl3 knockout mice, which are strongly but reciprocally expressed during spermatogenesis. In addition, the importance of UCHL1-dependent apoptosis for normal spermatogenesis and sperm quality control is discussed.     

Bcl-w forms complexes with Bax and Bak, and elevated ratios of Bax/Bcl-w and Bak/Bcl-w correspond to spermatogonial and spermatocyte apoptosis in the testis

Bcl-w, a prosurvival member of the Bcl-2 family, is essential for spermatogenesis. However, the mechanisms by which Bcl-w participates in the regulation of apoptosis in the testis are largely unknown. To explore the potential role of Bcl-w in the regulation of apoptosis in the testis, the expression of Bcl-w mRNA and protein during testicular development and spermatogenesis, the dimerization with the proapoptosis members of the Bcl-2 family, and the responses to hormonal stimulation in vitro and apoptosis-inducing signals in vivo were investigated. Both Bcl-w mRNA and protein were detected in Sertoli cells, spermatogonia, and spermatocytes, as well as in Leydig cells. The steady-state levels of Bcl-w mRNA and protein were much higher in Sertoli cells than in spermatogonia and spermatocytes. In the adult rat testis, both Bcl-w mRNA and protein in Sertoli cells displayed a stage-specific expression pattern. Bcl-w could form complexes with Bax and Bak but not with Bad. Bax and Bak were immunohistochemically localized to the same cell types as Bcl-w, but with higher expression levels in spermatocytes and spermatogonia than in Sertoli cells. FSH could up-regulate Bcl-w mRNA levels in the seminiferous tubules cultured in vitro, whereas no effect was observed when testosterone was applied. Three animal models that display spermatogonial apoptosis induced by blockade of stem cell factor/c-kit interaction by a function-blocking anti-c-kit antibody, spermatocyte apoptosis induced by methoxyacetic acid, and apoptosis of spermatogonia, spermatocytes, and spermatids induced by testosterone withdrawal after ethylene dimethane sulfonate treatment were employed to check the changes of Bcl-w, Bax, and Bak protein levels during apoptosis of specific germ cells. In all three models, the ratios of Bax/Bcl-w and Bak/Bcl-w were significantly elevated. The present study suggests that Bcl-w is an important prosurvival factor of Sertoli cells, spermatogonia, and spermatocytes and participates in the regulation of apoptosis by binding proapoptotic factors Bax and Bak. The ratios of Bax/Bcl-w and Bak/Bcl-w may be decisive for the survival of Sertoli cells, spermatogonia, and spermatocytes.     

Spermatogenesis and related plasma androgen levels in Atlantic halibut (Hippoglossus hippoglossus L.)

Spermatogenesis in male Atlantic halibut (Hippoglossus hippoglossus L.) was investigated by sampling blood plasma and testicular tissue from 15-39-month-old fish. The experiment covered a period in which all fish reached puberty and completed sexual maturation at least once. The germinal compartment in Atlantic halibut testis appears to be organized in branching lobules of the unrestricted spermatogonial type, because spermatocysts with spermatogonia were found throughout the testis. Spermatogenesis was characterized histologically, and staged according to the most advanced type of germ cell present: spermatogonia (Stage I), spermatogonia and spermatocytes (Stage II), spermatogonia, spermatocytes and spermatids (Stage III), spermatogonia, spermatocytes, spermatids and spermatozoa (Stage IV), and regressing testis (Stage V). Three phases could be distinguished: first, an initial phase with low levels of circulating testosterone (T; quantified by RIA) and 11-ketotestosterone (11-KT; quantified by ELISA), spermatogonial proliferation, and subsequently the initiation of meiosis marked by the formation of spermatocytes (Stage I and II). Secondly, a phase with increasing T and 11-KT levels and with haploid germ cells including spermatozoa present in the testis (Stage III and IV). Thirdly, a phase with low T and 11-KT levels and a regressing testis with Sertoli cells displaying signs of phagocytotic activity (Stage V). Circulating levels of 11-KT were at least four-fold higher than those of T during all stages of spermatogenesis. Increasing plasma levels of T and 11-KT were associated with increasing testicular mass throughout the reproductive cycle. The absolute level of, or the relation between, testis growth and circulating androgens were not significantly different in first time spawners compared to fish that underwent their second spawning season. These results provide reference levels for Atlantic halibut spermatogenesis.     

Characterization of high mobility group protein levels during spermatogenesis in the rat

The distribution, quantitation, and synthesis of high mobility group (HMG) proteins during spermatogenesis in the rat have been determined. HMG1, -2, -14, and -17 were isolated from rat testes by Bio-Rex 70 chromatography combined with preparative gel electrophoresis. Amino acid analysis revealed that each rat testis HMG protein was similar to its calf thymus analogue. Tryptic peptide maps of somatic and testis HMG2 showed no differences and, therefore, failed to detect an HMG2 variant. Testis levels of HMG proteins, relative to DNA content, were equivalent to other tissues for HMG1 (13 micrograms/mg of DNA), HMG14 (3 micrograms/mg of DNA), and HMG17 (5 micrograms/mg of DNA). The testis was distinguished in that it contained a substantially higher level of HMG2 than any other rat tissue (32 micrograms/mg of DNA). HMG protein levels were determined from purified or enriched populations of testis cells representing the major stages of spermatogenesis; spermatogonia and early primary spermatocytes, pachytene spermatocytes, early spermatids, and late spermatids; and testicular somatic cells. High levels of HMG2 in the testis were due to pachytene spermatocytes and early spermatids (56 +/- 4 and 47 +/- 6 micrograms/mg of DNA, respectively). Mixtures of spermatogonia and early primary spermatocytes showed lower levels of HMG2 (12 +/- 3 micrograms/mg of DNA) similar to proliferating somatic tissues, whereas late spermatids had no detectable HMG proteins. The somatic cells of the testis, including isolated populations of Sertoli and Leydig cells, showed very low levels of HMG2 (2 micrograms/mg of DNA), similar to those in nonproliferating somatic tissues. HMG proteins were synthesized in spermatogonia and primary spermatocytes, but not in spermatids. Rat testis HMG2 exhibited two bands on acid-urea gels. A "slow" form comigrated with somatic cell HMG2, while the other "fast" band migrated ahead of the somatic form and appeared to be testis-specific. The "fast" form of HMG2 accounted for the large increase of HMG2 levels in rat testes. These results show that the very high level of HMG2 in testis is not associated with proliferative activity as previously hypothesized.     

Expression, immunolocalization and processing of fertilins ADAM-1 and ADAM-2 in the boar (sus domesticus) spermatozoa during epididymal maturation

Background

The ubiquitin proteasome system (UPS) is a key player in regulating many cellular processes via proteasomal degradation of ubiquitinated proteins. Recently published data show that Jab1/CSN5 interacts with p97/VCP and controls the ubiquitination status of proteins bound to p97/VCP in mouse and human cells. However, coexpression of p97/VCP and Jab1/CSN5 in the developing rat testis and epididymis has not previously been studied.

Methods

Testicular and epididymal tissues from 5-, 15-, 30-, and 60-day-old rats were examined by immunohistochemistry and Western blotting. Colocalisation of proteins was determined by immunofluorescence microscopy.

Results

In the 5-day-old rat testis, p97/VCP and Jab1/CSN5 were specifically expressed in gonocytes. The expression of p97/VCP and Jab1/CSN5 significantly increased at day 15 and was found in spermatogonia, Sertoli cells and spermatocytes. In 30- and 60-day-old rat testes, p97/VCP indicated moderate to strong expression in Sertoli cells, spermatogonia, round and elongating spermatids. However, moderate to weak expression was observed in spermatocytes. Jab1/CSN5 showed strong expression in spermatogonia and spermatocytes, while relatively moderate expression was observed in round and elongating spermatids in 30- and 60-day-old rat testes. In contrast, in the epididymis, the expression of both proteins gradually increased from 5 to 60 days of age. After rats reached 2 weeks of age, the expression of both proteins was mostly restricted to the basal and principal cells of the caput epididymis.

Conclusions

Our study suggests that p97/VCP and Jab1/CSN5 could be an important part of the UPS in the developing rat testis and epididymis and that both proteins may be involved in the regulation of spermatogenesis and epididymal epithelial functions.     

Asymmetric Distribution of UCH‐L1 in Spermatogonia Is Associated With Maintenance and Differentiation of Spermatogonial Stem Cells

Asymmetric division of germline stem cells in vertebrates was proposed a century ago; however, direct evidence for asymmetric division of mammalian spermatogonial stem cells (SSCs) has been scarce. Here, we report that ubiquitin carboxy‐terminal hydrolase 1 (UCH‐L1) is expressed in type A (A_s, A_pr, and A_al) spermatogonia located at the basement membrane (BM) of seminiferous tubules at high and low levels, but not in differentiated germ cells distant from the BM. Asymmetric segregation of UCH‐L1 was associated with self‐renewal versus differentiation divisions of SSCs as defined by co‐localization of UCH‐L1^high and PLZF, a known determinant of undifferentiated SSCs, versus co‐localization of UCH‐L1^low/? with proteins expressed during SSC differentiation (DAZL, DDX4, c‐KIT). In vitro, gonocytes/spermatogonia frequently underwent asymmetric divisions characterized by unequal segregation of UCH‐L1 and PLZF. Importantly, we could also demonstrate asymmetric segregation of UCH‐L1 and PLZF in situ in seminiferous tubules. Expression level of UCH‐L1 in the immature testis where spermatogenesis was not complete was not affected by the location of germ cells relative to the BM, whereas UCH‐L1‐positive spermatogonia were exclusively located at the BM in the adult testis. Asymmetric division of SSCs appeared to be affected by interaction with supporting somatic cells and extracelluar matrix. These findings for the first time provide direct evidence for existence of asymmetric division during SSCs self‐renewal and differentiation in mammalian spermatogenesis. J. Cell. Physiol. 220: 460–468, 2009. © 2009 Wiley‐Liss, Inc.     

大鼠睾丸特异表达基因Ube1的分离鉴定及生物学特征

本研究采用抑制性消减杂交(suppression subtracfive hybridization, SSH)和cDNA快速扩增(rapid amplification of cDNA ends, RACE)技术从大鼠A型精原细胞和粗线期精母细胞中成功克隆出大鼠泛素激活酶(ubiquitin-activating enzyme)基因Ube1 (GenBank登录号EF690356).该基因序列全长3433 bp,其中开放阅读框有3171 bp,编码一个含1057个氨基酸的蛋白质.Blast比对显示,Ube1与小鼠泛素激活酶基因Ubely1的同源性为93%,与人泛素激活酶基因UBE1的同源性为82%.Ube1基因编码的蛋白质含泛素激活酶信号位点和泛素激活酶活化位点,这些位点也存在于人类和小鼠的泛素激活酶1中.RT-PCR分析显示,Ube1在睾丸中大量表达,而在心、肝、脾、肺、肾、肌肉、脑、卵巢中没有表达.荧光定量PCR分析不同生精细胞中Ube1的表达,显示Ube1在A型精原细胞中大量表达,在粗线期精母细胞、圆形精子细胞和支持细胞中微弱表达.以上结果提示,Ube1是大鼠睾丸特异表达基因,可能通过参与泛素/蛋白酶体途径来影响精子发生.     

Cellular expression of retinal dehydrogenase types 1 and 2: effects of vitamin A status on testis mRNA

We examined expression of retinal dehydrogenase (RALDH) types 1 and 2 in liver and lung, and the effect of vitamin A status on testis expression by in situ hybridization. Liver expressed RALDH1 and RALDH2 only in stellate cells and hepatocytes, respectively. Lung expressed RALDH1 and RALDH2 throughout the epithelia of the airways, from the principal bronchi to the respiratory bronchiole. Vitamin A-sufficient rats expressed RALDH1 in spermatocytes, with less intense expression in spermatogonia and spermatids, and expressed RALDH2 in interstitial cells, spermatogonia, and spermatocytes. Neither Sertoli nor peritubular cells showed detectable RALDH1 or RALDH2 mRNA. Vitamin A deficiency produced a sevenfold increase in RALDH1 and a 70-fold decrease in RALDH2 mRNA in testis. In each case, the net change reflected extensive loss of germ cells, increased intensity of expression in residual germ cells, and expression in Sertoli and peritubular cells. Low-dose RA relatively early during vitamin A depletion supported spermatogenesis and affected expression of both RALDHs, but did not reinstate "vitamin A normal" expression patterns. These results show that: RALDH1 and RALDH2 have distinct mRNA expression patterns in multiple cell types in three vitamin A target tissues; RALDH expression occurs in cell types that express cellular retinol-binding protein and retinol dehydrogenase isozymes (except stellate cells, for which retinol dehydrogenase expression remains unknown); vitamin A deficiency and RA supplementation affects the loci and intensity of RALDH mRNAs in testis; and low-dose RA does not substitute completely for retinol. Overall, these data provide insight into the unique functions of RALDH1 and RALDH2 in retinoid metabolism.     

Gonadotropin-Activated Androgen-Dependent and Independent Pathways Regulate Aquaporin Expression during Teleost (Sparus aurata) Spermatogenesis

The mediation of fluid homeostasis by multiple classes of aquaporins has been suggested to be essential during spermatogenesis and spermiation. In the marine teleost gilthead seabream (Sparus aurata), seven distinct aquaporins, Aqp0a, -1aa, -1ab, -7, -8b, -9b and -10b, are differentially expressed in the somatic and germ cell lineages of the spermiating testis, but the endocrine regulation of these channels during germ cell development is unknown. In this study, we investigated the in vivo developmental expression of aquaporins in the seabream testis together with plasma androgen concentrations. We then examined the in vitro regulatory effects of recombinant piscine gonadotropins, follicle-stimulating (rFsh) and luteinizing (rLh) hormones, and sex steroids on aquaporin mRNA levels during the spermatogenic cycle. During the resting phase, when plasma levels of androgens were low, the testis exclusively contained proliferating spermatogonia expressing Aqp1ab, whereas Aqp10b and -9b were localized in Sertoli and Leydig cells, respectively. At the onset of spermatogenesis and during spermiation, the increase of androgen plasma levels correlated with the additional appearance of Aqp0a and -7 in Sertoli cells, Aqp0a in spermatogonia and spermatocytes, Aqp1ab, -7 and -10b from spermatogonia to spermatozoa, and Aqp1aa and -8b in spermatids and spermatozoa. Short-term in vitro incubation of testis explants indicated that most aquaporins in Sertoli cells and early germ cells were upregulated by rFsh and/or rLh through androgen-dependent pathways, although Aqp1ab in proliferating spermatogonia was also activated by estrogens. However, expression of Aqp9b in Leydig cells, and of Aqp1aa and -7 in spermatocytes and spermatids, was also directly stimulated by rLh. These results reveal a complex gonadotropic control of aquaporin expression during seabream germ cell development, apparently involving both androgen-dependent and independent pathways, which may assure the fine tuning of aquaporin-mediated fluid secretion and absorption mechanisms in the seabream testis.     

The cloning of cyclin B3 and its gene expression during hormonally induced spermatogenesis in the teleost, Anguilla japonica

We cloned cyclin B1, B2, and B3 cDNAs from the eel testis. Northern blot analysis indicated that these cyclin B mRNAs were expressed and increased from day 3 onward after the hormonal induction of spermatogenesis, and that cyclin B3 was most dominantly expressed during spermatogenesis. In situ hybridization showed that cyclin B1 and B2 were present from the spermatogonium stage to the spermatocyte stage. On the other hand, cyclin B3 mRNA was present only in spermatogonia. Although mouse cyclin B3 is expressed specifically in the early meiotic prophase, these results indicate that eel cyclin B3 expression is limited during spermatogenesis to spermatogonia, but is not present in spermatocytes. These facts together suggest that eel cyclin B3 is specifically involved in spermatogonial proliferation (mitosis), but not in meiosis.     

Changes in mRNA length accompany translational regulation of the somatic and testis-specific cytochrome c genes during spermatogenesis in the mouse

The mouse testis contains two isotypes of cytochrome c, which differ in 14 of 104 amino acids: cytochrome cs is present in all somatic tissues and cytochrome cT is testis specific. The regulation of cytochrome cS and cytochrome cT gene expression during spermatogenesis was examined by Northern blot analysis using specific cDNA probes. Total RNA was isolated from adult tissues, enriched germinal cell populations and polysomal gradients of total testis and isolated germinal cells. Three cytochrome cS mRNAs were detected averaging 1.3 kb, 1.1 kb and 0.7 kb in all tissues examined; an additional 1.7 kb mRNA was observed in testis. Isolated germinal cells through prepuberal pachytene spermatocytes contained only the three smaller mRNAs; the 1.7 kb mRNA was enriched in round spermatids. All three smaller cytochrome cS mRNAs were present on polysomes; the 1.7 kb mRNA was non-polysomal. Cytochrome cT mRNA of 0.6-0.9 kb was detected in testis; mRNA levels were low in early spermatogonia and peaked in prepuberal pachytene spermatocytes. In adult pachytene spermatocytes, a subset of the cytochrome cT mRNAs, 0.7-0.9 kb, was present on polysomes; a shortened size class, 0.6-0.75 kb, was non-polysomal. A distinct, primarily non-polysomal, cytochrome cT 0.7 kb mRNA was present in round spermatids. These results indicate that (1) both cytochrome cS and cytochrome cT mRNAs are present in early meiotic cells, (2) a 1.7 kb cytochrome cS mRNA is post-meiotically expressed and non-polysomal and (3) cytochrome cS and cytochrome cT mRNAs are each developmentally and translationally regulated during spermatogenesis.