兔精子发生过程中cyclin B1基因表达和蛋白定位的发育阶段依赖性

利用原位杂交和免疫组化等方法,研究兔精子发生过程中生精细胞cyclin B1 mRNA的表达和蛋白定位特点,结果显示,兔生精上皮中Cyclin B1 mRNA的主要分布在初级精母细胞中,直至圆形精子细胞仍然存在,于精子细胞的变态过程中逐渐消失,在伸长的精子细胞和精子中未检测出cyclin B1 mRNA,Cyclin B1蛋白在进入分裂期的精原细胞和精母细胞中表达,在圆形精子细胞和伸长的精子细胞中呈现大量的cyclin B1蛋白,上述结果表明,在兔精子发生过程中,cyclin B1 mRNA表达和蛋白定位具有发育阶段依赖性的特征。     

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 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.     

Regulation of meiosis during mammalian spermatogenesis: the A-type cyclins and their associated cyclin-dependent kinases are differentially expressed in the germ-cell lineage

To begin to examine the function of the A-type cyclins during meiosis in the male, we have examined the developmental and cellular distribution of the cyclin A1 and cyclin A2 proteins, as well as their candidate cyclin-dependent kinase partners, Cdk1 and Cdk2, in the spermatogenic lineage. Immunohistochemical localization revealed that cyclin A1 is present only in male germ cells just prior to or during the first, but not the second, meiotic division. By contrast, cyclin A2 was expressed in spermatogonia and was most abundant in preleptotene spermatocytes, cells which will enter the meiotic pathway. Immunohistochemical detection of Cdk1 was most apparent in early pachytene spermatocytes, while staining intensity diminished in diplotene and meiotically dividing spermatocytes, the cells in which cyclin A1 expression was strongest. Cdk2 was highly expressed in all spermatocytes. Notably, in cells undergoing the meiotic reduction divisions, Cdk2 appeared to localize specifically to the chromatin. This was not the case for spermatogonia undergoing mitotic divisions. In the testis, cyclin A1 has been shown to bind both Cdk1 and Cdk2 but we show here that cyclin A2 binds only Cdk2. These results indicate that the A-type cyclins and their associated kinases have different functions in the initiation and passage of male germ cells through meiosis.     

Expression and phosphorylation of TOPK during spermatogenesis

Among normal organs and tissues, the MAPKK-like mitotic protein kinase TOPK is expressed exclusively in the testis. We analyzed the expression and phosphorylation of TOPK to address the functional role of this kinase during spermatogenesis. TOPK protein is expressed mainly in the cytosol of spermatocytes and spermatids, but not in spermatids and spermatogonia in situ. TOPK-Thr-9, a cdk1/cyclin B target residue, was specifically phosphorylated during mitotic and meiotic phases, while TOPK-Thr-198, a key amino acid for the ATP pocket, was constantly phosphorylated irrespective of the cell cycle. These data indicate that spermatogenic germ cells with vital proliferation activity express TOPK. As TOPK-Thr-9 was phosphorylated during both mitosis and meiosis, TOPK was indicted to play a role in cytokinesis and/or chromosomal segregation but not in DNA replication.     

Mammalian E-type Cyclins Control Chromosome Pairing,Telomere Stability and CDK2 Localization in Male Meiosis

Loss of function of cyclin E1 or E2, important regulators of the mitotic cell cycle, yields viable mice, but E2-deficient males display reduced fertility. To elucidate the role of E-type cyclins during spermatogenesis, we characterized their expression patterns and produced additional deletions of Ccne1 and Ccne2 alleles in the germline, revealing unexpected meiotic functions. While Ccne2 mRNA and protein are abundantly expressed in spermatocytes, Ccne1 mRNA is present but its protein is detected only at low levels. However, abundant levels of cyclin E1 protein are detected in spermatocytes deficient in cyclin E2 protein. Additional depletion of E-type cyclins in the germline resulted in increasingly enhanced spermatogenic abnormalities and corresponding decreased fertility and loss of germ cells by apoptosis. Profound meiotic defects were observed in spermatocytes, including abnormal pairing and synapsis of homologous chromosomes, heterologous chromosome associations, unrepaired double-strand DNA breaks, disruptions in telomeric structure and defects in cyclin-dependent-kinase 2 localization. These results highlight a new role for E-type cyclins as important regulators of male meiosis.     

Requirement of cyclin B2, but not cyclin B1, for bipolar spindle formation in frog (Rana japonica) oocytes

Cyclin B, the regulatory subunit of maturation-promoting factor (MPF), comprises several subtypes that are presumed to confer different functions on MPF although no direct evidence has been provided to date. To clarify the difference in the roles of cyclins B1 and B2, we used frog (Rana japonica) oocytes in which MPF is formed only after progesterone stimulation because it is possible to produce oocytes containing either cyclin B1-MPF or cyclin B2-MPF by antisense RNA-mediated translational inhibition of each mRNA. Using this advantage, we investigated the functions of cyclins B1 and B2 and obtained the following results: (a) oocytes synthesizing cyclin B2-MPF underwent meiosis I and II with formation of a bipolar spindle at each metaphase; (b) oocytes synthesizing cyclin B1-MPF formed a monopolar spindle at metaphase I and extruded an abnormal polar body; and (c) both oocytes underwent germinal vesicle breakdown (GVBD) and chromosome condensation. Immunocytochemical observations also revealed continuous localization of cyclin B2 on the spindle during meiosis. These results provide evidence of the requirement of cyclin B2, but not cyclin B1, for organizing the bipolar spindle, though either cyclin B1 or B2 is redundant for inducing GVBD and chromosome condensation.     

Gamma-ray exposure accelerates spermatogenesis of medaka fish,Oryzias latipes

To examine the spermatogenesis (and spermiogenesis) cell population kinetics after gamma-irradiation, the frequency and fate of BrdU-labeled pre-meiotic spermatogenic cells (spermatogonia and pre-leptotene spermatocytes) and spermatogonial stem cells (SSCs) of the medaka fish (Oryzias latipes) were examined immunohistochemically and by BrdU-labeling. After 4.75 Gy of gamma-irradiation, a statistically significant decrease in the frequency of BrdU-labeled cells was detected in the SSCs, but not in pre-meiotic spermatogenic cells. The time necessary for differentiation of surviving pre-meiotic spermatogenic cells without delay of germ cell development was shortened. More than 90% of surviving pre-meiotic spermatogenic cells differentiated into haploid cells within 5 days after irradiation, followed by a temporal spermatozoa exhaust in the testis. Next, spermatogenesis began in the surviving SSCs. However, the outcome was abnormal spermatozoa, indicating that accelerated maturation process led to morphological abnormalities. Moreover, 35% of the morphologically normal spermatozoa were dead at day 6. Based on these results, we suggest a reset system; after irradiation most surviving spermatogenic cells, except for the SSCs, are prematurely eliminated from the testis by spermatogenesis (and spermiogenesis) acceleration, and subsequent spermatogenesis begins with the surviving SSCs, a possible safeguard against male germ cell mutagenesis.     

MPF localization is controlled by nuclear export.

In eukaryotes, mitosis is initiated by M phase promoting factor (MPF), composed of B-type cyclins and their partner protein kinase, CDK1. In animal cells, MPF is cytoplasmic in interphase and is translocated into the nucleus after mitosis has begun, after which it associates with the mitotic apparatus until the cyclins are degraded in anaphase. We have used a fusion protein between human cyclin B1 and green fluorescent protein (GFP) to study this dynamic behaviour in real time, in living cells. We found that when we injected cyclin B1-GFP, or cyclin B1-GFP bound to CDK1 (i.e. MPF), into interphase nuclei it is rapidly exported into the cytoplasm. Cyclin B1 nuclear export is blocked by leptomycin B, an inhibitor of the recently identified export factor, exportin 1 (CRM1). The nuclear export of MPF is mediated by a nuclear export sequence in cyclin B1, and an export-defective cyclin B1 accumulates in interphase nuclei. Therefore, during interphase MPF constantly shuttles between the nucleus and the cytoplasm, but the bulk of MPF is retained in the cytoplasm by rapid nuclear export. We found that a cyclin mutant with a defective nuclear export signal does not enhance the premature mitosis caused by interfering with the regulatory phosphorylation of CDK1, but is more sensitive to inhibition by the Wee1 kinase.     

Changes in the expression and localization of cohesin subunits during meiosis in a non-mammalian vertebrate, the medaka fish

Until the onset of anaphase, sister chromatids are bound to each other by a multi-subunit protein complex called cohesin. Since chromosomes in meiosis behave differently from those in mitosis, the cohesion and separation of homologous chromosomes and sister chromatids in meiosis are thought to be regulated by meiosis-specific cohesin subunits. Actually, several meiosis-specific cohesin subunits, including Rec8, STAG3 and SMC1beta, are known to exist in mammals; however, there are no reports of meiosis-specific cohesin subunits in other vertebrates. To investigate the protein expression and localization of cohesin subunits during meiosis in non-mammalian species, we isolated cDNA clones encoding SMC1alpha, SMC1beta, SMC3 and Rad21 in the medaka and produced antibodies against recombinant proteins. Medaka SMC1beta was expressed solely in gonads, while SMC1alpha, SMC3 and Rad21 were also expressed in other organs and in cultured cells. SMC1beta forms a complex with SMC3 but not with Rad21, in contrast to SMC1alpha, which forms complexes with both SMC3 and Rad21. SMC1alpha and Rad21 were mainly expressed in mitotically dividing cells in the testis (somatic cells and spermatogonia), although their weak expression was detected in pre-leptotene spermatocytes. SMC1beta was expressed in spermatogonia and spermatocytes. SMC1beta was localized along the chromosomal arms as well as on the centromeres in meiotic prophase I, and its existence on the chromosomes persisted up to metaphase II, a situation different from that reported in the mouse, in which SMC1beta is lost from the chromosome arms in late pachytene despite its universal presence in vertebrates.     

Annual reproductive cycle and rate of the spermatogenic process in male mosquitofish <Emphasis Type="Italic">Gambusia affinis</Emphasis>

The present study investigates the relationship between the annual cycle of testicular development and external environment and the rate of spermatogenesis in the mosquitofish Gambusia affinis based on histological observations of testes. The annual reproductive cycle of the mosquitofish was divided into two periods, i.e., the spermatogenic period (May–October) and resting period (October–April). In the spermatogenic period, the transition from spermatogonia to spermatocytes begins and meiosis actively progresses. In the resting period, the transition from spermatogonia to spermatocytes ceases, meiosis of spermatocytes that already shifted by this period gradually progresses, and a considerable number of sperm balls are produced. Onset of spermatogenesis seems to be related to both a rise in water temperature and a prolonged photoperiod. 5-bromo-2-deoxyuridine (BrdU) was a useful in vivo marker of DNA synthesizing spermatogenic cells. The results of immunohistochemical detection of injected BrdU indicated that 5 days are needed for the conversion of spermatocytes to spermatids, 5 days for spermatids to spermatozoa, and 10 days for spermatozoa to sperm balls.     

Phosphorylation of Xenopus cyclins B1 and B2 is not required for cell cycle transitions.

The cdc2 kinase and B-type cyclins are known to be components of maturation- or M-phase-promoting factor (MPF). Phosphorylation of cyclin B has been reported previously and may regulate entry into and exit from mitosis and meiosis. To investigate the role of cyclin B phosphorylation, we replaced putative cdc2 kinase phosphorylation sites in Xenopus cyclins B1 and B2 by using oligonucleotide site-directed mutagenesis. We found that Ser-90 of cyclin B2 and Ser-94 or Ser-96 of cyclin B1 are the main phosphorylation sites both in functional Xenopus egg extracts and after phosphorylation with purified MPF in vitro. Microtubule-associated protein (MAP) kinase from Xenopus eggs phosphorylated cyclin B1 significantly at Ser-94 or Ser-96, whereas it was largely inactive against cyclin B2. The substitutions that ablated phosphorylation at these sites, however, resulted in no functional differences between mutant and wild-type cyclin, as judged by the kinetics of M-phase degradation, induction of mitosis in egg extracts, or induction of oocyte maturation. These results indicate that the phosphorylation of Xenopus B-type cyclins by cdc2 kinase or MAP kinase is not required for the hallmark functions of cyclin.     

The testicular cycle of Gambusia affinis holbrooki (Teleostei: Poeciliidae)

Fifteen male mosquito fish ( Gambusia affinis holbrooki ) were collected in 1989 on the 15th of each month to perform a quantitative histologic study of the annual testicular cycle including a calculation of the gonadosomatic index, testicular volume, and the total volume per testis occupied by each germ cell type. The cycle comprises two periods: spermatogenesis and quiescence. The spermatogenic period begins in April with the development of primary spermatogonia into secondary spermatogonia, spermatocytes and round spermatids. In May, the first spermatogenic wave is completed and the testicular volume begins to increase up to June when the maximum testicular volume and gonadosomatic index are reached. Germ cell proliferation with successive spermatogenetic waves continues until August. In September germ cell proliferation ceases and neither secondary spermatogonia nor spermatocytes are observed. However, spermiogenesis continues until October. In November, spermiogenesis has stopped and the testis enters the quiescent period up to April. During this period only primary spermatogonia and spermatozoa are present in the testis. In addition, a few spermatids whose spermiogenesis was arrested in November are observed. Testicular release of spermatozoa is continuous during the entire spermatogenesis period. The spermatozoa formed at the end of this period (September-October) remain in the testis during the quiescent period and are released at the beginning of the next spermatogenesis period in April. Developed Leydig cells appear all year long in the testicular interstitium, mainly around both efferent ducts and the testicular tubule sections showing S₄ spermatids.     

RNA interference during spermatogenesis in mice

Spermatogenesis consists of complex cellular and developmental processes, such as the mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and morphogenesis of haploid spermatids. In this study, we show that RNA interference (RNAi) functions throughout spermatogenesis in mice. We first carried out in vivo DNA electroporation of the testis during the first wave of spermatogenesis to enable foreign gene expression in spermatogenic cells at different stages of differentiation. Using prepubertal testes at different ages and differentiation stage-specific promoters, reporter gene expression was predominantly observed in spermatogonia, spermatocytes, and round spermatids. This method was next applied to introduce DNA vectors that express small hairpin RNAs, and the sequence-specific reduction in the reporter gene products was confirmed at each stage of spermatogenesis. RNAi against endogenous Dmc1, which encodes a DNA recombinase that is expressed and functionally required in spermatocytes, led to the same phenotypes observed in null mutant mice. Thus, RNAi is effective in male germ cells during mitosis and meiosis as well as in haploid cells. This experimental system provides a novel tool for the rapid, first-pass assessment of the physiological functions of spermatogenic genes in vivo.     

Abnormal spermatogenesis at low temperatures in the Japanese red-bellied newt,Cynops pyrrhogaster: possible biological significance of the cessation of spermatocytogenesis

In newt testis, spermatocytes never appear during winter, because secondary spermatogonia die by apoptosis just before meiosis. In the current study, we examined the effect of low temperatures on spermatogenesis. Incubation of newts at low temperatures (8, 12, 15 degrees C) induced defects in spermatogenesis in a temperature-dependent manner. At 8 degrees C, multinucleated giant cells (MGCs) were observed in spermatocytes and spermatogenesis never proceeded beyond meiosis. Although spermatocytes completed meiotic divisions at 12 degrees C, severe cell death was observed in the spermatids. At 15 degrees C both normal and abnormal spermiogenesis were observed. Under these conditions, impaired meiotic synapsis/recombination and down-regulation of the expression of the DMC1 protein, which play pivotal roles in meiotic pairing in eukaryotes, were also observed. Furthermore, to examine the quality of the sperm produced at low temperature for supporting development, artificial insemination was performed. The eggs inseminated with spermatozoa derived from newts kept at 15 degrees C demonstrated a restricted developmental capacity, even though these spermatozoa had an equal capacity for carrying out fertilization to those kept at 22 degrees C. These results suggest that meiosis at low temperatures cause the production of abnormal spermatozoa. Conservation and the significance of this phenomenon in poikilothermic vertebrates living in the temperate zones are also discussed.