Flow-cytometric isolation of testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions

There is a need to isolate different populations of spermatogenic cells to investigate the molecular events that occur during spermatogenesis. Here we developed a new method to identify and purify testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions (pvasa-GFP) at various stages of spermatogenesis. Rainbow trout piwi-like (rtili), rainbow trout scp3 (rt-scp3), and rainbow trout shippo1 (rt-shippo1) were identified as molecular markers for spermatogonia, spermatocytes, and spermatids, respectively. The testicular cells were separated into five fractions (A-E) by flow cytometry (FCM) according to their GFP intensities. Based on the molecular markers, fractions A and B were found to contain spermatogonia, while fractions C and D contained spermatocytes, and fraction E contained spermatids. We also classified the spermatogonia into type A, which contained spermatogonial stem cells (SSCs), and type B, which did not. As none of the molecular markers tested could distinguish between the two types of spermatogonia, we subjected them to a transplantation assay. The results indicated that cells with strong GFP fluorescence (fraction A) colonized the recipient gonads, while cells with weaker GFP fluorescence (fraction B) did not. As only SSCs could colonize the recipient gonads, this indicated that fraction A and fraction B contained mainly type A and type B spermatogonia, respectively. These findings confirmed that our system could identify and isolate various populations of testicular cells from rainbow trout using a combination of GFP-dependent FCM and a transplantation assay.     

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。     

Slow increase of Sertoli cell efficiency and daily sperm production causes delayed establishment of full sexual maturity in the rodent Chinchilla lanigera

Puberty in the male Andean rodent Chinchilla lanigera occurred approximately 3 mo after birth, whereas full sexual maturity was established much later. The objective of the present study was to investigate testis function in postpubertal chinchillas, with an emphasis on the estimation of seminiferous epithelium cycle length (n=6) and Sertoli cell (SC) and spermatogenic efficiencies (n=26). Samples of testes were collected between May and November. Each spermatogenic cycle lasted 10.2d and the total duration of spermatogenesis was approximately 46 d. The SC efficiency (spermatids/SC) and the daily sperm production per testis gram increased markedly (P<0.05) from 5 to 17-22 mo of age, whereas the conversion rates of type A1 spermatogonia to preleptone and the number of spermatids per pachytene remained stable (P>0.05) from 5 to 30 mo. Therefore, efficiency of the spermatogenic process increased equally during all phases of spermatogenesis. In conclusion, based on the gradual and striking postpubertal increases for SC and sperm production, we inferred that more undifferentiated spermatogonia and/or spermatogonial stem cells were produced and therefore, that the chinchilla might represent a good experimental model to investigate regulation of this crucial aspect of spermatogenesis.     

Differential Expression of Cyclins B1 and B2 during Medaka (Oryzias latipes) Spermatogenesis

The Cdc2-cyclin B complex (named the M-phase-promoting factor, MPF) is well known to be a key regulator of G2-M transition in both mitosis and meiosis. However, MPF may have functions other than the cell cycle regulation, since its activity is detectable in post-mitotic (or post-meiotic) non-dividing cells. Cyclin B comprises several subtypes, but their functional differences are still unknown. Despite the established function of MPF during oocyte maturation, its role during spermatogenesis, where spermatogenic cells undergo drastic morphological changes after meiosis, remains to be elucidated. To address these issues, we have isolated cDNA clones encoding cyclins B1 and B2 from medaka testis and raised polyclonal antibodies against their products. Using these as probes, we examined the expression patterns of cyclins B1 and B2 in medaka testis at both mRNA and protein levels. Cyclin B1 and B2 mRNAs were expressed in all stages of spermatogenic cells except for spermatozoa, although the expression levels varied according to the spermatogenic stages. Cyclin B1 protein was expressed only in spermatogonia and spermatocytes at prophase and metaphase with a transient disappearance at anaphase. On the other hand, cyclin B2 protein was continuously expressed throughout spermatogenesis, even in spermatogonia and spermatocytes at anaphase and in post-meiotic spermatids and spermatozoa. The difference in their expression patterns suggests that cyclins B1 and B2 have distinct roles in medaka spermatogenesis; i.e., cyclin B1 controls the meiotic cell cycle, whereas cyclin B2 is involved in process(es) other than meiosis.     

Quantitative analysis of spermatogenesis and apoptosis in the common marmoset (Callithrix jacchus) reveals high rates of spermatogonial turnover and high spermatogenic efficiency

Spermatogenesis is characterized by the succession in time and space of specific germ cell associations (stages). There can be a single stage (e.g., rodents and some macaques) or more than one stage (e.g., chimpanzee and human) per tubular cross section. We analyzed the organization of the seminiferous epithelium and quantified testicular germ cell production and apoptosis in a New World primate, the common marmoset (Callithrix jacchus). Tubule cross sections contained more than one stage, and the human six-stage system could be applied to marmoset spermatogenesis. Stereological (optical disector) analysis (n = 5) revealed high spermatogenic efficiency during meiosis and no loss of spermatids during spermiogenesis. The conversion of type A to type B spermatogonia was several-fold higher than that reported for other primates. Highest apoptotic rates were found for S-phase cells (20%) and 4C cells (15%) by flow cytometric analysis (n = 6 animals); histological analysis confirmed spermatogonial apoptosis. Haploid germ cell apoptosis was <2%. Marmoset spermatogenesis is very efficient and involves substantial spermatogonial proliferation. The prime determinants of germ cell production in primates appear to be proliferation and survival of spermatogonia rather than the efficiency of meiotic divisions. Based on the organizational similarities, common marmosets could provide a new animal model for experimental studies of human spermatogenesis.     

Selective partitioning of plasma membrane antigens during mouse spermatogenesis

The selective partitioning of cell membrane components during mouse spermatogenesis has been examined using a heterologous antibody raised against isolated type B spermatogonia. The anti-type B spermatogonia rabbit IgG (ATBS) binds to isolated populations of mouse primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene spermatocytes, leptotene/zygotene spermatocytes, pachytene spermatocytes, round spermatids, residual bodies, and mature spermatozoa. Although immunofluorescent labeling is uniformly distributed on the cell surface of early spermatogenic cells, a discrete topographical localization of IgG is observed on testicular, epididymal, and vas deferens spermatozoa. The convex surface of the acrosome, postacrosomal region, and tail are labeled. Antibody does not bind to a broad area corresponding to the concave region of the acrosome. The antibody also binds to mouse somatic cells including Sertoli cells, Leydig cells, thymocytes, and splenocytes, but not to mature spermatozoa of the vole, rat, hamster, guinea pig, rabbit, or human. ATBS, after absorption with mouse splenocytes or thymocytes, does not react with any somatic cells examined by fluorescence except with Sertoli cells. In addition, all reactivity with testicular, epididymal, and was deferens spermatozoa is abolished. However, spermatogenic cells at earlier stages of differentiation, including residual bodies, still react strongly with the absorbed antibody. The number of surface receptor sites per cell for absorbed ATBS ranges from approximately 3 million on primitive type A spermatogonia to 1 million on round spermatids and on residual bodies. Spermatozoa, however, have only 0.003 million binding sites for absorbed ATBS, in contrast to 10 million sites for the unabsorbed antibody. It appears that receptor sites for absorbed ATBS are not masked by components of epididymal secretions. These data imply, therefore, that specific mechanisms operate at the level of the cell membrane during spermiogenesis to insure that some surface components, not required in the mature spermatozoon, are removed selectively by partitioning to that portion of the spermatid membrane destined for the residual body.     

The cycle of the seminiferous epithelium in the Japanese quail (Coturnix coturnix japonica) and estimation of its duration

A regular, well defined spermatogenic cycle was found in the Japanese quail by examining thin sections of isolated lengths of seminiferous tubules embedded in epoxy resin to resolve the structure of developing spermatids. The stages of the cycle initially were identified in studies using a preparatory method for fixation which separated adjacent cellular associations. The cycle was divided into 10 stages with relative frequencies (%) of Stages I to X respectively of: 11.9, 14.8, 24.1, 10.3, 8.2, 6.4, 9.4, 5.5, 3.8 and 5.4. The duration of one cycle was 2.69 +/- 0.08 days (mean +/- s.e.m.) as determined by intraventricular injection of [3H]thymidine and autoradiographic examination of the testes 1-4 days later. It was estimated that lifespans were 2.01 days for type B spermatogonia, 3.86 days for primary spermatocytes, 0.15 days for secondary spermatocytes, and 4.54 days for spermatids. The results suggest that the kinetics of spermatogenesis in the quail are fundamentally similar to the pattern in mammals.     

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.     

Histology of salmonid testes during maturation

The commonly applied classification systems of fish gonad maturity divide the maturation process into certain stages. However, the scales do not entirely reflect the continuity of the maturation process. Based on light microscope observations, the paper describes a comprehensive pattern of testicular transformations during maturation. The study was carried out on precocious underyearling and 1-year-old males of sea trout (Salmo trutta m. trutta L.), 1-year-old males of salmon (Salmo salar L.), and males of brown trout (Salmo trutta m. fario L.) aged from 7 months to 4 years. A total of 821 gonads collected during all seasons of the year were examined. The fish were fixed in Bouin's fluid. Histological slides of the mid-part of the gonad were made using the standard paraffin technique. The 3-6 microm sections were stained with Heidenhain haematoxylin. Histological changes of testes during maturation were similar in the three species studied. Immature and resting gonads contained type A spermatogonia in lobules only. The appearance of cystic structures containing type B spermatogonia in the lobules signalled the beginning of the sexual cycle in male gonads. Type B spermatogonia underwent synchronous mitotic divisions resulting in an increase in the total number of spermatogonia. As the spermatogenesis continued, the gonads showed a gradual increase in the number of cysts containing cells at all the spermatogenetic stages: type B spermatogonia, primary and secondary spermatocytes, spermatids, and spermatozoa. The well-formed spermatozoa were released to the lobule lumen once the Sertoli cells and spermatozoa connections broke up and the cyst disappeared. This was a continuous process observed throughout the spawning season. The spermatozoa were moved to the efferent duct. While some of the germ cells were completing spermatogenesis, the lobules contained less and less cysts with type B spermatogonia, primary and secondary spermatocytes, and spermatids; eventually all the cells completed spermatogenesis. At the end of maturation, vacuoles, up to 18.9 microm in final diameter (brown trout), appeared in the Sertoli cells. The vacuoles were visible in the lobule wall epithelium for a prolonged period of time. In most salmonid individuals examined, the reproductive cycles were observed to overlap. In some fish, the preparation for another cycle began very early, i.e., at the and of preceding spermatogenesis, which had not been observed before. Gonad maturation in some males was incomplete.     

The origin of the synchronization of the seminiferous epithelium in vitamin A-deficient rats after vitamin A replacement

In seminiferous tubules of vitamin A-deficient rats, the remaining spermatogonia were A spermatogonia. These cells were topographically arranged as single and paired cells and clones of 4, 8, or more cells. The bromodeoxy-uridine-labeling index and the mitotic index of these cells were found to be 9% and 1%, respectively, indicating that these cells were slowly proliferating. Administration of vitamin A (retinol-acetate) resulted in a reinitiation of spermatogenesis in such a way that the epithelium became stage-synchronized. The rate of development of the spermatogenic cells between 7 and 21 days after vitamin A replacement was found to be similar to that in normal rats. At 24-30 h after administration of vitamin A, a 4- to 6-fold increase in the labeling index was found. In contrast, after 2 days, the labeling index was low, while the mitotic index was elevated (10%). A high labeling index was found again after 3 days. Assuming that during the first 7 days after vitamin A replacement the rate of development of the spermatogenic cells also was normal, it could be deduced that the spermatogonia labeled 24-30 h after vitamin A administration were A1 spermatogonia. These cells would then divide into A2 spermatogonia after about 2 days, which in turn would traverse their S phase after about 3 days. Hence, spermatogenesis in vitamin A-deficient rats would be arrested shortly before the S phase of the A1 spermatogonia. After administration of vitamin A, the spermatogonia synchronously start the series of six divisions leading to the formation of spermatocytes and, ultimately, they develop into mature spermatids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endocrinology of spermatogenesis in the hypophysectomized ram.

Adult rams were hypophysectomized and treated for 20 days with testosterone (2 X 0.25 g/day), PMSG (2 X 300 i.u./day) or hCG (2 X 250 i.u./day), or for 40 days with testosterone (2 X 0.25 g/day). All treatments maintained a normal concentration of testosterone within the seminiferous tubules. Quantitative histological analysis showed that (1) the differentiation from A0 to A1 spermatogonia was maintained by PMSG or hCG but not completely by testosterone; (2) the transition from intermediate spermatogonia to primary spermatocytes was maintained only by PMSG but not by testosterone or hCG; (3) meiotic prophase and spermiogenesis were maintained by the three hormones but there were qualitative abnormalities in the spermatids. These results suggest that in the ram, the differentiation of renewing stem spermatogonia is under LH control and that the last stages of spermatogonial multiplication, from intermediate to B spermatogonia and to primary spermatocytes, are under the control of the FSH-like activity of PMSG.     

Temporal expression of membrane antigens during mouse spermatogenesis.

The temporal expression of cell surface antigens during mammalian spermatogenesis has been investigated using isolated populations of mouse germ cells. Spermatogenic cells at advanced stages of differentiation, including pachytene primary spermatocytes, round spermatids, and residual bodies of Regaud and mature spermatozoa, contain common antigenic membrane components which are not detected before the pachytene stage of the first meiotic prophase. These surface constituents are not detected on isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, or leptotene and zygotene primary spermatocytes. These results have been demonstrated by immunofluorescence microscopy, by complement-mediated cytotoxicity, and by quantitative measurements of immunoglobulin (Ig) receptors on the plasma membrane of all cell populations examined. The cell surface antigens detected on germ cells are not found on mouse thymocytes, erythrocytes, or peripheral blood lymphocytes as determined by immunofluorescence and by cytotoxicity assays. Furthermore, absorption of antisera with kidney and liver tissue does not reduce the reactivity of the antibody preparations with spermatogenic cells, indicating that these antigenic determinants are specific to germ cells. This represents the first direct evidence for the ordered temporal appearance of plasma membrane antigens specific to particular classes of mouse spermatogenic cells. It appears that at late meiotic prophase, coincident with the production of pachytene primary spermatocytes, a variety of new components are inserted into the surface membranes of developing germ cells. The further identification and biochemical characterization of these constituents should facilitate an understanding of mammalian spermatogenesis at the molecular level.     

Expression and localization of DNA topoisomerase II during rat spermatogenesis

The potential role(s) of DNA topoiosmerase II (topo II) during chromatin changes that characterize different stages of spermatogenesis was investigated in the rat by an analysis of the expression and localization of topo II mRNA and protein in individual spermatogenic cells. Expression of topo II was restricted to spermatogonia, spermatocytes, and round and early-elongating spermatids. Two protein bands of 177 and 170 kDa were detected in immunoblots of spermatocytes and round spermatids, while bands of 148 and 142 kDa were prominent in preparations of elongating spermatids. Topo II levels and distribution patterns, as observed by immunofluorescent microscopy, exhibited cell type-specific variations. Differences in topo II staining patterns were also apparent when nuclear matrices of spermatogenic cells were prepared with different extraction conditions. In addition to its possible function as a structural component, topo II, associated with nuclear matrix preparations from spermatogenic cells, possessed catalytic activity. These observations indicate that both the 177 and 170 kDa and the 148 and 142 kDa forms of topo II share similar structural and functional properties. Topo IIβ mRNA was transcribed in rat spermatogenic cells at 6.2 kb. Relative levels of topo IIβ mRNA were high in spermatogonia and spermatocytes, and decreased in both round and early-elongating spermatids. Changes in topo II expression levels and localization patterns represent distinct stage-specific markers for the maturation of spermatogenic cells, and are consistent with the involvement of topo II in mediating DNA modifications and chromatin changes during spermatogenesis. © 1996 Wiley-Liss, Inc.     

Kinetics of spermatogenesis in the wild squirrel Funambulus palmarum (Linnaeus).

The paper describes in detail the morphology and kinetics of germ cell associations, pattern of mitotic divisions, frequency distribution of different cellular associations (stages) and percent degeneration of various germ cells in the squirrel in which spermatogenesis in adults occurs all year round. Eighteen steps of spermiogenesis were identified based on the development of the acrosomal system using PAS-haematoxylin. These were appropriately divided into Golgi, acrosome, cap and maturation phases. Thirteen types of cellular associations or stages (I-XIII) were characterized along the length of the seminiferous tubule which repeated itself in space and time constituting the seminiferous epithelial cycle (CSE). Of the 18 steps of spermiogenesis, the first 13 were associated with stages I-XIII, respectively, and the rest with the first 9 stages. Spermiation occurred in stage IX. Seven types of spermatogonia [A0, A1, A2, A3, A4, intermediate (In) and B type] were identified based on their shape, size and nuclear morphology. A0 spermatogonia are pale in appearance with homogeneously distributed chromatin surrounded by a thin nuclear membrane. These are present in all stages. A1 are oval in shape and possess a thicker nuclear membrane. They are found in stages VI-X. The chromatin material undergoes progressive condensation from A1 to A4 making the last generation of spermatogonia appear darker. The In spermatogonia which are derived from A4 are morphologically similar to them but smaller in size. The B-type spermatogonia derived from the In types possess a typically round nucleus with uniformly condensed chromatin material underneath the nuclear membrane. The spermatogonia divide mitotically at fixed stages of the CSE giving rise to their next generations. Thus, A-type spermatogonia divide at stages X, XIII/I, IV and V, while In divide at stage VI. During each CSE of the squirrel, 5 peaks of mitosis occur. There is a single generation of B-type spermatogonia. These differentiate into primary spermatocytes and undergo meiosis or maturation divisions which enter to form spermatids. The A4 which divide differentially in stage VI give rise to In- and A1-type spermatogonia. Therefore, A4 spermatogonia form renewing stem cells. Based on the above pattern of spermatogonial mitosis a model for stem cell renewal in the squirrel is proposed. The percentage degeneration of germ cells varied with the cell type. During a single CSE of the squirrel, a total of 42.09% germ cells were found to degenerate. An attempt is made to compare and contrast the kinetics of spermatogenesis in the wild squirrel with that of the other rodents studied so far.     

Sequential activation of Notch family receptors during mouse spermatogenesis

The expression pattern of Notch family receptors during mouse spermatogenesis was examined by immunohistochemistry. The entire cytoplasm of spermatogonia, spermatocytes and spermatids showed staining with antibodies against extracellular domains of Notch1, 2 and 4. In contrast, the nuclei of spermatogonia showed staining with an antibody against the intracellular domain of Notch3, and the nuclei of spermatocytes and spermatids showed staining with antibodies against the intracellular domains of Notch1 and 4. During regeneration of spermatogonia in busulfan-treated mice, the nuclei of all proliferating cells showed staining for the intracellular domain of Notch3. Western blot analysis showed that the molecular weights of the intracellular domains of Notch1 and 3 localizing in the nuclear fraction were smaller than those in the cytoplasmic fraction. This was consistent with the theory that the intracellular domain of Notch was cleaved in the cytoplasm and translocated to the nucleus. These results suggest that different Notch signals are sequentially activated during mouse spermatogenesis and control the proliferation and differentiation of spermatogenic stem cells.