Identification of a molecular marker for type A spermatogonia by microarray analysis using gonadal cells from pvasa-GFP transgenic rainbow trout (Oncorhynchus mykiss)

The spermatogonia of fish can be classified as being either undifferentiated type A spermatogonia or differentiated type B spermatogonia. Although type A spermatogonia, which contain spermatogonial stem cells, have been demonstrated to be a suitable material for germ cell transplantation, no molecular markers for distinguishing between type A and type B spermatogonia in fish have been developed to date. We therefore sought to develop a molecular marker for type A spermatogonia in rainbow trout. Using GFP-dependent flow cytometry (FCM), enriched fractions of type A and type B spermatogonia, testicular somatic cells, and primordial germ cells were prepared from rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa regulatory regions (pvasa-GFP rainbow trout). The gene-expression profiles of each cell fraction were then compared with a microarray containing cDNAs representing 16,006 genes from several salmonid species. Genes exhibiting high expression for type A spermatogonia relative to above-mentioned other types of gonadal cells were identified and subjected to RT-PCR and quatitative PCR analysis. Since only the rainbow trout notch1 homologue showed significantly high expression in the type A spermatogonia-enriched fraction, we propose that notch1 may be a useful molecular marker for type A spermatogonia. The combination of GFP-dependent FCM and microarray analysis of pvasa-GFP rainbow trout can therefore be applied to the identification of potentially useful molecular markers of germ cells in fish.     

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.     

Spermatogenesis in pharate adult testes of Drosophila in tissue cultures without ecdysones

The process of spermatogenesis in explanted testicular fragments from pharate adults (48 hr after puparium formation) of Drosophila melanogaster was examined under in vitro conditions without any added ecdysone substances. In the anterior fragments, which contained spermatogonia, no or only slight changes were found. In the middle fragments which contained germ cells at more advanced stages of spermatogenesis, spermatocytes, and spermatids, a slight increase in the number of spermatocytes or spermatids was observed. In the posterior fragments, which contained sperms at early stages of spermiogenesis, there was a marked elongation of the sperm bundles along their long axis.     

Enrichment of transplantable germ cells in salmonids using a novel monoclonal antibody by magnetic‐activated cell sorting

In the fish germ cell transplantation system, only type A spermatogonia (ASGs) and oogonia are known to be incorporated into the recipient genital ridges, where they undergo gametogenesis. Therefore, high colonization efficiency can be achieved by enriching undifferentiated germ cells out of whole testicular cells. In this study, we used magnetic‐activated cell sorting (MACS) for enriching undifferentiated germ cells of rainbow trout using a monoclonal antibody that recognizes a specific antigen located on the germ cell membrane. We screened the antibodies to be used for MACS by performing immunohistochemistry on rainbow trout gonads. Two antibodies, nos. 172 and 189, showed strong signals for ASGs and oogonia. Next, we performed MACS with antibody no. 172 using gonadal cells isolated from vasa‐gfp rainbow trout showing GFP in undifferentiated germ cells. We found that GFP‐positive cells are highly enriched in antibody no. 172‐positive fractions. Finally, to examine the transplantability of MACS‐enriched cells, we intraperitoneally transplanted sorted or unsorted cells into recipient larvae. We observed that transplantability of sorted cells, particularly ovarian cells, were significantly higher than that of unsorted cells. Therefore, MACS with antibody no. 172 could enrich ASGs and oogonia and become a powerful tool to improve transplantation efficiency in salmonids.     

DNA polymerases in mouse spermatogenic cells separated by sedimentation velocity.

Activity levels of DNA polymerase alpha and DNA polymerase beta have been measured in mouse spermatogenic cells separated by sedimentation velocity. Testes from prepuberal (17 day old) and sexually mature mice were dissociated and separated by unit gravity sedimentation into 6 populations of cells. Phase contrast microscopy and [3H]thymidine labeling kinetics revealed that at least 85% of the cells in fraction A were pachytene-stage primary spermatocytes, fraction B was enriched for primary spermatocytes and round spermatids, fraction C contained spermatogonia and/or pre-leptotene primary spermatocytes and later stages of spermatids (no spermatids were present in fraction C from the testes of 17 day old mice) and fractions D to F contained mixed populations of cells, many in later stages of spermiogenesis. When expressed as activity in 10(6) cells or as a specific activity, fractions A, B, and C from mature animals population initially loaded onto the gradient while fractions D, E and F had activity levels similar to or below the population of dissociated cells. The ratio of activity between the DNA polymerases was constant in fractions A, B, and C, but in fractions D, E, and F, the ratio decreased due to a more rapid decline of activity of polymerase alpha. A comparison of activity levels in fraction C from prepuberal and sexually mature mice revealed an increase in DNA polymerase alpha activity and a decrease in the activity of DNA polymerase beta in the cells from the 17 day old animals.     

Effects of a highly purified antiserum to FSH on testicular function in immature rats

Effects of highly purified antiserum (AS) to follicle stimulating hormone (FSH) on testicular function was studied in immature rats. Treatment with FSHAS for 10 days, from 25-34, decreased weights of the testis (p .001) and increased weights of the epididymis (p .05). Numbers of the cell types in the seminiferous epithelium, particularly Type A spermatogonia pachytene spermatocytes and spermatids, were markedly reduced, possibly due to: 1) decreased division of the initial stem cells, 2) impairment of division of Type B spermatogonia and their transformation to pachytene spermatocytes, and 3) desquamation and degeneration of pachytene spermatocytes and spermatids. FSHAS also affected the sertoli cell function which was reflected in the decreased binding of androgens to supernatant fraction of the testis and epididymides. Treatment with luteinizing hormone-AS for 5 days did not affect testicular function but the binding of androgens to the supernatants of the caput and cauda epididymides and ventral prostate was significantly reduced (p .001). These data indicate that FSH is necessary for the maintenance of the cellular integrity of the seminiferous epithelium during the completion of the 1st wave of spermatogenesis.     

The seminiferous epithelial cycle and spermat ogenesis in rams (ovis aries )

The efficiency of spermatogenesis and degenerations of different spermatogenic cells under normal conditions of the environment have been investigated in rams. The meiotic divisions and the position of first-generation spermatids in haematoxylin-eosin stained testicular preparations were used to identify eight stages of the seminiferous epithelial cycle (SEC). The stages of relatively long duration (i.e., 1,2,3,4,8) were sub-divided. The percent-ages of frequency for the 14 stages reported were also studied. Three generations of type A (A(1), A(2), A(3)), one generation of type intermediate (In) and two generations of type B (B(1), B(2)) spermatogonia were recognized. A(2) and B(2) spermatogonia as well as primary and secondary spermatocytes did not degenerate. Contrarily, A(1), A(2), A(3), In and B(1) spermatogonia showed 25, 13.7, 27.3 and 21.2% degenerations respectively. We concluded that compared with the previously used eight-stage classification, subdividing stages with long durations as done in this study facilitates investigating the degenerations of spermatogenic cells. The efficiency of spermatogenesis in rams was 47.58% since one A(3) spermatogonium produces 30.45 spermatids/spermatozoa against the expected number of 64.     

Changes in the testicular acid and alkaline phosphatase activities at different durations following prostatectomy: a correlative study with spermatogenesis.

The purpose of this investigation was to make a correlative study between spermatogenesis and testicular acid and alkaline phosphatase activities in mature prostatectomized animals at different post-operative periods. The results demonstrate that there was a significant augmentation in the activity of testicular acid and alkaline phosphatases subsequent to 14 and 21 days of prostatectomy. A parallel quantitative study of spermatogenesis at stage VII of the seminiferous cycle, namely, type A spermatogonia (ASg), preleptotene spermatocytes (pLSc), mid-pachytene spermatocytes (mPSc) and step 7 spermatids (7sd), revealed that there was a significant reduction in the number of step 7 spermatids after 14 and 21 days. No change was observed in the above testicular enzymes and spermatogenesis after 7 days of prostatectomy. Therefore, it is concluded that prostatectomy can alter the above testicular enzyme activities and spermatogenesis in chronic prostatectomized state.     

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.     

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.     

Culture conditions for maintaining the survival and mitotic activity of rainbow trout transplantable type A spermatogonia

Germ-cell transplantation is a powerful tool for studying gametogenesis in many species. We previously showed that spermatogonia transplanted into the peritoneal cavity of trout hatchlings were able to colonize recipient gonads, and produced fully functional sperm and eggs in synchrony with the germ cells of the recipient. An in vitro-culture system enabling spermatogonia to expand, when combined with transplantation, would be valuable in both basic and applied biology. To this end, we optimized culture conditions for type A spermatogonia in the present study using immature rainbow trout at 8-10 month of age. Spermatogonial survival and mitotic activity were improved during culture in Leibovitz's L-15 medium (pH 7.8) supplemented with 10% fetal bovine serum at 10 degrees C compared with culture under standard conditions for salmonids (Hank's MEM (pH 7.3) supplemented with 25 mM HEPES and 5% FBS, and culture at 20 degrees C). Elimination of testicular somatic cells promoted spermatogonial mitotic activity. In addition, insulin, trout embryonic extract, and basic fibroblast growth factor promoted the mitosis of purified spermatogonia in an additive manner. Mitotic activity increased nearly sevenfold over 19 days of culture compared with growth factor-free conditions and was maintained for >1 month. Furthermore, the cultured spermatogonia could colonize and proliferate in recipient gonads following transplantation. This study represents the first step towards establishing a cell line that can be transplanted for use in surrogate broodstock technology and cell-mediated gene-transfer systems.     

Gene expression during mammalian spermatogenesis. II. Evidence for stage-specific differences in mRNA populations

Gene expression during murine spermatogenesis has been studied using highly enriched populations of cells obtained by velocity sedimentation at unit gravity and further purified by density gradient centrifugation through Percoll. Polypeptides whose synthesis was directed by total cytoplasmic RNA from round spermatids, pachytene spermatocytes, primitive type A spermatogonia, and Sertoli cells in cell-free translation systems have been compared by two-dimensional polyacrylamide gel electrophoresis, followed by fluorography. At the level of detection provided by the electrophoretic methods used, each population of cells contained mRNAs encoding over 200 polypeptides, many of which were present in high abundance in all four cell types. However, for each cell type examined, a minimum of 5-10% of these polypeptides appear to be either specific to or greatly enriched within a particular cell type. Analysis of the polysomal and nonpolysomal cell fractions from pachytene spermatocytes and round spermatids revealed that the two compartments share many identical mRNAs but specific mRNAs are selectively compartmentalized between the cell fractions and between the two cell types. Movement between compartments was seen; e.g., some polypeptides encoded by mRNA found primarily in the nonpolysomal fraction of pachytene cells were later seen in the polysomal fraction from round spermatids. Virtually every other combination was also observed. These results suggest that the control of gene expression at the level of selective production of mRNA and selective utilization of mRNA are among the mechanisms involved in regulation of spermatogenic cell differentiation.     

Testicular phenotype in luteinizing hormone receptor knockout animals and the effect of testosterone replacement therapy

The LH receptor knockout model, developed in our laboratory, was used in determining what FSH alone can do in the absence of LH signaling and whether any of the testicular LH actions are not mediated by androgens. The results revealed that null animals contained smaller seminiferous tubules, which contained the same number of Sertoli cells, spermatogonia, and early spermatocytes as wild-type siblings. The number of late spermatocytes, on the other hand, was moderately decreased, the number of round spermatids was dramatically decreased, and elongated spermatids were completely absent. These changes appear to be due to an increase in apoptosis in spermatocytes. While the number of Leydig cells progressively increased from birth to 60 days of age in wild-type animals, they remained unchanged in null animals. Consequently, 60-day-old null animals contained only a few Leydig cells of fetal type. The age-dependent increase in testicular macrophages lagged behind in null animals compared with wild-type siblings. Orchidopexy indicated that -/- testicular phenotype was not due to abdominal location. Rather, it was mostly due to androgen deficiency, as 21-day testosterone replacement therapy stimulated the growth of seminiferous tubules, decreased apoptosis, and increased the number of late spermatocytes and round spermatids and their subsequent differentiation into mature sperm. The therapy, however, failed to restore adult-type Leydig cells and testicular macrophage numbers to the wild-type levels. In summary, our data support the concept that FSH signaling alone can maintain the proliferation and development of Sertoli cells, spermatogonia, and early spermatocytes. LH actions mediated by testosterone are required for completion of spermatogenesis, and finally, androgen-independent actions of LH are required for the formation of adult-type Leydig cells and recruitment of macrophages into the testes.     

Expression patterns of gdnf and gfrα1 in rainbow trout testis

In mice, glial cell line-derived neurotrophic factor (GDNF) is essential for normal spermatogenesis and in vitro culture of spermatogonial stem cells. In murine testes, GDNF acts as paracrine factor; Sertoli cells secrete it to a subset of spermatogonial cells expressing its receptor, GDNF family receptor α1 (GFRα1). However, in fish, it is unclear what types of cells express gdnf and gfrα1. In this study, we isolated the rainbow trout orthologues of these genes and analyzed their expression patterns during spermatogenesis. In rainbow trout testes, gdnf and gfrα1 were expressed in almost all type A spermatogonia (ASG). Noticeably, unlike in mice, the expression of gdnf was not observed in Sertoli cells in rainbow trout. During spermatogenesis, the expression levels of these genes changed synchronously; gdnf and gfrα1 showed high expression in ASG and decreased dramatically in subsequent developmental stages. These results suggested that GDNF most likely acts as an autocrine factor in rainbow trout testes.