Morphology and cell population kinetics of primary spermatogonia in the frog (Rana esculenta) (Amphibia: Anura)

Two morphologically distinct primary spermatogonial cell types were observed in the frog testis and distinguished on the basis of nuclear characteristics. They have been designated the pale and dark types of primary spermatogonia. On the basis of a kinetic analysis, it is proposed that the pale spermatogonia possess the faculty of self-renewal as well as that of forming dark spermatogonia; they are thus bipotential stem cells comparable to the undifferentiated type of mammalian spermatogonia. The dark spermatogonia, in contrast, are committed to a single pathway, i.e. to form secondary sperrnatogonia, and can be defined as differentiated or committed elements of the primary spermatogonial population. The number of stem cell spermatogonia and differentiated spermatogonia vary according to the period of the year, as does the rate of turnover of stem cells, with nearly 60–90% of cells temporarily out of the cell cycle at any given time. It is indicated that the spermatogonial population represents a 'cell renewal system' in a steady state for appreciably long periods of time, however, changing with season in as far as the magnitude of yield of spermatogonial cells is concerned. This implies that an equality should exist between the rate at which stem cells enter cell-cycling and the rate at which daughter cells change their morphological identity.     

性成熟前食蟹猴生精细胞的发育进程

目的阐明性成熟前食蟹猴生精细胞的发育进程。方法分别采集性成熟前不同年龄（0岁、0.5岁、1岁、1.5岁、2岁、2.5岁、3岁、3.5岁、4岁）食蟹猴睾丸,制作石蜡切片,进行HE染色和PAS/H染色。根据生精细胞的染色特性,分析性成熟前食蟹猴生精细胞的发育进程,并对食蟹猴精原干细胞进行初步鉴定。结果 HE染色结果显示,1岁及以下食蟹猴生精上皮上生精细胞仅有精原干细胞（包括Ad、At及Ap型精原细胞）,1.5岁食蟹猴生精上皮上开始出现B型精原细胞,3岁食蟹猴生精上皮上出现精母细胞,4岁食蟹猴生精上皮上出现从精原干细胞到精子的所有生殖细胞。PAS/H染色结果显示,1～2.5岁食蟹猴Ad型精原细胞胞质呈PAS阳性,At型精原细胞胞质呈PAS弱阳性,Ap型精原细胞胞质呈PAS阴性;其他生精细胞及支持细胞胞质呈阴性;0.5岁及以下,3岁及以上食蟹猴生精细胞的胞质PAS/H染色特性与前者存在差异。结论本文详细阐述了性成熟前食蟹猴生精细胞随年龄增长的渐次性发育模式,并建立了性成熟前食蟹猴精原干细胞原位鉴定的一种新方法,这些研究结果为食蟹猴精原干细胞的其他相关研究奠定了基础。     

Response of the human testis to long-term estrogen treatment: Morphology of Sertoli cells,Leydig cells and spermatogonial stem cells

Summary The present investigation is concerned with the morphological changes observed in human testicular tissue following prolonged estrogen administration. Testicular material obtained from 11 transsexual patients who had been submitted to long-term estrogen treatment prior to sex-reversal surgery was studied by means of light- and electron microscopy.The testes of all patients examined present a more or less uniform appearance: There are narrow seminiferous cords surrounded by an extensively thickened lamina propria. They contain Sertoli cells and spermatogonia exclusively. There is no evidence of typical Leydig cells.The persisting spermatogonia show the characteristic features of pale type-A spermatogonia, whereas dark type-A spermatogonia are almost completely eliminated from the epithelium. In view of the fact that spermatogonia that survived radiotherapy and treatment with various noxious agents have recently been regarded as the stem cells of the human testis, it is suggested that also the majority of those spermatogonial types that are less sensitive to disturbances of the endocrine balance may consist of stem cells. The present results, therefore, corroborate the concept that the stem cells of the human testis may be derived from pale type-A spermatogonia or the variants of this cell type.Sertoli cells display two types of ovoid nuclei. In contrast to untreated material the nuclei lie adjacent to the basal lamina, and organelles and telolysosomes are confined to the apical cytoplasm. The apico-basal differentiation of mature cells, therefore, is not observed. Moreover, typical organelles and inclusions of mature cells are absent, as are the junctional specializations. Thus, Sertoli cells have transformed into immature cells, resembling precursors prior to puberty.Fibroblast-like cells in the interstitial tissue, which display strongly lobulated nuclei, a well-developed smooth endoplasmic reticulum, lipid droplets, and numerous inclusions are assumed to represent dedifferentiated Leydig cells.Since after estrogen treatment serum testosterone and gonadotropin levels are known to be reduced, it appears that the morphological changes correlate well with the endocrine status.     

Gfra1 silencing in mouse spermatogonial stem cells results in their differentiation via the inactivation of RET tyrosine kinase

Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate into sperm. The role of growth factor receptors in regulating self-renewal and differentiation of spermatogonial stem cells remains largely unclear. This study was designed to examine Gfra1 receptor expression in immature and adult mouse testes and determine the effects of Gfra1 knockdown on the proliferation and differentiation of type A spermatogonia. We demonstrated that GFRA1 was expressed in a subpopulation of spermatogonia in immature and adult mice. Neither Gfra1 mRNA nor GFRA1 protein was detected in pachytene spermatocytes and round spermatids. GFRA1 and POU5F1 (also known as OCT4), a marker for spermatogonial stem cells, were co-expressed in a subpopulation of type A spermatogonia from 6-day-old mice. In addition, the spermatogonia expressing GFRA1 exhibited a potential for proliferation and the ability to form colonies in culture, which is a characteristic of stem cells. RNA interference assays showed that Gfra1 small interfering RNAs (siRNAs) knocked down the expression of Gfra1 mRNA and GFRA1 protein in type A spermatogonia. Notably, the reduction of Gfra1 expression by Gfra1 siRNAs induced a phenotypic differentiation, as evidenced by the elevated expression of KIT, as well as the decreased expression of POU5F1 and proliferating cell nuclear antigen (PCNA). Furthermore, Gfra1 silencing resulted in a decrease in RET phosphorylation. Taken together, these data indicate that Gfra1 is expressed dominantly in mouse spermatogonial stem cells and that Gfra1 knockdown leads to their differentiation via the inactivation of RET tyrosine kinase, suggesting an essential role for Gfra1 in spermatogonial stem cell regulation.     

Basic features of bovine spermatogonial culture and effects of glial cell line-derived neurotrophic factor

Spermatogonial stem cells (SSC) are a small self-renewing subpopulation of type A spermatogonia, which for the rest are composed of differentiating cells with a very similar morphology. We studied the development of primary co-cultures of prepubertal bovine Sertoli cells and A spermatogonia and the effect of glial cell line-derived neurotropic factor (GDNF) on the numbers and types of spermatogonia, the formation of spermatogonial colonies and the capacity of the cultured SSC to colonize a recipient mouse testis. During the first week of culture many, probably differentiating, A spermatogonia entered apoptosis while others formed pairs and chains of A spermatogonia. After 1 week colonies started to appear that increased in size with time. Numbers of single (A(s)) and paired (A(pr)) spermatogonia were significantly higher in GDNF treated cultures at Days 15 and 25 (P < 0.01 and 0.05, respectively), and the ratio of A(s) to A(pr) and spermatogonial chains (A(al)) was also higher indicating enhanced self-renewal of the SSC. Furthermore, spermatogonial outgrowths in the periphery of the colonies showed a significantly higher number of A spermatogonia with a more primitive morphology under the influence of GDNF (P < 0.05). Spermatogonial stem cell transplantation experiments revealed a 2-fold increase in stem cell activity in GDNF treated spermatogonial cultures (P < 0.01). We conclude that GDNF rather than inducing proliferation, enhances self-renewal and increases survival rates of SSC in the bovine spermatogonial culture system.     

A critical stage in spermatogenesis for radiation-induced cell death in the medaka fish, Oryzias latipes

To ensure the high-fidelity transmission by reproductive cells of genetic information from generation to generation, cells have evolved surveillance systems to eliminate genomic lesions by inducing cell suicide and/or DNA repair. In this report, gamma-ray-induced cell death was investigated using the medaka fish, Oryzias latipes, because of the ease with which the differentiation stages of its spermatogenic cells can be identified. After 4.75 Gy gamma irradiation, the maximum rate of death of spermatogonial stem cells was observed at 18 h, and that of differentiating spermatogonia was at 12 h, followed by a peak in the extent of DNA fragmentation detected by the TUNEL assay. Dose-response curves for the death rate showed an obvious increase in the death rate for early-differentiating spermatogonia even after 0.11 Gy irradiation, whereas there were no such increases for spermatogonial stem cells and late-differentiating spermatogonia. In the male germ cells of this fish, the stage during spermatogenesis most sensitive to radiation-induced cell death is in early-differentiating spermatogonia, the immediate descendants of the stem cells. These spermatogonia may have a rigorous surveillance system for genomic lesions induced in spermatogonial stem cells.     

Proliferation and differentiation of spermatogonial stem cells in the w/wv mutant mouse testis

Mutations in the dominant-white spotting (W; c-kit) and stem cell factor (Sl; SCF) genes, which encode the transmembrane tyrosine kinase receptor and its ligand, respectively, affect both the proliferation and differentiation of many types of stem cells. Almost all homozygous W or Sl mutant mice are sterile because of the lack of differentiated germ cells or spermatogonial stem cells. To characterize spermatogenesis in c-kit/SCF mutants and to understand the role of c-kit signal transduction in spermatogonial stem cells, the existence, proliferation, and differentiation of spermatogonia were examined in the W/Wv mutant mouse testis. In the present study, some of the W/Wv mutant testes completely lacked spermatogonia, and many of the remaining testes contained only a few spermatogonia. Examination of the proliferative activity of the W/Wv mutant spermatogonia by transplantation of enhanced green fluorescent protein (eGFP)-labeled W/Wv spermatogonia into the seminiferous tubules of normal SCF (W/Wv) or SCF mutant (Sl/Sld) mice demonstrated that the W/Wv spermatogonia had the ability to settle and proliferate, but not to differentiate, in the recipient seminiferous tubules. Although the germ cells in the adult W/Wv testis were c-kit-receptor protein-negative undifferentiated type A spermatogonia, the juvenile germ cells were able to differentiate into spermatogonia that expressed the c-kit-receptor protein. Furthermore, differentiated germ cells with the c-kit-receptor protein on the cell surface could be induced by GnRH antagonist treatment, even in the adult W/Wv testis. These results indicate that all the spermatogonial stem cell characteristics of settlement, proliferation, and differentiation can be demonstrated without stimulating the c-kit-receptor signal. The c-kit/SCF signal transduction system appears to be necessary for the maintenance and proliferation of differentiated c-kit receptor-positive spermatogonia but not for the initial step of spermatogonial cell differentiation.     

Effects of stem cell factor and granulocyte macrophage-colony stimulating factor on survival of porcine type A spermatogonia cultured in KSOM.

Spermatogenesis is initiated with the divisions of the type A spermatogonial stem cells; however, the regulation of this stem cell population remains unknown. In order to obtain a better understanding of the biology of these cells, type A spermatogonia were isolated from 80-day-old pig testes by sedimentation velocity at unit gravity. The cells were cultured for up to 120 h in Dulbecco's modified Eagle's medium/Ham's F-12 medium (DMEM/F12) or a potassium-rich medium derived by the simplex optimization method (KSOM). At the end of the 120-h culture period, 30-50% of the spermatogonia were viable in KSOM, whereas in DMEM/F12 very few cells survived. Using KSOM as the culture medium, the effects of stem cell factor (SCF) and granulocyte macrophage-colony stimulating factor (GM-CSF) were studied. SCF significantly enhanced the percentage of cell survival at 100 ng/ml but not at lower concentrations. In comparison, GM-CSF promoted survival at relatively low concentrations (0.01, 0.1, and 1 ng/ml). At a higher dose (10 ng/ml), a significant reduction in percentage of cell survival was observed. The combination of SCF with GM-CSF had no significant effect on the percentage survival of type A spermatogonial cells. These data indicate that SCF and GM-CSF play a role in the regulation of survival and/or proliferation of type A spermatogonia.     

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.     

Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF

To study self-renewal and differentiation of spermatogonial stem cells, we have transplanted undifferentiated testicular germ cells of the GFP transgenic mice into seminiferous tubules of mutant mice with male sterility, such as those dysfunctioned at Steel (Sl) locus encoding the c-kit ligand or Dominant white spotting (W) locus encoding the receptor c-kit. In the seminiferous tubules of Sl/Sl(d) or Sl(17H)/Sl(17H) mice, transplanted donor germ cells proliferated and formed colonies of undifferentiated c-kit (-) spermatogonia, but were unable to differentiate further. However, these undifferentiated but proliferating spermatogonia, retransplanted into Sl (+) seminiferous tubules of W mutant, resumed differentiation, indicating that the transplanted donor germ cells contained spermatogonial stem cells and that stimulation of c-kit receptor by its ligand was necessary for maintenance of differentiated type A spermatogonia but not for proliferation of undifferentiated type A spermatogonia. Furthermore, we have demonstrated that their transplantation efficiency in the seminiferous tubules of Sl(17H)/Sl(17H) mice depended upon the stem cell niche on the basement membrane of the recipient seminiferous tubules and was increased by elimination of the endogenous spermatogonia of mutant mice from the niche by treating them with busulfan.     

Expansion of murine spermatogonial stem cells through serial transplantation

Mammalian male germ cells might be generally thought to have infinite proliferative potential based on their life-long production of huge numbers of sperm. However, there has been little substantial evidence that supports this assumption. In the present study, we performed serial transplantation of spermatogonial stem cells to investigate if they expand by self-renewing division following transplantation. The transgenic mouse carrying the Green fluorescent protein gene was used as the donor cell source that facilitated identification and recollection of colonized donor germ cells in the recipient testes. The established colonies of germ cells in the recipient testes were collected and transplanted to new recipients. This serial transplantation of spermatogonial stem cells repopulated the recipient testes, which were successfully performed sequentially up to four times from one recipient to the next. The incubation periods between two sequential transplantations ranged from 55 to 373 days. During these passages, the spermatogonial stem cells showed constant activity to form spermatogenic colonies in the recipient testis. They continued to increase in number for more than a year following transplantation. Colonization efficiency of spermatogonial stem cells was determined to be 4.25% by using Sl/Sl(d) mice as recipients that propagated only undifferentiated type A spermatogonia in their testes. Based on the colonization efficiency, one colony-forming activity was assessed to equate to about 20 spermatogonial stem cells. The spermatogonial stem cells were estimated to expand over 50-fold in 100 days in this experiment.     

EFFECT OF A SPERMATOGONIAL CHALONE ON THE GROWING RAT TESTIS

Immature rats were used in an experiment to test the possible influence of a spermatogonial chalone on the expanding spermatogonial population in their developing testes. An extract from adult rat testes was injected intraperitoneally into 33-day-old rats and control animals were injected with an equal amount of saline. Two groups of normal adult rats similarly injected with the testicular extract and saline solutions served as additional controls. Following these injections, all animals were administered a dose of ³H-thymidine 10 hr before sacrifice. An analysis of the labeling indices of the various types of spermatogonia revealed that in young rats injected with testicular extract the percentage of labeled type A spermatogonia was significantly lower than in control animals. In contrast, the labeling indices of Intermediate and type B spermatogonia were similar in the two groups of young rats. In the two groups of adult animals, there was no difference in the labeling indices of type A or of other types of spermatogonia. These data indicated that the saline extract of adult testes contained a substance, a spermatogonial chalone, inhibiting specifically the proliferation of some type A spermatogonia. The results also support the concept that a spermatogonial chalone may intervene, through its action on the spermatogonial stem cell population, to arrest the growth of the seminiferous tubules as the animal reaches maturity.     

The role of the tumor suppressor p53 in spermatogenesis

The p53 protein appeared to be involved in both spermatogonial cell proliferation and radiation response. During normal spermatogenesis in the mouse, spermatogonia do not express p53, as analyzed by immunohistochemistry. However, after a dose of 4 Gy of X-rays, a distinct p53 staining was present in spermatogonia, suggesting that, in contrast to other reports, p53 does have a role in spermatogonia. To determine the possible role of p53 in spermatogonia, histological analysis was performed in testes of both p53 knock out C57BL/6 and FvB mice. The results indicate that p53 is an important factor in normal spermatogonial cell production as well as in the regulation of apoptosis after DNA damage. First, p53 knock out mouse testes contained about 50% higher numbers of A1 spermatogonia, indicating that the production of differentiating type spermatogonia by the undifferentiated spermatogonia is enhanced in these mice. Second, 10 days after a dose of 5 Gy of X-rays, in the p53 knock out testes, increased numbers of giant sized spermatogonial stem cells were found, indicating disturbance of the apoptotic process in these cells. Third, in the p53 knock out testis, the differentiating A2-B spermatogonia are more radioresistant compared to their wild-type controls, indicating that p53 is partly indispensable in the removal of lethally irradiated differentiating type spermatogonia. In accordance with our immunohistochemical data, Western analysis showed that levels of p53 are increased in total adult testis lysates after irradiation. These data show that p53 is important in the regulation of cell production during normal spermatogenesis either by regulation of cell proliferation or, more likely, by regulating the apoptotic process in spermatogonia. Furthermore, after irradiation, p53 is important in the removal of lethally damaged spermatogonia.     

Proliferation and differentiation of bovine type A spermatogonia during long-term culture

The present study was aimed at developing a method for long-term culture of bovine type A spermatogonia. Testes from 5-mo-old calves were used, and pure populations of type A spermatogonia were isolated. Cells were cultured in minimal essential medium (MEM) or KSOM (potassium-rich medium prepared according to the simplex optimization method) and different concentrations of fetal calf serum (FCS) for 2-4 wk at 32 degrees C or 37 degrees C. Culture in MEM resulted in more viable cells and more proliferation than culture in KSOM, and better results were obtained at 37 degrees C than at 32 degrees C. After 1 wk of culture in the absence of serum, only 20% of the cells were alive. However, in the presence of 2.5% FCS, approximately 80% of cells were alive and proliferating. Higher concentrations of FCS only enhanced numbers of somatic cells. In long-term culture, spermatogonia continued to proliferate, and eventually, type A spermatogonial colonies were formed. The majority of colonies consisted mostly of groups of cells connected by intercellular bridges. Most of the cells in these colonies underwent differentiation because they were c-kit positive, and ultimately, cells with morphological and molecular characteristics of spermatocytes and spermatids were formed. Occasionally, large round colonies consisting of single, c-kit-negative, type A spermatogonia (presumably spermatogonial stem cells) were observed. For the first time to our knowledge, a method has been developed to allow proliferation and differentiation of highly purified type A spermatogonia, including spermatogonial stem cells during long-term culture.     

Bcl-2 inhibits apoptosis of spermatogonia and growth of spermatogonial stem cells in a cell-intrinsic manner

The growth, differentiation, and death/survival of spermatogonia are precisely regulated for the proper production of spermatozoa. We have previously shown that Bcl-2 ectopically expressed in spermatogonia caused the inhibition of normal spermatogonial apoptosis and the subsequent failure of differentiation in transgenic mice. In addition, the growth of spermatogonial stem cells seemed to be temporally arrested in the transgenic mice. In the present study, we attempted to examine whether the abnormality of spermatogonia described above was caused by Bcl-2 misexpression in the spermatogonia or by an abnormal spermatogenic environment of the transgenic mice. We transplanted testicular cells of transgenic mice to seminiferous tubules of W/Wv mice in which transplanted normal testicular cells can undergo spermatogenesis. We found that the transplanted spermatogonia of the transgenic mice reproduced a series of abnormal changes including temporal growth arrest of spermatogonial stem cells and abnormal accumulation of spermatogonia in tubules, which were also observed in the testes of the transgenic mice. The results indicated that Bcl-2 inhibited apoptosis of spermatogonia and growth of spermatogonial stem cells in a cell-intrinsic manner. We also cultured testicular cells of transgenic mice and found that the spermatogonia of the transgenic mice were better able to survive than were those of wild-type mice but that their differentiation was not affected. The result suggested that failure of differentiation of the accumulated spermatogonia in the transgenic testes is not due to the abnormality of the bcl-2 misexpressing spermatogonia, but may be caused by extrinsic problems including improper interaction of spermatogonia with supporting cells.     

H3K27 Demethylase,JMJD3, Regulates Fragmentation of Spermatogonial Cysts

The spermatogonial stem cell (SSC) compartment is maintained by self-renewal of stem cells as well as fragmentation of differentiating spermatogonia through abscission of intercellular bridges in a random and stochastic manner. The molecular mechanisms that regulate this reversible developmental lineage remain to be elucidated. Here, we show that histone H3K27 demethylase, JMJD3 (KDM6B), regulates the fragmentation of spermatogonial cysts. Down-regulation of Jmjd3 in SSCs promotes an increase in undifferentiated spermatogonia but does not affect their differentiation. Germ cell-specific Jmjd3 null male mice have larger testes and sire offspring for a longer period compared to controls, likely secondary to increased and prolonged maintenance of the spermatogonial compartment. Moreover, JMJD3 deficiency induces frequent fragmentation of spermatogonial cysts by abscission of intercellular bridges. These results suggest that JMJD3 controls the spermatogonial compartment through the regulation of fragmentation of spermatogonial cysts and this mechanism may be involved in maintenance of diverse stem cell niches.     

Spermatogenetic clones developing from repopulating stem cells surviving a high dose of an alkylating agent.

We investigated stem cell renewal and differentiation in 10- and 15-days-old spermatogonial clones developing in mouse seminiferous epithelium after an extremely large cell loss, inflicted by high doses of the alkylating agent Myleran. The spermatogonial clones arise from cells that resemble the Ais spermatogonia but have a larger nuclear diameter. In spite of their mitotic activity these 'repopulating stem cells' lie mainly isolated or in pairs. This explained by migration and differentiation. Migration appeared to occur at random in all directions along the basement membrane of the seminiferous tubule. After one or more divisions of the stem cells, a second type of cell appears, which is called the 'differentiating spermatogomium'. The time elapsing before this type of cell appears, depends on the dose of Myleran: the larger the dose the later differentiation starts. A relation could be demonstrated between the stage of the cycle of the seminiferous epithelium and the start of differentiation. Differentiating cells were found isolated or in groups of two, four, eight or sixteen cells. Hence we concluded that at least up to their fourth division differentiating cells divide synchronously without degenerations. Three types of division of repopulating stem cells were distinguished, producing (1) two repopulating stem cells, (2) one repopulating stem cell and one cell starting spermatogonial differentiation, or (3) two differentiating cells. Type 1 divisions were found most frequently.     

Isolation,in vitro study,and stem cell markers for type A spermatogonia in a Characiformes species

The objective of this study was to establish a protocol for the characterization, isolation, and culture of type A spermatogonia using specific molecular markers for these cells in fish. To this end, adult Prochilodus lineatus testes were collected and digested enzymatically and the resulting testicular suspension was separated using a discontinuous Percoll gradient, followed by differential plating. The cell cultures obtained were monitored for 15 days and analyzed using the immunofluorescence method with anti‐Vasa, anti‐GFRα1, and anti‐OCT4 antibodies. Spermatogonial enrichment was also performed using flow cytometry. Although discontinuous Percoll gradient centrifugation followed by differential plating enabled the removal of differentiated germ cells and somatic cells, enriching the pool of type A spermatogonia, the enrichment of type A spermatogonia through flow cytometry of samples without Percoll proved to be more efficient. Prominent cell agglomerates that were characterized according to different stem cell markers as type A spermatogonia were observed during the 15 days of the cell culture. The use of immunoperoxidase and western blot analysis methods confirmed the specificity of the markers for type A spermatogonia of P. lineatus. When combined with specific cell culture conditions, the positive characterization of these molecular markers clarified certain aspects of spermatogonial regulation, such as survival and proliferation. Finally, understanding the regulation of the in vitro germ cell maintenance process may contribute to the enhancement of in vivo and in vitro reproduction techniques of endangered or aquaculture fish species.