Identification of the starting point for spermatogenesis and characterization of the testicular stem cell in adult male rhesus monkeys

BACKGROUND: Spermatogonial expansion in man and non-human primates has been studied for decades. Controversy persists about the cell type representing the testicular stem cell and the exact kinetics of spermatogonial proliferation. We recently determined the starting point of spermatogenesis and proposed a model for clonal expansion of spermatogonia in adult macaques. Here we want to confirm the initiation event, study and compare the details of the kinetics of spermatogonial expansion in vivo and in vitro, and characterize a population of A spermatogonia acting as testicular stem cells. METHODS and RESULTS: We localized BrdU-positive spermatogonia in whole mounts and sections of adult rhesus monkey testes. Culture of testicular tissue was used to determine the expansion and differentiation of premeiotic germ cells. We confirm that A(pale) spermatogonia divide equally at stage VII and produce two types of progeny after mitosis at stage IX of the seminiferous cycle following defined clonal patterns. Small numbers of proliferating single A spermatogonia exist which present a population of label-retaining cells. CONCLUSIONS: In the rhesus monkey the population of A(pale) spermatogonia cycle continuously and initiate spermatogenesis by a self-renewing division at stage VII of the seminiferous epithelial cycle. Rarely dividing single A spermatogonia exist which potentially are the male germline stem cells in the primate testis.     

Spermatogonial transplantation: the principle and possible applications

Spermatogenesis is the process of male germ cell proliferation and differentiation that begins at puberty and lasts throughout life. Spermatogonia, especially stem spermatogonia, are the cells essential for the continued maintenance of spermatogenesis. Although many studies of spermatogonia have been performed with morphological methods, the very nature of spermatogonia still remains unknown. The technique of spermatogonial transplantation, developed in 1994, made it possible to study functional aspect of spermatogonial stem cells. Many new developments, such as cryopreservation, xenotransplantation, purification, and culturing of spermatogonial stem cells, have been achieved and are still under investigation. The techniques could be used not only for basic research but also for medicine and other disciplines.     

Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity

The nucleostemin (NS) gene encodes a nucleolar protein found at high levels in several types of stem cells and tumor cell lines. The function of NS is unclear but it may play a critical role in S-phase entry by stem/progenitor cells. Here we characterize NS expression in murine male germ cells. Although NS protein was highly expressed in the nucleoli of all primordial germ cells, only a limited number of gonocytes showed NS expression in neonatal testes. In adult testes, NS protein was expressed at high levels in the nucleoli of spermatogonia and primary spermatocytes but at only low levels in round spermatids. To evaluate the properties of cells expressing high levels of NS, we generated transgenic reporter mice expressing green fluorescent protein (GFP) under the control of the NS promoter (NS-GFP Tg mice). In adult NS-GFP Tg testes, GFP and endogenous NS protein expression were correlated in spermatogonia and spermatocytes but GFP was also ectopically expressed in elongated spermatids and sperm. In testes of NS-GFP Tg embryos, neonates, and 10-day-old pups, however, GFP expression closely coincided with endogenous NS expression in developing germ cells. In contrast to a previous report, our results support the existence in neonatal testes of spermatogonial stem cells with long-term repopulating capacity. Furthermore, our data show that NS expression does not correlate with cell-cycle status during prepuberty, and that strong NS expression is essential for the maintenance of germline stem cell proliferation capacity. We conclude that NS is a marker of undifferentiated status in the germ cell lineage during prepubertal spermatogenesis.     

The pluripotency factor LIN28 in monkey and human testes: a marker for spermatogonial stem cells?

Mammalian spermatogenesis is maintained by spermatogonial stem cells (SSCs). However, since evidentiary assays and unequivocal markers are still missing in non-human primates (NHPs) and man, the identity of primate SSCs is unknown. In contrast, in mice, germ cell transplantation studies have functionally demonstrated the presence of SSCs. LIN28 is an RNA-binding pluripotent stem cell factor, which is also strongly expressed in undifferentiated mouse spermatogonia. By contrast, two recent reports indicated that LIN28 is completely absent from adult human testes. Here, we analyzed LIN28 expression in marmoset monkey (Callithrix jacchus) and human testes during development and adulthood and compared it with that in mice. In the marmoset, LIN28 was strongly expressed in migratory primordial germ cells and gonocytes. Strikingly, we found a rare LIN28-positive subpopulation of spermatogonia also in adult marmoset testis. This was corroborated by western blotting and quantitative RT-PCR. Importantly, in contrast to previous publications, we found LIN28-positive spermatogonia also in normal adult human and additional adult NHP testes. Some seasonal breeders exhibit a degenerated (involuted) germinal epithelium consisting only of Sertoli cells and SSCs during their non-breeding season. The latter re-initiate spermatogenesis prior to the next breeding-season. Fully involuted testes from a seasonal hamster and NHP (Lemur catta) exhibited numerous LIN28-positive spermatogonia, indicating an SSC identity of the labeled cells. We conclude that LIN28 is differentially expressed in mouse and NHP spermatogonia and might be a marker for a rare SSC population in NHPs and man. Further characterization of the LIN28-positive population is required.     

Proliferation of spermatogonia and Sertoli cells in maturing mice

Summary Spermatogonial proliferation was studied in mice from day 13 p.p. when the seminiferous epithelium is incomplete, until week 12 p. p. when a steady state at adult levels has been attained. Counts of undifferentiated, A ₁ and intermediate spermatogonia and primary spermatocytes in stages IV and IX of the cycle of the seminiferous epithelium were made in whole mounted seminiferous tubules. Sertoli cell proliferation was studied in a separate series from 6 to 14 days p.p. employing the ³H-thymidine labeling index.It appeared that 1. Sertoli cell proliferation stops at day 12 whereafter the cells obtain their adult appearance; 2. The numbers of stem cell spermatogonia and the production of differentiating A ₁ spermatogonia increase almost twofold between day 13 and week 12; 3. The efficiency of the divisions of the differentiating A ₁-B spermatogonia is similar to that in the adult throughout this period; 4. At all ages studied, the cell counts revealed an almost constant numerical relationship between Sertoli cells and germ cells, which suggests a function of Sertoli cells in the regulation of spermatogonial proliferation.     

GDNF-mediated signaling via RET tyrosine 1062 is essential for maintenance of spermatogonial stem cells

Well-organized spermatogenesis, including the maintenance of spermatogonial stem cells (SSCs), is indispensable for continuous male fertility. Signaling by glial cell line-derived neurotrophic factor (GDNF) via the RET/GDNF family receptor α1 (GFRα1) receptor complex is essential for self-renewal of murine SSCs and may also regulate their differentiation. When phosphorylated, tyrosine 1062 in RET presents a binding site for the phosphotyrosine-binding domains of several adaptor and effector proteins that are important for activation of a variety of intracellular signaling pathways. In this study, we investigated the role of signaling via RET tyrosine 1062 in spermatogenesis using RET Y1062F knockin mice (Y1062F mice), in which tyrosine 1062 was replaced with phenylalanine. Homozygous Y1062F mice showed marked atrophy of testes due to reduced production of germ cells. RET-expressing spermatogonia in seminiferous tubules of homozygous Y1062F mice decreased after postnatal day (P) 7 and germ cells were almost undetectable by P21. These phenomena appeared to be due to a lack of SSC self-renewal and inability to maintain the undifferentiated state. Our findings suggest that RET signaling via tyrosine 1062 is essential for self-renewal of SSCs and regulation of their differentiation.     

NANOS2 acts downstream of glial cell line-derived neurotrophic factor signaling to suppress differentiation of spermatogonial stem cells

Stem cells are maintained by both stem cell-extrinsic niche signals and stem cell-intrinsic factors. During murine spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) signal emanated from Sertoli cells and germ cell-intrinsic factor NANOS2 represent key regulators for the maintenance of spermatogonial stem cells. However, it remains unclear how these factors intersect in stem cells to control their cellular state. Here, we show that GDNF signaling is essential to maintain NANOS2 expression, and overexpression of Nanos2 can alleviate the stem cell loss phenotype caused by the depletion of Gfra1, a receptor for GDNF. By using an inducible Cre-loxP system, we show that NANOS2 expression is downregulated upon the conditional knockout (cKO) of Gfra1, while ectopic expression of Nanos2 in GFRA1-negative spermatogonia does not induce de novo GFRA1 expression. Furthermore, overexpression of Nanos2 in the Gfra1-cKO testes prevents precocious differentiation of the Gfra1-knockout stem cells and partially rescues the stem cell loss phenotypes of Gfra1-deficient mice, indicating that the stem cell differentiation can be suppressed by NANOS2 even in the absence of GDNF signaling. Taken together, we suggest that NANOS2 acts downstream of GDNF signaling to maintain undifferentiated state of spermatogonial stem cells.     

Establishment of the paternal methylation imprint of the human H19 and MEST/PEG1 genes during spermatogenesis

Parental-specific epigenetic modifications are imprinted on a subset of genes in the mammalian genome during germ cell maturation. However, the precise timing of their establishment remains to be determined. Methylation of CpG dinucleotides has been shown to be a part of the parental imprint. We have examined how the methylation pattern characteristic of the paternal allele in germ cells are established during human spermatogenesis. Two representative imprinted genes, H19 and MEST/PEG1, were studied. The experiments were performed using the bisulphite sequencing method on microdissected individual cells at different stages of male germ cell differentiation. We show that both genes are unmethylated in fetal spermatogonia, suggesting that all pre-existing methylation imprints are already erased by this stage. The MEST/PEG1 gene remains unmethylated at all subsequent post-pubertal stages of spermatogenesis, including mature spermatozoa. The methylation of H19 typical of the paternal allele first appears in a subset of adult spermatogonia and then is maintained in spermatocytes, spermatids and mature spermatozoa. Our results suggest that the methylation imprint inherited from the parents is first erased in the male germ line at an early fetal stage. The paternal-specific imprint is re-established only later, during spermatogonial differentiation in the adult testis.     

Characterization, cryopreservation, and ablation of spermatogonial stem cells in adult rhesus macaques

Spermatogonial stem cells (SSCs) are at the foundation of mammalian spermatogenesis. Whereas rare A(single) spermatogonia comprise the rodent SSC pool, primate spermatogenesis arises from more abundant A(dark) and A(pale) spermatogonia, and the identity of the stem cell is subject to debate. The fundamental differences between these models highlight the need to investigate the biology of primate SSCs, which have greater relevance to human physiology. The alkylating chemotherapeutic agent, busulfan, ablates spermatogenesis in rodents and causes infertility in humans. We treated adult rhesus macaques with busulfan to gain insights about its effects on SSCs and spermatogenesis. Busulfan treatment caused acute declines in testis volume and sperm counts, indicating a disruption of spermatogenesis. One year following high-dose busulfan treatment, sperm counts remained undetectable, and testes were depleted of germ cells. Similar to rodents, rhesus spermatogonia expressed markers of germ cells (VASA, DAZL) and stem/progenitor spermatogonia (PLZF and GFRalpha1), and cells expressing these markers were depleted following high-dose busulfan treatment. Furthermore, fresh or cryopreserved germ cells from normal rhesus testes produced colonies of spermatogonia, which persisted as chains on the basement membrane of mouse seminiferous tubules in the primate to nude mouse xenotransplant assay. In contrast, testis cells from animals that received high-dose busulfan produced no colonies. These studies provide basic information about rhesus SSC activity and the impact of busulfan on the stem cell pool. In addition, the germ cell-depleted testis model will enable autologous/homologous transplantation to study stem cell/niche interactions in nonhuman primate testes.     

GDNF-induced seminomatous tumours in mouse--an experimental model for human seminomas?

Glial-cell-line-derived neurotrophic factor (GDNF) is a distant member of the transforming growth factor superfamily. It binds to and activates a receptor complex consisting of GFR-alpha1 and Ret receptor tyrosine kinase. In testis, GDNF is expressed by Sertoli cells. We have shown by transgenic loss- and gain-of-function mouse models that GDNF regulates the cell fate decision of undifferentiated spermatogonia. In the GDNF +/- mice, the spermatogonia differentiate in excess leading to the depletion of germ cells. In the mice overexpressing GDNF in testes, undifferentiated spermatogonia accumulate in the tubules, no sperm is produced, and the mice are infertile. After a year, the GDNF overexpressing mice frequently (89%) develop testicular tumours, and most of them are bilateral (56%). All these tumours show the same histological pattern. They are composed of round spermatogonial/gonocytic cells with only a scant cytoplasm. The tumours are locally invasive but do not metastasise. They express germ line markers, are positive for alkaline phosphatase, and aneuploid with a triploid peak. Thus, by several histological, molecular, and histochemical characteristics, the GDNF-induced tumours mimic classical seminomas in men, but the precursor lesions are apparently different in mouse and man.     

Human spermatogenesis: basic research and clinical issues]

Histological evaluation of human spermatogenesis suffers from the hazy border line between normal and pathological germ cell development. This border line needs better definition for histological fertility diagnosis and the early detection of germ cell tumors. Testicular biopsies from more than 2,900 patients with fertility disturbances and more than 1,900 patients with testicular tumors were investigated by means of semithin sectioning, different immunocytochemical methods and transmission electron microscopy. Cellular systems of the human testes possess a degree of autonomy from the body. Their morphological and functional heterogeneity reveals characteristics of cells that are not terminally differentiated. In the testis of an adult, fertile man not only the proliferation of spermatogonia, maturation divisions of spermatocytes and differentiation of spermatids take place, but also abortive germ cells, as well as apoptotic and degenerative cells appear. Disturbances of spermatogenesis are defined by the evaluation of quantity and quality of germ cell alterations. Compensatory and non compensatory defects of spermatogenesis may be distinguished. Deficiency of spermatogonial cell types, multilayered spermatogonia, megalospermatocytes, malformed spermatids and single tumor cells in the face of sufficient development of mature spermatids are considered compensatory defects of spermatogenesis. Dominating malformed germ cells or tumor cells accompanied by an arrest or lack of spermatogenesis, however, represent non-compensatory defects of spermatogenesis. In addition, normal organization and function of the microvasculature, Leydig cells and compartmentalizing cells in the intertubular space are prerequisites for spermatogenesis. The neuroendocrine function of Leydig cells may be responsible for regulating the blood flow rate and the permeability to hormones and nutritive substances. Finally, for patients a successful definition of the border line between normal and pathological events of germ cell development may be essential for early detection of germ cell tumors. Therefore, anatomical sciences not only contribute to basic research, advanced diagnostics and therapeutic concepts related to diseases of the male gonad, but also to the improvement of assisted reproduction.     

COMMENTS

Glial-cell-line-derived neurotrophic factor (GDNF) is a distant member of the transforming growth factor superfamily. It binds to and activates a receptor complex consisting of GFR-α1 and Ret receptor tyrosine kinase. In testis, GDNF is expressed by Sertoli cells. We have shown by transgenic loss- and gain-of-function mouse models that GDNF regulates the cell fate decision of undifferentiated spermatogonia. In the GDNF +/− mice, the spermatogonia differentiate in excess leading to the depletion of germ cells. In the mice overexpressing GDNF in testes, undifferentiated spermatogonia accumulate in the tubules, no sperm is produced, and the mice are infertile. After a year, the GDNF overexpressing mice frequently (89%) develop testicular tumours, and most of them are bilateral (56%). All these tumours show the same histological pattern. They are composed of round spermatogonial/gonocytic cells with only a scant cytoplasm. The tumours are locally invasive but do not metastasise. They express germ line markers, are positive for alkaline phosphatase, and aneuploid with a triploid peak. Thus, by several histological, molecular, and histochemical characteristics, the GDNF-induced tumours mimic classical seminomas in men, but the precursor lesions are apparently different in mouse and man.     

Regulation of the density of spermatogonia in the seminiferous epithelium of the Chinese hamster: I. Undifferentiated spermatogonia

The topographical arrangement of the clones of A single, A paired, and A aligned (As, Apr, and Aal) spermatogonia on the basement membrane of seminiferous tubules of the Chinese hamster was studied. It was found that at least some of these clones are not distributed at random as clones of similar cell number were often seen in clusters. Areas were found with few or many As spermatogonia. Also, clusters of Apr spermatogonia were found, indicating that in such an area many As spermatogonia more or less synchronously formed Apr spermatogonia. Since clusters of clones of 16 Aal spermatogonia were observed, it can be concluded that these clusters of Apr spermatogonia may proliferate in at least a roughly synchronous way. It was found that over large areas the densities of undifferentiated spermatogonia may be very low or high in comparison to the mean density in the animal. Whether the ratio of self-renewal and differentiation of the stem cells changed locally in response to the high or low density of undifferentiated spermatogonia in particular areas was investigated. No indications for a regulatory mechanism to keep the density of stem cells and/or the density of undifferentiated spermatogonial clones at a certain level could be detected in the normal Chinese hamster. This lack of regulation was at least partly responsible for the widely different numbers of A1 spermatogonia that were formed in the various areas studied in stage IX.     

Stem cell factor/c-kit system in spermatogenesis

One of the major unresolved questions with male infertility is the identification of the molecular origin of a great majority of the spermatogenetic arrests currently diagnosed as idiopathic male infertility. During the past years, several families of regulating factors have been implicated in spermatogenesis defects observed essentially in animal models. Among these factors are signalling molecules, and particularly the stem cell factor (SCF)/c-kit system. The SCF and its receptor c-kit are an appropriate example to illustrate the role of signalling molecules in the physiology and pathology of spermatogenesis. The SCF/c-kit regulates primordial germ cell migration, proliferation and apoptosis during fetal gonadal development. The SCF/c-kit also regulates spermatogonia proliferation in the adult animal. In mutant mice, abnormalities of the SCF/c-kit gene expression, such as gene deletion, point mutation, alternative splicing defect, lead to different types of spermatogenesis alterations (e.g. decrease in primordial germ cell migration, decrease in spermatogonia proliferation). More recently, defects in SCF/c-kit gene expression have also been shown in human testicular dysfunctions. Indeed, a reduction in SCF/c-kit expression has been evidenced in oligozoospermia/azoospermia associated with an increase in the germ cell apoptosis process. In addition, c-kit seems to be a good marker of seminoma testicular tumours. This review reports a large number of data--obtained essentially in animal models--that suggest an important role for the SCF/c-kit system in spermatogenesis and, as a corollary, its potential involvement in spermatogenic defects.     

The genetic control and germ cell kinetics of the female and male germ line in mammals including man.

The female germ line (germ cell lineage, Keimbahn) is provided with only one proliferation wave, the oogenic, whereas male gametogenesis involves two successive waves: prespermatogenic, which corresponds to the female proliferation wave, and spermatogenesis, which is responsible for the immense number of male gametes produced in mature testes. Both male proliferation systems are linked by the transitional or T prospermatogonia. Using the reverse percentage of labelled metaphases method, it has been shown that the first differences between female and male germ cells can be identified by the end of the first wave, when oogonia and multiplying or M prospermatogonia are proliferating. This prenatal first wave of proliferation of male germ cells was also demonstrated in man and ceases around the 22nd week of pregnancy. Spermatogenesis involves a stock of stem cells (stem spermatogonia), a flexibly reacting pool of undifferentiated spermatogonia and several generations of differentiating spermatogonia, which proliferate almost exponentially. Furthermore, it consists of spermatocytes and haploid spermatids transforming into spermatozoa. The oocytes pass through the preleptotene stage, synthesizing DNA, and thereafter traverse the meiotic prophase up to the diplotene stage. In mammals they act as 'pre-embryos' in a similar but to a lesser degree than oocytes of amphibia and insects. The maternal chromosomes are largely responsible for the development of the embryo, the paternal genome for the development of the extra-embryonic tissue. The synthesis of transgenic animals is a powerful weapon in the armoury of geneticists, as has recently been demonstrated: a 14 kb genomic DNA fragment (Sry) is sufficient to induce testis differentiation and subsequent male development when introduced into chromosomally female mouse embryos.     

Identification and regulation of a stage-specific stem cell niche enriched by Nanog-positive spermatogonial stem cells in the mouse testis

The ability of spermatogonial stem cells to acquire embryonic stem cell (ESC) properties in vitro has recently been of great interest. However, studies focused on the in vivo regulation of testicular stem cells have been hampered because the exact anatomical location of these cells is unknown. Moreover, no specialized stem cell niche substructure has been identified in the mammalian testis thus far. It has also been unclear whether the adult mammalian testis houses pluripotent stem cells or whether pluripotency can be induced only in vitro. Here, we demonstrate, for the first time, the existence of a Nanog-positive spermatogonial stem cell subpopulation located in stage XII of the mouse seminiferous epithelial cycle. The efficiency of the cells from seminiferous tubules with respect to prolonged pluripotent gene expression was correlated directly with stage-specific expression levels of Nanog and Oct4, demonstrating the previously unknown stage-specific regulation of undifferentiated spermatogonia (SPG). Testicular Nanog expression marked a radioresistant spermatogonial subpopulation, supporting its stem cell nature. Furthermore, we demonstrated that p21 acts as an upstream regulator of Nanog in SPG and mouse ESCs, and our results demonstrate that promyelocytic leukemia zinc finger is a specific marker of progenitor SPG. Additionally, we describe a novel method to cultivate Nanog-positive SPG in vitro. This study demonstrates the existence and location of a previously unknown stage-specific spermatogonial stem cell niche and reports the regulation of radioresistant spermatogonial stem cells.