Overexpression of ubiquitin carboxyl-terminal hydrolase L1 arrests spermatogenesis in transgenic mice

Ubiquitin carboxyl-terminal hydrolase 1 (UCH-L1) can be detected in mouse testicular germ cells, mainly spermatogonia and somatic Sertoli cells, but its physiological role is unknown. We show that transgenic (Tg) mice overexpressing EF1alpha promoter-driven UCH-L1 in the testis are sterile due to a block during spermatogenesis at an early stage (pachytene) of meiosis. Interestingly, almost all spermatogonia and Sertoli cells expressing excess UCH-L1, but little PCNA (proliferating cell nuclear antigen), showed no morphological signs of apoptosis or TUNEL-positive staining. Rather, germ cell apoptosis was mainly detected in primary spermatocytes having weak or negative UCH-L1 expression but strong PCNA expression. These data suggest that overexpression of UCH-L1 affects spermatogenesis during meiosis and, in particular, induces apoptosis in primary spermatocytes. In addition to results of caspases-3 upregulation and Bcl-2 downregulation, excess UCH-L1 influenced the distribution of PCNA, suggesting a specific role for UCH-L1 in the processes of mitotic proliferation and differentiation of spermatogonial stem cells during spermatogenesis.     

Impaired spermatogenesis in the Japanese eel, Anguilla japonica: Possibility of the existence of factors that regulate entry of germ cells into meiosis

In the cultivated male Japanese eel, spermatogonia are the only germ cells present in the testis. Weekly injections of human chorionic gonadotropin (HCG) can induce complete spermatogenesis from proliferation of spermatogonia to spermiogenesis. In some cases, however, HCG injection fails to induce complete spermatogenesis. Testicular morphological observations revealed that HCG-injected eels could be classified into three types based on their testicular conditions. Type 1 eels had a well-developed testis and the milt could be acquired by hand-stripping. In type 2 eels, spermatogenesis was also induced by HCG injection, but testicular size was remarkably smaller than that of type 1 eels, and the milt could not be hand-stripped. At the end of the experiment, type 2 fish had only spermatogonia and a small amount of spermatozoa, but no spermatocytes or spermatids, in their testis. Type 3 eels had thready testis, which did not develop any germ cells during the experimental period. These results suggest that, despite elevations of plasma 11–ketotestosterone levels, HCG injections were not successful in inducing the completion of spermatogenesis in type 2 and type 3 eels. In most spermatogonia of type 2 eels, meiosis was not induced by HCG injections. Furthermore, only few mitotic divisions had occurred as evidenced by the presence of 2³ to 2⁶ late type B spermatogonia in most cysts. This suggests that spermatogonial stem cells undergo four or five, and occasionally six, mitotic divisions before the interruption of spermatogenesis in type 2 eels. It is proposed that those numbers of mitotic divisions are related to a mediator that regulates entry of spermatogonia of the Japanese eel into meiosis.     

Proteomic analysis of the spermatogonial stem cell compartment in dogfish Scyliorhinus canicula L

In the dogfish (Scyliorhinus canicula L.) the testicular germinative zone (GZ), composed of large isolated spermatogonia surrounded by elongating pre-Sertoli cells, is located between the albuginea and the ventrolateral intratesticular vessel. During the spermatogenic wave, cysts radiate in maturational order forming distinct testicular zones. In this study, soluble proteins of the GZ and of the zone containing cysts with spermatocytes were separated by two-dimensional electrophoresis. Gel images were matched and then evaluated for GZ-specific proteins. From the1400 protein spots identified, 680 were found to be apparently specific to this zone. Using MALDI-TOF/TOF mass spectrometry, de novo sequences were obtained for 33 proteins out of the 169 selected for identification by mass spectrometry, but only 16 of these 169 proteins were identified. One of them, proteasome subunit alpha-6, was analyzed further by immunohistochemistry. This study demonstrates the utility of the dogfish as a model for proteome analysis of the spermatogonial stem cell niche, even if it remains restricted by the lack of genomic data available on Elasmobranchs.     

Fetal bovine serum simultaneously stimulates apoptosis and DNA synthesis in premeiotic stages of spermatogenesis in spiny dogfish (Squalus acanthias) in vitro: modulation by androgen and spermatogenic activity status

Using the simple cystic spermatogenesis in the shark testis as a model, we previously reported the relative resistance of immature spermatogonia (stem cell and early-stage spermatogonia) to apoptosis in the normal testis and after spermatoxicant exposure in vivo. Apoptosis was monitored by fluorescence image analysis of living cysts, using the validated acridine orange (AO) vital staining technique. Findings show that FBS simultaneously stimulates both apoptosis and [³H]thymidine incorporation in immature spermatogonial clones in a concentration-dependent manner in vitro. Furthermore, androgen inhibits apoptosis and increases cyst viability, more so with 10% FBS than with 1% FBS. All the effects were as a function of spermatogenic activity status but were distinct in early-stage spermatogonial cysts isolated from testes awakening from the previous winter spermatogenic arrest period. Results are discussed in the context of the alternating germ–Sertoli cell population kinetics of early-stage spermatogonial cysts in Squalus acanthias’s protracted testicular cycle.     

Magnetic activated cell sorting allows isolation of spermatogonia from adult primate testes and reveals distinct GFRa1‐positive subpopulations in men

Background Isolation of spermatogonial stem cells (SSCs) could enable in vitro approaches for exploration of spermatogonial physiology and therapeutic approaches for fertility preservation. SSC isolation from adult testes is difficult due to low cell numbers and lacking cell surface markers. Glial cell‐derived neurotrophic factor family receptor alpha‐1 (GFRα1) plays a crucial role for the maintenance of SSCs in rodents and is expressed in monkey spermatogonia. Methods Magnetic activated cell sorting was employed for the enrichment of GFRα1+ spermatogonia from adult primate testes. Results Magnetic activated cell sorting of monkey cells enriched GFRα1+ cells threefold. 11.4% of GFRα1+ cells were recovered. 42.9% of GFRα1+ cells were recovered in sorted fractions of human testicular cells, representing a fivefold enrichment. Interestingly, a high degree of morphological heterogeneity among the GFRα1+ cells from human testes was observed. Conclusions Magnetic activated cell sorting using anti‐GFRα1 antibodies provides an enrichment strategy for spermatogonia from monkey and human testes.     

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.     

Functional demonstration of the ability of a primary spermatogonium as a stem cell by tracing a single cell destiny in Xenopus laevis

In Xenopus, although primary spermatogonium (PG), the largest cell in the testis, is believed to be spermatogonial stem cell by histological observations, functional evidence has never been obtained. In the present study, we first indicated that culture of juvenile testis in a medium supplemented with follicle stimulating hormone resulted in no proliferation of PG. In this culture system, early secondary spermatogonia could undergo mitotic divisions with a concomitant decrease in their size, so that they became distinguishable in size from PG. Because the subcutaneous environment of juveniles permitted aggregates of the dissociated testicular cells to reconstruct the normal testis structure, we next inserted a genetically marked PG isolated from cultured testes into the aggregate and transplanted it subcutaneously. In this system, 73.9% of the aggregates contained a marked PG. When we observed the aggregates 12 weeks after transplantation, most aggregates (70.0%) contained marked PG that had self-renewed. Among these, fully growing aggregates contained many spermatogenic cells at the later developmental stage. These results suggested that isolated PG from the cultured testes had the ability as stem cells, and that purification of the spermatogenic stem cells became reliable in Xenopus.     

Involvement of GnRH neuron in the spermatogonial proliferation of the scallop, Patinopecten yessoensiss

The aim of this study was to quantitatively analyze a pattern of proliferation of gonial cells and to demonstrate neural involvement in spermatogonial proliferation of the scallop by the in vitro experiment. Immunocytochemistry for incorporated BrdU was used to identify mitotically active gonial cells. The pattern of proliferation of gonial cells was divided into two phases: phase I; oogonia and spermatogonia slowly proliferate through the growing stage: phase II; oogonia develop into oocytes and spermatogonia start to proliferate rapidly from the mature stage through the spawning stage. The neurons detected with anti-mammalian (m)GnRH antibody were distributed sparsely in the pedal ganglion and predominantly in the cerebral ganglion of both sexes at the growing stage. The extracts from the cerebral and pedal ganglion (CPG) of both sexes collected at the growing stage promoted proliferation of spermatogonia in the in vitro culture of the testicular tissue as well as mGnRH. However, CPG extract had no effect on oogonial proliferation. The increased mitotic activity induced by CPG and mGnRH was abolished by the addition of mGnRH antagonists and anti-mGnRH antibody, suggesting that the spermatogonial proliferation is regulated by GnRH-like peptide in CPG of the scallop. The same mitotic activity as CPG extract and mGnRH was observed in the hemocyte lysate, but not in the serum. These findings suggest that the spermatogonial proliferation at phase II in the scallop may be under the neuroendocrine control by GnRH neuron in CPG.     

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.     

The spermatogonial stem cell: from basic knowledge to transgenic technology

Differentiation of germ cells in the testis originates from a constantly renewed small pool of stem cells. They give rise to the first differentiated spermatogenic cells (spermatogonia). These committed cells will then follow a strictly defined succession of steps, starting with six synchronized mitotic cycles before reaching the first meiotic stages. Following a first identification of the spermatogonial stem cells on morphological and cytological criteria, a functional assay was devised, based on their ability to repopulate the testis of a sterile recipient. Purification and characterization of the stem fraction is in progress. Fundamental knowledge of the biology of the germ line and preclinical studies in several important fields will benefit of these advances, while gene transfer prior to reimplantation opens a new approach in transgenic technology.     

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.     

The Fate of Spermatogonial Stem Cells in the Cryptorchid Testes of RXFP2 Deficient Mice

The environmental niche of the spermatogonial stem cell pool is critical to ensure the continued generation of the germ cell population. To study the consequences of an aberrant testicular environment in cryptorchidism we used a mouse model with a deletion of Rxfp2 gene resulting in a high intra-abdominal testicular position. Mutant males were infertile with the gross morphology of the cryptorchid testis progressively deteriorating with age. Few spermatogonia were identifiable in 12 month old cryptorchid testes. Gene expression analysis showed no difference between mutant and control testes at postnatal day 10. In three month old males a decrease in expression of spermatogonial stem cell (SSC) markers Id4, Nanos2, and Ret was shown. The direct counting of ID4+ cells supported a significant decrease of SSCs. In contrast, the expression of Plzf, a marker for undifferentiated and differentiating spermatogonia was not reduced, and the number of PLZF+ cells in the cryptorchid testis was higher in three month old testes, but equal to control in six month old mutants. The PLZF+ cells did not show a higher rate of apoptosis in cryptorchid testis. The expression of the Sertoli cell FGF2 gene required for SSC maintenance was significantly reduced in mutant testis. Based on these findings we propose that the deregulation of somatic and germ cell genes in the cryptorchid testis, directs the SSCs towards the differentiation pathway. This leads to a depletion of the SSC pool and an increase in the number of PLZF+ spermatogonial cells, which too, eventually decreases with the exhaustion of the stem cell pool. Such a dynamic suggests that an early correction of cryptorchidism is critical for the retention of the SSC pool.     

Neuregulins are essential for spermatogonial proliferation and meiotic initiation in neonatal mouse testis

The transition from mitosis to meiosis is unique to germ cells. In murine embryonic ovaries and juvenile testes, retinoic acid (RA) induces meiosis via the stimulated by retinoic acid gene 8 (Stra8), but its molecular pathway requires elucidation. We present genetic evidence in vivo and in vitro that neuregulins (NRGs) are essential for the proliferation of spermatogonia and the initiation of meiosis. Tamoxifen (TAM) was injected into 14-day post-partum (dpp) Sertoli cell-specific conditional Nrg1(Ser-/-) mutant mice. TAM induced testis degeneration, suppressed BrdU incorporation into spermatogonia and pre-leptotene primary spermatocytes, and decreased and increased the number of STRA8-positive and TUNEL-positive cells, respectively. In testicular organ cultures from 5-6 dpp wild-type mice and cultures of their re-aggregated spermatogonia and Sertoli cells, FSH, RA [all-trans-retinoic acid (ATRA), AM580, 9-cis-RA] and NRG1 promoted spermatogonial proliferation and meiotic initiation. However, TAM treatment of testicular organ cultures from the Nrg1(Ser-/-) mutants suppressed spermatogonial proliferation and meiotic initiation that was promoted by FSH or AM580. In re-aggregated cultures of purified spermatogonia, NRG1, NRG3, ATRA and 9-cis-RA promoted their proliferation and meiotic initiation, but neither AM580 nor FSH did. In addition, FSH, RAs and NRG1 promoted Nrg1 and Nrg3 mRNA expression in Sertoli cells. These results indicate that in juvenile testes RA and FSH induced meiosis indirectly through Sertoli cells when NRG1 and NRG3 were upregulated, as NRG1 amplified itself and NRG3. The amplified NRG1 and NRG3 directly induced meiosis in spermatogonia. In addition, ATRA and 9-cis-RA activated spermatogonia directly and promoted their proliferation and eventually meiotic initiation.     

Sterile Testis Complementation with Spermatogonial Lines Restores Fertility to DAZL-Deficient Rats and Maximizes Donor Germline Transmission

Despite remarkable advances in assisted reproductive capabilities ∼4% of all couples remain involuntarily infertile. In almost half of these cases, a lack of conception can in some measure be attributed to the male partner, wherein de novo Y-chromosomal deletions of sperm-specific Deleted-in-Azoospermia (DAZ) genes are particularly prevalent. In the current study, long-term cultures of rat spermatogonial stem cells were evaluated after cryo-storage for their potential to restore fertility to rats deficient in the DAZ-like (DAZL) gene. Detailed histological analysis of DAZL-deficient rat testes revealed an apparently intact spermatogonial stem cell compartment, but clear failure to produce mature haploid gametes resulting in infertility. After proliferating >1 million-fold in cell number during culture post-thaw, as few as 50,000 donor spermatogonia transplanted into only a single testis/recipient effectively restored fecundity to DAZL-deficient rats, yielding 100% germline transmission to progeny by natural mating. Based on these results, the potency and efficacy of this donor stem cell line for restoring fertility to azoospermic rodents is currently unprecedented. Prospectively, similar successes in humans could be directly linked to the feasibility of obtaining enough fully functional spermatogonial stem cells from minimal testis biopsies to be therapeutically effective. Thus, regeneration of sperm production in this sterile recipient provides an advanced pre-clinical model for optimizing the efficacy of stem cell therapies to cure a paradoxically increasing number of azoospermic men. This includes males that are rendered infertile by cancer therapies, specific types of endocrine or developmental defects, and germline-specific de novo mutations; all of whom may harbor healthy sources of their own spermatogonial stem cells for treatment.     

LY6A/E (SCA-1) expression in the mouse testis

Recently, it was found by two research groups that LY6A, known widely in the stem cell community as stem cell antigen-1 or SCA-1, is expressed on testicular side population (SP) cells. Whether these SP cells are spermatogonial stem cells is a point of disagreement and, therefore, the identity of the LY6A-positive cells as well. We studied the expression pattern of LY6A in testis by immunohistochemistry and found it to be expressed in the interstitial tissue on peritubular myoid, endothelial, and spherical-shaped peritubular mesenchymal cells. To address the question whether LY6A has a function in spermatogenesis or testis development, we studied the testis of Ly6a(-/-) mice (allele Ly6a(tm1Pmf)). We found no morphological abnormalities or differences in numbers of spermatogonia, spermatocytes, Leydig cells, or macrophages in relation to the number of Sertoli cells. Therefore, we conclude that LY6A expression does not influence testis development or spermatogenesis and that spermatogonial stem cells are LY6A negative.     

Developmental expression of the translocator protein 18 kDa (TSPO) in testicular germ cells

Translocator protein (TSPO) is a high affinity 18 kDa drug- and cholesterol-binding protein strongly expressed in steroidogenic tissues where it mediates cholesterol transport into mitochondria and steroid formation. Testosterone formation by Leydig cells in the testis is critical for the regulation of spermatogenesis and male fertility. Male germ cell development comprises two main phases, the pre-spermatogenesis phase occurring from fetal life to infancy and leading to spermatogonial stem cell (SSC) formation, and spermatogenesis, which consists of repetitive cycles of germ cell mitosis, meiosis and differentiation, starting with SSC differentiation and ending with spermiogenesis and spermatozoa formation. Little is known about the molecular mechanisms controlling the progression from one germ cell phenotype to the next. Here, we report that testicular germ cells express TSPO from neonatal to adult phases, although at lower levels than Leydig cells. TSPO mRNA and protein were found at specific steps of germ cell development. In fetal and neonatal gonocytes, the precursors of SSCs, TSPO appears to be mainly nuclear. In the prepubertal testis, TSPO is present in pachytene spermatocytes and dividing spermatogonia. In adult testes, it is found in a stage-dependent manner in pachytene spermatocyte and round spermatid nuclei, and in mitotic spermatogonia. In search of TSPO function, the TSPO drug ligand PK 11195 was added to isolated gonocytes with or without the proliferative factors PDGF and 17β-estradiol, and was found to have no effect on gonocyte proliferation. However, TSPO strong expression in dividing spermatogonia suggests that it might play a role in spermatogonial mitosis. Taken together, these results suggest that TSPO plays a role in specific phases of germ cell development.