Propagation of human spermatogonial stem cells in vitro.

CONTEXT: Young boys treated with high-dose chemotherapy are often confronted with infertility once they reach adulthood. Cryopreserving testicular tissue before chemotherapy and autotransplantation of spermatogonial stem cells at a later stage could theoretically allow for restoration of fertility. OBJECTIVE: To establish in vitro propagation of human spermatogonial stem cells from small testicular biopsies to obtain an adequate number of cells for successful transplantation. DESIGN, SETTING, AND PARTICIPANTS: Study performed from April 2007 to July 2009 using testis material donated by 6 adult men who underwent orchidectomy as part of prostate cancer treatment. Testicular cells were isolated and cultured in supplemented StemPro medium; germline stem cell clusters that arose were subcultured on human placental laminin-coated dishes in the same medium. Presence of spermatogonia was determined by reverse transcriptase polymerase chain reaction and immunofluorescence for spermatogonial markers. To test for the presence of functional spermatogonial stem cells in culture, xenotransplantation to testes of immunodeficient mice was performed, and migrated human spermatogonial stem cells after transplantation were detected by COT-1 fluorescence in situ hybridization. The number of colonized spermatogonial stem cells transplanted at early and later points during culture were counted to determine propagation. MAIN OUTCOME MEASURES: Propagation of spermatogonial stem cells over time. RESULTS: Testicular cells could be cultured and propagated up to 15 weeks. Germline stem cell clusters arose in the testicular cell cultures from all 6 men and could be subcultured and propagated up to 28 weeks. Expression of spermatogonial markers on both the RNA and protein level was maintained throughout the entire culture period. In 4 of 6 men, xenotransplantation to mice demonstrated the presence of functional spermatogonial stem cells, even after prolonged in vitro culture. Spermatogonial stem cell numbers increased 53-fold within 19 days in the testicular cell culture and increased 18,450-fold within 64 days in the germline stem cell subculture. CONCLUSION: Long-term culture and propagation of human spermatogonial stem cells in vitro is achievable.

Stra8 and its inducer, retinoic acid, regulate meiotic initiation in both spermatogenesis and oogenesis in mice.

In eukaryotes, diploid cells give rise to haploid cells via meiosis, a program of two cell divisions preceded by one round of DNA replication. Although key molecular components of the meiotic apparatus are highly conserved among eukaryotes, the mechanisms responsible for initiating the meiotic program have diverged substantially among eukaryotes. This raises a related question in animals with two distinct sexes: Within a given species, are similar or different mechanisms of meiotic initiation used in the male and female germ lines? In mammals, this question is underscored by dramatic differences in the timing of meiotic initiation in males and females. Stra8 is a vertebrate-specific, cytoplasmic factor expressed by germ cells in response to retinoic acid. We previously demonstrated that Stra8 gene function is required for meiotic initiation in mouse embryonic ovaries. Here we report that, on an inbred C57BL/6 genetic background, the same factor is also required for meiotic initiation in germ cells of juvenile mouse testes. In juvenile C57BL/6 males lacking Stra8 gene function, the early mitotic development of germ cells appears to be undisturbed. However, these cells then fail to undergo the morphological changes that define meiotic prophase, and they do not display the molecular hallmarks of meiotic chromosome cohesion, synapsis and recombination. We conclude that, in mice, Stra8 regulates meiotic initiation in both spermatogenesis and oogenesis. Taken together with previous observations, our present findings indicate that, in both the male and female germ lines, meiosis is initiated through retinoic acid induction of Stra8.

Expression of the pluripotency marker UTF1 is restricted to a subpopulation of early A spermatogonia in rat testis.

The population of early A spermatogonia includes stem cells that possess spermatogonial stem cell properties. Recent reports suggest that these cells have the ability to regain pluripotent properties. Here, we show that expression of the pluripotency marker undifferentiated embryonic cell transcription factor 1 (UTF1) is restricted to distinct germ cells within the testis. In embryonic and neonatal testes, all gonocytes were found to strongly express UTF1. During further testicular development, expression of UTF1 was restricted to a subset of A spermatogonia and with the increase in age the number of cells expressing UTF1 decreased even more. Ultimately, in the adult rat testis, only a small subset of the A spermatogonia expressed UTF1. Remarkably, even in testes of vitamin A-deficient rats, in which the early A spermatogonia (A(s), A(pr), and A(al)) are the only type of spermatogonia, only a subset of the spermatogonia expressed UTF1. In the adult rat testis, expression of UTF1 is restricted to a subpopulation of the ZBTB16 (PLZF)-positive early A spermatogonia. Furthermore, the observed distribution pattern of UTF1-expressing cells over the different stages of the cycle of the seminiferous epithelium suggests that the expression of UTF1 is restricted to those A(s), A(pr), and short chains of A(al) spermatogonia that are in the undifferentiated state and therefore maintain the ability to differentiate into A1 spermatogonia in the next round of the epithelial cycle or possibly even in other directions when they are taken out of their testicular niche.

Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary.

The sex of an individual is determined by the fate of the gonad. While the expression of Sry and Sox9 is sufficient to induce male development, we here show that female differentiation requires activation of the canonical beta-catenin signaling pathway. beta-catenin activation is controlled by Rspo1 in XX gonads and Rspo1 knockout mice show masculinized gonads. Molecular analyses demonstrate an absence of female-specific activation of Wnt4 and as a consequence XY-like vascularization and steroidogenesis. Moreover, germ cells of XX knockout embryos show changes in cellular adhesions and a failure to enter XX specific meiosis. Sex cords develop around birth, when Sox9 becomes strongly activated. Thus, a balance between Sox9 and beta-catenin activation determines the fate of the gonad, with Rspo1 acting as a crucial regulator of canonical beta-catenin signaling required for female development.

Intercellular bridges and apoptosis in clones of male germ cells.

When an As spermatogonium divides to form a pair of Apr spermatogonia the two daughter cells stay interconnected by an intercellular bridge. These cytoplasmic bridges form after every subsequent division leading to large clones of interconnected germ cells. Cohorts of spermatogonia maintain synchronous development throughout spermatogenesis, which has been attributed to the presence of these intercellular bridges. To examine whether apoptotic signals are transduced through the intercellular bridges we studied germ cell apoptosis in whole mounts of seminiferous tubules from non-irradiated and irradiated mouse testes, using whole mount seminiferous tubules and confocal microscopy. This allowed us to use TUNEL staining of apoptotic germ cells and at the same time to study these apoptotic germ cells in their topographical context. Our results show that in response to ionizing radiation single spermatogonia within a clone can undergo apoptosis without affecting their neighboring cells. Additionally, also early spermatocytes were shown to undergo apoptosis individually. Both radiation-induced spermatogonial apoptosis and spontaneous apoptosis of spermatocytes are caused by DNA damage of individual cells. Degeneration of healthy spermatogonia because of regulatory signals, however, follows other death inducing mechanisms, which lead to apoptosis of chains of interconnected spermatogonia.

Function of DNA-protein kinase catalytic subunit during the early meiotic prophase without Ku70 and Ku86.

All components of the double-stranded DNA break (DSB) repair complex DNA-dependent protein kinase (DNA-PK), including Ku70, Ku86, and DNA-PK catalytic subunit (DNA-PKcs), were found in the radiosensitive spermatogonia. Although p53 induction was unaffected, spermatogonial apoptosis occurred faster in the irradiated DNA-PKcs-deficient scid testis. This finding suggests that spermatogonial DNA-PK functions in DNA damage repair rather than p53 induction. Despite the fact that early spermatocytes lack the Ku proteins, spontaneous apoptosis of these cells occurred in the scid testis. The majority of these apoptotic spermatocytes were found at stage IV of the cycle of the seminiferous epithelium where a meiotic checkpoint has been suggested to exist. Meiotic synapsis and recombination during the early meiotic prophase induce DSBs, which are apparently less accurately repaired in scid spermatocytes that then fail to pass the meiotic checkpoint. The role for DNA-PKcs during the meiotic prophase differs from that in mitotic cells; it is not influenced by ionizing radiation and is independent of the Ku heterodimer.

DNA double-strand breaks and gamma-H2AX signaling in the testis.

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated at serine 139 and forms gamma-H2AX foci at the sites of damage. These foci then play a role in recruiting DNA repair and damage-response factors and changing chromatin structure to accurately repair the damaged DNA. These gamma-H2AX foci appear in response to irradiation and genotoxic stress and during V(D)J recombination and meiotic recombination. Independent of irradiation, gamma-H2AX occurs in all intermediate and B spermatogonia and in preleptotene to zygotene spermatocytes. Type A spermatogonia and round spermatids do not exhibit gamma-H2AX foci but show homogeneous nuclear gamma-H2AX staining, whereas in pachytene spermatocytes gamma-H2AX is only present in the sex vesicle. In response to ionizing radiation, gamma-H2AX foci are generated in spermatogonia, spermatocytes, and round spermatids. In irradiated spermatogonia, gamma-H2AX interacts with p53, which induces spermatogonial apoptosis. These events are independent of the DNA-dependent protein kinase (DNA-PK). Irradiation-independent nuclear gamma-H2AX staining in leptotene spermatocytes demonstrates a function for gamma-H2AX during meiosis. gamma-H2AX staining in intermediate and B spermatogonia, preleptotene spermatocytes, and sex vesicles and round spermatids, however, indicates that the function of H2AX phosphorylation during spermatogenesis is not restricted to the formation of gamma-H2AX foci at DNA double-strand breaks.

Establishment of cell lines with rat spermatogonial stem cell characteristics.

Spermatogonial cell lines were established by transfecting a mixed population of purified rat A(s) (stem cells), A(pr) and A(al) spermatogonia with SV40 large T antigen. Two cell lines were characterized and found to express Hsp90alpha and oct-4, specific markers for germ cells and A spermatogonia, respectively. Expression of c-kit, normally expressed in A spermatogonia from late A(al) spermatogonia onwards, could not be detected in either cell line. Furthermore, no expression of vimentin (Sertoli cell marker) and alpha-smooth muscle actin (peritubular cell marker) could be found. Upon transplantation of these cell lines into recipient mice, the cells were found to be able to migrate to the basement membrane and to colonize seminiferous tubules. Taken together, we conclude that our cell lines have spermatogonial stem cell characteristics. These first spermatogonial cell lines with stem cell characteristics can now be used to study spermatogonial gene expression in comparison with more advanced germ cells.