SUMMARY Spermatogenesis is a complex and dynamic cellular differentiation process critical to male reproduction and sustained by spermatogonial stem cells (SSCs). Although patterns of gene expression have been described for aggregates of certain spermato- genic cell types, the full continuum of gene expression patterns underlying ongoing spermatogenesis in steady state was previously unclear. Here, we catalog single-cell transcriptomes for >62,000 individual spermatogenic cells from immature (postnatal day 6) and adult male mice and adult men. This allowed us to resolve SSC and progenitor spermatogonia, elucidate the full range of gene expression changes during male meiosis and spermiogenesis, and derive unique gene expression signatures for multiple mouse and human spermatogenic cell types and/or subtypes. These transcriptome datasets provide an information-rich resource for studies of SSCs, male meiosis, testicular cancer, male infertility, or contraceptive development, as well as a gene expression roadmap to be emulated in efforts to achieve spermatogenesis in vitro.

BACKGROUND The genetic basis of nonobstructive azoospermia is unknown in the majority of infertile men. METHODS We performed array comparative genomic hybridization testing in blood samples obtained from 15 patients with azoospermia, and we performed mutation screening by means of direct Sanger sequencing of the testis-expressed 11 gene (TEX11) open reading frame in blood and semen samples obtained from 289 patients with azoospermia and 384 controls. RESULTS We identified a 99-kb hemizygous loss on chromosome Xq13.2 that involved three TEX11 exons. This loss, which was identical in 2 patients with azoospermia, predicts a deletion of 79 amino acids within the meiosis-specific sporulation domain SPO22. Our subsequent mutation screening showed five novel TEX11 mutations: three splicing mutations and two missense mutations. These mutations, which occurred in 7 of 289 men with azoospermia (2.4%), were absent in 384 controls with normal sperm concentrations (P = 0.003). Notably, five of those TEX11 mutations were detected in 33 patients (15%) with azoospermia who received a diagnosis of azoospermia with meiotic arrest. Meiotic arrest in these patients resembled the phenotype of Tex11-deficient male mice. Immunohistochemical analysis showed specific cytoplasmic TEX11 expression in late spermatocytes, as well as in round and elongated spermatids, in normal human testes. In contrast, testes of patients who had azoospermia with TEX11 mutations had meiotic arrest and lacked TEX11 expression. CONCLUSIONS In our study, hemizygous TEX11 mutations were a common cause of meiotic arrest and azoospermia in infertile men. (Funded by the National Institutes of Health and others.)

Not applicable.

In western countries, 60% of all malignancies diagnosed in men between 17-45 years of age are germ cell tumors (GCT). GCT arise from the common precursor lesion carcinoma in situ, which transforms within an average of 9 years into invasive Type-II GCTs. Seminomas are considered to be the default developmental pathway of carcinoma in situ cells and the seminoma-like cell line TCam-2 has been used to study seminoma biology in vitro. However, the generation of an animal model, which would allow for the in vivo analysis of seminoma formation, remained elusive. We applied transplantation approaches using TCam-2 cell transfer into ectopic (skin, brain) and orthopic (testis) sites of immunodeficient mice. We demonstrate that a transplantation into the seminiferous tubules results in formation of a carcinoma in situ/seminoma. In contrast, TCam-2 cells adopt an embryonal carcinoma-like fate when grafted to the flank or corpus striatum and display downregulation of the seminoma marker SOX17 and upregulation of the embryonal carcinoma markers SOX2 and CD30. Grafted TCam-2 cells reduce AKT-, ERK-, EphA3-, and Tie2/TEK-signaling to levels comparable to embryonal carcinoma cells. Hence, TCam-2 cell transplantation into the testis generated a carcinoma in situ/seminoma mouse model, which enables addressing the biology of these tumors in vivo. The fact that TCam-2 cells give rise to a carcinoma in situ/seminoma or embryonal carcinoma in a transplantation site specific manner implies that conversion of carcinoma in situ/seminoma to an embryonal carcinoma does not require additional genetic aberrations but relies on signals from the tumor-microenvironment.

Immature testicular tissue of a wide variety of mammalian species continues growth and maturation when ectopically grafted under the dorsal skin of adult nude mouse recipients. Tissues from most donor species fully mature, exhibiting complete spermatogenesis within months. The connection to the recipient's vascular system is mandatory for graft development, and failure of vascularization leads to necrosis in the grafted tissue. In the present study, we analyze to what extent 1) the xenografted immature donor tissue and 2) the recipient's cells and tissues contribute to the functional recovery of a "testicular xenograft." We address whether recipient cells migrate into the testicular parenchyma and whether the circulatory connection between the donor testicular tissue and the recipient is established by ingrowing host or outgrowing donor blood vessels. Although this issue has been repeatedly discussed in previous xenografting studies, so far it has not been possible to unequivocally distinguish between donor and recipient tissues and thus to identify the mechanisms by which the circulatory connection is established. To facilitate the distinction of donor and recipient tissues, herein we used immature green fluorescent protein-positive rat testes as donor tissues and adult nude mice as graft recipients. At the time of graft recovery, donor tissues could be easily identified by the GFP expression in these tissues, allowing us to distinguish donor- and recipient-derived blood vessels. We conclude that the circulatory connection between graft and host is established by a combination of outgrowing small capillaries from the donor tissue and formation of larger vessels by the host, which connect the graft to subcutaneous blood vessels.