Single-Cell Atlas of Adult Testis in Protogynous Hermaphroditic Orange-Spotted Grouper, Epinephelus coioides

Spermatogenesis is a process of self-renewal and differentiation in spermatogonial stem cells. During this process, germ cells and somatic cells interact intricately to ensure long-term fertility and accurate genome propagation. Spermatogenesis has been intensely investigated in mammals but remains poorly understood with regard to teleosts. Here, we performed single-cell RNA sequencing of ~9500 testicular cells from the male, orange-spotted grouper. In the adult testis, we divided the cells into nine clusters and defined ten cell types, as compared with human testis data, including cell populations with characteristics of male germ cells and somatic cells, each of which expressed specific marker genes. We also identified and profiled the expression patterns of four marker genes (calr, eef1a, s100a1, vasa) in both the ovary and adult testis. Our data provide a blueprint of male germ cells and supporting somatic cells. Moreover, the cell markers are candidates that could be used for further cell identification.

ZDHHC19 Is Dispensable for Spermatogenesis, but Is Essential for Sperm Functions in Mice

Spermatogenesis is a complicated process involving mitotically proliferating spermatogonial cells, meiotically dividing spermatocytes, and spermatid going through maturation into spermatozoa. The post-translational modifications of proteins play important roles in this biological process. S-palmitoylation is one type of protein modifications catalyzed by zinc finger Asp-His-His-Cys (ZDHHC)-family palmitoyl S-acyltransferases. There are 23 mammalian ZDHHCs that have been identified in mouse. Among them, Zdhhc19 is highly expressed in adult testis. However, the in vivo function of Zdhhc19 in mouse spermatogenesis and fertility remains unknown. In this study, we knocked out the Zdhhc19 gene by generating a 2609 bp deletion from exon 3 to exon 6 in mice. No differences were found in testis morphology and testis/body weight ratios upon Zdhhc19 deletion. Spermatogenesis was not disrupted in Zdhhc19 knockout mice, in which properly developed TRA98+ germ cells, SYCP3+ spermatocytes, and TNP1+ spermatids/spermatozoa were detected in seminiferous tubules. Nevertheless, Zdhhc19 knockout mice were male infertile. Zdhhc19 deficient spermatozoa exhibited multiple defects including abnormal morphology of sperm tails and heads, decreased motility, and disturbed acrosome reaction. All of these led to the inability of Zdhhc19 mutant sperm to fertilize oocytes in IVF assays. Taken together, our results support the fact that Zdhhc19 is a testis enriched gene dispensable for spermatogenesis, but is essential for sperm functions in mice.

P–017 The maintenance of testicular architecture and germ cell in adult testis tissue under organ culture condition based on the gas-liquid interface method

Study question Can the gas-liquid interface organ culture system that achieved in vitro spermatogenesis in mice also support in vitro spermatogenesis in human adult testis? Summary answer Although the progression of spermatogenesis was not observed, germ cells were maintained without the degeneration of the architecture in both fresh and cryopreserved testicular tissues. What is known already Although the research on in vitro spermatogenesis have been conducted for 100 years, only the organ culture system using gas-liquid interface method achieved in vitro spermatogenesis in mice. It has not been verified whether this culture system can be applied to other mammals including humans and induce spermatogenesis. Study design, size, duration Testicular tissue was obtained from the transgender patients receiving sex reassignment surgery. Testicular specimens were either immediately processed for cultivation or cryopreserved, using a vitrification freezing protocol. Organ culture of testicular fragments was performed in three different media for a maximum period of 3 weeks to evaluate the short-term changes in the cultured tissues (viability, proliferation and maintenance of germ and somatic cells). Participants/materials, setting, methods Fresh and cryopreserved-thawed testis fragments (1–2 mm3) were cultured using the organ culture system in alpha-MEM with knock-out serum replacement (K group), alpha-MEM with lipid-rich BSA (A group) and DMEM with FBS (D group). Luteinizing hormone, follicle stimulating hormone and testosterone were supplemented. The number of germ cells (using DDX4), proliferative activity of germ cells (using EdU assay) and intratubular cell apoptosis (by TdT-mediated dUTP Nick End Labeling) were evaluated by immunohistochemical staining weekly. Main results and the role of chance The architecture of the seminiferous tubules was maintained until the second week of culture in both the fresh and the cryopreserved culture group. The number of DDX4-positive germ cells per seminiferous tubule in groups D, K, and A was 49 ± 24, 55 ± 21, 50 ± 26 cells/tubule in 1 day, 32 ± 13, 42 ± 7, 36 ± 21 cells/tubule in 1week, respectively. The numbers gradually decreased to 26 ± 8, 24 ± 6 and 27 ± 18 cells/tubule, in 2 weeks, respectively, with no difference among the groups. The number of intratubular EdU-positive cells of groups D, K, and A was 0.2 ± 0.2, 2.8 ± 2.1, 1.1 ± 0.8 cells/tubule at 1 day, 0.1 ± 0.2, 0.5 ± 0.6, 0.3 ± 0.6 cells/tubule at 1 week, respectively. The values were 0.01, 0.05, and 0.03 at 2 weeks. Thus, EdU-positive cells drastically decreased from the first week of culture. The number of DDX4-positive germ cells and the intratubular EdU-positive cells in the cryopreserved culture group was not different from that in the fresh culture group. Limitations, reasons for caution Current organ culture systems are incomplete, being unable to induce human in vitro spermatogenesis. Further research is needed to improve culture condition with the aim of producing fertile sperm of infertile adult male patients. Wider implications of the findings: Our organ culture system could maintain testis structure and germ cells. By using the testis tissues of the transgender patients, which are available with their consent, we will promote the investigation of the culture condition necessary for germ cell proliferation and differentiation. Trial registration number Grant-in-Aid for Scientific Research on Innovative Areas 18H05546, Grant-in-Aid for Young Scientists (A) 17H05098 and Takeda Science Foundation

TCF21+ mesenchymal cells contribute to testis somatic cell development, homeostasis, and regeneration in mice

Testicular development and function rely on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity declines in aging and disease. Whether the adult testis maintains a reserve progenitor population remains uncertain. Here, we characterize a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that TCF21lin cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as potential reserve somatic progenitors following injury. In vitro, TCF21lin cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, TCF21+ cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be potentially leveraged in development of future therapies for hypoandrogenism and/or infertility.

Differentiation of Fetal Sertoli Cells in the Adult Testis

Sertoli cells proliferate and construct seminiferous tubules during fetal life, then undergo differentiation and maturation in the prepubertal testes. In the adult testes, mature Sertoli cells maintain spermatogonia and support spermatogenesis during the entire lifetime. Although Sertoli-like cells have been derived from iPS cells, they tend to remain immature. To investigate whether Sertoli cells can spontaneously acquire the ability to support spermatogenesis when transferred into the adult testis, we transplanted mouse fetal testicular cells into a Sertoli-depleted adult testis. We found that donor E12.5, E14.5 and E16.5 Sertoli cells colonized adult seminiferous tubules and supported host spermatogenesis two months after transplantation, demonstrating that immature fetal Sertoli cells can undergo sufficient maturation in the adult testis to become functional. This technique will be useful to analyze the developmental process of Sertoli cell maturation, and to investigate the potential of iPS-derived Sertoli cells to colonize, undergo maturation, and support spermatogenesis within the testis environment.

Overexpression of DAZL, STRA8, and BOULE Genes and Treatment With BMP4 or Retinoic Acid Modulate the Expression of MSC Overexpressing Germ Cell Genes

In vitro gamete derivation from stem cells has potential applications in animal reproduction as an alternative method for the dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Mesenchymal stem cells (MSCs) may be suitable candidates for in vitro gamete derivation considering their differentiative capacity and their potential for cell therapy. Due to its relevance in gametogenesis, it has been reported that retinoic acid (RA) and bone morphogenetic protein (BMP) 4 are able to upregulate the expression of specific markers associated to the early stages of germ cell (GCs) differentiation in bovine fetal MSCs (bfMSCs). In the present study, we used polycistronic vectors containing combinations of GC genes DAZL, STRA8, and BOULE followed by exposure to BMP4 or RA to induce GC differentiation of bovine fetal adipose tissue-derived MSC (AT-MSCs). Cells samples at Day 14 were analyzed according to the expression of pluripotent genes NANOG and OCT4 and GC genes DAZL, STRA8, BOULE, PIWI, c-KIT, and FRAGILIS using Q-PCR. Fetal and adult testis and AT-MSCs samples were also analyzed for the expression of DAZL, STRA8, and NANOG using immunofluorescence. Increased gene expression levels in the adult testis and cell-specific distribution of DAZL, STRA8, and NANOG in the fetal testis suggest that these markers are important components of the regulatory network that control the in vivo differentiation of bovine GCs. Overexpression of DAZL and STRA8 in bi-cistronic and DAZL, STRA8, and BOULE in tri-cistronic vectors resulted in the upregulation of OCT4, NANOG, and PIWIL2 in bovine fetal AT-MSCs. While BMP4 repressed NANOG expression, this treatment increased DAZL and c-KIT and activated FRAGILIS expression in bovine fetal AT-MSCs. Treatment with RA for 14 days increased the expression of DAZL and FRAGILIS and maintained the mRNA levels of STRA8 in bovine fetal AT-MSCs transfected with bi-cistronic and tri-cistronic vectors. Moreover, RA treatment repressed the expression of OCT4 and NANOG in these cells. Thus, overexpression of DAZL, STRA8, and BOULE induced the upregulation of the pluripotent markers and PIWIL2 in transfected bovine fetal AT-MSCs. The partial activation of GC gene expression by BMP4 and RA suggests that both factors possess common targets but induce different gene expression effects during GC differentiation in overexpressing bovine fetal AT-MSCs.

Expression of Wnt5a defines the major progenitors of fetal and adult Leydig cells

SummaryLeydig cells (LCs) are the major androgen-producing cells in the testes. They arise from steroidogenic progenitors, whose origins, maintenance and differentiation dynamics remain largely unknown. Here, we identified Wnt5a as a specific marker of steroidogenic progenitors, whose expression begins at around E11.5-E12.5 in interstitial cells of the fetal mouse testis. In vivo lineage tracing indicates that Wnt5a-expressing progenitors are initially present in large numbers in the fetal testis and then progressively decrease as development progresses. We provide evidence that Wnt5a-expressing cells are bona fide progenitors of peritubular myoid cells as well as fetal and adult LCs, contributing to most of the LCs present in the fetal and adult testis. Additionally, we show in the adult testis that Wnt5a expression is restricted to a subset of LCs exhibiting a slow but noticeable clonal expansion, revealing hitherto unappreciated proliferation of fully differentiated LCs as a contribution to the adult LC pool.

Tcf21+ mesenchymal cells contribute to testis somatic cell development, homeostasis, and regeneration

SummaryTesticular development and function relies on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity decline in aging and disease. Whether the adult testis maintains a reserve progenitor population with repair or regenerative capacity remains uncertain. Here, we characterized a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that Tcf21+ cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as reserve somatic progenitors following injury. In vitro, Tcf21+ cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, Tcf21+ cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be leveraged in development of future therapies for hypoandrogenism and/or infertility.HighlightsMultipotent Tcf21+ MPs can differentiate into somatic testis cell typesTcf21+ cells contribute to testis and ovary somatic cells during gonadal developmentTcf21+ cells replenish somatic cells of the aging testis and in response to tissue injuryTestis Tcf21 cells resemble resident fibroblast populations in multiple organs