Zijue Zhu

Dynamics of the Transcriptome during Human Spermatogenesis: Predicting the Potential Key Genes Regulating Male Gametes Generation.

Many infertile men are the victims of spermatogenesis disorder. However, conventional clinical test could not provide efficient information on the causes of spermatogenesis disorder and guide the doctor how to treat it. More effective diagnosis and treating methods could be developed if the key genes that regulate spermatogenesis were determined. Many works have been done on animal models, while there are few works on human beings due to the limited sample resources. In current work, testis tissues were obtained from 27 patients with obstructive azoospermia via surgery. The combination of Fluorescence Activated Cell Sorting and Magnetic Activated Cell Sorting was chosen as the efficient method to sort typical germ cells during spermatogenesis. RNA Sequencing was carried out to screen the change of transcriptomic profile of the germ cells during spermatogenesis. Differential expressed genes were clustered according to their expression patterns. Gene Ontology annotation, pathway analysis, and Gene Set Enrichment Analysis were carried out on genes with specific expression patterns and the potential key genes such as HOXs, JUN, SP1, and TCF3 which were involved in the regulation of spermatogenesis, with the potential value serve as molecular tools for clinical purpose, were predicted.

Establishment and Characterization of Human Germline Stem Cell Line with Unlimited Proliferation Potentials and no Tumor Formation.

Spermatogonial stem cells (SSCs) have significant applications in both reproductive and regenerative medicine. However, primary human SSCs are very rare, and a human SSC line has not yet been available. In this study, we have for the first time reported a stable human SSC line by stably expressing human SV40 large T antigen. RT-PCR, immunocytochemistry, and Western blots revealed that this cell line was positive for a number of human spermatogonial and SSC hallmarks, including VASA, DAZL, MAGEA4, GFRA1, RET, UCHL1, GPR125, PLZF and THY1, suggesting that these cells are human SSCs phenotypically. Proliferation analysis showed that the cell line could be expanded with significant increases of cells for 1.5 years, and high levels of PCNA, UCHL1 and SV40 were maintained for long-term culture. Transplantation assay indicated that human SSC line was able to colonize and proliferate in vivo in the recipient mice. Neither Y chromosome microdeletions of numerous genes nor tumor formation was observed in human SSC line although there was abnormal karyotype in this cell line. Collectively, we have established a human SSC line with unlimited proliferation potentials and no tumorgenesis, which could provide an abundant source of human SSCs for their mechanistic studies and translational medicine.

VEGF/VEGFR2 Signaling Regulates Germ Cell Proliferation in vitro and Promotes Mouse Testicular Regeneration in vivo

Vascular endothelial growth factor (VEGF) plays fundamental roles in testicular development; however, its function on testicular regeneration remains unknown. The objective of this study was to explore the roles VEGF/VEGFR2 signaling plays in mouse germ cells and in mouse testicular regeneration. VEGF and the VEGFR2 antagonist SU5416 were added to culture medium to evaluate their effects on spermatogonial stem cell line (C18-4 cells) proliferation. Testicular cells obtained from newborn male ICR mice were grafted into the dorsal region of male BALB/c nude mice. VEGF and SU5416 were injected into the graft sites to assess the effects of the VEGF and VEGFR2 signaling pathways on testicular reconstitution. The grafts were analyzed after 8 weeks. We found that VEGF promoted C18-4 proliferation in vitro, indicating its role in germ cell survival. HE staining revealed that seminiferous tubules were reconstituted and male germ cells from spermatogonia to spermatids could be observed in testis-like tissues 8 weeks after grafting. A few advantaged male germ cells, including spermatocytes and spermatids, were found in SU5416-treated grafts. Moreover, VEGF enhanced the expression of genes specific for male germ cells and vascularization in 8-week grafts, whereas SU5416 decreased the expression of these genes. SU5416-treated grafts had a lower expression of MVH and CD31, indicating that blockade of VEGF/VEGFR2 signaling reduces the efficiency of seminiferous tubule reconstitution. Collectively, these data suggest that VEGF/VEGFR2 signaling regulates germ cell proliferation and promotes testicular regeneration via direct action on germ cells and the enhancement of vascularization.

Decreased Expression of KIFC1 in Human Testes with Globozoospermic Defects

Globozoospermia is a rare (prevalence of <0.1%) but severe male infertility condition. In our previous study, we found that robust KIFC1 immunostaining was detected in the human elongating/elongated spermatids during human acrosomogenesis. However, the relationship between the decreased expression of KIFC1 and human globozoospermia remains largely unknown. Testicular biopsies of 30 globozoospermia and 30 obstructive azoospermia patients who underwent infertility evaluation and treatment were utilized in this study. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blots, immunohistochemistry, an in vivo model, and intratesticular injection of small inhibitory RNA (siRNA) against the Kifc1 gene were employed, and sperm abnormalities were evaluated by hematoxylin and eosin (H&E) staining and immunocytochemistry. We revealed that the testicular level of KIFC1 mRNA in globozoospermia was significantly reduced compared with that in obstructive azoospermia, and the KIFC1 protein was barely detectable in testicular specimens in 30% (9 of 30) of patients with globozoospermia. Furthermore, knockdown of the Kifc1 gene in mice increased the percentage of sperm with globozoospermic defects (26.5%). Decreased KIFC1 expression was mainly observed in the testes of patients with globozoospermia at the spermatid stage, which may be useful for counseling and management of such patients.

VEGFC/VEGFR3 Signaling Regulates Mouse Spermatogonial Cell Proliferation via the Activation of AKT/MAPK and Cyclin D1 Pathway and Mediates the Apoptosis by affecting Caspase 3/9 and Bcl-2

ABSTRACT We have previously shown that the transcript levels of Vegfc and its receptor Vegfr3 were high in spermatogonia and extremely low in spermatocytes and spermatids. However, it remains unknown about the functions and the mechanisms of VEGFC/VEGFR3 signaling in regulating the fate determinations of spermatogonia. To this end, here we explored the role and signaling pathways of VEGFC/VEGFR3 by using a cell line derived from immortalized mouse spermatogonia retaining markers of mitotic germ cells, namely GC-1 cells. VEGFR3 was expressed in mouse primary spermatogonia and GC-1 cells. VEGFC stimulated the proliferation and DNA synthesis of GC-1 cells and enhanced the phosphorylation of PI3K-AKT and MAPK, whereas LY294002 (an inhibitor for AKT) and CI-1040 (an inhibitor for MAPK) blocked the effect of VEGFC on GC-1 cell proliferation. Furthermore, VEGFC increased the transcripts of c-fos and Egr1 and protein levels of cyclin D1, PCNA and Bcl-2. Conversely, the blocking of VEGFC/VEGFR3 signaling by VEGFR3 knockdown reduced the phosphorylation of AKT/MAPK and decreased the levels of cyclin D1 and PCNA. Additionally, VEGFR3 knockdown not only resulted in more apoptosis of GC-1 cells but also led to a decrease of Bcl-2 and promoted the cleavage of Caspase-3/9 and PARP. Collectively, these data suggested that VEGFC/VEGFR3 signaling promotes the proliferation of GC-1 cells via the AKT /MAPK and cyclin D1 pathway and it inhibits the cell apoptosis through Caspase-3/9, PARP and Bcl-2. Thus, this study sheds a novel insight to the molecular mechanisms underlying the fate decisions of mammalian spermatogonia.

BMP4 promotes mouse iPS cell differentiation to male germ cells via Smad1/5, Gata4, Id1 and Id2.

Generation of male germ cells from pluripotent cells could provide male gametes for treating male infertility and offer an ideal model for unveiling molecular mechanisms of spermatogenesis. However, the influence and exact molecular mechanisms, especially downstream effectors of BMP4 signaling pathways, in male germ cell differentiation of the induce pluripotent stem (iPS) cells, remain unknown. This study was designed to explore the role and mechanism of BMP4 signaling in the differentiation of mouse iPS cells to male germ cells. Embryoid body (EB) formation and recombinant BMP4 or Noggin were utilized to evaluate the effect of BMP4 on male germ cell generation from mouse iPS cells. Germ cell-specific genes and proteins as well as the downstream effectors of BMP4 signaling pathway were assessed using real-time PCR and Western blots. We found that BMP4 ligand and its multiple receptors, including BMPR1a, BMPR1b and BMPR2, were expressed in mouse iPS cells. Real-time PCR and Western blots revealed that BMP4 could upregulate the levels of genes and proteins for germ cell markers in iPS cells-derived EBs, whereas Noggin decreased their expression in these cells. Moreover, Smad1/5 phosphorylation, Gata4 transcription and the transcripts of Id1 and Id2 were enhanced by BMP4 but decreased when exposed to Noggin. Collectively, these results suggest that BMP4 promotes the generation of male germ cells from iPS cells via Smad1/5 pathway and the activation of Gata4, Id1 and Id2 This study thus offers novel insights into molecular mechanisms underlying male germ cell development.

Transcriptome research on spermatogenic molecular drive in mammals

It is known that spermatogenic disorders are associated with genetic deficiency, although the primary mechanism is still unclear. It is difficult to demonstrate the molecular events occurring in testis, which contains germ cells at different developmental stages. However, transcriptomic methods can help us reveal the molecular drive of male gamete generation. Many transcriptomic studies have been performed on rodents by utilizing the timing of the first wave of spermatogenesis, which is not a suitable strategy for research in fertile men. With the development of separation methods for male germ cells, transcriptome research on the molecular drive of spermatogenesis in fertile men has seen great progress, and the results could be ultimately applied to improve the diagnosis and treatment for male infertility.

AB199. Human germ cell secreting factor Nodal regulates Sertoli cell functions

Objectives To explore the regulatory effects of germ cells and germ cells secreting factor Nodal on the function of Sertoli cells derived from obstructive azoospermia and non-obstructive azoospermia patients. Design Comparative and controlled study. Materials and methods Human Sertoli cells and germ cells were isolated using two-steps enzymatic digestions from the testes of obstructive azoospermia and non-obstructive azoospermia patients respectively. Expressions of Nodal signaling components in human Sertoli cells and germ cells were identified by PCR and immunochemistry. Human germ cells and Sertoli cells were cocultured in vitro to evaluate their effects on Sertoli cells. Human recombinant nodal and its receptor inhibitor SB431542 were added in the Sertoli cells culture medium to study their effects on Sertoli cell functions. CCK8 measurement was used to evaluate the proliferative activity. Q-PCR and western blot were applied to assess the expression of functional Sertoli cell genes. Results Human germ cells down-regulated blood-testis-barrier associated genes (CLDN11, OCLN) expressions of Sertoli cells in co-culture system. Nodal was expressed in germ cells but not in Sertoli cells, whereas its receptors ALK4, ALK7, and ActR-IIB were detected on Sertoli cells, which indicated Nodal signaling pathway, may play roles in the regulation of germ cells to Sertoli cells. Human recombinant nodal could promote the proliferation of human Sertoli cells, while the proliferative activity was inhibited by SB431542. Nodal could enhance the expressions of functional Sertoli cell genes (GDNF, SCF, BMP4, and ABP), while SB431542 decreased their expressions. In contrast, Nodal decreased the expression of blood-testis-barrier associated genes (CLDN11, OCLN), while SB431542 increase their expressions. Conclusions Human Sertoli cell functions could be regulated by germ cells via paracrine pathway. Human germ cells secrete Nodal which could regulate Sertoli cell functions.

Effect of Kallikrein-related Peptidase KLK1 on Ameliorating Spermatogenesis Regeneration in Busulfan-induced Azoospermic Mice and Promoting Mouse Spermatogonial Stem Cell Proliferation In Vitro

OBJECTIVES To investigate the effect of kallikrein-related peptidase KLK1 on azoospermic mice induced by busulfan and mouse spermatogonial stem cell. METHODS Mice were treated with a single intraperitoneal injection of busulfan, and 4 weeks later, they received a daily intraperitoneal injection of KLK1 at different doses for another 4 weeks. Eight weeks after the busulfan treatment, all mice were sacrificed and their testes were collected for histological evaluation, immunostaining and protein extraction. In vitro, immortalized mouse spermatogonial stem cells, namely C18-4 cells, were treated with KLK1 for proliferation assays. RESULTS Histological evaluation of testes, epididymis and epididymal fluid showed that KLK1-treated mice had better spermatogenesis than the control group. Immunostaining showed that tissue samples from testes of KLK1-treated mice had more PLZF- and SCP3-positive cells per seminiferous tubule as well as more PNA-positive cells in the seminiferous tubules. Western blots revealed higher expression levels of PCNA in KLK1-treated mice than in control mice. C18-4 cells treated with KLK1 had a higher proliferation rate and higher expression levels of PCNA, Cyclin A and Cyclin E, and the level of phosphorylated ERK2 were increased after KLK1 treatment. CONCLUSION Collectively, KLK1 can improve spermatogenesis in azoospermic mice, and KLK1 can promote the proliferation of mouse spermatogonial stem cells via activating ERK1/2 and cell cycle proteins Cyclin A and Cyclin E. This study could offer novel approach and provide new targets for the treatment of azoospermia.

In Vitro Modeling of Human Germ Cell Development Using Pluripotent Stem Cells

Summary Due to differences across species, the mechanisms of cell fate decisions determined in mice cannot be readily extrapolated to humans. In this study, we developed a feeder- and xeno-free culture protocol that efficiently induced human pluripotent stem cells (iPSCs) into PLZF+/GPR125+/CD90+ spermatogonium-like cells (SLCs). These SLCs were enriched with key genes in germ cell development such as MVH, DAZL, GFRα1, NANOS3, and DMRT1. In addition, a small fraction of SLCs went through meiosis in vitro to develop into haploid cells. We further demonstrated that this chemically defined induction protocol faithfully recapitulated the features of compromised germ cell development of PSCs with NANOS3 deficiency or iPSC lines established from patients with non-obstructive azoospermia. Taken together, we established a powerful experimental platform to investigate human germ cell development and pathology related to male infertility.