Expression profiling of stemness markers in testicular germline stem cells from neonatal and adult Swiss albino mice during their transdifferentiation in vitro.

BACKGROUND: Spermatogonial stem cells (SSCs) were considered to be stem cells with limited potencies due to their existence in adult organisms. However, the production of spermatogonial stem cell colonies with broader differentiation capabilities in primary germ cell cultures from mice of select genetic backgrounds (C57BL6/Tg14, ddY, FVB and 129/Ola) indicated that SSCs from these strains were pluripotent. METHODS: We established primary cultures of SSCs from neonatal and adult Swiss 3T3 Albino mice. Stemness of SSC colonies were evaluated by performing real-time PCR and immunofluorescence analysis for a panel of chosen stemness markers. Differentiation potentials of SSCs were examined by attempting the generation of embryoid bodies and evaluating the expression of ectodermal, mesodermal and endodermal markers using immunofluorescence and real-time PCR analysis. RESULTS: Spermatogonial stem cells from neonatal and mature mice testes colonised in vitro and formed compact spermatogonial stem cell colonies in culture. The presence of stem cell markers ALPL, ITGA6 and CD9 indicated stemness in these colonies. The differentiation potential of these SSC colonies was demonstrated by their transformation into embryoid bodies upon withdrawal of growth factors from the culture medium. SSC colonies and embryoid bodies formed were evaluated using immunofluorescence and real-time PCR analysis. Embryoid body like structures derived from both neonatal and adult mouse testis were quite similar in terms of the expression of germ layer markers. CONCLUSION: These results strongly suggest that SSC-derived EB-like structures could be used for further differentiation into cells of interest in cell-based therapeutics.

Expression of Cnnm1 and Its Association with Stemness, Cell Cycle, and Differentiation in Spermatogenic Cells in Mouse Testis.

Cyclin M1 (CNNM1) functions as a copper storage protein in neuronal cells. We report that Cnnm1 is expressed in mouse testis and brain and has a coding sequence of 1761 bp that encodes a 586 amino acid protein with a molecular weight of 66 kDa. Cnnm1 is expressed in the testes of mice from neonatal to adult stages with relatively higher levels in neonates. CNNM1 expression appeared to be restricted to c-KIT- and OCT3/4-positive cells in the testis, indicating that they are early spermatogonial cells. Spermatogonial stem cells in primary culture expressed Cnnm1, and their differentiation into embryoid body-like clusters in vitro resulted in the loss of Cnnm1 expression. Silencing of Cnnm1 in GC1-spg cells resulted in a significant reduction in the number of cells in G1 phase with concomitant increase in the numbers of cells in both S and G2/M phases. Further, retinoic acid downregulated the expression of Cnnm1 in GC1-spg cells. We conclude that CNNM1 is associated with stemness and self-renewal, and its downregulation triggers differentiation in spermatogonial cells in mouse.

Aberrant Expression of Dynein light chain 1 (DYNLT1) is Associated with Human Male Factor Infertility.

DYNLT1 is a member of a gene family identified within the t-complex of the mouse, which has been linked with male germ cell development and function in the mouse and the fly. Though defects in the expression of this gene are associated with male sterility in both these models, there has been no study examining its association with spermatogenic defects in human males. In this study, we evaluated the levels of DYNLT1 and its expression product in the germ cells of fertile human males and males suffering from spermatogenic defects. We screened fertile (n = 14), asthenozoospermic (n = 15), oligozoospermic (n = 20) and teratozoospermic (n = 23) males using PCR and Western blot analysis. Semiquantitative PCR indicated either undetectable or significantly lower levels of expression of DYNLT1 in the germ cells from several patients from across the three infertility syndrome groups, when compared with that of fertile controls. DYNLT1 was localized on head, mid-piece, and tail segments of spermatozoa from fertile males. Spermatozoa from infertile males presented either a total absence of DYNLT1 or its absence in the tail region. Majority of the infertile individuals showed negligible levels of localization of DYNLT1 on the spermatozoa. Overexpression of DYNLT1 in GC1-spg cell line resulted in the up-regulation of several cytoskeletal proteins and molecular chaperones involved in cell cycle regulation. Defective expression of DYNLT1 was associated with male factor infertility syndromes in our study population. Proteome level changes in GC1-spg cells overexpressing DYNLT1 were suggestive of its possible function in germ cell development. We have discussed the implications of these observations in the light of the known functions of DYNLT1, which included protein trafficking, membrane vesiculation, cell cycle regulation, and stem cell differentiation.

Dramatic changes in 67 miRNAs during initiation of first wave of spermatogenesis in Mus musculus testis: global regulatory insights generated by miRNA-mRNA network analysis.

We mapped global changes in miRNA and mRNA profiles spanning the first wave of spermatogenesis using prepubertal (Postnatal Day 8 [P8]), pubertal (P16), and adolescent (P24) Mus musculus testes and identified the differential expression of 67 miRNAs and 8226 mRNAs. These two data sets were integrated into miRNA-dependent regulatory networks based on miRWalk predictions. In a network representing the P8 to P16 transition, downregulation of four miRNAs and upregulation of 19 miRNAs were linked with 81 upregulated target mRNAs and 228 downregulated target mRNAs, respectively. Furthermore, during the P16 to P24 transition, two miRNAs were downregulated, and eight miRNAs were upregulated, which linked with 64 upregulated mRNAs and 389 downregulated mRNAs, respectively. Only three of the miRNAs present in the network (miR-34b-5p, miR-34c, and miR-449a) showed a progressive increase from P8 through P16 to P24, while the remaining miRNAs in the network showed statistically significant changes in their levels either during the P8 to P16 transition or during the P16 to P24 transition. Analysis of the chromosomal location of these differentially expressed miRNAs showed that 14 out of 25 miRNAs upregulated from P8 to P16, and 18 out of 40 miRNAs upregulated from P8 to P24 were X-linked. This is suggestive of their escape from meiotic sex chromosome inactivation and postmeiotic sex chromatin. This integrated network of miRNA-level and mRNA-level changes in mouse testis during the first wave of spermatogenesis is expected to build a base for evaluating the role of miRNA-mediated gene expression regulation in maturing mammalian testis.