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.

Autoimmune Regulator Enhanced the Expression of Caspase-3 and Did Not Induce Massive Germ Cell Apoptosis in GC1-Spg Cells.

BACKGROUND/AIMS: AIRE is known for its involvement in autoreactive T-cell deletion in thymic epithelium. Though extrathymic expression of AIRE is well documented, the functional relevance of AIRE in non-thymus tissues is emerging. AIRE is expressed in neonatal and adult testis, and has been implicated in sporadic germ cell apoptosis in developing testis. In this study we examined whether AIRE has any role in inducing apoptosis in cultured spermatogonial cells. METHODS: We over-expressed AIRE or CARD domain of AIRE in GC1-spg cells and evaluated its impact on cell cycle using fluorescence activated cell sorting following Hoechst 33342 staining. Apoptosis was assayed using Annexin-V staining. Caspase-3 cleavage was assessed on western blots and caspase-3 expression was quantitated using realtime PCR. RESULTS: We report that C18-4 cells which are derived from Type A spermatogonia expressed AIRE, while GC1-spg which is closer to Type B spermatogonia was negative for AIRE expression. Overexpression of AIRE or CARD domain of AIRE induced Caspase-3 expression in GC1-spg cells. Silencing of AIRE in C18-4 cells inhibited Caspase-3 expression. When overexpressed, AIRE and CARD brought about a very negligible increase in germ cell death and resulted in altered cell cycle pattern with a reduction in G1 phase. This was not associated with any increase in activation of Caspase-3. CONCLUSION: We conclude that the CARD domain of AIRE enhances caspase-3 expression through possible direct DNA binding and triggers non-apoptotic downstream signaling in cultured spermatogonial cells.

Autoimmune Regulator (AIRE) Is Expressed in Spermatogenic Cells, and It Altered the Expression of Several Nucleic-Acid-Binding and Cytoskeletal Proteins in Germ Cell 1 Spermatogonial (GC1-spg) Cells.

Autoimmune regulator (AIRE) is a gene associated with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE is expressed heavily in the thymic epithelial cells and is involved in maintaining self-tolerance through regulating the expression of tissue-specific antigens. The testes are the most predominant extrathymic location where a heavy expression of AIRE is reported. Homozygous Aire-deficient male mice were infertile, possibly due to impaired spermatogenesis, deregulated germ cell apoptosis, or autoimmunity. We report that AIRE is expressed in the testes of neonatal, adolescent, and adult mice. AIRE expression was detected in glial cell derived neurotrophic factor receptor alpha (GFRα)(+) (spermatogonia), GFRα(-)/synaptonemal complex protein (SCP3)(+) (meiotic), and GFRα(-)/Phosphoglycerate kinase 2 (PGK2)(+) (postmeiotic) germ cells in mouse testes. GC1-spg, a germ-cell-derived cell line, did not express AIRE. Retinoic acid induced AIRE expression in GC1-spg cells. Ectopic expression of AIRE in GC1-spg cells using label-free LC-MS/MS identified a total of 371 proteins that were differentially expressed. 100 proteins were up-regulated, and 271 proteins were down-regulated. Data are available via ProteomeXchange with identifier PXD002511. Functional analysis of the differentially expressed proteins showed increased levels of various nucleic-acid-binding proteins and transcription factors and a decreased level of various cytoskeletal and structural proteins in the AIRE overexpressing cells as compared with the empty vector-transfected controls. The transcripts of a select set of the up-regulated proteins were also elevated. However, there was no corresponding decrease in the mRNA levels of the down-regulated set of proteins. Molecular function network analysis indicated that AIRE influenced gene expression in GC1-spg cells by acting at multiple levels, including transcription, translation, RNA processing, protein transport, protein localization, and protein degradation, thus setting the foundation in understanding the functional role of AIRE in germ cell biology.

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.