Abstract
In the postnatal testis, undifferentiated Type-A Spermatogonial Stem Cells (SSCs) differentiate into Type-B Spermatogonial Cells (SGCs) in the presence of c-Kit, which is expressed in a cell/stage specific manner during spermatogenesis in mammals. c-Kit is subjected to tight transcriptional control as it is essential not only for germ cells but also for the development of hematopoietic stem cells and melanocytes. Although, c-Kit expression is a dynamically regulated process, the molecular basis for its transcriptional regulation during spermatogenesis remains mostly unidentified. The aim of the study is to determine whether or not introduction of c-Kit expressing transgene construct into c-Kit negative SSCs isolated from Wv/Wv mutant mice will differentiate and colonize in the testis of recipient mice. For this purpose, a 7.4 kb pG1-Kit-ORF-GFP transgene construct that contains a 4.4 kb c-Kit-ORFGFP expression cassette was designed and its in vitro expression was confirmed by immunofluorescence and flowcytometry analysis. For comparison of the results, three control-GFP constructs, having 1.1 kb (lacking both c-Kit promoter and ORF), 4.1kb (lacking c-Kit promoter) and 1.4 kb (lacking c-kit-ORF) were also designed and introduced into SSCs by electroporation (EP). These SSCs were introduced into the testis of busulfan treated recipient infertile mice via rete using Germ Cell Transplantation (GCT) approach. It was observed that SSCs carrying c-Kit-ORF-GFP transgene insert were able to colonized and differentiated into other germ cell lineages in the seminiferous tubules of recipient mice as compared to controls. In conclusion, the testis Wv/Wv mutant mice have the population of SSCs, wherein the function of c-Kit signaling is impaired for the first time we report that SSCs function in the recipient infertile mice could be resumed with the introduction of c-Kit-ORF-GFP transgene insert. Thus the present findings may have significant impact in understanding reproductive physiology and potential for novel future therapies in patients with testicular failure.
Highlights
Spermatogenesis is highly complex, well-orchestrated and efficient biological process in which Spermatogonial Stem Cells (SSCs) undergo self-renewal, proliferation and differentiation to produce virtually unlimited number of spermatozoa [1]
This has been confirmed by functional assay wherein SSCs carrying 4.4 kb c-KitORF-GFP insert was introduced into recipient infertile mice by Germ Cell Transplantation (GCT)
The results revealed that 85-90% of P815 cells and ES-E14TG2α cells showed c-Kit expression as compared to the cells transfected with 4.3 kb control-GFP construct, indicating 302 bp c-Kit promoter is able to drive specific c-Kit expression (Figure 1)
Summary
Spermatogenesis is highly complex, well-orchestrated and efficient biological process in which Spermatogonial Stem Cells (SSCs) undergo self-renewal, proliferation and differentiation to produce virtually unlimited number of spermatozoa [1] During this process, several genes (c-myc, c-Kit, Oct-4, Androgen Binding Protein (ABP), CyclicAMP Responsive Modulator (CREM), Protamine-1, Heat Shock Protein-70 (HSP-70), Phosphogylcerate Kinase-2 (PGK-2), Transition Nuclear Protein-2 (TNP-2) and Azoospermic Factor-1 (AZF-1) etc.) are known to be expressed in cell and stage specific manner in the testis [1,2]. The molecular basis of male infertility arising from defects in c-Kit remains poorly understood
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