Characterization of germ cells from pre-pubertal bull calves in preparation for germ cell transplantation.
Although methods to assess testis cell populations are established in mice, the detailed validation of similar methods for bovine testis cells is necessary for the development of emerging technologies such as male germ cell transplantation. As young calves provide donor cells for germ cell transplantation, we characterized cell populations from three key pre-pubertal stages. Nine Angus bull calves were selected to represent three stages of testis development at ages (and testis weights) of 2-3 months (Stage 1, 10 g), 4-5 months (Stage 2, 35 g), and 6-7 months (Stage 3, 70 g). The proportion and absolute numbers of germ and somatic cells in fixed sections and from enzymatically dissociated seminiferous tubules were assessed. Germ cells were identified by DBA and PGP9.5 staining, and Sertoli cells by vimentin and GATA-4 staining. Staining of serial sections confirmed that DBA and PGP9.5 identified similar cells, which were complementary to those stained for vimentin and GATA-4. In fixed tubules, the proportion of cells within tubules that were positive for DBA and PGP9.5 increased nearly three-fold from Stage 1 to Stage 2 with no further increase at Stage 3. Absolute numbers of spermatogonia also increased between Stages 1 and 2. After enzymatic dissociation of tubules, three times more DBA- and PGP9.5-positive cells were isolated from Stage 3 testes than from either Stage 1 or 2 testes. A higher proportion of spermatogonia was observed after enzymatic isolation than were present in seminiferous tubules. These data should help to predict the yield and expected proportions of spermatogonia from three distinct stages of testis development in pre-pubertal bull calves.
Herrid
Muren
M
CSIRO Food Futures National Research Flagship, CSIRO Livestock Industries, Armidale, NSW, 2350, Australia.
Davey
Rhonda J
RJ
Hill
Jonathan R
JR
D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D002417:Cattle; D002454:Cell Differentiation / Q000502:physiology; D002469:Cell Separation / Q000379:methods; D005306:Fertilization / Q000502:physiology; D050991:GATA4 Transcription Factor / Q000378:metabolism; D005854:Germ Cells / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D007315:Insemination, Artificial / Q000379:methods*; D008297:Male; D012671:Seminiferous Tubules / Q000166:cytology / Q000378:metabolism; D012708:Sertoli Cells / Q000166:cytology / Q000378:metabolism; D012741:Sexual Maturation / Q000502:physiology; D013091:Spermatogenesis / Q000502:physiology; D013093:Spermatogonia / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D013737:Testis / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism; D046509:Tissue Culture Techniques; D016378:Tissue Transplantation / Q000379:methods*; D043222:Ubiquitin Thiolesterase / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
D015415:Biomarkers; D050991:GATA4 Transcription Factor; C482860:UCHL1 protein, human; D014746:Vimentin; D043222:Ubiquitin Thiolesterase