Characterization of germ cells from pre-pubertal bull calves in preparation for germ cell transplantation

Abstract

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.

Enrichment of CD9<sup>+</sup> spermatogonial stem cells from goat (<i>Capra aegagrus hircus</i>) testis using magnetic microbeads

“…The isolation of this small spermatogonial population is technically challenging because of their small number, so magnetic beads are specifically useful for their isolation including stem cells from various tissues [23][24][25] and organ such as bone marrow, muscle and liver [26][27][28]. Many molecular markers have been used to identify and study undifferentiated spermatogonia and gonocytes such as promyelocytic leukemia zinc finger protein (PLZF) in rodents, non human primates and pigs [29][30][31][32], ubiquitin carboxyl esterase L1 (UCHL1) in the bull [33] and boar [32,34,35], VASA in many species including bulls [36], boars [37], primates [31] & mice [38], CD9 in mouse and rat [5]. The sorting efficiency of intact cells mainly rely on the availability of specific or particular surface markers on the membrane of stem cells.…”

Characterization and In Vitro Culture of Male Germ Cells from Developing Bovine Testis

“…Gonocytes and type A spermatogonia were identified using lectin DBA and rabbit anti-UCHL1 (PGP9.5; 1:1000 dilution; Biomol, Exeter, UK) [25][26][27]30]. Testis sections were also stained with an antibody to another germ cell marker, rabbit anti-VASA (DDX4/MVH; 1:200 dilution; Abcam, Cambridge, UK), and antibodies to two stem cell markers, rabbit anti-NANOG (1:100 dilution; PeproTech, Rocky Hill, NJ, USA) and rabbit anti-OCT3/4 (1:100 dilution; Millipore, Billerica, MA, USA).…”

THY1 is a conserved marker of undifferentiated spermatogonia in the pre-pubertal bull testis

“…Promyelocytic leukemia zinc finger protein (PLZF) is expressed exclusively by the undifferentiated spermatogonial population in rodents, nonhuman primates, and pigs (Buaas et al 2004, Costoya et al 2004, Hermann et al 2007, Luo et al 2009). Ubiquitin carboxyl-terminal esterase L1 (UCHL1) is a general marker of spermatogonia in the bull (Herrid et al 2007) and boar (Frankenhuis et al 1982, Luo et al 2006. Additionally, expression of VASA (DDX4) has been localized to several sub-types of spermatogonia in many species including bulls (Bartholomew & Parks 2007), boars (Lee et al 2005), primates (Hermann et al 2007), and mice (Toyooka et al 2000).…”