Identification of conditions supporting in vitro maintenance of bovine undifferentiated spermatogonia
Open Access
- Author:
- Crouse, Rebecca Ann
- Graduate Program:
- Animal Science
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- May 21, 2012
- Committee Members:
- Dr Jon Oatley, Thesis Advisor/Co-Advisor
- Keywords:
- Spermatogonial stem cells
undifferentiated spermatogonia
bovine
culture
testis - Abstract:
- Proliferation and differentiation of the undifferentiated spermatogonial population provides the foundation for spermatogenesis, and spermatogonial stem cells (SSCs) represent a sub-population capable of infinite self-renewal. Long-term culture of SSCs in conditions supporting self-renewal and subsequent transplantation into testes of recipient males for reestablishment of spermatogenesis provides a means to preserve and expand male germlines. Moreover, these capabilities provide a platform for direct genetic modification of the male germline to produce transgenic animals. For cattle, these capabilities could provide a novel avenue for enhancing production efficiency and generation of genetically modified food animals. Studies with rodents revealed that culture of undifferentiated spermatogonia in serum-free media conditions promotes formation of germ cell clumps that contain SSCs. In addition, supplementation with the growth factor glial cell line-derived neurotrophic factor (GDNF) is essential for long-term expansion of germ cell clumps including self-renewal of SSCs, and fibroblast growth factor 2 (FGF2) enhanced theses effects. To date, conditions supporting formation and expansion of bovine undifferentiated spermatogonial clumps that are similar in morphology to those in rodent cultures have not been reported. In this study, we examined several conditions that support rodent undifferentiated spermatogonia in vitro and identified conditions which supported formation of undifferentiated spermatogonial clumps from pre-pubertal bull testes that are identical in morphology to those in rodent cultures. These conditions included mitotically inactivated bovine embryonic fibroblast (BEF) feeder cells in DMEM/F12 medium containing StemPro supplement and addition of recombinant human forms of GDNF and FGF2. We determined these cells express the transcription factor Plzf, confirming an undifferentiated spermatogonial phenotype. Upon long-term culture we found that the number of clumps declines in these conditions beginning with the first week and the cells could not be maintained for greater than 3 weeks indicating lack of key factors that promote their survival and proliferation. Thus, we conducted microarray analyses of isolated bovine undifferentiated spermatogonia to identify enriched expression of growth factor receptors whose corresponding ligands may enhance their maintenance in vitro. From these analyses we determined that expression of colony stimulating factor 1 receptor (Csf-1r) and chemokine (c-x-c motif) receptor 4 (Cxcr4) are highly enriched in the bovine undifferentiated spermatogonial fraction. Addition of recombinant human CSF-1, along with GDNF and FGF2, significantly enhanced the maintenance of undifferentiated spermatogonia during the first week of culture by greater than 2-fold compared to cultures receiving GDNF and FGF2 only. Supplementation of recombinant human SDF-1 in addition to the GDNF, FGF2, and CSF-1 increased the number of undifferentiated spermatogonia that were maintained by 1.75-fold compared with CSF-1, GDNF, and FGF2 alone. These results implicate CSF-1 and SDF-1 as additional extrinsic factors that promote survival and proliferation of bovine undifferentiated spermatogonia. Furthermore, a Sertoli-enriched feeder cell monolayer was tested and found to improve support of undifferentiated spermatogonia significantly when compared to the BEF feeders. Collectively, these studies have identified conditions that enhance the short-term maintenance of bovine undifferentiated spermatogonia in vitro and serve as an important step towards discovering conditions which promote long-term expansion of the cells including self-renewal of SSCs.