Factors supporting long-term culture of bovine male germ cells

Abstract

Spermatogonial stem cells (SSCs) are unipotent in nature, but mouse SSCs acquire pluripotency under the appropriate culture conditions. Although culture systems are available for rodent and human germ-cell lines, no proven culture system is yet available for livestock species. Here, we examined growth factors, matrix substrates and serum-free supplements to develop a defined system for culturing primitive germ cells (gonocytes) from neonatal bovine testis. Poly-L-lysine was a suitable substrate for selective inhibition of the growth of somatic cells and made it possible to maintain a higher gonocyte:somatic cell ratio than those maintained with gelatin, collagen or Dolichos biflorus agglutinin (DBA) substrates. Among the serum-free supplements tested in our culture medium, knockout serum replacement (KSR) supported the proliferation and survival of gonocytes better than the supplements B-27 and StemPro-SFM after sequential passages of colonies. Under our optimised culture conditions consisting of 15% KSR supplement on poly-L-lysine-coated dishes, the stem-cell and germ-cell potentials of the cultured gonocytes were maintained with normal karyotype for more than 2 months (over 13 passages). The proposed culture system, which can maintain a population of proliferating bovine germ stem cells, could be useful for studying SSC biology and germline modifications in livestock animals.

p53 inhibits the proliferation of male germline stem cells from dairy goat cultured on poly‐L‐lysine

“…GDNF has a critical role in mGSCs self‐renewal, mediated by GFRa1 and C‐Ret (Meng et al, ; Naughton, Jain, Strickland, Gupta, & Milbrandt, ; Oatley et al, ), through the activation of SFK and AKT pathways (He, Jiang, Hofmann, & Dym, ; Lee et al, ; Oatley et al, ). However, whether PL regulates mGSCs self‐renewal and differentiation through any of the aforementioned pathways is yet to be determined (He et al, ; Sahare et al, ). In the dairy goat, studies on mGSCs were mainly based on cell culture and focused on description of biological characteristics (Hua et al, ).…”

“…Poly‐L‐lysine, a synthetic molecule with a highly positively charged homopolypeptide, enhances cell adhesion by imparting a positive charge to the culture surface and is often used as a coating agent in different applications (Lam & Longaker, ). In addition, PL was reported to benefit the differentiation of neural stem cells, mesenchymal stem cells, pluripotent stem cells (PSCs), spermatogonia stem cells (SSCs), as well as in tissue engineering (He, Lu, Zhang, Hao, & Yang, ; Lam & Longaker, ; Lee, Wu, Cheng, & Yang, ; Malik et al, ; Sahare et al, ; Zhang et al, ). However, the commonly used extracellular matrices in cell culture, including gelatin, laminin, Matrigel, fibronectin and poly‐lysine, have rarely been compared for their effects on mGSC cultures.…”

The effects of growth factors on proliferation of spermatogonial stem cells from Guangxi Bama mini‐pig

“…In domestic animals, EGF and FGF had positive effect on the number and size of SSC‐like colony of pig (Kuijk et al, ). Bovine mGSCs could propagate for 2 months in medium supplemented with GDNF, bFGF and EGF (Sahare et al, ). However, no growth factor had been previously reported to be able to support long‐term proliferation of SSCs of pig.…”

Recent advances of in vitro culture systems for spermatogonial stem cells in mammals

“…Defining the SSC populations by using biochemical markers that distinguish them from spermatogonia in other stages of differentiation was a great tool for the isolation of potential SSCs and the development of culture systems in rodents. In recent years, several molecular markers have been identified for SSCs in rodents (Table ) . Most of these markers are expressed in progenitor SSCs, including As spermatogonia and undifferentiated spermatogonia (Apr and Aal spermatogonia).…”

“…These systems also could be an alternative for pronuclear microinjection and somatic cell cloning . Although several attempts have been made to develop a culture system for livestock species, as shown in Table , most of these studies achieved only short‐term SSC cultures. The culture system for bovine SSCs has been demonstrated in the pre‐pubertal testis and the neonatal testis .…”