Establishment of in vitro culture system of pig spermatogonial stem cells

doi:10.3969/j.issn.1004-1524.2021.12.05

Acta Agriculturae Zhejiangensis ›› 2021, Vol. 33 ›› Issue (12): 2254-2263.DOI: 10.3969/j.issn.1004-1524.2021.12.05

• Animal Science • Previous Articles Next Articles

Establishment of in vitro culture system of pig spermatogonial stem cells

ZHANG Mao¹(), ZHAO Xin², CAI Gengyuan², YANG Huaqiang²^,^*()

1. Henry Fok College of Biology and Agriculture, Shaoguan University, Shaoguan 512005, China
2. National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China

Received:2020-09-01 Online:2021-12-25 Published:2022-01-10
Contact: YANG Huaqiang

Abstract

Abstract:

Spermatogonial stem cells (SSCs) are the guarantee of spermatogenesis and fertility in male mammals. In vitro culture of spermatogonial stem cells not only provides materials for the study of spermatogenesis, but also helps to develop new methods of livestock conservation and animal genetic modification. In order to find out the better culture system of porcine spermatogonial stem cells in vitro, two step enzymatic method of collagenase Ⅳ-trypsin was used to digest the testis of 3-7 day Yorkshire piglets to obtain single cell suspension. And the spermatogonial stem cells were purified using differential adherent with different time programs. In addition, different cytokines were added to study the best culture system for the proliferation of spermatogonial stem cells. The results showed that the highest percentage of UCHL-1 positive germ cells was 18.59%±0.94%, and the result of cytokine addition showed that spermatogonial stem cells added with 20 ng·mL ^-1 GDNF, 10 ng·mL^-1 IGF and 20 ng·mL^-1 bFGF had the best effect of proliferation. Spermatogonial stem cells were cultured in DMEM/F12 with 1% FBS and the cytokines described above, taking Sertoli cells as feeder laye. After 15 days, a large number of spermatogonial stem cell colonies were observed. The proliferation of spermatogonial stem cells was confirmed by immunofluorescence, AKP staining and fluorescent quantitative PCR detcetion. In brief, we initially established the culture system of porcine spermatogonial stem cells in vitro, through which a large number of spermatogonial stem cells can be cultured in vitro, laying the foundation for subsequent research on spermatogonial stem cells.

Key words: pig, spermatogonial stem cells, cytokines, in vitro culture

CLC Number:

S828

ZHANG Mao, ZHAO Xin, CAI Gengyuan, YANG Huaqiang. Establishment of in vitro culture system of pig spermatogonial stem cells[J]. Acta Agriculturae Zhejiangensis, 2021, 33(12): 2254-2263.

Figures/Tables 5

Table 1 Primers design

基因 Gene	引物 Primer (5'→3')	产物 Product/bp	Tm/ ℃
GAPDH	F:AGGGCTGCTTTTAACTCTGGCAA	180	56
	R:GATGGTGATGGCCTTTCCATTG
UCHL-1	F:TCCGGAAGACAGAGCAAAATGC	150	56
	R:AGTTCATAGAGGTGGCCATCCA
Thy-1	F:GTGCTCTTGGGCACTGTGGG	178	57
	R:TCTTGCTGGAGATGCTGGGC
Gfra-1	F:GAACGGAGGCGGCAGACCAT	242	57
	R:AAGCCCAGAGTAGGCGAGGAG
C-kit	F:GATGCCTTCAAGGATTTGGA	181	53
	R:ATGGAATCTGAGGCCTTCCT
Oct4	F:CGCGAAGCTGGACAAGGAGA	151	56
	R:CAAAGTGAGCCCCACATCGG
Nanog	F:AACCAAACCTGGAACAGCCAGAC	152	56
	R:GTTTCCAAGACGGCCTCCAAAT
Dazl	F:ACAGTGGCCTGCTGGGGAAC	153	56
	R:TGTGGGCCATTTCCAGAGGA

Fig. 1 Detection of purified SSCs A, Immunofluorescence detection (200×), a1-a3, Immunofluorescence after purification by method A; b1-b3, Immunofluorescence after purification by method B; c1-c3, Immunofluorescence after purification by method C. B, AKP staining, the red arrow indicates AKP staining positive cells (400×). C, Quantitative PCR analysis, ** represented the difference was significant at P<0.01.

Fig. 2 Effect of different concentrations of mitomycin C on Sertoli cells (up) and effect of different cytokine combinations on proliferation of spermatogonial stem cells (down) Bars marked without the same letters indicated that the difference was significant at P<0.05.

Fig. 3 In vitro culture of SSCs A, Inoculated spermatogonial stem cells (200×); B, Spermatogonial stem cells cultured for 6 days(200×); C, Spermatogonial stem cells cultured for 15 days(100×); D,Spermatogonial stem cell colonies cultured for 15 days(200×).

Fig. 4 Detection of proliferating SSCs A, Immunofluorescence of UCHL-1 (200×), a1, white light, a2, UCHL-1 antibody staining. B, AKP staining (200×). C, Quantitative PCR detection, ** meaned the difference is extremely significant. D, RT-PCR detection, M, DL2000; lanes 1-7 are GAPDH, UCHL-1, Gfra-1, C-kit, Thy-1, Oct-4, Nanog genes, respectively.

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Establishment of in vitro culture system of pig spermatogonial stem cells

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