The role of signaling pathways on proliferation and self‐renewal of cultured bovine primitive germ cells

Sahare, Mahesh; Otomo, Ayagi; Komatsu, Kana; Minami, Naojiro; Yamada, Masao; Imai, Hiroshi

doi:10.1007/s12522-014-0189-x

Cited by 6 publications

(8 citation statements)

References 48 publications

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“…The established cell lines from immature testes had mouse ES cell‐like morphology in serum‐free medium supplemented with GDNF and bLIF, and they expressed both germ‐cell‐specific ( UCHL‐1 and DBA ) and pluripotent stem cell markers ( OCT4 , SOX2 , KLF4 ). Consistent with our results, other studies have reported that in vitro culture of gonocytes from neonatal bovine testes yields ES cell‐like colonies (Sahare et al, ) that express cell surface markers of ES cells (Li et al, ). In contrast, isolated undifferentiated spermatogonia from adult testes could be maintained in the medium only in the presence of BIO and formed colonies with botryoidally aggregated cell morphology, similar to typical mouse GS cells.…”

Section: Discussionsupporting

confidence: 92%

“…GDNF is produced by Sertoli cells and supports mouse SSC proliferation in vivo (Meng et al, ) and in vitro (Hasegawa, Namekawa, & Saga, ). In cattle, the addition of GDNF to culture medium activates the mitogen‐activated protein kinase (MAPK) signaling pathway and supports long‐term proliferation of gonocyte isolated from neonatal and immature testes (Sahare et al, ). However, when the neonatal culture system was applied to culture adult bovine spermatogonia, they could not be maintained for a long time (Fujihara et al, ).…”

Section: Introductionmentioning

confidence: 99%

“…Several growth factors including GDNF are employed to derive germ cell lines from immature bovine testes (Oatley et al, 2016;Sahare et al, 2015). GDNF is produced by Sertoli cells and supports mouse SSC proliferation in vivo (Meng et al, 2000) and in vitro (Hasegawa, Namekawa, & Saga, 2013).…”

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confidence: 99%

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Long‐term culture of undifferentiated spermatogonia isolated from immature and adult bovine testes

Suyatno

Kitamura

Ikeda

et al. 2018

Molecular Reproduction Devel

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Undifferentiated spermatogonia eventually differentiate in the testis to produce haploid sperm. Within this cell population, there is a small number of spermatogonial stem cells (SSCs). SSCs are rare cells in the testis, and their cellular characteristics are poorly understood. Establishment of undifferentiated cell line would provide an indispensable tool for studying their biological nature and spermiogenesis/spermatogenesis in vitro. However, there have been few reports on the long-term culture of undifferentiated spermatogonia in species other than rodents. Here, we report the derivation and long-term in vitro culture of undifferentiated spermatogonia cell lines from immature and adult bovine testes. Cell lines from immature testes were maintained in serum-free culture conditions in the presence of glial-cell-line-derived neurotropic factor (GDNF) and bovine leukemia inhibitory factor (bLIF). These cell lines have embryonic stem (ES)-like cell morphology, express pluripotent-stem-cell-specific and germ-cell-specific markers at the protein and mRNA levels, and contributed to the inner cell mass (ICM) of embryos in the blastocyst stage. Meanwhile, cell lines established from adult testes were maintained in low-serum media in the presence of 6-bromoindirubin-3'-oxime (BIO). These cell lines have characteristics resembling those of previously reported male mouse germ cell lines as confirmed by their botryoidally aggregated morphology, as well as the expression of germ-cell-specific markers and pluripotent stem cell markers. These findings could be useful for the development of long-term culture of undifferentiated spermatogonia, which could aid in conservation of species and improvement of livestock production through genome editing technology.

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Section: Discussionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

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Long‐term culture of undifferentiated spermatogonia isolated from immature and adult bovine testes

Suyatno

Kitamura

Ikeda

et al. 2018

Molecular Reproduction Devel

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“…These results indicate that mouse and cattle germ cells need different factors for cell growth. Similarly, our previous findings reveal that mitogenactivated protein kinase (MAPK) signalling plays a crucial role in maintaining self-renewal and colony formation of bovine gonocytes in culture, whereas cultured mouse germ cells require the crosstalk between MAPK and phosphoinositide 3-kinase (PI3K) signalling pathways for self-renewal (Sahare et al 2015). These different signalling pathways are activated under the influence of different growth factors.…”

Section: Discussionmentioning

confidence: 83%

Factors supporting long-term culture of bovine male germ cells

Sahare

Kim

Otomo

et al. 2016

Reprod. Fertil. Dev.

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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.

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“…However, no long‐term culture system for livestock species has been developed. In the authors’ laboratory, growth factors, matrix substrates for culture dishes, and serum‐free supplements have been examined in order to develop a defined system for culturing primitive GCs (gonocytes) from the neonatal bovine testis . Poly‐L‐lysine is a suitable substrate for the selective inhibition of the growth of somatic cells and makes it possible to maintain gonocytes.…”

Section: In Vitro Culture Of the Spermatogonial Stem Cellsmentioning

confidence: 99%

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

Sahare

Suyatno

Imai

2018

Reprod Medicine & Biology

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BackgroundSpermatogonial stem cells (SSCs) in the mammalian testis are unipotent stem cells for spermatozoa. They show unique cell characteristics as stem cells and germ cells after being isolated from the testis and cultured in vitro. This review introduces recent progress in the development of culture systems for the establishment of SSC lines in mammalian species, including humans.MethodsBased on the published reports, the isolation and purification of SSCs, identification and characteristics of SSCs, and culture system for mice, humans, and domestic animals have been summarized.ResultsIn mice, cell lines from SSCs are established and can be reprogrammed to show pluripotent stem cell potency that is similar to embryonic stem cells. However, it is difficult to establish cell lines for animals other than mice because of the dearth of understanding about species‐specific requirements for growth factors and mechanisms supporting the self‐renewal of cultured SSCs. Among the factors that are associated with the development of culture systems, the enrichment of SSCs that are isolated from the testis and the combination of growth factors are essential.ConclusionProviding an example of SSC culture in cattle, a rational consideration was made about how it can be possible to establish cell lines from neonatal and immature testes.

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The role of signaling pathways on proliferation and self‐renewal of cultured bovine primitive germ cells

Abstract: These results indicate activation of MAPK plays a critical role in self-renewal and survival of bovine gonocytes via cyclin D1 and CDK2.

Cited by 6 publications

References 48 publications

Long‐term culture of undifferentiated spermatogonia isolated from immature and adult bovine testes

Long‐term culture of undifferentiated spermatogonia isolated from immature and adult bovine testes

Factors supporting long-term culture of bovine male germ cells

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

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