Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Aug;81(2):258-66.
doi: 10.1095/biolreprod.108.075200. Epub 2009 Apr 15.

Loss of Etv5 decreases proliferation and RET levels in neonatal mouse testicular germ cells and causes an abnormal first wave of spermatogenesis

Gaurav Tyagi  1 , Kay Carnes, Carla Morrow, Natalia V Kostereva, Gail C Ekman, Daryl D Meling, Chris Hostetler, Michael Griswold, Kenneth M Murphy, Rex A Hess, Marie-Claude Hofmann, Paul S Cooke
Affiliations

Loss of Etv5 decreases proliferation and RET levels in neonatal mouse testicular germ cells and causes an abnormal first wave of spermatogenesis

Gaurav Tyagi et al. Biol Reprod. 2009 Aug.

Abstract

Mice that are ets variant gene 5 (ETV5) null (Etv5(-/-)) undergo the first wave of spermatogenesis but lose all spermatogonial stem cells (SSCs) during this time. The SSC loss in Etv5(-/-) mice begins during the neonatal period, suggesting a role for ETV5 in SSC self-renewal during this period. Herein, we show that Etv5 mRNA was present in perinatal mouse testis and that ETV5 was expressed in fetal Sertoli cells and by germ cells and Sertoli cells during the neonatal period. Transplantation of Etv5(-/-) germ cells failed to establish spermatogenesis in W/W(v) mice testes, indicating that germ cell ETV5 has a key role in establishment or self-renewal of transplanted SSCs. The SSC self-renewal is stimulated by glial cell-derived neurotrophic factor (GDNF) acting through the RET/GDNF family receptor alpha 1 (GFRA1) receptor complex in SSCs. Immunohistochemistry, quantitative PCR, and laser capture microdissection revealed decreased RET mRNA and protein expression in spermatogonia of neonatal Etv5(-/-) mice by Postnatal Days 4-8, indicating that disrupted GDNF/RET/GFRA1 signaling may occur before initial spermatogonial stem/progenitor cell decrease. Etv5(-/-) spermatogonia had reduced proliferation in vivo and in vitro. Decreased cell proliferation may cause the observed decreases in the number of type A spermatogonia (Postnatal Day 17) and daily sperm production (Postnatal Day 30) in Etv5(-/-) mice, indicating quantitative impairments in the first wave of spermatogenesis. In conclusion, ETV5 is expressed beginning in fetal Sertoli cells and can potentially have effects on neonatal Sertoli cells and germ cells. In addition, ETV5 has critical effects on neonatal spermatogonial proliferation, which may involve impaired signaling through the RET receptor.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Temporal and cellular expression of ETV5 in WT testes. The expression of Etv5 mRNA by real-time PCR in neonatal testes (A) was approximately half of that in adult (120-day-old) brain, which has the highest Etv5 mRNA level in mice, and is comparable to that in adult testis. Results are the mean ± SEM. Values with different lowercase letters are significantly different from each other (P < 0.05); n = 4 for each time point except the adult brain sample. B–D) Immunohistochemistry for ETV5 in WT testis at Fetal Day 16 (FD16) and Postnatal Days (P) 0 and 4. B) At FD16, ETV5 expression in the seminiferous cords was limited to Sertoli cell nuclei (Sc), whereas the gonocytes (G−) were negative. Some interstitial cells (Is) were also positive. C and D) In contrast to fetal testis, the pattern of expression was different at P0 and P4, when Sertoli cells and some gonocyte nuclei (G+) showed positive staining, but other gonocytes were negative (G−). Bar = 25 μm.
FIG. 2.
FIG. 2.
Etv5−/− germ cells fail to initiate spermatogenesis after transplantation into W/Wv testes. Establishment of spermatogenesis (Sp) was observed in W/Wv testes 60 days after transplantation with WT germ cells (A) but not with Etv5−/− germ cells (B). Bar = 25 μm.
FIG. 3.
FIG. 3.
Ret mRNA is reduced in neonatal Etv5−/− testes. Results are presented as the mean ± SEM and are given as the percentage of WT controls at each age (n = 4) for WT and Etv5−/− testes at all ages. Values with different lowercase letters are significantly different (P < 0.05) compared with WT controls.
FIG. 4.
FIG. 4.
GCNA1-stained microdissected spermatogonia from 5-day-old Etv5−/− testes have decreased levels of Ret mRNA relative to those of WT controls (n = 6). Data are presented as the mean ± SEM. *Significantly different at P < 0.05.
FIG. 5.
FIG. 5.
RET immunohistochemistry in WT and Etv5−/− testes (n = 4). The intensity of RET staining per spermatogonia is markedly reduced in Etv5−/− testis (B), although the numbers of cells staining for RET (arrows) are comparable to those of the WT testis (A). Insets are higher magnification of spermatogonia showing differences in staining intensity. Bar = 50 μm (10 μm in insets).
FIG. 6.
FIG. 6.
Etv5−/− spermatogonia showed decreased proliferation in vitro. The WT and Etv5−/− spermatogonia were cultured for 6 days in various combinations of GDNF, GFRA1-Fc fusion protein, and FGF2, and the number of proliferating colonies was counted. Data are expressed as the mean ± SEM. *Significant difference between the WT control and corresponding Etv5−/− culture (P < 0.05).
FIG. 7.
FIG. 7.
Neonatal Etv5−/− (B) spermatogonia produce markedly smaller colonies than WT (A) cultures. Cells were cultured in the presence of GDNF, GFRA1-Fc fusion protein, and FGF2. Bar = 25 μm.
FIG. 8.
FIG. 8.
Spermatogonial stem/progenitor cells from Etv5−/− testes have decreased proliferation in vivo at Postnatal Day 8. Data are shown as the percentage of RET-positive spermatogonia that are proliferating as determined by RET and MKI67 immunostaining in serial Postnatal Day 8 WT (n = 4) and Etv5−/− (n = 3) testis sections. Results are the mean ± SEM. *Significant difference (P < 0.05).
FIG. 9.
FIG. 9.
At Postnatal Day 17, the number of type A spermatogonia was significantly reduced in Etv5−/− testes relative to that in WT testes (n = 3 for both groups). *Significant difference (P < 0.05).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Guan K, Nayernia K, Maier LS, Wagner S, Dressel R, Lee JH, Nolte J, Wolf F, Li M, Engel W, Hasenfuss G.Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 2006; 440: 1199–1203. - PubMed
    1. Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, Baba S, Kato T, Kazuki Y, Toyokuni S, Toyoshima M, Niwa O, et al. Generation of pluripotent stem cells from neonatal mouse testis. Cell 2004; 119: 1001–1012. - PubMed
    1. Griswold MD.The central role of Sertoli cells in spermatogenesis. Semin Cell Dev Biol 1998; 9: 411–416. - PubMed
    1. Ogawa T, Ohmura M, Ohbo K.The niche for spermatogonial stem cells in the mammalian testis. Int J Hematol 2005; 82: 381–388. - PubMed
    1. Hofmann MC.Gdnf signaling pathways within the mammalian spermatogonial stem cell niche. Mol Cell Endocrinol 2008; 288: 95–103. - PMC - PubMed

Publication types

MeSH terms

Substances