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Targeting the Gdnf Gene in Peritubular Myoid Cells Disrupts Undifferentiated Spermatogonial Cell Development

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Targeting the Gdnf Gene in Peritubular Myoid Cells Disrupts Undifferentiated Spermatogonial Cell Development Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development Liang-Yu Chena, William D. Willisa, and Edward M. Eddya,1 aGamete Biology Group, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709 Edited by John J. Eppig, The Jackson Laboratory, Bar Harbor, ME, and approved December 16, 2015 (received for review September 9, 2015) Spermatogonial stem cells (SSCs) are a subpopulation of undiffer- type A-aligned (Aal) spermatogonia, and these in turn divide to entiated spermatogonia located in a niche at the base of the sem- form longer chains of differentiating (type A1,A2,A3, intermedi- iniferous epithelium delimited by Sertoli cells and peritubular myoid ate, and B) spermatogonia. (PM) cells. SSCs self-renew or differentiate into spermatogonia that Although SSCs are single cells, not all As spermatogonia are ∼ proliferate to give rise to spermatocytes and maintain spermato- likely to be SSCs. There are 35,000 As spermatogonia in the genesis. Glial cell line-derived neurotrophic factor (GDNF) is essential testes of adult mice (8), but only about 3,000 of these have the for this process. Sertoli cells produce GDNF and other growth factors ability to regenerate spermatogenesis when transplanted to and are commonly thought to be responsible for regulating SSC germ cell-depleted testes (9). Although there are no generally development, but limited attention has been paid to the role of PM accepted molecular markers specific for SSCs, potential candidates cells in this process. A conditional knockout (cKO) of the androgen are inhibitor of DNA binding 4 (ID4) and paired box 7 (PAX7), – receptor gene in PM cells resulted in male infertility. We found that which are expressed in minor subsets of As spermatogonia (10 12). testosterone (T) induces GDNF expression in mouse PM cells in vitro However, it remains to be reported if ID4 and PAX7 are coex- and neonatal spermatogonia (including SSCs) co-cultured with pressed in the same subset of As spermatogonia. SSCs also share molecular markers with undifferentiated spermatogonia, in- T-treated PM cells were able to colonize testes of germ cell-depleted – mice after transplantation. This strongly suggested that T-regulated cluding Nanos2, Gfra1, Zbtb16, Bcl6b, and THY1(13 17). In addition, differentiating spermatogonia have characteristic mo- production of GDNF by PM cells is required for spermatogonial de- – velopment, but PM cells might produce other factors in vitro that are lecular markers, including Ngn3, Nanos3, Spo11,andKIT(18 22). responsible. In this study, we tested the hypothesis that production These molecular markers have been proven to be valuable tools for monitoring the presence or absence of different populations of of GDNF by PM cells is essential for spermatogonial development by spermatogonia. generating mice with a cKO of the Gdnf gene in PM cells. The cKO A conditional knockout (cKO) of the androgen receptor (Ar) males sired up to two litters but became infertile due to collapse of gene in PM cells resulted in progressive loss of spermatogonia spermatogenesis and loss of undifferentiated spermatogonia. These beginning at postnatal d 21, leading to disorganization of the studies show for the first time, to our knowledge, that the produc- seminiferous epithelium and infertility (23). This strongly sug- tion of GDNF by PM cells is essential for undifferentiated spermato- gested that androgens regulate genes in PM cells whose products gonial cell development in vivo. are essential for SSC maintenance. In other studies, mice hetero- zygous for a global mutation in the gene for glial cell-derived spermatogonial stem cell | stem cell niche | male fertility | neurotrophic factor (Gdnf) had reduced stem cell reserves, whereas spermatogenesis | conditional gene targeting Significance he seminiferous epithelium is separated by tight junctions Tbetween Sertoli cells into a luminal compartment containing Glial cell line-derived neurotrophic factor (GDNF) growth factor spermatocytes and spermatids and a basal compartment con- induces spermatogonial stem cells to proliferate in culture to taining spermatogonial stem cells (SSCs) and spermatogonia. produce progenitor spermatogonia. Sertoli cell GDNF was The basal compartment is bounded above and on the sides by thought responsible for this in the testis. We showed that tes- Sertoli cells and below by the basement membrane of the sem- tosterone triggers GDNF secretion by peritubular myoid (PM) iniferous tubule and a layer of peritubular myoid (PM) cells. cells in culture and that undifferentiated spermatogonia (sper- SSCs are thought to reside in a microenvironmental niche in the matogonial stem cells and progenitors) co-cultured with testos- basal compartment, where extrinsic cues influence their decision terone-treated PM cells and transplanted to sterile mice restored to either self-renew or enter the pathway of spermatogonial de- spermatogenesis. To determine if PM cell GDNF is essential to velopment (1, 2). They are a minor fraction of the undifferentiated maintain the undifferentiated spermatogonial population, we spermatogonia in the basal compartment. The other undifferen- made mice lacking the Gdnf gene in PM cells. The number of tiated spermatogonia (progenitors) give rise to differentiating undifferentiated spermatogonia was severely depleted in 2-wk- spermatogonia that proliferate mitotically to progress on a de- old mice and adults were infertile. This is the first study, to our velopmental pathway toward becoming spermatocytes (3, 4). Our knowledge, to show that the undifferentiated spermatogonial current understanding of the progression of SSCs to differentiat- pool cannot be maintained without GDNF from PM cells. ing spermatogonia comes mainly from cell kinetic studies, germ cell transplantation assays, and the use of molecular markers that Author contributions: L.-Y.C. and E.M.E. designed research; L.-Y.C. and W.D.W. performed identify different populations of spermatogonia. research; L.-Y.C., W.D.W., and E.M.E. analyzed data; and L.-Y.C. and E.M.E. wrote The leading model for spermatogonial development specifies the paper. that when SSCs divide, they either self-renew by becoming two The authors declare no conflict of interest. type A-single (As) spermatogonia or give rise to type A-paired This article is a PNAS Direct Submission. 1 (Apr) spermatogonia connected by an intercellular bridge to To whom correspondence should be addressed. Email: [email protected]. BIOLOGY – become undifferentiated spermatogonia (5 7). The pairs continue This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. DEVELOPMENTAL to divide to form short chains of bridge-connected undifferentiated 1073/pnas.1517994113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1517994113 PNAS | February 16, 2016 | vol. 113 | no. 7 | 1829–1834 Downloaded by guest on September 25, 2021 and cKO males were mated continuously for 6 mo with one WT female each, and the numbers of litters sired by WT males (6.17 ± 0.71) and Het males (5.82 ± 0.98) were not significantly different, whereas the number of litters sired by cKO males was significantly lower (1.5 ± 0.85) (Fig. 1A). The WT and Het males sired 4–7 litters and were fertile throughout the mating trial, whereas the cKO males sired 0–2 litters and were fertile only during the first 3 mo (Fig. 1B), suggesting an age-dependent loss of fertility in cKO males. However, the sizes of the litters sired by WT (6.54 ± 0.48), Het (7.43 ± 0.52), and cKO (5.8 ± 1.05) males were not significantly different. To identify possible underlying causes for the loss of fertility in cKO males, other male reproductive system parameters were examined. At 8 wk, testis weights were significantly lower in cKO than in Het and WT males (Fig. 1C), but there were no differences in body weights (Fig. S1D). In addition, the sperm numbers in the cauda epididymis of 8-, 12-, and 24-wk-old WT, Het, and cKO males were determined (Fig. 1D). There were no significant differences at 8 wk, but by 12 wk sperm numbers were significantly lower in cKO males Fig. 1. Effects of targeted disruption of the Gdnf gene in PM cells on male than in WT and Het males. By 24 wk, the sperm numbers were reproductive function. (A) The number of litters sired by WT, Het, and cKO dramatically lower for cKO males and significantly lower for males in a 6 mo breeding study. (B) The average number of litters sired by Het males than for WT males. These results led us to hypoth- WT, Het, and cKO males in the first and second 3-mo period of the esize that the loss of fertility with age was due to disruption of breeding study. (C) The weights of testes from 8-wk-old WT, Het, and cKO spermatogonial development. mice. (D) The number of sperm released from the cauda epididymides of 8-, To test this hypothesis, sections of testes from 1- to 12-wk-old 12-, and 24-wk-old WT, Het, and cKO males. Each bar and data point repre- ± < < WT, Het, and cKO males were immunostained for ZBTB16 sents the mean SEM. *P 0.05, **P 0.01. (Table S1), a marker for undifferentiated spermatogonia (15). Differences were not apparent at 1 wk (Fig. S2A), but at 2 wk some seminiferous tubules in cKO males lacked spermatogonia mice with a transgene overexpressing GDNF experienced an B C overaccumulation of undifferentiated spermatogonia (24). GDNF (Fig. S2 ). This increased at 4 wk (Fig. S2 ) and was more pro- also was reported to be critical for development of SSCs in vitro and nounced at 8 wk (Fig. S2D). By 12 wk, some tubules in Het mice their ability to restore spermatogenesis after transplantation to the lacked undifferentiated spermatogonia, and spermatogenesis was testes of germ-cell-depleted mice (25, 26). This led us to hypothe- severely defective in most tubules in cKO mice (Fig.
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