Testicular spermatogonial stem cells (SSCs) are a heterogeneous population of stem cells, and definitive marker for the most primitive subset that undergoes asymmetric cell division remains to be identified. A novel subpopulation of pluripotent, very small embryonic-like stem cells (VSELs) has been reported in both human and mouse testes. Follicle-stimulating hormone (FSH) receptors (FSHRs) are expressed on Sertoli cells in testis and on granulosa cells in ovary, but recently FSHRs are reported on VSELs in ovaries, bone marrow, and cord blood. The present study was aimed to investigate whether FSHRs are also expressed on testicular stem cells (VSELs and SSCs) and their possible modulation by FSH using intact and chemoablated (25 mg/kg busulfan) mice. Chemoablated testis was a better model to study stem cell biology since quiescent stem cells survive along with the Sertoli cells in the tubules. Proliferating cell nuclear antigen-positive, small-sized cells presumed to be VSELs were clearly visualized, and flow cytometry analysis revealed an increase in LIN−/CD45−/SCA-1+ VSELs from 0.045±0.008% to 0.1±0.03% of total cells in chemoablated testis after FSH treatment. Very small embryonic-like stem cells expressing nuclear octamer-binding transcription factor 4 (OCT-4) and SSCs with cytoplasmic OCT-4 were detected. Very small embryonic-like stem cells (Oct-4A, Sca-1, Nanog), SSCs (Oct-4), and proliferation (Pcna) specific transcripts were upregulated on FSH treatment. Stem cells expressed FSHR and were stimulated by FSH, and Fshr3 was the predominant transcript maximally modulated by FSH. Nuclear OCT-4 and SCA-1 (stem cell antigen 1) positive VSELs are the most primitive stem cells in testis, and FSH stimulates them to undergo asymmetric cell division including self-renewal and give rise to SSCs, which in turn proliferate rapidly and undergo clonal expansion and further differentiation.
ParadisiRNataliFFabbriRBattagliaCSeracchioliRVenturoliS. Evidence for a stimulatory role of high doses of recombinant human follicle-stimulating hormone in the treatment of male-factor infertility. Andrologia. 2014;46(9):1067–1072.
3.
ThemmenAPNHuhtaniemiIT. Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function. Endocr Rev. 2000;21(5):551–583.
4.
SriramanVRaoAJ. FSH, the neglected sibling: evidence for its role in regulation of spermatogenesis and Leydig cell function. Indian J Exp Biol. 2005;43(11):993–1000.
KumarTR.The quest for male germ-line stem cell markers: PAX7 gets ID’d. J Clin Invest. 2014;124(10):4219–4222.
7.
AloisioGMNakadaYSaatciogluHD. PAX7 expression defines germline stem cells in the adult testis. J Clin Invest. 2014;124(9):3929–3944.
8.
ChanFOatleyMJKaucherAV. Functional and molecular features of the Id4+ germline stem cell population in mouse testes. Genes Dev. 2014;28(12):1351–1362.
9.
RatajczakMZ. A novel view of the adult bone marrow stem cell hierarchy and stem cell trafficking. Leukemia. 2015;29(4):776–782.
10.
LimJJSungSYKimHJ. Long-term proliferation and characterization of human spermatogonial stem cells obtained from obstructive and non-obstructive azoospermia under exogenous feeder-free culture conditions. Cell Prolif. 2010;43(4):405–417.
11.
IzadyarFWongJMakiC. Identification and characterization of repopulating spermatogonial stem cells from the adult human testis. Hum Reprod. 2011;26(6):1296–1306.
12.
BhartiyaDParteSPatelHAnandSSriramanKGunjalP. Pluripotent very small embryonic-like stem cells in adult mammalian gonads. In: Ratajczak, ed. “Adult Stem Cell Therapies: Alternatives to Plasticity,” Stem Cell Biology and Regenerative Medicine. New York: Springer; 2014:191–209. ISBN: 978-1-4939-1000-7. doi:10.1007/978-1-4939-1001-4_1
13.
BhartiyaDUnniSParteSAnandS. Very small embryonic-like stem cells: implications in reproductive biology. Biomed Res Int. 2013;2013:682326.
14.
ParteSPatelHSriramanKBhartiyaD. Isolation and characterization of stem cells in the adult mammalian ovary. Methods Mol Biol. 2015;1235:203–229.
15.
BhartiyaDKasiviswanathanSUnniSK. Newer insights into premeiotic development of germ cells in adult human testis using Oct-4 as a stem cell marker. J Histochem Cytochem. 2010;58(12):1093–1106.
16.
AnandSBhartiyaDSriramanKPatelHManjramkarDD. Very small embryonic-like stem cells survive and restore spermatogenesis after busulphan treatment in mouse testis. J Stem Cell Res Ther. 2014;4:216.
17.
KurkurePPrasadMDhamankarVBakshiG. Very small embryonic-like stem cells (VSELs) detected in azoospermic testicular biopsies of adult survivors of childhood cancer. Reprod Biol Endocrinol. 2015;13:122.
18.
StimpfelMSkutellaTKubistaMMalicevEConradSVirant-KlunI. Potential stemness of frozen-thawed testicular biopsies without sperm in infertile men included into the in vitro fertilization programme. J Biomed Biotechnol. 2012;2012:291038.
19.
Virant-KlunIStimpfelMCvjeticaninBVrtacnik-BokalESkutellaT. Small SSEA-4-positive cells from human ovarian cell cultures: related to embryonic stem cells and germinal lineage?J Ovarian Res. 2013;6:24.
20.
PesceMSchölerHR. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells. 2001;19(4):271–278.
MierzejewskaKBorkowskaSSuszynskaE. Hematopoietic stem/progenitor cells express several functional sex hormone receptors-novel evidence for a potential developmental link between hematopoiesis and primordial germ cells. Stem Cells Dev. 2015;24(8):927–937.
23.
Abdelbaset-IsmailASuszynskaMBorkowskaS. Human hematopoietic stem/progenitor cells express several functional sex hormone receptors. J Cell Mol Med. 2016;20:134–46.
24.
SriramanKBhartiyaDAnandSBhutdaS. Mouse ovarian very small embryonic-like stem cells resist chemotherapy and retain ability to initiate oocyte-specific differentiation. Reprod Sci. 2015;22(7):884–903.
25.
SimoniMNieschlagEGromollJ. Isoforms and single nucleotide polymorphisms of the FSH receptor gene: implications for human reproduction. Hum Reprod Update. 2002;8(5):413–421.
26.
SairamMRBabuPS. The tale of follitropin receptor diversity: a recipe for fine tuning gonadal responses?Mol Cell Endocrinol. 2007;260-262:163–171.
27.
Zuba-SurmaEKKuciaMWuW. Very small embryonic-like stem cells are present in adult murine organs: ImageStream-based morphological analysis and distribution studies. Cytometry A. 2008;73A(12):1116–1127.
28.
LiYGantaSChengCCraigRGantaRRFreemanLC. FSH stimulates ovarian cancer cell growth by action on growth factor variant receptor. Mol Cell Endocrinol. 2007;267(1-2):26–37.
29.
XuGYangLZhangWWeiX. All the tested human somatic cells express both Oct4A and its pseudogenes but express Oct4A at much lower levels compared with its pseudogenes and human embryonic stem cells. Stem Cells Dev. 2015;24(13):1546–1557.
30.
JezMAmbadySKashpurO. Expression and differentiation between OCT4A and its pseudogenes in human ESCs and differentiated adult somatic cells. PLoS One. 2014;9(2):e89546.
31.
LiedtkeSStephanMKöglerG. Oct4 expression revisited: potential pitfalls for data misinterpretation in stem cell research. Biol Chem. 2008;389(7):845–850.
32.
SamardzijaCQuinnMFindlayJKAhmedN. Attributes of Oct4 in stem cell biology: perspectives on cancer stem cells of the ovary. J Ovarian Res. 2012;5(1):37.
33.
Mays-HoopesLLBolenJRiggsADSinger-SamJ. Preparation of spermatogonia, spermatocytes, and round spermatids for analysis of gene expression using fluorescence-activated cell sorting. Biol Reprod. 1995;53(5):1003–1011.
34.
DahiaCLRaoAJ. Regulation of FSH receptor, PKIbeta, IL-6 and calcium mobilization: possible mediators of differential action of FSH. Mol Cell Endocrinol. 2006;247(1-2):73–81.
35.
KubotaHAvarbockMRBrinsterRL. Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells. Proc Natl Acad Sci U S A. 2003;100(11):6487–6492.
36.
van BragtMPCilibertiNStanfordWLde RooijDGvan PeltAM. LY6A/E (SCA-1) expression in the mouse testis. Biol Reprod. 2005;73(4):634–638.
37.
AnandSBhartiyaDSriramanKMallickA. Underlying mechanisms that restore spermatogenesis from very small embryonic-like stem cells on transplanting healthy niche cells in busulphan treated mouse testis [published online ahead of print Sep 23, 2016]. Stem Cell Rev. 2016. doi:10.1007/s12015-016-9685-1.
38.
ShaikhANagvenkarPPethePHindujaIBhartiyaD. Molecular and phenotypic characterization of CD133 and SSEA4 enriched very small embryonic-like stem cells in human cord blood. Leukemia. 2015;29(9):1909–1917.
39.
VassenaREguizabalCHeindryckxB. Stem cells in reproductive medicine: ready for the patient?Hum Reprod. 2015;30(9):2014–2021.
40.
BhartiyaD. Stem cells, progenitors & regenerative medicine: a retrospection. Indian J Med Res. 2015;141(2):154–161.
41.
BhartiyaDSinghJ. FSH-FSHR3-stem cells in ovary surface epithelium: basis for adult ovarian biology, failure, aging, and cancer. Reproduction. 2015;149(1):R35–R48.
42.
BhartiyaDSriramanKGunjalPModakH. Gonadotropin treatment augments postnatal oogenesis and primordial follicle assembly in adult mouse ovaries?J Ovarian Res. 2012;5(1):32.
43.
ParteSBhartiyaDManjramkarDDChauhanAJoshiA. Stimulation of ovarian stem cells by follicle stimulating hormone and basic fibroblast growth factor during cortical tissue culture. J Ovarian Res. 2013;6(1):20.
44.
OrthJChristensenAK. Autoradiographic localization of specifically bound 125I-labeled follicle-stimulating hormone on spermatogonia of the rat testis. Endocrinol. 1978;103(5):1944–1951.
45.
BaccettiBCollodelGCostantino-CeccariniE. Localization of human follicle-stimulating hormone in the testis. FASEB J. 1998;12(11):1045–1054.
46.
KulkarniSAGardeSVShethAR. Immunocytochemical localization of bioregulatory peptides in marmoset testes. Arch Androl. 1992;29(1):87–102.
47.
BoitaniCPolitiMGMennaT. Spermatogonial cell proliferation in organ culture of immature rat testis. Biol Reprod. 1993;48(4):761–767.
48.
YarneyTAFahmyMHSairamMRKhanHMacdonaldEA. Ontogeny of FSH receptor messenger ribonucleic acid transcripts in relation to FSH secretion and testicular function in sheep. J Mol Endocrinol. 1997;18(2):113–125.
49.
SairamMRJiangLGYarneyTAKhanH. Alternative splicing converts the G-protein coupled follitropin receptor gene into a growth factor type I receptor: implications for pleiotropic actions of the hormone. Mol Reprod Dev. 1997;48(4):471–479.
50.
SimoniMGromollJHöppnerW. Mutational analysis of the follicle-stimulating hormone (FSH) receptor in normal and infertile men: identification and characterization of two discrete FSH receptor isoforms. J Clin Endocrinol Metab. 1999;84(2):751–755.
51.
SongGJParkYSLeeYSLeeCCKangIS. Alternatively spliced variants of the follicle-stimulating hormone receptor gene in the testis of infertile men. Fertil Steril. 2002;77(3):499–504.
52.
SullivanRRFarisBREbornDGriegerDMCino-OzunaAGRozellTG. Follicular expression of follicle stimulating hormone receptor variants in the ewe. Reprod Biol Endocrinol. 2013;11:113–20.
53.
CrepieuxPMarionSMartinatN. The ERK-dependent signalling is stage-specifically modulated by FSH, during primary Sertoli cell maturation. Oncogene. 2001;20(34):4696–4709.
54.
SimoniMCasariniL. Mechanisms in endocrinology: Genetics of FSH action: a 2014-and-beyond view. Eur J Endocrinol. 2014;170(3):R91–R107.
55.
O’ShaughnessyPJ. Hormonal control of germ cell development and spermatogenesis. Semin Cell Dev Biol. 2014;29:55–65.
56.
ThemmenAP. An update of the pathophysiology of human gonadotrophin subunit and receptor gene mutations and polymorphisms. Reproduction. 2005;130(3):263–274.
57.
DesaiSSRoyBSMahaleSD. Mutations and polymorphisms in FSH receptor: functional implications in human reproduction. Reproduction. 2013;146(6):R235–R248.
58.
McLachlanRIWrefordNGde KretserDMRobertsonDM. The effects of recombinant follicle-stimulating hormone on the restoration of spermatogenesis in the gonadotropin-releasing hormone-immunized adult rat. Endocrinology. 1995;136(9):4035–4043.
59.
ForestaCBettellaAFerlinAGarollaARossatoM. Evidence for a stimulatory role of follicle-stimulating hormone on the spermatogonial population in adult males. Fertil Steril. 1998;69(4):636–642.
60.
GarollaASeliceREnglB. Spermatid count as a predictor of response to FSH therapy. Reprod Biomed Online. 2014;29(1):102–112.
RaoAJRamachandraSGRameshV. Induction of infertility in adult male bonnet monkeys by immunization with phage-expressed peptides of the extracellular domain of FSH receptor. Reprod Biomed Online. 2004;8(4):385–391.
63.
MoudgalNRSairamMRKrishnamurthyHNSridharSKrishnamurthyHKhanH. Immunization of male bonnet monkeys (M. radiata) with a recombinant FSH receptor preparation affects testicular function and fertility. Endocrinology. 1997;138(7):3065–3068.
64.
Kanatsu-ShinoharaMShinoharaT. Spermatogonial stem cell self-renewal and development. Annu Rev Cell Dev Biol. 2013;29:163–187.
65.
WaheebRHofmannMC. Human spermatogonial stem cells: a possible origin for spermatocytic seminoma. Int J Androl. 2011;34(4 pt 2):e296–e305.
66.
O’ShaughnessyPJMonteiroAVerhoevenGDe GendtKAbelMH. Effect of FSH on testicular morphology and spermatogenesis in gonadotrophin-deficient hypogonadal mice lacking androgen receptors. Reproduction. 2010;139(1):177–184.
67.
AbelMHBabanDLeeSCharltonHMO’ShaughnessyPJ. Effects of FSH on testicular mRNA transcript levels in the hypogonadal mouse. J Mol Endocrinol. 2009;42(4):291–303.
68.
TakashimaSKanatsu-ShinoharaMTanakaT. Functional differences between GDNF-dependent and FGF2-dependent mouse spermatogonial stem cell self-renewal. Stem Cell Reports. 2015;4(3):489–502.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
