Underlying Mechanisms that Restore Spermatogenesis on Transplanting Healthy Niche Cells in Busulphan Treated Mouse Testis.
Very small embryonic-like stem cells (VSELs) exist among spermatogonial stem cells and survive chemotherapy in both mice and human testes because of their relatively quiescent nature. Our earlier study revealed that inter-tubular transplantation of niche (Sertoli or bone marrow derived mesenchymal) cells can restore spermatogenesis from endogenous surviving VSELs. Present study was undertaken to delineate the effect of busulphan on testicular stem/germ/Sertoli cells and to comprehend the underlying mechanisms of how transplanted niche cells restore spermatogenesis. Ploidy analysis showed an increase in diploid cells on D7 and VSELs (2-6 μm; LIN-/CD45-/SCA-1+) were detected at all time-points studied and were maximum on D15 after busulphan treatment. They were visualized in cell smears, expressed nuclear NANOG and SOX2 and BrdU uptake on D15 suggested they were proliferating in response to stress induced by busulphan. Verapamil-sensitive side population detected comprised SCA-1 positive stem cells (5 ± 0.02 % in normal and 8.6 ± 2.02 % in chemoablated testis). Adverse effects of busulphan on Sertoli cells by transcriptome analysis included altered expression of Gdnf, Scf, Fgf, Bmp4, androgen binding protein, components of blood-testis-barrier and also stem cells related signaling pathways including Wnt. GFP positive transplanted cells aligned themselves as 'neo-tubules' and were visualized adjacent to 'native' germ cells depleted tubules. 'Neo-tubules' provide paracrine support to endogenous VSELs to undergo spermatogenesis. Quantitative analysis was done to track proliferation (PCNA) and differentiation (MVH) of stem cells by immuno-localization studies at different time intervals. Results provide an alternative strategy to restore spermatogenesis in cancer survivors from endogenous stem cells which needs to be further researched.
Stem Cell Biology Department, National Institute for Research in Reproductive Health, JM Street, Parel, Mumbai, 400 012, India.
Sandhya
S
Anand
Stem Cell Biology Department, National Institute for Research in Reproductive Health, JM Street, Parel, Mumbai, 400 012, India. bhartiyad@nirrh.res.in.
Deepa
D
Bhartiya
Stem Cell Biology Department, National Institute for Research in Reproductive Health, JM Street, Parel, Mumbai, 400 012, India.
Kalpana
K
Sriraman
Stem Cell Biology Department, National Institute for Research in Reproductive Health, JM Street, Parel, Mumbai, 400 012, India.
Alpna
A
Mallick
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000166:cytology; D002066:Busulfan / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000378:metabolism* / Q000637:transplantation; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008297:Male; D000071317:Nanog Homeobox Protein / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D012708:Sertoli Cells / Q000166:cytology; D013091:Spermatogenesis; D055153:Stem Cell Niche; D013737:Testis / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism
D018906:Antineoplastic Agents, Alkylating; D000071317:Nanog Homeobox Protein; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D002066:Busulfan
Azoospermia; Busulphan; Infertility; Niche; Sertoli cells; Spermatogenesis; Stem cells; Testis; VSELs
Biol Reprod. 2008 Sep;79(3):475-85
18480464
J Histochem Cytochem. 2010 Dec;58(12):1093-106
20805580
Nat Med. 2014 Aug;20(8):857-69
25100531
J Biomed Biotechnol. 2012;2012:291038
22431916
Reproduction. 2005 Jul;130(1):15-28
15985628
Biol Reprod. 2003 Mar;68(3):1064-71
12604661
Mol Cell Endocrinol. 2000 Mar 30;161(1-2):117-20
10773400
J Ovarian Res. 2013 Apr 09;6:24
23570331
Stem Cells. 2006 Jun;24(6):1505-11
16456131
Tissue Cell. 1999 Oct;31(5):461-72
10612257
Reproduction. 2009 Mar;137(3):497-508
19036951
Hum Reprod. 2014 May;29(5):989-96
24522758
Reprod Biol Endocrinol. 2015 Nov 09;13:122
26553338
Hum Reprod. 2005 Sep;20(9):2376-82
15919776
Int J Mol Med. 2006 Jan;17 (1):21-8
16328007
Cell Rep. 2014 May 22;7(4):1284-97
24794432
Leukemia. 2006 May;20(5):857-69
16498386
Proc Natl Acad Sci U S A. 2003 May 27;100(11):6487-92
12738887
Stem Cells. 2006 Jan;24(1):3-12
16449630
Reprod Biol Endocrinol. 2015 Apr 18;13:33
25903688
FASEB J. 2004 Feb;18(2):376-8
14688197
Biol Reprod. 2005 Oct;73(4):634-8
15930324
Reprod Sci. 2016 Nov;23 (11):1493-1508
27189070
Biol Reprod. 2016 May;94(5):112
27053363
Development. 2013 Jan 15;140(2):255-65
23250203
Hum Reprod Update. 2016 Dec;23 (1):41-76
27614362
Biol Reprod. 2010 Feb;82(2):422-32
19794154
PLoS One. 2012;7(1):e29764
22253774
J Cell Physiol. 2007 Apr;211(1):149-58
17167785