The Influence Of Sertol Cell Dependant Microenvironment On Germ Cell Development

Spermatogenesis is a complex and ordered process,during which spermatogonial stem cells(SSCs)undergo self-renewal and differentiation into spermatocytes and then generate haploid spermatids through two successive meiosis.This highly orchestrated process is controlled by complex regulatory programs.Disturbing this process will cause failure in spermatogenesis and lead to male infertility.The microenvironment in seminiferous tubules play an important role in mammalian spermatogenesis.In mouse,the niche are formed by Sertoli cells,Leydig cells,and hematopoietic endothelia,etc.Tight junctions formed between Sertoli cells create the blood-testis barrier(BTB),which divides seminiferous tubules into basal and adluminal compartments.Therefore,proper functions of Sertoli cells are key to sustain normal spermatogenesis.Brg1 is a catalytic subunit of the mammalian Swi/Snf chromatin-remodeling complex and participates in transcriptional regulation and repair of double-strand DNA breaks caused by genotoxic agents.It is of great importance to understand how Brg1-SWI/SNF complex play a unique role in maintaining proper functions of Sertoli cells,and in turn to sustain appropriate microenvironment for postnatal germ cell development.In addition,it has been reported that bioenergtics in Sertoli cells has unique characteristics.As we know,mitochondria are the major metabolic site for production of energy and reactive oxygen species(ROS).Mitochondrial functions are largely influenced by their dynamically changing structures.In different cell types,mitochondria adapt distinct morphology and structures which are largely regulated via mitochondrial fusion and fission events.Mfn1 and Mfn2 are two key GTPases in mitochondrial fusion,and Nrf1 is a transcription factor known for its role in mitochondrial biogenesis.Therefore,investigating genes that are important for mitochondrial fusion and biogenesis will contribute to our understanding how metabolic regulation impacts the functions of sertoli cell-dependent microenvironment.In this study,to understand the physiological functions of these genes in Sertoli cells and their regulatory effects on the spermatogenesis,we specifically deleted Brg1,Mfn1,Mfn2,or Nrf1 in Sertoli cells by crossing conditional knockout mice and an Amh-cre line.We found that specific deletion of Brg1 in Sertoli cells had no apparent influence on spermatogenesis and fertility in mice.The lack of influence may result from functional redundancy of the Brg1 homolog,Brm,which is also expressed in Sertoli cells.Either Mfn1 or Mfn2 deficiency in Sertoli cells does not affect spermatogenesis.However,upon deletion of both Mfn1 and Mfn2 in Sertoli cells,male infertility was observed.In Mfn1 and Mfn2 double knockout mice,the blood-testis barrier(BTB)was disrupted,together with a reduction in Sertoli cell number.This in turn led to germ cell falling-off from seminiferous tubules and decreased numbers of germ cells due to increased apoptosis.These results thus suggest that Mfn1 and Mfn2 have essential but redundant roles in maintaining Sertoli cell functions in mice.Finally,Nrf1 deletion in embryonic Sertoli cells leads to complete loss of structure of seminiferous tubules,by which Sertoli cells and germ cells quickly depleted.Our previous studies showed that embryonic germ cells with deletion of Mfn1 resulted in a meiotic defect in male mice.We observed that mitochondria from Mfn1 deficient spermatogonia were larger in diameter,round and swollen.However,we could not exclude the possibility that the defects in spermatocyte development upon Mfn1 knockout was a secondary effect from spermatogonial deficiency.Also,it remains unknown whether Mfn2,the other mitochondrial fusion protein,is required for meiosis in mice.Here we further explored the intrinsic roles of Mfn1 and Mfn2 in germ cell development,by conditional deletions of Mfn1 and Mfn2 in neonatal spermatogonia,via a Stra8-Cre mouse model.Compared to littermate controls,a significant decrease in testis size was observed in 5-week mice upon Mfn1 deletion by Stra8-Cre.Germ cells were meiotic arrested in these Mfn1 mutant mice,supporting that the Mfn1 is required for meiosis.By contrast,when Mfn2 was similarly ablated,partial meiotic arrest of germ cells were observed,and enlongated sperms were seen in part of seminiferous tubules in 5-week mice.Nevertheless,we did not observe any mature spermatozoa in the epididymides in those Mfn2 mutant mice.In addition,more severe phenotype was seen upon double knockout of Mfn1 and Mfn2 with fewer spermatocytes than those due to deletion of either.These results thus indicated that Mfn1 and Mfn2 have complimentary but non-redundant roles during meiosis in spermatogenesis.In summary,the present studies supported the notion that Sertoli cells play an important role during spermatogenesis.In addition,through different gene knockout mouse models,we revealed the crtical functions of mitochondrial dynamics and biogenesis in maintaining Sertoli cell functions.We further confirmed that Mfn1 and Mfn2 were both indispensable for meiosis in mice.