Abstract: PD08-07
Sources of Funding: This work was supported by: P50 HD076210, U1 1U01HD074542-01, Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust, the Mr. Robert S. Dow Foundation, Irena and Howard Laks Foundation; Urology Care Foundation Research Scholar Award Program and AUA New York Section Research Scholar Fund

Introduction

Sertoli cell only (SCO) syndrome is the most severe form of male factor infertility. We have previously demonstrated loss of expression of miRNA202-5p in Sertoli cells in men with SCO; however, it is unknown if such loss of miRNA-202-5p in Sertoli cells is due to the loss of germ cells or is a primary event. Our objective was to evaluate if loss of germ cells will lead to secondary loss of miRNA202-5p expression. An inducible SCO animal model, Lecithin: Retinol Acyltransferase (LRAT) knockout (KO) mouse was used to answer our question.

Methods

9 LRAT KO animals were fed either Vitamin A sufficient (ASuf) or Vitamin A deficient (ADef) diets for 8wks, with the latter known to produce SCO. H&E of testicular slides was used to assess histology. Immunofluorescence (IF) with antibodies against SYCP3 (marker of spermatocytes) was used to confirm loss of meiotic cells in LRAT KO Adef mice. RNAseq and smallRNA sequencing was performed using total RNA extracted from testes. Sequencing results were processed using JMP Genomics. Expression levels of GFRa1, PLZF, SCYP3, PRM1, TNP, CLU, and VIM were used to evaluate loss of different germ cell populations in the Adef group, and to normalize the data to the number of Sertoli cells. MicroRNA202-5p expression (Sertoli specific), miR-34c (germ cell specific) and let-7 (ubiquitous) were measured from sequencing data and further confirmed using QRT-PCR. Results were statistically significant at FRD=0.01

Results

3 mice were evaluated in each respective group, LRAT Asuf and ADef conditions, at both 6 and 8wk time points. Histology, IF, and sequencing data demonstrated that spermatogenesis was present in all groups except for LRAT ADef mice at 8 weeks. Histology revealed heterogeneity, with most tubules resembling SCO in LRAT ADef testes. Normal spermatogenesis was observed in LRAT KO Asuf mice, and hypo spermatogenesis was observed inLRAT KO ADef mice at 6wks. There was no significant change in expression levels of miR202-5p between LRAT ASuf an ADef groups at 8wks. However, expression of miR34c was significantly decreased in the LRAT Adef group. Let-7 expression remained same.

Conclusions

Loss of meiotic germ cells in LRAT KO ADef mice did not result in loss of miRNA-202-5p expression when compared to controls despite development of predominantly SCO histology. Our results provide strong evidence that the observed loss of expression of miRNA202-5p in men with SCO is not due to the primary loss of germ cells but is a result of primary miRNA-202-5p dysfunction in Sertoli cells. _x000D_

Funding

This work was supported by: P50 HD076210, U1 1U01HD074542-01, Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust, the Mr. Robert S. Dow Foundation, Irena and Howard Laks Foundation; Urology Care Foundation Research Scholar Award Program and AUA New York Section Research Scholar Fund