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A nuclear role for the Argonaute protein AGO2 in mammalian gametogenesis

ABSTRACT: The reduced sperm count observed in Ago2 cKO mice implies that AGO2 has non-redundant functions in the male germ line. Because AGO2 is a key protein in the RNA interference (RNAi) pathway, we first postulated that AGO2 loss disrupts normal transcriptional and translational dynamics of target mRNAs relevant to sperm maturation. To address this hypothesis, we took advantage of the apparently normal spermatogenesis in Ago2 cKO mice, which allowed us to purify matched meiotic and post-meiotic germ cells from Ago2 cKO and wild type controls. We examined changes in the transcriptome and proteome of these two spermatogenic stages in Ago2 cKO relative to control mice using RNA-seq and quantitative mass spectrometry (MS). To further examine if the changes in mRNA and protein levels detected in Ago2 cKO germ cells was due to a loss of regulation by the canonical AGO2-miRNA pathway, we characterized AGO2 protein interactors by AGO2 immunoprecipitation-mass spectrometry (IP-MS) in cytoplasmic and nuclear fractions of post-meiotic cells

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: TuKiet Lam

LAB HEAD: TuKiet Lam

PROVIDER: PXD027935 | Pride | 2022-08-01

REPOSITORIES: Pride

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Project description:Mammalian spermatogenesis is a complex biological process that occurs within a highly organized tissue, the seminiferous epithelium. The coordinated maturation of spermatogonia, spermatocytes and spermatids suggests the existence of precise programs of gene expression in these cells as well as in their neighboring somatic Sertoli cells. The objective of this study was to elucidate genes encoding the proteins that execute these programs. Rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX-XI, XII, XIII-XIV of the cycle were isolated by microdissection and Sertoli cells, spermatogonia plus early spermatocytes, pachytene spermatocytes and spermatids were purified from enzymatically-dispersed testes. Microarray analysis using Rat Genome 230 2.0 arrays identified a total of 16,971 probe sets that recognized transcripts. A comparison with the transcriptome of other tissues identified 398 testis-specific probe sets, which therefore are potential targets for the development of new contraceptives. Sequential waves of cell and stage-specific gene expression are associated with progression of germ cells through the stages of the cycle of the seminiferous epithelium and 1612 probe sets recognized transcripts whose expressions varied at least 4-fold across the stages of the cycle. Pathway analyses reveal that entire biological processes are regulated cyclically in testicular cells. Important among these are cell cycle and DNA repair. Thus, stage-specific gene expression is a widespread and fundamental characteristic of spermatogenic cells and Sertoli cells. Experiment Overall Design: Seminiferous tubules at the following stages or groups of stages were isolated by transillumination-assisted microdissection: I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX-XI, XII, XIII-XIV. Twenty-five segments of tubules, 2 m in length, were collected at each stage or group of stages from each rat. Tubules from two separate animals were pooled to form one sample from each stage or groups of stages. A total of 5 independent samples per stage or groups of stages were analyzed. Mature Sertoli cells were isolated to about 85% purity (n=4). Three groups of germ cells were isolated by centrifugal elutriation from enzyme-dispersed testes: round spermatids (n=4), pachytene spermatocytes (n=4) and spermatogonia plus early spermatocytes (n=3) . Purity of these cells has been shown to vary between 85% to 95%

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A nuclear role for the Argonaute protein AGO2 in mammalian gametogenesis

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