The piRNA pathway is developmentally regulated during spermatogenesis in Drosophila

Abstract

PIWI-interacting RNAs (piRNAs) are predominantly produced in animal gonads to suppress transposons during germline development. Our understanding about the piRNA biogenesis and function is predominantly from studies of the Drosophila female germline. piRNA pathway function in the male germline, however, remains poorly understood. To study overall and stage-specific features of piRNAs during spermatogenesis, we analyzed small RNAs extracted from entire wild-type testes and stage-specific arrest mutant testes enriched with spermatogonia or primary spermatocytes. We show that most active piRNA clusters in the female germline do not majorly contribute to piRNAs in testes, and abundance patterns of piRNAs mapping to different transposon families also differ between male and female germlines. piRNA production is regulated in a stage-specific manner during spermatogenesis. The piRNAs in spermatogonia-enriched testes are predominantly transposon-mapping piRNAs, and almost half of those exhibit a ping-pong signature. In contrast, the primary spermatocyte-enriched testes have a dramatically high amount of piRNAs targeting repeats like suppressor of stellate and AT-chX The transposon-mapping piRNAs in the primary spermatocyte stages lacking Argonaute3 expression also show a ping-pong signature, albeit to a lesser extent. Consistently, argonaute3 mutant testes also retain ping-pong signature-bearing piRNAs, suggesting that a noncanonical ping-pong cycle might act during spermatogenesis. Our study shows stage-specific regulation of piRNA biogenesis during spermatogenesis: An active ping-pong cycle produces abundant transposon-mapping piRNAs in spermatogonia, while in primary spermatocytes, piRNAs act to suppress the repeats and transposons.

Keywords: Drosophila; PIWI proteins; RNA silencing; germline; piRNA; spermatogenesis.

FIGURE 1.

Aub and Ago3 exhibit distinct expression patterns during spermatogenesis. (A) Schematic representation of a wild-type Drosophila testis. The asterisk denotes the anterior apex. Germline stem cells (GSCs, shown in red) located next to the hub divide asymmetrically to give rise to GSCs and differentiating gonialblasts. Gonialblasts undergo four rounds of mitotic division, producing germline cysts collectively known as spermatogonia (shown in pink) located in the germinal proliferation center (GPC). Spermatogonia further differentiate into primary spermatocytes (shown in blue). (B) y w testes stained for Aub and Ago3 showing apex region (top), magnified GPC (middle), and primary spermatocytes (bottom). Scale bars represent 50 µm in the top panels and 10 µm in the middle and bottom panels. While Aub was expressed from GSCs to primary spermatocytes, Ago3 was detected only in GPC. (C) Schematic representation of the purification method used to enrich testicular extract with spermatogonia or primary spermatocytes. bam and bgcn mutant testes were used to obtain GPCs containing GSCs and spermatogonia. The apex of can and sa mutant were manually removed to get rid of spermatogonia. (D) Western blots showing the expression of Aub, Ago3, and the loading control α-Tubulin in y w, bam, bgcn, can, and sa testes. Ago3 expression was observed in y w, bam, and bgcn, but not in can and sa testes devoid of GPCs.

FIGURE 2.

Distinct piRNA populations are produced during spermatogenesis. (A) The normalized numbers of 23- to 29-nt reads mapping to canonical transposons and clusters in bam, bgcn, can, sa, and y w libraries. Transposon and cluster-mapping piRNAs were globally more abundant in bam and bgcn testes. (B) Principal component analysis of transposon-mapping piRNAs in each different type of arrested mutant testes. While piRNAs in can and sa mutants, and those in bam and bgcn exhibit similarity, respectively, they are quite different compared with the other group. (C) Scatter-plot representing the expression of transposon-mapping piRNAs in spermatogonia versus primary spermatocytes. Log₂ of the mean number of reads of piRNAs mapping to each transposon families in bam and bgcn mutant testes are plotted on the x-axis, and those in can and sa mutant testes are plotted on the y-axis. piRNAs mapping to most of the transposon families are more abundant in spermatogonia (red dots) while some others are more abundant in primary spermatocytes (blue dots). (D) Heat-map representing the normalized expression levels of the most abundant transposon-mapping piRNAs (minimum in white, maximum in red) in bam, bgcn, can, sa, and y w libraries. Most of the transposon-mapping piRNAs were enriched in bam and bgcn libraries while some others show different trends. (E) The normalized number of AT-chX piRNAs in the indicated libraries. (F) The normalized number of Su(Ste) piRNAs in the indicated libraries. Both Su(Ste) and AT-chX piRNAs were highly enriched in can and sa libraries.

FIGURE 3.

piRNAs with ping-pong signature are more abundant in spermatogonia but still detected in primary spermatocytes. (A) Bar graph showing the normalized numbers of transposon-mapping piRNAs in the indicated libraries. Black and gray bars represent those without a ping-pong signature and with a ping-pong signature, respectively. The pie charts represent the percentage of those piRNAs in the total population of transposon-mapping piRNAs. piRNAs with ping-pong signature were more abundant in bam and bgcn libraries but still detected in can and sa libraries. (B) Heat-map representing the ping-pong ratios of the transposon-mapping piRNAs (minimum in white, maximum in green) in the indicated libraries. For most transposon-mapping piRNA families, the ping-pong ratios are higher in spermatogonia (bam and bgcn libraries) compared with primary spermatocytes (can and sa libraries).

FIGURE 4.

A noncanonical ping-pong amplification cycle takes place in Drosophila testes. (A) Immunostaining of endogenous Aub and Ago3 in Drosophila testis of the indicated genotype. The asterisk denotes the anterior apex. Scale bars represent 50 µm. Insets show magnified cells from the GPC. In aub mutant testes, Aub and Ago3 expressions were significantly reduced. In ago3 mutant testes, Ago3 was lost but Aub was comparably expressed and localized to perinuclear nuage. (B) Bar graph showing the normalized numbers of transposon-mapping piRNAs in the indicated libraries. Black and gray bars represent those without a ping-pong signature and with a ping-pong signature, respectively. The pie charts represent the percentage of those piRNAs in the total population of transposon-mapping piRNAs. Transposon-mapping piRNAs with a ping-pong signature were totally lost in aub mutant testes, while a small fraction of them was still present in ago3 mutants. (C) Heat-map representing the ping-pong ratios of the transposon-mapping piRNAs (minimum in white, maximum in green) in the indicated libraries. In aub mutant testes, piRNAs containing ping-pong signatures are significantly reduced for almost all transposon families. Ago3 loss did not cause as severe a loss in ping-pong signatures for most transposon-mapping piRNAs as observed for that in aub mutants.