Major spliceosome defects cause male infertility and are associated with nonobstructive azoospermia in humans

Abstract

Processing of pre-mRNA into mRNA is an important regulatory mechanism in eukaryotes that is mediated by the spliceosome, a huge and dynamic ribonucleoprotein complex. Splicing defects are implicated in a spectrum of human disease, but the underlying mechanistic links remain largely unresolved. Using a genome-wide association approach, we have recently identified single nucleotide polymorphisms in humans that associate with nonobstructive azoospermia (NOA), a common cause of male infertility. Here, using genetic manipulation of corresponding candidate loci in Drosophila, we show that the spliceosome component SNRPA1/U2A is essential for male fertility. Loss of U2A in germ cells of the Drosophila testis does not affect germline stem cells, but does result in the accumulation of mitotic spermatogonia that fail to differentiate into spermatocytes and mature sperm. Lack of U2A causes insufficient splicing of mRNAs required for the transition of germ cells from proliferation to differentiation. We show that germ cell-specific disruption of other components of the major spliceosome manifests with the same phenotype, demonstrating that mRNA processing is required for the differentiation of spermatogonia. This requirement is conserved, and expression of human SNRPA1 fully restores spermatogenesis in U2A mutant flies. We further report that several missense mutations in human SNRPA1 that inhibit the assembly of the major spliceosome dominantly disrupt spermatogonial differentiation in Drosophila. Collectively, our findings uncover a conserved and specific requirement for the major spliceosome during the transition from spermatogonial proliferation to differentiation in the male testis, suggesting that spliceosome defects affecting the differentiation of human spermatogonia contribute to NOA.

Keywords: GWAS; NOA; spermatogenesis; spermatogonia; spliceosome.

Fig. 1.

The locus 15q26.3 (rs7166401) is susceptible to male infertility. (A) Regional association plot of SNP rs7166401. SNP rs716640 was identified by genome-wide association study (GWAS) in Han Chinese men. SNPs are shown in purple, and r² values for other SNPs are indicated in color. Genes within the region of interest (1 Mb) are annotated with direction of transcription marked by arrows. (B) Schematic diagram of spermatogenesis in Drosophila. At the blind apical end of the testis, germline stem cells that surround stromal hub cells divide asymmetrically to self-renew and to produce goniablasts. Goniablasts undergo four rounds of transient amplifying divisions to form 16 spermatogonia; these enter meiosis and undergo differentiation into spermatocytes, spermatids, and mature sperm.

Fig. 2.

U2A is required for spermatogonial differentiation in the Drosophila testis. (A) Western blot of testis lysates from control and U2A mutant flies confirms the absence of U2A protein (∼30 kDa). Exogenous expression of U2A in U2A¹; hs-U2A flies was induced by heat-shock treatment (U2A¹; hs-U2A^ON) until adulthood. In the absence of heat-shock treatment (U2A¹; hs-U2A^OFF), adults were converted to null mutants with complete absence of U2A protein in the testis. (B) In contrast to WT (w¹¹¹⁸) controls, seminal vesicles from mutant U2A¹ (U2A¹; hs-U2A^OFF) flies did not contain mature sperm. (Scale bar: 20 μm.) (C) Distribution of germ cells in the apex of the testis of control (w¹¹¹⁸) and mutant U2A¹ (U2A¹; hs-U2A^OFF) flies. Immunostaining using anti-Vasa (green), anti-Hts (red), and Hoechst 33342 (blue) (Upper; scale bar: 20 μm), and anti-pMAD and anti-Fas3 (Lower; scale bar: 20 μm). The arrow indicates a cyst with >16 cells. (D) Absence of spermatocyte cysts in the apical third of the testis of U2A mutant flies (U2A¹; hs-U2A^OFF). Shown is the percentage of testes not containing spermatocyte cysts in the apical third; 100 testes were assessed per genotype. Spermatocyte cysts were identified by Hoechst staining. (E) Distribution of spermatogonia (Bam⁺) and cells in S phase (EdU) in the apical tip of testes from w¹¹¹⁸ and U2A mutant flies (U2A¹; hs-U2A^OFF). (Scale bar: 20 μm.)

Fig. 3.

U2A is required for mei-p26 pre-mRNA processing during spermatogonial differentiation. (A) Immunostaining of WT (w¹¹¹⁸) and U2A mutant (U2A¹; hs-U2A^OFF) testis with anti–Mei-p26 reveals reduced levels in the mutant. (Scale bar: 20 μm.) (B) Real-time qPCR analysis of mei-p26 and ote mRNA levels in testes. Values were normalized to WT. w¹¹¹⁸ (blue); bam knockdown is shown in green; U2A¹/hs-U2A^OFF, in red. (C) Diagram of mei-p26 pre-mRNA fragments detected by PCR. Amplicons may include exon 6, exon 7, and intron 6–7 sequences. (Exon numbering refers to transcript RE; SI Appendix, Fig. S3A.) (D) Partially processed mei-p26 transcripts containing intron 6–7 sequences in U2A mutant and bam > U2A RNAi testis. (E) Simultaneous knockdown of U2A and mei-p26 causes a more severe phenotype compared with individual RNAi-mediated knockdown. Anti-Vasa is shown in green; anti-Hts, in red; and Hoechst 33342, in blue. (Scale bar: 20 μm.)

Fig. 4.

The major spliceosome plays a role during the transition from spermatogonial proliferation to differentiation in Drosophila. (A) Germ cell-specific RNAi-mediated knockdown of smb, U1-70K, sf3a1, sf3b5, prp8, and brr2 in adult flies phenocopies the U2A mutant. (Upper) Immunostaining of testes with anti-Vasa (green) and anti-Hts (red). (Lower) Counterstaining of nuclei in testes and seminal vesicles using Hoechst 33342. (Scale bar: 20 μm.) (B) Reduced level of processed mei-p26 mRNA in the testes of adult flies with knockdown of snRNP factors. The intronless ote transcript served as a control. (C) Partially processed mei-p26 transcripts containing intron 6–7 sequences were detected in testes of flies with knockdown of snRNP factors.

Fig. 5.

Human SNRPA1 restores spermatogonial development in Drosophila U2A mutant and knockdown flies. (A) Human SNRPA1 interacts with mei-p26 RNA in Drosophila S2 cells. Shown in qRT-PCR analysis of RNA isolated by immunoprecipitation of Flag-U2A (red), Flag-SNRPA1 (green), and Flag-GFP (blue). (B) Expression of hSNRPA1 rescues germ cell development in U2A knockdown flies. Shown is normal germ cell development in the testis apex of bam > U2A RNAi; bam > hSNRPA1 flies (Upper; anti-Vasa in green, anti-Hts in red, and Hoechst 33342 in blue) and the presence of mature sperm in the seminal vesicles (Lower; Hoechst 33342 staining). (Scale bar: 20 μm.) (C) Expression of hSNRPA1 restores mei-p26 mRNA processing in U2A knockdown flies. Incompletely spliced mei-p26 transcripts were present in bam > U2A RNAi flies, but not in bam > U2A RNAi; bam > hSNRPA1 flies. (D) Expression of hSNRPA1 restores spermatogenesis in U2A¹ null mutant flies. Shown is immunostaining of testis with anti-Vasa (green) and anti-Hts (red); nuclei were counterstained with Hoechst 33342. In U2A mutant flies (U2A¹; hs-U2A^OFF), the testes are filled with small spermatogonial cysts (Upper), and seminal vesicles lack mature sperm (Lower). In the presence of U2A or hSNRPA1, mutant flies undergo normal germ cell maturation (Upper) and produce mature sperm (Lower). (Scale bar: 20 μm.)

Fig. 6.

A human SNRPA1 missense mutation deregulates major spliceosome assembly and disrupts spermatogenesis dominantly. (A) Location of the mutated amino acids (shown in magenta) in the crystal structure of the spliceosomal U2B-hSNRPA1-hairpin IV of the U2 snRNA complex (Protein Data Bank ID code 1A9N). (B) Gel-retardation analysis of hU2B binding to 6-carboxyfluorescein (FAM)-labeled U2hpIV RNA in the presence of hSNRPA1 or hSNRPA1 missense mutants. (C) Overexpression of hSNRPA1 (Q148R) in germ cells, but not in somatic cells, phenocopies the U2A mutant. Staining was done with anti-Vasa (green), anti-Hts (red), and Hoechst 33342. (Scale bar: 20 μm.) (D) Incomplete processing of mei-p26 pre-mRNA in flies expressing hSNRPA1 (Q148R). Shown is qRT-PCR amplification of fragments containing intron 6–7 sequences (Fig. 3C).