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.2020 Aug 19;18(1):103.
doi: 10.1186/s12915-020-00826-z.

Large-scale discovery of male reproductive tract-specific genes through analysis of RNA-seq datasets

Affiliations

Large-scale discovery of male reproductive tract-specific genes through analysis of RNA-seq datasets

Matthew J Robertson et al. BMC Biol..

Abstract

Background: The development of a safe, effective, reversible, non-hormonal contraceptive method for men has been an ongoing effort for the past few decades. However, despite significant progress on elucidating the function of key proteins involved in reproduction, understanding male reproductive physiology is limited by incomplete information on the genes expressed in reproductive tissues, and no contraceptive targets have so far reached clinical trials. To advance product development, further identification of novel reproductive tract-specific genes leading to potentially druggable protein targets is imperative.

Results: In this study, we expand on previous single tissue, single species studies by integrating analysis of publicly available human and mouse RNA-seq datasets whose initial published purpose was not focused on identifying male reproductive tract-specific targets. We also incorporate analysis of additional newly acquired human and mouse testis and epididymis samples to increase the number of targets identified. We detected a combined total of 1178 genes for which no previous evidence of male reproductive tract-specific expression was annotated, many of which are potentially druggable targets. Through RT-PCR, we confirmed the reproductive tract-specific expression of 51 novel orthologous human and mouse genes without a reported mouse model. Of these, we ablated four epididymis-specific genes (Spint3, Spint4, Spint5, and Ces5a) and two testis-specific genes (Pp2d1 and Saxo1) in individual or double knockout mice generated through the CRISPR/Cas9 system. Our results validate a functional requirement for Spint4/5 and Ces5a in male mouse fertility, while demonstrating that Spint3, Pp2d1, and Saxo1 are each individually dispensable for male mouse fertility.

Conclusions: Our work provides a plethora of novel testis- and epididymis-specific genes and elucidates the functional requirement of several of these genes, which is essential towards understanding the etiology of male infertility and the development of male contraceptives.

Keywords: Contraception; Drug target; Male reproductive tract; Paralog; Sperm maturation; Spermatid; Spermatozoa.

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Conflict of interest statement

All authors have no competing interests.

Figures

Fig. 1
Fig. 1
Summary of the human and mouse RNA-seq samples used in the identification of novel male reproductive tract-specific drug targets. The RNA-seq samples used in the human (a) and mouse (b) analyses are schematically shown. Principal component analysis was performed on the human (c) and mouse (d) non-reproductive and reproductive samples separately. The colors of the circles next to the tissues listed ina andb correspond to the colors used in the circles for the PCA inc andd. Sample size (N) values in red and/or black denote the number of new (red) and previously published (black) samples included in our analysis.
Fig. 2
Fig. 2
Identification of candidate drug male reproductive gene targets.a Diagrammatic representation of overall methodology used to identify reproductive tract-specific candidate genes in humans (720 genes) and in mice (1062 genes). The maximum gene expression was determined across all the non-reproductive tissue samples for each gene for a reproductive tissue or cell sample of interest. Genes were then filtered for significance using a false discovery rate (FDR) of less than or equal to 0.05 based on the differential gene expression analysis for the non-reproductive tissue with maximum gene expression and reproductive tissue or cell sample of interest. Genes that passed the FDR filter were filtered such that the average TPM expression value of the maximum expressing non-reproductive tissue was less than or equal to 1.0 TPM and the average TPM expression value of the reproductive tissue or cell of interest was greater than or equal to 10.0 TPM.b Diagrammatic representation of the number of human and mouse candidate genes in terms of (1) the number of orthologs in the opposite species, (2) the number of genes previously or not previously identified in a prior transcriptomics-based drug target report, (3) the availability and phenotypic outcome of any reported mouse models, and (4) the number of novel genes without a reported mouse model congruent across both species. The main value in each bubble represents the total number of candidate genes identified regardless of tissue or cell identified in. The numbers in parentheses comprise the total number of candidate genes that are either epididymis-specific or specific to testis and epididymis, but not testis and/or testis cell-specific only.
Fig. 3
Fig. 3
Two hundred and thirty-three novel human reproductive tract-specific genes that each have mouse orthologous genes but with no reported knockout mouse models. The listed genes were identified in one or more datasets as indicated in the Venn diagram. Underlined genes were also identified in our studies as reproductive tract-specific in mouse (109 genes). Genes written in blue encode either enzymes, kinases, GPCRs, oGPCRs, transporters, transcription factors, or proteins involved in epigenetic regulation (74 genes). Genes written in dark red were identified in both testis (testis and/or testis cell) and epididymis (10 genes).
Fig. 4
Fig. 4
Representative novel reproductive tract-specific human (a) and mouse (b) genes without a reported mouse model. The listed genes were identified through our studies as reproductive tract-specific in both humans and mice. The digital PCR (heatmap) depicts the average transcripts per million (TPM) value per tissue per gene from the indicated human and mouse RNA-seq datasets as processed in parallel through our bioinformatics pipeline. The data was obtained from 264 published and newly acquired datasets. White = 0 TPM, Black ≥ 30 TPM. The expression profile of the human and mouse housekeeping genes,GAPDH andEif3l, is included as reference. For data obtained from published datasets, superscript values succeeding the sample names reference the publications as follows: 1 (Djureinovic et al. [9]; Fagerberg et al. [30]), 2 (Guo et al. [24]), 3 (Zhu et al. [23]), 4 (Kumar et al. [26]), 5 (Browne et al. [21]), 6 (Consortium et al. [19]), 7 (Helsel et al. [25]), 8 (da Cruz et al. [22]), and 9 (Zimmermann et al. [20]).
Fig. 5
Fig. 5
RT-PCR confirmation of reproductive tract specificity in both humans (a) and mice (b). The genes listed in this figure are novel as identified through our studies and without a reported mouse model. Humans do not have an equivalent protein-coding equivalent to mouseSpint5.GAPDH andHprt are included as housekeeping genes.
Fig. 6
Fig. 6
Three hundred and two novel mouse genes with human orthologs without a reported mouse model. The listed genes were identified in one or more mouse datasets as indicated in the Venn diagram. Underlined genes were also identified through our studies as reproductive tract-specific in human (111 genes). Genes written in blue encode either enzymes, kinases, GPCRs, oGPCRs, transporters, transcription factors, or proteins involved in epigenetic regulation (60 genes). Genes written in dark red were identified in both testis (testis and/or testis cell) and epididymis (14 genes).
Fig. 7
Fig. 7
Novel reproductive tract-specific mouse genes with human reproductive tract enrichment, without a reported mouse model. The digital PCR (heatmap) depicts the average transcripts per million (TPM) value per tissue per gene from the indicated human (a) and mouse (b) RNA-seq datasets. The data was obtained from 264 published and newly acquired datasets. White = 0 TPM, Black ≥ 30 TPM. The expression profile of the human and mouse housekeeping genes,GAPDH andEif3l, is included as reference. For data obtained from published datasets, superscript values are as previously mentioned.
Fig. 8
Fig. 8
Generation of knockout mice for functional validation.a–e Genomic structure and knockout strategy for mouseSpint3 (a),Spint4/5 (b),Ces5a (c),Pp2d1 (d), andSaxo1 (e). Single or dual sgRNAs were designed to target the indicated exons in each gene. Each founder animal’s deletion mutation is indicated with red hash lines. For all butSpint4/5, F1 and R1 are wild-type primers and F2 and R2 are mutant primers. ForSpint4/5, F1 ofSpint4 was combined with R2 ofSpint5 to detect the mutant allele.f–j Representative Sanger sequence result of mutant mice depicted ina–e, respectively.k–o Representative genotype result of mutant mice using the indicated primers ina–e, respectively.
Fig. 9
Fig. 9
Phenotype analysis of CRISPR/Cas9 generated null mice for determining the contraceptive potential of the selected genes.Spint4/5 andCes5a null mice show significant fertility defects; meanwhile,Spint3,Pp2d1, andSaxo1 null mice appear normal. Fertility (a–c), body and reproductive organ weights (d–f), and sperm parameters (g–i) were all measured between knockout (−/−) and littermate control [wild-type (+/+) and heterozygous (+/−)] mice as indicated. Bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.005. ns, not significant.
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