Identification and characterization of testis-specific gene expressions in mouse tissues

2.1 Ethics statement and sample collection

The ethics committee of King Abdullah International Medical Research Center ethically approved this study under institutional review board reference No. IRB/1201/22 (study No. SP22R/076/04). Mature male and female SWR/J mice aged 10–12 weeks that weighed 30–35 g were obtained from the Experimental Animal Care Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. Tissues were isolated from the testis, brain, spinal cord, heart, spleen, liver, stomach, small intestine, colon, kidney, urinary bladder, and prostate of male mice and from the breast, ovary, and uterus of female mice.

2.2 RNA isolation and complementary DNA (cDNA) synthesis

Approximately 30 mg of each mouse tissue was used to isolate the total RNA using an All-Prep DNA/RNA Mini Kit (80204; Qiagen). A total of 2 μg of RNA was reverse transcribed into cDNA from each sample using a High-Capacity cDNA Reverse Transcription Kit (4368814; Applied Biosystems).

2.3 Primer design for reverse transcription PCR (RT-PCR)

Within the coding sequences (CDSs) of the selected genes (available at https://www.ncbi.nlm.nih.gov/), all RT-PCR primers were designed manually by following the optimal criteria, including a primer length of 20 nucleotides, 50% G-C content for each primer, intron-spanning primers, and designing the forward and reverse primers without intermolecular or intramolecular interactions. The sequences of all the designed primers are listed with their amplicon sizes in Table 1.

2.4 RT-PCR amplification and agarose gel electrophoresis

Each RT-PCR reaction for each gene contained 10 μL of BioMix Red (BIO-25006; BioLine), 8.4 µL of nuclease-free water, 0.8 µL of each forward and reverse primers (10 μM), and 0.8 µL of diluted cDNA. RT-PCR cycle setup and gel electrophoresis were performed as described in previous paper (Almutairi et al., 2022b). A mouse Actb was used to confirm the cDNA efficiency and the testis cDNA was used to determine the primer efficiency of all tested genes.

2.5 Sequence analysis of the RT-PCR products

The sequences of the RT-PCR amplification products were analyzed to confirm the specificity of the selected genes as described in previous work (Almutairi et al., 2022a).

2.6 Building a phylogenetic tree of the testis-specific genes

Initially, the amino acid sequences of the testis-specific genes in the different selected eutherian species were retrieved from the NCBI database. Multiple protein sequence alignments were then performed using the T-Coffee tool to determine the percent identity. The MEGA-11 online software (https://www.megasoftware.net/) was then used to generate phylogenetic trees and conduct evolutionary studies (Tamura et al., 2021).

2.7 Construction of the gene-gene interaction network

The testis-specific gene-gene interaction network and functional connections were constructed using algorithms from the GeneMANIA prediction service (University of Toronto, Toronto, Canada) to perform a network analysis of shared genes and forecast related genes (Warde-Farley et al., 2010).

2.8 Genotype-tissue expression (GTEx) database

The GTEx database (Carithers et al., 2015) was applied to validate the findings using publicly accessible data. By using the gene symbols or IDs of interest, the GTEx portal was queried, and the gene expressions were retrieved from the normalized transcripts per million (TPM) data as a heatmap. The heatmap was created using the R programming language and rendered with the heatmap package.

3.1 Expression profiles of the selected genes in the GenBank database

In the present study, 20 testis-restricted expression genes from the Embank database were selected: Adam29, Cabs1, Cpxcr1, Fthl17a, Sycp1, Caber, Stra8, Tex101, Odf3, Tex48, Ldhc, Odf1, Spata19, Tmprss12, Adad1, Dmrtc2, Luzp4, Prm2, Prss54, and Syce1. These genes were exclusively expressed in the adult testis and were not detected in any of the other 11 mouse tissues, as shown in Table 2.

3.2 RT-PCR expression profiles of the ubiquitously expressed genes in the different adult mouse tissues

The selected genes from the GenBank database that showed testis-specific expressions were then investigated the testis specificity using RT-PCR analysis and 15 different tissues isolated from adult mice. These tissues are described in Section 2.1. Our results showed that the Adam29, Cabs1, Cpxcr1, Fthl17a, and Sycp1 genes were expressed in all the tested tissues, including the testis (Fig. 1). The strengths of their expressions were facultative among the mouse tissues. In other words, their expressions differed in pace between the studied tissues, which means that both thick and faint bands were observed for each gene in the different mouse tissues. The Adam29, Cabs1, Cpxcr1, Fthl17a, and Sycp1 genes showed excessive expression not only in the testis but also in other tissues. For example, the expression of the Adam29 gene was stronger in 6 of the 14 tissues other than the testis. It was also expressed as fainter bands in the brain, spinal cord, heart, liver, stomach, colon, kidney, and breast tissues. Cabs1 gene expression was stronger in the spleen, small intestine, prostate, breast, ovary, and uterus tissues than in the brain, spinal cord, heart, liver, stomach, kidney, and urinary bladder tissues. For the Cpxcr1 gene, the strongest bands in the tissues other than the testis were observed in the spleen, prostate, and breast tissue samples, while the bands in the other tissue samples were relatively within the same range. The RT-PCR amplification results of the Fthl17a and Sycp1 genes showed that they were also expressed strongly in the spleen and small intestine tissues. Overall, the Adam29, Cabs1, Cpxcr1, Fthl17a, and Sycp1 genes are non-testis specific, as indicated in the GenBank database, because they were detected in all mouse tissues. Therefore, they should be reclassified as ubiquitously expressed genes according to their expression patterns observed in all types of examined tissues.

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Fig 1

RT-PCR expression profiles of the ubiquitously expressed genes in the different mouse tissues. The agarose gel images show the RT-PCR analysis results for the Adam29, Cabs1, Cpxcr1, Fthl17a, and Sycp1 genes. To the right of the agarose gel images are the official symbols for the genes, followed by the predicted sizes of the resulting products in brackets.

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3.3 RT-PCR expression profiles of the testis-predominant genes in the different adult mouse tissues

A total of 9 genes (Cabyr, Stra8, Tex101, Odf3, Tex48, Ldhc, Odf1, Spata19, and Tmprss12) were expressed strongly in the testis and weakly in other mouse tissues (Fig. 2). Thus, these genes cannot be considered as testis specific, as described in the GenBank database (Table 2). They were categorized as testis-predominant genes because their expressions were not observed in all tissues and showed strong expressions in the testis and weaker expressions in a few other mouse tissues. In detail, the gel electrophoresis result of the Cabyr gene showed a strong band in the testis and 3 faint bands in the brain, liver, and stomach. For the Stra8 gene in the testis cDNA, the anticipated RT-PCR product size was 724 bp (Table 1), but the PCR results in the stomach, small intestine, and urinary bladder revealed a band approximately 800 bp in size. However, this larger band based on the sequence analysis was related to the Stra8 gene (Table 3), which may indicate a new splice variant. Thus, the Stra8 gene was categorized as testis predominant. In the RT-PCR analysis for Tex101, a strong signal was observed in the testis and 4 weak signals were detected in the stomach, small intestine, kidney, urinary bladder, and prostate; however, no significant signal was observed in the other mice tissues. Moreover, the expression patterns of the Odf3 and Tex48 genes was found in 33% of the mouse tissues However, the strongest expressions of both genes were observed only in the testis. Likewise, Ldhc was expressed in 60% of the mouse tissues, but its expression was stronger in the testis. Furthermore, the detection frequency of the Spata19 and Odf1 genes was 60% and 53%, respectively in the mouse tissues. Spata19 was expressed in the testis, brain, heart, stomach, small intestine, kidney, urinary bladder, and prostate. However, Odf1 was expressed in the same tissues, except the brain. Tmprss12 was expressed in 60% of the mouse tissues.

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Fig. 2

RT-PCR expression profiles of the testis-predominant genes in the different mouse tissues. The agarose gel images show the RT-PCR analysis results for the Cabyr, Stra8, Tex101, Odf3, Tex48, Ldhc, Odf1, Spata19, and Tmprss12 genes. To the right of the agarose gel images are the official symbols for the genes, followed by the predicted sizes of the resulting products in brackets.

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To verify the RT-PCR findings, we used DNA sequence analysis on a subset of RT-PCR products for 4 testis-predominant genes. When compared with the reference sequences in the NCBI database using the BLAST program, the DNA sequencing results for the Cabyr, Odf3, Tex101, and Tex48 PCR products in the tissues showed sequence similarity to the Cabyr, Odf3, Tex101, and Tex48 genes (Table 3). Therefore, these results are consistent with the RT-PCR results, and these genes can no longer be considered testis specific and should be included among the testis-predominant genes in mice.

3.4 RT-PCR expression profiles of the testis-specific genes in the different adult mouse tissues

By contrast, the RT-PCR screening results of the last 6 of our selected genes indicated that the expression patterns of the Adad1, Dmrtc2, Luzp4, Prm2, Prss54, and Syce1 genes in the 15 different mouse tissues were restricted to the testis, and no indication of RT-PCR expression was found in the other tissue samples (Fig. 3). The RT-PCR analysis for Adad1 analysis in the small intestine and colon tissues revealed an unexpectedly large faint band (approximately 730 bp), compared with the anticipated band in the testis (664 bp). However, the sequence of this large band was not related to the Adad1 gene (Table 3). Moreover, the sequence results of the RT-PCR amplification product of Luzp4 confirmed that the unexpected small band of approximately 420 bp that appeared in the prostate and breast tissues was not related to the Luzp4 gene compared with the anticipated band in the testis (517 bp). The RT-PCR amplification results of the Prss54 gene showed that the small faint bands of the PCR products had an unexpected size of approximately 550 bp compared with the anticipated size of 764 bp. However, these bands were not related to the Prss54 gene according to the sequence outcome. Thus, this gene is testis specific because the anticipated band was only observed in the testicular tissue, and not in the other mouse tissues. Finally, the study findings regarding the testis specificity of the Adad1, Dmrtc2, Luzp4, Prm2, Prss54, and Syce1 genes are consistent with those reported in the NCBI database. In this study, by using the RT-PCR technique, the Adad1, Dmrtc2, Luzp4, Prm2, Prss54, and Syce1 genes were identified to be the only testis-specific genes among the 20 genes selected. Thus, these testis-specific genes could have potential usefulness in several medical applications such as the diagnosis of reproductive diseases.

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Fig. 3

RT-PCR expression profiles of the testis-specific genes in the different mouse tissues. The agarose gel images show the RT-PCR analysis results for the Adad1, Dmrtc2, Luzp4, Prm2, Prss54, and Syce1 genes. To the right of the agarose gel images are the official symbols for the genes, followed by the predicted sizes of the resulting products in brackets.

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3.5 Phylogenetic trees of the identified testis-specific genes

For phylogeny, multiple protein sequence alignments of different placental mammals for each individual testis-specific gene (Adad1, Dmrtc2, Luzp4, Prm2, Prss54, and Syce1) were applied by using ClustalW. The phylogenetic analysis of the testis-specific genes in the mouse tissues and their orthologs showed a high level of sequence resemblance, which implies that the proteins are conserved in mice and other various eutherian species. Moreover, the generated phylogenetic trees showed that the mouse and rat shared the closest evolutionary relationship among the testis-specific genes, indicating that they may have shared a common ancestor (Fig. 4).

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Fig. 4

Conservation of testis-specific genes in eutherians. Multiple protein sequence alignments were carried out with the T-Coffee program, and phylogenetic trees were built using the MEGA-11 software. The phylogenies of (A) Adad1, (B) Dmrtc2, (C) Luzp4, (D) Prss54, (E) Prm2, and (F) Syce1 are shown. The percent identity of each eutherian species sequence relative to the reference sequence (mouse:1) is shown in parentheses.

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3.6 Gene-gene interaction network of the identified testis-specific genes

The GeneMANIA program was used to construct the gene-gene interaction network to analyze the co-expression of the testis-specific genes that were overexpressed in the mouse testis but not in the other tissues and to identify the testis-specific genes that play critical roles in the spermatogenesis process (Fig. 5). The central network is the reference network, whereas the surrounding networks are representative of the testis-specific genes. Each network contained the core node that represented the testis-specific gene members and the 15 surrounding nodes that exemplified the most frequently adjacent genes that were strongly connected with the testis-specific genes. The testis-specific genes' interactions to their neighbors are shown in purple. The red circle indicates the spermatogenic gene that is only found in the testes.

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Fig. 5

GeneMANIA database-based network of gene-gene interactions for testis-specific genes. Each central node represents the testis-specific gene (shown as circles). The top 15 most frequent adjacent genes highly connected to testis-specific genes are shown. The edges represent the interactions between the testis-specific genes and other genes (shown as lines). Genes involved in spermatogenesis are colored red.

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The results of the bioinformatic analyses showed that the Prm2, Dmrtc2, and Adad1 genes of the 6 identified testis-specific genes are involved in spermatogenesis in mice, whereas the remaining 3 genes (Syce1, Prss54, and Luzp4) are not. Moreover, only Pdit among the 15 neighboring genes plays a role in spermatogenesis. Regarding the functional testis-specific genes in spermatogenesis, GeneMANIA revealed that Prm2 was co-expressed only with the Prss54 of the characterized testis-specific genes and was co-expressed with 10 of the 15 adjacent genes, including Pdit, Spz1, Nt5c1b, Odf1, Akap4, 1700113H08Rik, Cetn1, Spes1, 1700012A03Rik, and Spact1l (Fig. 5, top left). The Dmrtc2 gene was highlighted to be co-expressed with two testis-specific genes, namely Prss54 and Adad1, and 10 neighboring genes, namely Pdit, odf1, 1700113H08Rik, Spdya, G6pd2, Fam47c, Cetn1, Spam1, 1700012A03Rik, and Spact1l (Fig. 5, top right). Moreover, the co-expression network of the Adad1 gene (Fig. 5, top middle) showed that the gene-gene interaction between Adad1 and 2 testis-specific genes (Dmrtc2 and Syce1), and between Adad1 and 11 other genes (Tmbim7, Spz1, Nt5c1b, Odf1, Akap4, 1700113H08Rik, Spdya, Fam47c, Spesp1, Spam1, and 1700012A03Rik).

The GeneMANIA analysis revealed that the Syce1 gene is co-exprressed with the Press54 and Adad1 testis-specific genes and with other neighboring genes, namely Tmbim7, Spz1, Nt5c1b, Akap4, Spdya, G6pd2, Fam47c, Spam1, and 1700012A03Rik (Fig. 5, bottom left). Furthermore, the interconnected network of Prss54 confirmed that it was the most testis-specific gene with co-expression correlation with 3 testis-specific genes (Prm2, Syce1, and Dmrtc2) and 13 adjacent genes (Pdit, Tmbim7, Spz1, Nt5c1b, Odf1, Akap4, 1700113H08Rik, G6pd2, Cetn1, Spesp1, Spam1, 1700012A03Rik, and Spact1l) (Fig. 5, bottom right). However, the last testis-specific gene identified (Luzp4) did not form a co-expression network with the other testis-specific genes and the other 15 neighboring genes (Fig. 5, bottom middle). Thus, no gene-gene interaction was found between Luzp4 and the other genes according to the GeneMANIA database.