Whole transcriptome sequencing of testis and epididymis reveals genes associated with sperm development in roosters

Abstract

Background

Chickens play a crucial role as the primary global source of eggs and poultry, and the quality of rooster semen significantly impacts poultry reproductive efficiency. Therefore, it is imperative to comprehend the regulatory mechanisms underlying sperm development.

Results

In this study, we established transcriptome profiles of lncRNAs, miRNAs, and mRNAs in 3 testis tissues and 3 epididymis tissues from “Jing Hong No.1” roosters at 24, 35, and 64 weeks of age. Using the data, we conducted whole transcriptome analysis and constructed a ceRNA network. We detected 10 differentially expressed mRNAs (DEmRNAs), 33 differentially expressed lncRNAs (DElncRNAs), and 10 differentially expressed miRNAs (DEmiRNAs) in the testis, as well as 149 DEmRNAs, 12 DElncRNAs, and 10 DEmiRNAs in the epididymis. These genes were found to be involved in cell differentiation and development, as well as various signaling pathways such as GnRH, MAPK, TGF-β, mTOR, VEGF, and calcium ion pathways. Subsequently, we constructed two competing endogenous RNA (ceRNA) networks comprising DEmRNAs, DElncRNAs, and DEmiRNAs. Furthermore, we identified four crucial lncRNA-mRNA-miRNA interactions that govern specific biological processes in the chicken reproductive system: MSTRG.2423.1-gga-miR-1563-PPP3CA and MSTRG.10064.2-gga-miR-32-5p-GPR12 regulating sperm motility in the testis; MSTRG.152556.1-gga-miR-9-3p-GREM1/THYN1 governing immunomodulation in the epididymis; and MSTRG.124708.1-gga-miR-375-NDUFB9/YBX1 controlling epididymal sperm maturation and motility.

Conclusions

Whole transcriptome sequencing of chicken testis and epididymis screened several key genes and ceRNA regulatory networks, which may be involved in the regulation of epididymal immunity, spermatogenesis and sperm viability through the pathways of MAPK, TGF-β, mTOR, and calcium ion. These findings contribute to our comprehensive understanding of the intricate molecular processes underlying rooster spermatogenesis, maturation and motility.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-024-10836-8.

Introduction

The quality of semen directly impacts male fertility, with sperm development playing a crucial role in determining semen quality. The testis serves as the primary site for the origin and development of spermatozoa [1], while the epididymis plays a pivotal role in facilitating their transportation, maturation, and storage within the reproductive system [2]. Notably, although avian sperm exhibit low motility in the testis, their viability significantly increases upon reaching the epididymis [3].

Whole-transcriptome sequencing enables the identification and quantification of RNA molecules within a biological sample, making it a valuable technique for investigating gene expression patterns and discovering new RNA transcripts. Numerous studies have demonstrated the pivotal role of mRNA in regulating sperm motility through processes such as PI3K-PKA signaling [4], steroid hormone biosynthesis [5], and oxidative phosphorylation [6]. DNAH5 is an essential protein involved in sperm movement [7], and mutations in its genetic sequence can result in complete loss of sperm motility [8]. The genes MMP1, SLN, WT1, PLIN1, and LRRIQ146 are likely to play crucial roles in governing epididymal sperm motility in male chickens [9]. MiRNAs are small, single-stranded, non-coding RNA molecules in length from 21 to 25 nucleotides. During germ cell development, miR-31 regulates the expression of Stra8, thereby controlling meiosis [10]. MiR-26a modulates PDHX, a target gene involved in the sugar metabolism pathway, impacting porcine spermatozoa viability [11]. Additionally, miR-205 serves as a marker for human azoospermia [12, 13]. LncRNAs, which are non-coding nucleotides exceeding 200 bases in length, exert regulatory effects on sperm motility through their influence on testicular development [14]as well as flagellar function and structure [15]. A comprehensive transcriptome analysis of chicken testes revealed that the network involving MSTRG.3077.3/MSTRG.9085.1-gga-miR-138-5p-CADM1 and MSTRG.2290.1-gga-miR-142-3p-GNAQ/PPP3CA plays a pivotal role in governing sperm motility regulation [16].

The genetic regulation of reproductive and productive performance in chickens has been extensively investigated. Studies have demonstrated the involvement of genes in skeletal muscle regulation. For instance, TMEM182 gene has been identified as a suppressor of skeletal muscle growth and development [17], while miR-460b-5p has been shown to enhance the proliferation and differentiation of adult chicken muscle cells by targeting RBM19 [18]. ACE, CALM1, and DRD1 are three pivotal genes associated with enhancing chicken egg production [19–21]. Although current research primarily focuses on regulating genes related to chicken reproductive and production performance, there is a dearth of studies concerning chicken sperm development.

The JingHong No.1 breeder rooster exhibits exceptional production performance, high fertilisation rate, and good environmental adaptability. Reproductive performance of breeder roosters follows an age-dependent pattern, with sexual maturity achieved at 20 weeks and reaching its peak around 30 weeks [22]. In this study, the developmental stage (24 weeks), maturity stage (35 weeks), and decline stage (64 weeks) were considered for selecting the JingHong No. 1 breeder roosters as test subjects. Then, the expression profiles of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) were evaluated and compared at distinct developmental stages in testicular and epididymal tissues derived from Jing Hong No. 1 breeder roosters using whole transcriptome sequencing. Subsequently, the differentially expressed mRNAs (DEmRNAs), lncRNAs (DElncRNAs), and mRNAs (DEmRNAs) were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Based on these findings, our objective was to construct a competing endogenous RNA regulatory network identify genes, miRNAs, and lncRNAs associated with the testis and epididymis development as well as sperm quality in roosters.

Materials and methods

Experimental animals and sample preparation

Seventy purebred JingHong NO.1 roosters, provided by Beijing Huadu Group Co., were housed under standardized conditions with ad libitum access to food and water, following a 16-hour light and 8-hour dark cycle. The diets and nutritional formulas used in the study are presented in Additional file 1: Table S1. At 24, 35, and 64 weeks of age, semen quality was measured and recorded at different weeks of age (Additional file 2: Table S2) and three chickens were randomly selected for euthanasia. The chickens were anaesthetised with an intravenous injection of 0.2% sodium pentobarbital (40 mg/kg), and the carotid artery was severed and bled to death under deep anaesthesia. Testis and epididymis tissues were collected, rapidly frozen and stored in liquid nitrogen.

Semen quality measurement and analysis

The collected sperm was immediately analyzed using the Weili Color Sperm Quality Testing System (WLJY-9000, Beijing Weili New Century Science & Technology Development Co., Beijing, China). Statistical Product and Service Solutions (SPSS) (v24.0) was used to conduct comparative analyses of sperm motility, volume, density, and effective count between different development stages.

RNA extraction, library construction, and sequencing

RNA was extracted from the testis and epididymis using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The quality and concentration of RNA were accessed using the NanoDrop 2000 spectrophotometer (Implen, Westlake Village, CA, USA) and the Agilent 2100 RNA 6000 Pico kit (Agilent, Waldbronn, Germany). For mRNA and lncRNA sequencing, rRNA-depleted RNA was prepared from each sample with 5 µg of total RNA. VAHTS mRNA-seq v2 Library Prep Kit (Vazyme NR611-02) (NEB, Ipswich, MA, USA) was used to generate 18 libraries, which then sequenced on Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA), employing a paired-end protocol with read length of 150 bp. Small RNA library preparation involved use of Illumina NEBNext Multiplex Small RNA Library Prep Set (NEB) with 3 µg total RNA per sample followed by sequencing on the Illumina Hiseq X-ten platform (Illumina), utilizing a paired-end protocol with read length of 50 bp.

Quality assessment, alignment optimization, and transcriptome reconstruction of RNA-seq data

The raw RNA-seq reads underwent trimming using Fastp v0.39 [23] with default parameters. Subsequently, they were aligned to the reference chicken genome bGalGal1.mat.broiler. GRCg7b (https://www.ncbi.nlm.nih.gov/data-hub/genome/GCF_016699485.2/ ) using HISAT2 v2.2.0 [24] with default settings. The resulting SAM files from the alignment process were converted to BAM format using samtools v1.1.0 [25]. The aligned reads were then individually assembled against the reference genome using StringTie v2.1.7 [26]. Finally, all transcripts were merged together using StringTie v2.2.1 to generate a comprehensive dataset.

Identification and enrichment analyses of DEmRNAs

The quantification of protein-coding genes was performed using HTseq-count v2.0.2 [27]. Differential expression analysis of various developmental stages in the testis and epididymis was conducted using edgeR v3.36.0 [28] in R, with differentially expressed genes identified by applying a significance threshold at FDR < 0.05 and an absolute fold Change > 2. Enrichment analyses of the identified DEmRNAs were carried out using the ClusterProfiler package v4.2.2 [29] in R, incorporating GO [30] and KEGG [31].

Identification, prediction and functional enrichment analysis of DElncRNAs

During the identification of lncRNAs, we employed the GFFcompare tool v0.12.6 [32] to annotate the chicken reference genome with the assembled transcripts. Subsequently, transcripts that were shorter than 200 base pairs, had fewer than two exons, and exhibited low expression with less than three reads were excluded. Finally, the output transcripts were categorized based on class_code = “i""u""x” and identified using CPC2 v2.0 (Coding Potential Calculator 2) [33], CNCI v2.0 (Coding-Non-Coding Index) [34], and PLEK v1.2 (Predictor of Long non-coding RNAs and Messenger RNAs based on an Improved K-mer Scheme) [35]. The CPC2, CNCI, and PLEK are widely used for transcript coding capacity prediction.

The expression of lncRNAs was quantified using FeayureCounts v2.0.1 [36], and differential analysis of lncRNAs in testis and epididymal tissues across different developmental stages was conducted separately. The significance threshold was set at FDR < 0.05, with a fold change > 2 considered significant. Potential cis- and trans-targeting mRNAs of the DElncRNAs were predicted based on their chromosomal locations and gene expression levels. Subsequently, GO and KEGG enrichment analyses were performed on the target mRNAs using the ClusterProfiler package v4.2.2 in R.

Identification and target gene prediction of DEmiRNAs and functional enrichment analysis

The clean reads of miRNA were aligned to the chicken reference genome using Bowtie2 v2.3.5.1 [37]. The miRNA read counts were then quantified with Hetseq-count v2.0.2 [27]. Differential expression analysis of miRNAs between testis and epididymis at different developmental stages was cconducted using edgeR v3.36.0 [28], where significance thresholds were set at FDR < 0.05 and an absolute value of Fold Change > 2. The predicted target genes of DEmiRNAs were obtained using MirDB v6.0 [38] and Targectscan v8.0 [39]. Finally, GO term and KEGG pathway enrichment analyses for the target mRNAs were performed using ClusterProfiler v4.2.2 in R.

Construction of the lncRNA-miRNA-mRNA regulatory network

We constructed a competitive endogenous RNA (ceRNA) network involving lncRNAs, miRNAs, and mRNA through the following steps: (1) The interactions between lncRNAs and miRNAs were assessed using miranda v3.3a [40]. (2) Both miRDB and TargetScan v8.0 were employed to predict the associations between miRNAs and mRNAs. (3) By utilizing Cytoscape v3.9 [41], we identified overlapping DElncRNAs and DEmiRNAs to generate a comprehensive ceRNA network encompassing lncRNA-miRNA-mRNA interactions.

Real-time PCR analysis of DEmRNAs, DElncRNAs, and DEmiRNAs

The RNA-Seq results were validated using real-time PCR (qPCR). Following the manufacturer’s protocol, 1000 ng of total RNA was reverse transcribed using the GoScrip Reverse Transcription System kit (Promega, Madison, Wisconsin, USA). Individual qPCR reactions were performed using TransStart Top Green qPCR SuperMix (Trans, Beijing, China) and AriaMx Real-Time PCR (Agilent, Waldbronn, Germany). The reaction mixture volume was 20 µL consisting of 1.2 µL cDNA, 0.8 µL for each primer, 10 µL Mix solution, and 8 µL sterile water. The qPCR cycling conditions included an initial denaturation at 94 °C for 30 s; followed by amplification at 94 °C for 5 s and annealing/extension at 60 °C for 30 s for 40 cycles; finally concluding with a melting curve analysis from 65 °C to 95 °C. GAPDH served as the endogenous control. HNRNPAB, SPP1, PIPOX, HMGN1, THYN1, and MSTRG.152276.1 were randomly selected for qPCR assays, and the primers were designed and synthesized by Shanghai Bioengineering Co (https://www.sangon.com) (Additional file 3: Table S3). The quantitative results were analyzed using the 2^−ΔΔCt method [42].

Results

Analysis of RNA-Seq data

In this study, a total of 18 cDNA libraries were produced. The transcriptomes of the testis and epididymis generated approximately one billion (with an average of 55,792,587) and one billion fifty million (with an average of 58,703,529) high-quality reads, respectively. The GC content and Q30 percentages of the testis transcriptomes ranged from 45.56 to 53.50% and from 92.99 to 96.84%, respectively. Conversely, for the epididymis transcriptomes, the GC content and Q30 percentages were in the range of 45.59–54.16% and from 92.15 to 97.26%, respectively (Additional file 4: Table S4, Additional file 5: Table S5). Furthermore, over 90% of the clean data obtained from both testicular and epididymal tissues aligned with the reference genome.

Differentially expressed lncRNAs associated with spermatogenesis

The study compared the results of CNCI, CPC2, and PLEK for the identification of lncRNAs in testis and epididymis RNA samples, resulting in a total of 4869 and 3097 lncRNAs detected in testis and epididymal tissues, respectively (Fig. 1A and B). Differential expression analysis revealed a total of 26 DElncRNAs in testis tissues, with 21 up-regulated and 5 down-regulated lncRNAs observed in T_24W vs. T_35W comparison. Similarly, in T_35w vs. T_64w comparison, we identified 15 DElncRNAs consisting of 12 up-regulated and 3 down-regulated lncRNAs (Fig. 1C and D, Additional file 6: Table S6). In the epididymis tissues, 6 DElncRNAs were found to be differentially expressed between E_24W and E_35W samples; among them 5 were up-regulated while 1 was down-regulated lncRNAs. Furthermore, 10 DElncRNAs were detected when comparing E_35W vs. E_64W samples, with 5 being up-regulated and 5 being down-regulated (Fig. 1E and F, Additional file 7: Table S7). Notably, 8 DElncRNAs exhibited co-expression patterns in testis tissues whereas 4 DElncRNAs showed co-expression patterns specifically in epididymis tissues (Fig. 1G and H).

Fig. 1.

Identification and differential analysis of lncRNAs. (A) The numbers of lncRNAs detected by CNCI, CPC2, and PLEK software in testis. (B) The numbers of lncRNAs detected by CNCI, CPC2, and PLEK software in epididymis. (C) Volcano plot showing the differential expression of lncRNAs in T_24w vs. T_35w. (D) Volcano plot showing the differential expression of lncRNAs in T_35w vs. T_64w. (E) Volcano plot showing the differential expression of lncRNAs in E_24w vs. E _35w. (F) Volcano plot showing the differential expression of lncRNAs in E _35w vs. E _64w. (G) The co-expressed DElncRNAs in testis. (H) The co-expressed DElncRNAs in epididymis

To elucidate the functional roles of these DElncRNAs, further investigation was conducted resulting in the identification of potential target genes: namely 287 target genes for testicular DElncRNA candidates and 589 target genes for epididymal DElncRNAs candidates. The GO enrichment analysis, along with KEGG pathway analysis, revealed several significant GO terms and KEGG pathways associated with testis tissues, such as dendrite morphogenesis, positive regulation of cell differentiation, maturation performance, epididymal cell differentiation, motor properties, microtubule binding, microtubule protein binding, Wnt signaling pathway, and calcium signaling pathway. Additionally, the epididymis exhibited enrichment in various key biological processes, including positive regulation of the Wnt signaling pathway, chromatin assembly or disassembly, ribosomes, oxidative phosphorylation, apoptosis, and tight junctions (Fig. 2A and D). Among these, oxidative phosphorylation and Wnt signalling pathways are involved in the regulation of epididymal sperm maturation and sperm viability. Calcium signalling pathways are involved in the regulation of testicular immunity.

Fig. 2.

Enrichment analysis of DElncRNAs. (A) GO enrichment analysis of DElncRNAs in T_24w vs. T_35w. (B) GO enrichment analysis of DElncRNAs in T_35w vs. T_64w. (C) GO enrichment analysis of DElncRNAs in E_24w vs. E_35w. (D) GO enrichment analysis of DElncRNAs in E_35w vs. E_64w

Differentially expressed mRNAs associated with spermatogenesis

A total of 23,382 and 16,402 mRNAs were identified in the testis and epididymis, respectively. In the testis, we identified 68 DEmRNAs in T_24W vs. T_35W comparison, with 37 up-regulated and 31 down-regulated mRNAs. In T_35W vs. T_64W comparison, a total of 64 DEmRNAs were found, with 41 up-regulated and 23 down-regulated mRNAs (Fig. 3A and B, Additional file 8: Table S8). In the epididymis, we discovered a set of 106 DEmRNAs in E_24W vs. E_35W comparison, including 84 up-regulated and 22 down-regulated mRNAs. Additionally, a total of 80 DEmRNAs were found to be differentially expressed at 35 weeks of age in the comparison of E_35W with E_64W, including 73 up-regulated mRNAs and 7 down-regulated mRNAs (Fig. 3C and D, Additional file 9: Table S9). Notably, 22 DEmRNAs were co-expressed throughout all stages of testis development, while 37 DEmRNAs showed consistent expression across all stages of epididymal development, respectively (Fig. 3E and F). GNL3, HADH, HERC4, CAS8, LHX3, MYL6, RPL14, RPS27A and AvBD7 are involved in the regulation of spermatogenesis, sperm maturation, and sperm motility. The clustering analyses demonstrated a high degree reproducibility among the DEmRNA within the testis and epididymis tissues, while revealing substantial differences between the groups (Fig. 3G and H).

Fig. 3.

Identification and analysis of differentially expressed mRNAs. (A) Volcano plot showing the differential expression of mRNAs in T_24w vs. T_35w. (B) Volcano plot showing the differential expression of mRNAs in T_35w vs. T_64w. (C) Volcano plot showing the differential expression of mRNAs in E_24w vs. E _35w. (D) Volcano plot showing the differential expression of mRNAs in E _35w vs. E _64w. (E) The co-expressed DEmRNAs in testis. (F) The co-expressed DEmRNAs in epididymis. (G) Heatmap of all DEmRNAs expression in testis. (H) Heatmap of all DEmRNAs expression in epididymis

To gain insight into the biological functions of DEmRNAs, we performed GO and KEGG analyses. The results revealed significant enrichment of genes involved in activation of MAPK activity, cAMP-mediated signaling, positive regulation of developmental growth, male gonad development, as well as signaling pathways for fatty acid degradation, VEGF signaling pathway, ErbB signaling pathway, GnRH signaling pathway, and calcium signaling pathway in the testis. In contrast, in the epididymis, the enriched GO terms and KEGG pathways were primarily associated with defense response to bacteria, oxidative phosphorylation, negative regulation of apoptotic process, translation, Ribosome, Phagosome, beta-Alanine metabolism, and mTOR signaling pathway (Fig. 4A and H). Among these, the MAPK pathway, the mTOR pathway and the oxidative phosphorylation pathway are involved in the regulation of spermatogenesis, immunity and sperm viability.

Fig. 4.

(A) GO enrichment analysis of DEmRNAs in T_24w vs. T_35w. (B) GO enrichment analysis of DEmRNAs in T_35w vs. T_64w. (C) KEGG enrichment analysis of DEmRNAs in T_24w vs. T_35w. (D) KEGG enrichment analysis of DEmRNAs in T_35w vs. T_64w. (E) GO enrichment analysis of DEmRNAs in E_24w vs. E_35w. (F) GO enrichment analysis of DEmRNAs in E_35w vs. E_64w. (G) KEGG enrichment analysis of DEmRNAs in E_24w vs. E_35w. (H) KEGG enrichment analysis of DEmRNAs in E_35w vs. E_64w

Differentially expressed miRNA associated with spermatogenesis

In the testis and epididymis, a total of 594 and 769 miRNAs were detected, with the majority falling within the 21–27 nt range (Fig. 5A and B). In the testis, we identified 7 DEmiRNAs, with 5 up-regulated and 2 down-regulated in T_24W vs. T_35W comparison. Furthermore, 4 DEmiRNAs were identified in T_35W vs. T_64W comparison, with 1 up-regulated and 3 down-regulated (Fig. 5C and D, Additional file 10: Table S10). In the epididymis, 7 miRNAs were identified as differentially expressed in the E_24W vs. E_35W and T_35W vs. T_64W groups, respectively. All of these miRNAs exhibited down-regulation at 35 weeks of age (Fig. 5E and F, Additional file 11: Table S11). Interestingly, 1 DEmiRNAs and 4 DEmiRNAs were found to be co-expressed throughout all stages of testis and epididymis development, respectively (Fig. 5G and H). miR-142-3p, miR-137-3p, miR-10b-5p, miR-146-5p and miR-451 are involved in the regulation of spermatogenesis, sperm motility, and sperm maturation.

Fig. 5.

Identification and analysis of differentially expressed miRNAs. (A) Length distribution of small RNA reads in testis. (B) Length distribution of small RNA reads in epididymis. (C) Volcano plot showing the differential expression of miRNAs in T_24w vs. T_35w. (D) Volcano plot showing the differential expression of miRNAs in T_35w vs. T_64w. (E) Volcano plot showing the differential expression of miRNAs in E_24w vs. E _35w. (F) Volcano plot showing the differential expression of miRNAs in E _35w vs. E _64w. (G) The co-expressed DEmiRNAs in testis. (H) The co-expressed DEmiRNAs in epididymis

We predicted that these DEmiRNAs potentially regulate a total of 1390 in the testis and an additional set of 5315 target genes in the epididymis. Our analysis revealed that these target genes were mainly involved in biological processes such as negative regulation of canonical Wnt signaling pathway, RNA biosynthetic process, tissue development, cell differentiation, MAPK signalling pathway, Wnt signalling pathway, TGF-beta signalling pathway, mTOR signalling pathway, ErbB signalling pathway, and FoxO signalling pathway within the testicular tissue. Additionally, in case of epididymal tissue, the enrichment was observed for several key biological processes including regulation of cellular biosynthetic process, epithelial cell differentiation, male gonad development, cellular developmental process, Calcium signaling pathway, Tight junction, MAPK signaling pathway, Wnt signaling pathway, GnRH signaling pathway, and Regulation of actin cytoskeleton (Fig. 6A and H). The mTOR and TGF-beta signalling pathways are involved in the process of immune regulation. The MAPK pathway is involved in the regulation of spermatogenesis. Wnt signalling, actin cytoskeleton regulation and calcium signalling are involved in the regulation of sperm motility, and spermatid maturation.

Fig. 6.

(A) GO enrichment analysis of DEmiRNAs in T_24w vs. T_35w. (B) GO enrichment analysis of DEmiRNAs in T_35w vs. T_64w. (C) KEGG enrichment analysis of DEmiRNAs in T_24w vs. T_35w. (D) KEGG enrichment analysis of DEmiRNAs in T_35w vs. T_64w. (E) GO enrichment analysis of DEmiRNAs in E_24w vs. E_35w. (F) GO enrichment analysis of DEmiRNAs in E_35w vs. E_64w. (G) KEGG enrichment analysis of DEmiRNAs in E_24w vs. E_35w. (H) KEGG enrichment analysis of DEmiRNAs in E_35w vs. E_64w

Validation of RNA-Seq results

To ensure the accuracy of the RNA-Seq results, HNRNPAB, SPP1, PIPOX, HMGN1, THYN1, and MSTRG.152276.1 were randomly selected for subsequent qPCR validation analyses in this study. The expression patterns of all selected genes were found to be concordant between RNA-Seq and qPCR results (Fig. 7).

Fig. 7.

Validation of RNA-seq data using qPCR

Construction and analysis of ceRNAs regulatory network

Based on the regulatory relationship between DEmiRNA-DEmRNA and DEmiRNA-DElncRNA, we identified significantly differentially expressed lncRNAs and mRNAs that were co-regulated by the same miRNAs. In the testis, we discovered 8 lncRNA-miRNA-mRNA interactions involving 3 lncRNAs, 5 mRNAs, and 3 miRNAs (Fig. 8A and C). Additionally, in the epididymis, we found 6 lncRNA-miRNA-mRNA interactions comprising of 3 lncRNAs, 6 mRNAs, and 3 miRNAs (Fig. 8B and D). MSTRG.2423.1-gga-miR-1563-PPP3CA, MSTRG.10064.2-gga-miR-32-5p-GPR12, MSTRG.124708.1-gga-miR-375-NDUFB9/YBX1, MSTRG.152556.1-gga-miR-9-3p-GREM1/THYN1 has an important regulatory role in sperm development.

Fig. 8.

The ceRNA regulatory network. (A) lncRNA-miRNA-mRNA network of testis. (B) lncRNA-miRNA-mRNA network of epididymis. (C) Key ceRNA regulatory networks of testis. (D) Key ceRNA regulatory networks of epididymis. The shades of the colors indicate the lncRNAs (yellow), mRNAs (green) and miRNAs (blue)

Discussion

The reproductive efficiency of the rooster plays a crucial role in livestock breeding for enhancing breed genetics. Spermatogenesis occurs in the testis [1], while sperm maturation and storage take place in the epididymis [2]. The regulation of these organs significantly influences sperm development, which is a complex mechanism posing significant challenges in the understanding genetics control. In this study, we conducted a comprehensive transcriptome analysis of testis and epididymis tissues to identify candidate genes and their regulatory mechanisms. In the testis, a total of 68 DEmRNAs, 26 DElncRNAs, and 7 DEmiRNAs were identified between T_24W and T_35W stages. Similarly, between T_35W and T_64W stages, 64 mRNAs, 15 lncRNAs, and 4 miRNAs showed differential expression. In the epididymis, we identified a total of 106 differentially expressed mRNAs, 6 lncRNAs, and 7 miRNAs in E_24W vs. E_35W comparison. Similarly, we identified 80 differentially expressed mRNAs, 10 lncRNAs, and 7 microRNAs in T_35W vs. T_64W comparison.

In this research, differentially expressed genes GNL3, HADH, HERC4, CAS8, LHX3, MYL6, RPL14, RPS27A, and AvBD7 were identified in testis and epididymal tissues, potentially associated with spermatogenesis and spermatid motility. Previous studies have reported a positive correlation between GNL3 expression and sperm motility [43]. Furthermore, the essential role of GNL3 and LHX3 in organism growth and development have been elucidated. Knockdown of GNL3 induced growth arrest in larvae of Hidradenitis elegans, a small, rod-shaped nematode [44]. Additionally, LHX3 has demonstrated its ability to activate hormones associated with animal growth and reproductive processes, as well as the development and functioning of various target organs [45]. In our study, we observed low expression levels of GNL3 and LHX3 at 24 weeks of age which subsequently later increased at 35 and 64 weeks of age. These findings highlight the significant contributions of GNL3 and LHX3 to testicular development in chickens. Knockdown of HADH in the mouse testis resulted in reduced sperm concentrations, motility, and an increase in abnormal spermatozoa [46]. HERC4 is ubiquitously expressed but exhibited higher prevalence and activity in the testis during spermatogenesis [47]. Notably, decreased HERC4 expression was observed in mouse testes leading to decreased sperm count/viability along with an increased number of abnormal spermatozoa [48]. GAS8 primarily localized within adult mouse testes where it is regulated during late meiotic stages of male spermatocytes [49]. Moreover, GAS8 up-regulation has been implicated in regulating flagellar motility regulation within growth-arrested NIH3T3 cells [50]. RPL14 and RPS27A have been linked to various aspects related to bull sperm health including function, survival, as well as antioxidant stress response [51]. Additionally, AvBD7 plays a crucial role in innate host defense while also protecting avian sperm from bacterial infections occurring in both male and female reproductive tracts [52]. MYL6 may contribute to male infertility and azoospermia. A study utilizing the Prss55 knockout mouse model unveiled that MYL6 contributes to sperm structure differentiation, thereby potentially impacting sperm functionality and leading to azoospermia or aberrant sperm morpohology [53].

A total of 20 DEmiRNAs were identified in the testis and epididymis tissues, including miR-142-3p, miR-137-3p, miR-10b-5p, miR-146-5p, and miR-451, which exhibit potential regulatory roles in spermatogenesis and sperm motility. Moreover, gga-miR-142-3p targets PPP3CA, the catalytic subunit A of protein phosphatase 3 that plays a crucial role in growth and development processes [54]. In mice models, inhibiting PPP3CA inactivation impedes testicular development and significantly reduces mature sperm count [55]. Additionally, DNM2 regulation during meiosis and post-meiotic spermatogenesis affects spermatogenesis [56]. Our hypothesis is that miR-137-3p regulates the expression of NDUFB9, which has been demonstrated to enhance sperm viability and antioxidant abilities while reducing oxidative stress after thawing cryopreserved semen [57]. Notably, 9 epididymal samples displayed remarkably high expression levels of both miR-10b-5p and miR-146b-5p on average. Studies have shown that in mice models, miR-10b-5p has the potential to target SMAD3, WNT5A, MAPK8IP3, PDGFRB, CREBBP, PRLR, and LEP, and play a crucial role in regulating sperm motility through Wnt, MAPK, and Jak-STAT pathways [58]. Furthermore, research indicates a significant expression of miR-10b-5p in the semen of bulls exhibiting high sperm motility, suggesting its substantial regulatory impact on sperm motility [59]. In this study, miR-451 demonstrated the highest expression among all differentially expressed miRNAs in the epididymis. Previous studies have proposed that miR-451 competes for mRNA binding, thereby enhancing sperm count and motility in testis tissues of mice [60]. However, the regulatory mechanism of miR-451 in the epididymis remains unexplored, emphasizing the necessity for further investigation.

To investigate the role of DEmRNAs, DEmiRNAs, and DEmiRNAs in the testis and epididymis, we conducted GO and KEGG enrichment analyses on the identified differential genes. Our analysis revealed significant enrichment of microtubule protein binding, actin cytoskeleton reorganization, MAPK signaling pathway, mTOR signaling pathway, and calcium signaling pathway in the testicular tissue. Sperm flagellar motility is a result of microtubule sliding and ATP-driven kinesin activity in the axoneme [61]. Actin, on the other hand, exhibits distribution across the head, equatorial, posterior parietal, and caudal regions of spermatozoa. This distribution implies that actin plays a critical role in sperm energetics, parietal cytokinesis, and sperm motility [62]. Furthermore, the calcium cycle can enhance sperm motility by activating phospholipase A2, which regulates the release of endogenous fatty acids for β-oxidation within the mitochondria [63]. Intracellular calcium ions (Ca2+) and Ca2 + bound to calmodulin (CaM) have been identified as regulators of sperm motility [64]. The mTOR signaling pathway has been extensively studied due to its important role in maintaining and differentiating spermatogonial stem cells, germ cells, and support cells, including vital functions such as maintaining the blood-testis barrier during spermatogenesis [65, 66]. The MAPK signaling pathway plays a critical role in signal transitions in the organism and is associated with cell proliferation, differentiation, and apoptosis along with various processes including spermatogenesis [67], spermatid maturation [68], sperm capacitation, and acrosome reaction [69].

In epididymis tissue, we observed enrichment in the oxidative phosphorylation, bacterial defense response, Wnt signaling pathway, and TGF-β signaling pathway. The oxidative phosphorylation (OXPHOS) pathway is responsible for generating adenosine triphosphate (ATP), which is essential for energy acquisition and facilitates highly active motility in spermatozoa [70]. OXPHOS represents a more efficient ATP production pathway compared to glycolysis. Oxidative phosphorylation regulates the acrosome reaction and chromatin integrity in spermatozoa [71]. Sperm maturation in the epididymis is regulated by the Wnt signaling pathway [72], which also plays a role in growth and development. Studies have indicated that Wnt signaling can inhibit epididymis development in mice by affecting cell proliferation, as demonstrated using a mouse biological model [73]. Several investigations have explored the relationship between the epididymis and immune defense mechanisms [74, 75]. TGF-β plays a crucial role in maintaining effective peripheral immune tolerance of sperm in the epididymis for normal sperm development [76]. Additionally, the TGF-β signaling pathway regulates various cellular processes including differentiation, proliferation, cell adhesion, and apoptosis [77]. Disruption of TGF-β signaling leads to epididymal damage characterized by increased white blood cells with granuloma formation, production of antisperm antibodies (ASA), as well as elevated pro-inflammatory pathways [78].

Based on the unique regulatory relationships between DElncRNAs, DEmiRNAs, and DEmRNAs, we constructed ceRNA regulatory networks in both the testis and epididymis. Our study suggests that MSTRG.2423.1-gga-miR-1563-PPP3CA and MSTRG.10064.2-gga-miR-32-5p-GPR12 have the potential to regulate spermatogenesis and motility in chicken testis. The pathway MSTRG.124708.1-gga-miR-375-NDUFB9/YBX1 is crucial for regulating epididymal sperm maturation and motility. Similarly, the pathway MSTRG.152556.1-gga-miR-9-3p-GREM1/THYN1 plays a significant role in regulating immune response of epididymis sperm. It is predicted that gga-miR-1563 targets MSTRG.2423.1 and PPP3CA, which represents the catalytic subunit A of protein phosphatase 3. These identified genes play important roles in growth, development [54], and spermatogenesis [79], making them potential key candidates for modulating reproductive traits in domestic animals [80]. Previous studies have shown that dephosphorylation of PPP3CA inhibits its activity [81], while mice with PPP3CA inactivation exhibit impaired testis development [55]. Additionally, PPP3CA has been found to regulate DNM2 during meiosis and spermatogenesis within the acrosome, thereby influencing spermatogenesis process itself [56]. GPR12, a member of the orphan protein-coupled receptor superfamily, is regulated and targeted by gga-miR-32-5p. It has been reported to play a crucial role in maintaining meiotic prophase block in oocytes [82]. Furthermore, GPR12 exhibits abundant expression in the testis [83] and is involved in controlling cell proliferation [84]. Based on our hypothesis, we propose that gga-miR-32-5p may regulate testicular cell proliferation and development through modulation of its target gene GPR12, thereby influencing spermatogenesis and sperm motility. However, further investigation is required to fully elucidate the intricate mechanisms underlying this regulation. The involvement of YBX1 in energy metabolic pathways affecting sperm function and fertility has been demonstrated previously [85]. Additionally, NDUFB9 plays a crucial role as an important component of respiratory chain complex I for energy metabolism and electron transfer in the respiratory chain [86]. Our results indicate that NDUFB9 is enriched in the oxidative phosphorylation pathway, which aligns with our findings. Analysis of viability parameters and histology of frozen sheep semen revealed that melatonin can enhance sperm viability and antioxidant properties after thawing by regulating NDUFB9 [57]. Based on the targeting relationship, it is predicted that MSTRG.124708.1-gga-miR-375-NDUFB9/YBX1 is an important pathway in regulating epididymal sperm maturation and motility. GREM1 has been implicated in regulating the TGF-β pathway [87], which plays a vital role in maintaining effective immune tolerance during sperm maturation and development in the epididymis [76]. THYN1 serves as an important regulator of Pax5 expression [88], with Pax5 being a critical transcription factor involved in B cell development essential for proper immune system [89]. Our findings suggest the existence of a concomitant targeting interaction between gga-miR-9-3p, MSTRG.152556.1, THYN1, and GREM1, indicating the involvement of the MSTRG.152556.1-gga-miR-9-3p-THYN1/GREM1 pathway in modulating immune responses in the chicken epididymis.

Conclusion

In this study, whole transcriptome sequencing was conducted on the testis and epididymis of Jinghong No. 1 breeder roosters at different growth stages. The interaction network predictions indicated that MSTRG.2423.1-gga-miR-1563-PPP3CA and MSTRG.10064.2-gga-miR-32-5p-GPR12 have the potential to regulate spermatogenesis and motility processes in the testis. Moreover, it is predicted that MSTRG.124708.1-gga-miR-375-NDUFB9/YBX1 is involved in regulating epididymis sperm maturation and motility, while MSTRG.152556.1-gga-miR-9-3p-GREM1/THYN1 may play a role in immunomodulation of epididymal spermatozoa (Fig. 9). The findings provide valuable insights into the functional mechanism underlying lncRNA-miRNA-mRNA regulation during chicken testicular spermatogenesis, motility, and epididymal spermatid maturation. It also offers a theoretical basis for improving chicken fertility and sperm quality.

Fig. 9.

A map of the pattern of ceRNA regulation of sperm development

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

lncRNA

Long non-coding RNA

miRNA

MicroRNA

DEmRNA

Differentially expressed mRNA

DEmiRNA

Differentially expressed miRNA

DElncRNA

Differentially expressed lncRNA

T_24w

Testis tissue at 24 weeks of age

T_35w

Testis tissue at 35 weeks of age

T_64w

Testis tissue at 64 weeks of age

E_24w

Epididymal tissue at 24 weeks of age

E _35w

Epididymal tissue at 35 weeks of age

E _64w

Epididymal tissue at 64 weeks of age

ceRNA

Competing endogenous RNA

qPCR

Real-time PCR

GO

Gene Ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

Author contributions

SHG performed the experiments and the manuscript. LYZ and YZ performed the sample collection. SHG and BLC analysed the data. XLQ, XGW, and LFX suggested revisions to the manuscript. CL was involved in the design of the experiments. XXS and YXX designed the study and provided overall guidance. All authors read and approved the final version of the manuscript.

Funding

This study was supported by the Beijing Innovation Consortium of Agriculture Research System (BAIC04 − 2021).

Data availability

RNA-seq data from this study can be read from NCBI sequences (Accession Number: PRJNA1109558).

Declarations

Ethics approval and consent to participate

All experimental procedures and ethical treatment of animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006). The Animal Ethics Committee of Beijing University of Agriculture thoroughly reviewed and granted approval for all procedures under Approval ID: BUA-zc-20200073.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.