Long noncoding RNAs regulated spermatogenesis in varicocele‐induced spermatogenic dysfunction

Abstract Objectives To evaluate the expression, potential functions and mechanisms of long noncoding RNAs (lncRNAs) in the pathogenesis of varicocele (VC)‐induced spermatogenic dysfunction. Materials and Methods We established a rat model with left experimental VC and divided rats into the sham group, the VC group, and the surgical treatment group (each group, n = 10). Haematoxylin and eosin (HE) staining and sperm quality were analysed to evaluate spermatogenesis function. LncRNA expression profiles were analysed using lncRNA‐Seq (each group n = 3) and validated using quantitative real‐time polymerase chain reaction (each group n = 10). Correlation analysis and gene target miRNA prediction were used to construct competing endogenous RNA network. The regulated signalling pathway and spermatogenic dysfunction of differentially expressed lncRNAs (DE lncRNAs) were validated by Western blot. Results HE detection and sperm quality analysis showed that VC could induce spermatogenic dysfunction. Eight lncRNAs were upregulated and three lncRNAs were downregulated in the VC group compared with the sham group and surgical treatment group. The lncRNA of NONRATG002949.2, NONRATG001060.2, NONRATG013271.2, NONRATG022879.2, NONRATG023424.2, NONRATG005667.2 and NONRATG010686.2 were significantly negatively related to sperm quality, while NONRATG027523.1, NONRATG017183.2 and NONRATG023747.2 were positively related to sperm quality. The lncRNAs promote spermatogenic cell apoptosis and inhibit spermatogonia and spermatocyte proliferation and meiotic spermatocytes by regulating the PI3K–Akt signalling pathway. Conclusion DE lncRNAs may be potential biomarkers for predicting the risk of spermatogenic dysfunction in VC and the effect of surgical repair. These DE lncRNAs promote spermatogenic dysfunction by regulating the PI3K–Akt signalling pathway.


| INTRODUCTION
Varicocele (VC) is a common disease in male infertility in which the internal spermatic vein malformation is twisted, dilated, and elongated. 1 The prevalence of VC in the general population is approximately 15%, and in infertile males, it is approximately 35%. 2,3 VC is a common aetiology of male infertility. Studies have shown that VC plays a role in decreased testicular function, leading to spermatogenic dysfunction and diminished testosterone levels. 4,5 Some mechanisms may contribute to VC-induced spermatogenic dysfunction, including the ionic imbalance, high testicular temperature, neuroendocrine system dysfunction, hypoxia, chronic oxidative and disruption of the blood-testis barrier. [6][7][8][9][10] Those chronic stress may reduce the function of spermatogenesis and damage the structure of spermatogenic cells such as DNA, RNA, lipids and proteins resulting in poor sperm quality. 10 However, the exact mechanisms remain unclear and require more research.
Long noncoding RNAs (lncRNAs), a kind of noncoding RNA (ncRNA), are more than 200 nucleotides in length and lack functional protein-coding ability. 11 Some studies have shown that lncRNAs play an essential role in the adjustment of gene expression and have broad functions in many critical biological processes, such as genomic imprinting, differentiation, apoptosis, nuclear organization, alternative splicing and nuclear import. 11 LncRNAs participate in various disease processes, such as diabetes, cardiovascular disease and cancers. 12,13 Wen et al. showed that testis-specific lncRNAs play an important role in late Drosophila spermatogenesis. 14 Sanei-Ataabadi et al. found that oxidativerelated lncRNAs are related to VC-connected male sterility. 15 Although many lncRNAs exist in spermatogenesis, their expression and function in spermatogenic dysfunction induced by VC remain to be studied.
To understand the expression and function of lncRNAs in VC-induced spermatogenic dysfunction, in the present study, we performed RNA-seq to profile lncRNA expression in VC rats, and the results were validated for expression in the testis and relationships with sperm quality. The findings will contribute to understanding these mechanisms of spermatogenic dysfunction caused by VC and identifying new biomarkers for the diagnosis and treatment of VC-induced spermatogenic dysfunction. Based on Turner's previously published surgical protocol, we built a left experimental VC rat model. 16 The sham group received a similar treatment without left renal vein obstruction. The VC rat models tested the sperm vein diameter at 8 weeks after modelling. Compared with the sham group, a more than twofold increase in the outer diameter of the left spermatic vein is considered a successful VC model.
Meanwhile, the surgical treatment group received varicocelectomy at 8 weeks after modelling. The steps were as follows: open the abdomen layer by layer, separate the spermatic vein, ligate the vein with 4-0 silk thread, test the outer diameter of the left spermatic vein after ligating, and finally, close the abdomen layer by layer. Samples were obtained for next analysis at 4 weeks after varicocelectomy.

| Semen analysis
The left caudal epididymis was minced in phosphate-buffered saline, and the sperm were released after incubation for 5 min at 37 C. The sperm count and sperm motility, including progressive (PR) and nonprogressive (NP) motility, were analysed using a computer-aided analysis system (Weili). Total motility is defined as the percentage of PR and NR sperm motility.

| Histological examination
Fresh testicular tissues were immobilized with 4% formalin for 1 day, dehydrated in the presence of increased ethanol concentrations, and embedded in paraffin for sectioning. The sections were stained with haematoxylin and eosin (HE) dye and observed under a light microscope.

| Total RNA extraction and lncRNA sequencing
We extracted total RNA of fresh testicular tissues with TRIzol (Invitrogen) and qualified it with an Agilent 2100 Bioanalyzer (Agilent Technologies), a NanoDrop spectrophotometer (Thermo Fisher Scientific) and 1% (wt/vol) agarose gels. Three rats from each group were randomly selected for lncRNA sequencing. Highthroughput sequencing and subsequent data analysis were implemented by GENESKY Biotechnologies Inc. using the standard Illumina HiSeq 2500 platform.

| Identification of differentially expressed lncRNAs
The edgeR package 17

| Construction of lncRNA-miRNA-mRNA network
We predicted interactions between lncRNAs and miRNAs by miRanda software with a perfect seed match. Full-length sequences of lncRNA and miRNA were selected. miRNA-mRNA interactions were identified by combining RNAhybird and miRanda software. Then, we screened pairs of positive correlations between the expression of lncRNAs and mRNAs, and the lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) network was obtained and visualized with Cytoscape v3.7.1 software. 18

| Protein-protein interaction network
We constructed protein-protein interaction (PPI) networks based on predicted target genes using the interactive gene retrieval tools (STRING) database 19 and visualized them through Cytoscape v3.7.1. 19 The cytoHubba plug-in was used to rank target genes and identify hub genes.

| Functional enrichment analysis
To investigate the biological function of DE lncRNAs, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, including biological process, molecular function and cellular component analyses, for target genes and hub genes by Metascape. 20 Terms with p < 0.05 were considered statistically significant.

| Validation by quantitative real-time polymerase chain reaction
We extracted total RNA from testicular tissue samples using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Then, first-strand cDNA Synthesis Super Mix for qPCR (Yeasen) was used to amplify the cDNA. Subsequently, a SYBR Green PCR kit (Yeasen) was used for quantitative real-time polymerase chain reaction (RT-PCR) with a cDNA template on a real-time detector (MA-6000; Molarray).

| Statistical analysis
Data were represented by the mean ± SD. Unpaired Student's t-test was used to compare parameter data between groups. The Mann-Whitney rank-sum test was used for nonparametric data. We carried out a Pearson correlation analysis between DE lncRNA expression and semen parameters. We carried out all statistical analyses using GraphPad Prism 8.0 (GraphPad Software). A result of p < 0.05 was regarded with statistical significance.

| RESULTS
The research flow chart is summarized in Figure 1. As showed in  Figure 2B) and was not significantly different between the VC group and the surgical treatment group (p > 0.05, Figure 2B). The sperm count, total motility and PR motility of rats in the sham group were apparently higher than those in the VC group ( Figure 2C-E). The sperm count and total motility in rats after surgical treatment did not increase significantly ( Figure 2C, D), but the number of PR motility sperm increased significantly compared with the VC group (p < 0.01, Figure 2E).

| HE staining of rat testicular tissues
As shown in Figure 2A, HE staining analysis further reflected that the counts of spermatogonia, spermatocytes and round spermatids in the seminiferous tubules of the VC group were significantly reduced, while those in the surgical treatment group were significantly increased.

| Identification of the coexpression of DE lncRNAs in the VC group
To elucidate the mechanism of impaired sperm quality in the testes of VC rats in more detail, entire testis samples for lncRNA  Figure 3B,D). Venn diagram analysis showed that 11 DE lncRNAs overlapped between the two comparisons ( Figure 3E,F). Specifically, eight DE lncRNAs were upregulated and three DE lncRNAs were downregulated in the VC group (Table 3).

| Functional enrichment analysis of target genes
For further research, the biological processes and pathways based on the established ceRNA network were explored. The DE lncRNAs were closely related to inflammation or immune-associated biological processes, apoptosis and oxidative stress, such as immune effector processes, cytokine production, innate immune response, defensive reaction regulation, leukocyte apoptotic process and response to oxidative stress ( Figure 5 and Table 4). Similarly, several KEGG pathways were identified, including cytokine-cytokine receptor interactions, pathways in cancer and the PI3K-Akt signalling pathway ( Figure 6 and Table 5). Overall, these results suggest that the DE lncRNAs are correlated with the behaviour of VC.

| Construction of the PPI network and identification of hub genes
As shown in Figure 7, we predicted a PPI network to display the interactions of target genes through the STRING database. Then, we recognized the top 10 genes with the highest association in the PPI network by the cytoHubba plug-in ( Figure 8A and Table 6). to external stimulus (GO:0032103), and two pathways, including toxoplasmosis (rno05145) and innate immune system (R-RNO-168249), were significantly related to hub genes ( Figure 8B and Table 7).

| Validation of DE lncRNAs by RT-PCR
Furthermore, we verified the expression of DE lncRNAs in testicular tissues of VC rats by RT-PCR. As illustrated in Figure 9, the rela-

| Validation of regulated spermatogenic cell apoptosis by DE lncRNAs by TUNEL staining of rat testicular tissues
We performed TUNEL staining of rat testis tissue to investigate the regulation of DE lncRNAs on spermatogenic cell apoptosis. The TUNEL assay results showed that the percentage of apoptotic cells in the testicular tissue of VC rats increased but decreased after surgical intervention ( Figure 12A,B). The DE lncRNAs regulate spermatogenic cell apoptosis in VC, and surgical treatment significantly improves apoptosis.   LncRNAs are important mediators of the ceRNA regulatory network, and they can absorb miRNAs and regulate the expression of target genes. 24 We constructed a ceRNA network and suggested that 4 lncRNAs potentially interact with 2 miRNAs (miR-301a-5p and miR-328a-5p) and 12 mRNAs. We identified more VC-induced spermatogenic dysfunction and surgical repair-specific ceRNA pairs than previous studies. In previous studies, miRNAs as biomarkers for VC have been widely discussed. Xu et al. analysed the expression of miR-210-3p in patients with VC and found that the level of miR-210-3p in the seminal plasma of patients with VC was 2.18 times higher than that of healthy people. 25 Zhi et al. found that miR-192a could be a predictive factor for the spermatogenic status of patients after VC repair. 26 However, studies of the functions of miR-301a-5p and miR-328a-5p in VC or spermatogenesis are still lacking. Our study may provide two potential diagnostic and therapeutic candidates for VC-induced spermatogenic dysfunction. miR-301a-5p and miR-328a-5p participate in biological processes in many other diseases. Wang et al. found that lncRNA EPB41L4A-AS2 sponges miR-301a-5p and inhibits hepatocellular carcinoma development. 27 The expression of miR-301a-5p was certified in gastric cancer tissues, and high miR-301-5p expression was found to be associated with the aggressiveness of gastric cancer. 28  arded as a germ cell survival factor in the human testis. 43 Dube et al.
tested the epididymis tissues by RT-PCR, and found that decreased expression of epidermal growth factor increasing PI3K-Akt signalling pathway regulated the specific luminal microenvironment necessary for the creation of fertilizing-competent spermatozoa. 44 Wang et al. 45 used a VC rat model study and found that the PI3K-Akt signalling pathway plays a regulatory role in VC-induced spermatogenesis disorder. Zhao et al. 40 performed in vitro experiments and found that the PI3K-Akt signalling pathway participates in regulating sprmatogonial cell apoptosis and proliferation. A previous study indicated that lncRNAs also induce spermatogenic cell apoptosis in VC patients.
According to DE lncRNA functional enrichment, TUNEL and HE staining results, we selected the phenotypes of spermatogenic cell apoptosis, spermatogenic cell proliferation and meiotic spermatocytes to validate by Western blot. We found that the expression of the apoptosis-promoting proteins caspase-9 and Bax was increased, and the apoptosis-inhibiting protein Bcl-2 was decreased in the testes of VC rats. The TUNEL assay results indicated that the percentage of apoptotic spermatogenic cells in the testis tissue of VC rats increased and decreased after surgical intervention. Some apoptotic mechanisms were believed to be connected with VC, originating in the mitochondria of spermatocytes and working in the nucleus in Wu et al.'s study. 9 Zhao et al. found that lncRNA gadd7 was upregulated in the semen of VC patients, and an in vitro study indicated that overexpression of lncRNA gadd7 induced the apoptosis of spermatocytes and suppressed GC-1 and GC-2 cell proliferation. 46,47 We detected the expression of PCNA and PLZF, two biomarkers associated with spermatogonia and spermatocyte proliferation and differentiation. 48,49 We found that the expression of PCNA and PLZF was decreased in VC rats and restored after surgical repair. STRA8, REC8 and SYCP3 are biomarkers associated with meiotic spermatocytes. 49,50 Our results showed that the expression of STRA8 and REC8 was decreased in VC rats. STRA8 is a biomarker for entry of germ cells into meiotic prophase I, and REC8 is a meiotic marker gene. 49,50 These results indicated that DE lncRNAs promote spermatogenic cell apoptosis and inhibit spermatogonia and spermatocyte proliferation and meiotic spermatocytes via the PI3K-Akt signalling pathway, which may affect subsequent spermatogenesis processes and sperm quality in VC.
Our study provides a foundation for the expression signature of lncRNAs and preliminarily studies the functions and mechanisms of lncRNAs regulating the process of spermatogenesis in VCinduced spermatogenic dysfunction and surgical repair. Our study has some limitations. First, the sample size for lncRNA-seq was small.
Second, nosogenesis may vary in different VC periods. Third, the functions of lncRNAs were predicted from bioinformatics analyses. Spermatogenesis is a complex biological process, and the functions of lncRNAs at different stages of VC require more systemic investigations.
Therefore, more studies based on larger sample sizes and in different stages of VC are necessary in the future.

| CONCLUSION
Our study provides a foundation for the expression signature of