Zheng et al. Parasites Vectors (2019) 12:529 https://doi.org/10.1186/s13071-019-3783-2 Parasites & Vectors RESEARCH Open Access Transcriptomic analysis of reproductive damage in the epididymis of male Kunming mice induced by chronic infection of Toxoplasma gondii PRU strain Yu‑Xiang Zheng1,2†, Xiu‑Xiang Zhang3†, Jorge A. Hernandez4, Yasser S. Mahmmod5,6, Wan‑Yi Huang1,2, Gui‑Feng Li1,2, Ya‑Pei Wang1,2, Xue Zhou1,2, Xiu‑Ming Li7* and Zi‑Guo Yuan1,2* Abstract Background: Some researchers have reported that Toxoplasma gondii can cause serious reproductive impairment in male animals. Specifically, T. gondii destroy the quality of sperm in the epididymis, which affects their sexual ability. However, among such studies, none have investigated the male reproductive transcriptome. Therefore, to investigate the relationship between T. gondii and sperm maturation, we infected mice with T. gondii prugniaud (PRU) strain and performed transcriptome sequencing of the epididymis. Results: Compared with the control group, 431 upregulated and 229 downregulated differentially expressed genes (DEGs) were found (P-value < 0.05, false discovery rate (FDR) < 0.05 and |log2 (fold change)| ≥ 1). According to results of a bioinformatics analysis, Gene Ontology (GO) function is divided into three categories: cellular component, molecular function and biological process. Upon performing GO analysis, we found that some DEGs correlated with an integral part of membrane, protein complex, cell surface, ATP binding, immune system process, signal transduction and metabolic process which are responsible for the epididymal injury. DEGs were mapped to 101 unique KEGG path‑ ways. Pathways such as cytokine-cytokine receptor interaction, glycolysis/gluconeogenesis and apoptosis are closely related to sperm quality. Moreover, Tnfsf10 and spata18 can damage the mitochondria in sperm, which decreases sperm motility and morphology. Conclusions: We sequenced the reproductive system of male mice chronically infected with T. gondii, which provides a new direction for research into male sterility caused by Toxoplasma infection. This work provides valuable information and a comprehensive database for future studies of the interaction between T. gondii infection and the male reproductive system. Keywords: Toxoplasma gondii, Chronic infection, Reproductive impairment, DEGs, RNA-seq Background infection to their fetus through vertical transmission Toxoplasma gondii is an intracellular parasite that resulting in abortion, fetal abnormalities and death [3]. infects almost all warm-blooded animals [1, 2]. Preg- Additionally, Toxoplasma infection can cause damage nant women infected with T. gondii can transmit the to the male reproductive system, such as sexual dys- function and infertility. The epididymis is an impor- *Correspondence: lxm_nj@qq.com; ziguoyuan@scau.edu.cn tant accessory organ of the male reproductive system, † Yu-Xiang Zheng and Xiu-Xiang Zhang contributed equally to this work where sperm maturation and storage take place. Sperm 1 College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, Guangdong, People’s Republic of China maturation in the epididymis is a highly programmed 7 College of Animal Science and Technology, Heilongjiang BaYi process which may be affected by the epididymis micro- Agricultural University, Daqing 163319, People’s Republic of China environment. Therefore, studying the characteristics of Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zheng et al. Parasites Vectors (2019) 12:529 Page 2 of 11 the epididymis is important for identification of fac- Methods tors affecting sperm maturation. Previous studies have Study population and experiment set‑up shown that reproductive pipeline damage [4] and hypo- Thirty specific-pathogen-free eight-week-old Kunming gonadism [5] are associated with T. gondii infection. male mice were purchased from the Laboratory Ani- Furthermore, a large number of tachyzoites in semen mal Center of Guangdong Province. Fifteen mice were of infertile patients and anti-sperm antibodies (AsAb) subjected as the experiment groups, and 15 mice were were detected in T. gondii infection [6]. Other studies subjected as the control group (to reduce individual dif- have reported more advanced pathological changes in ferences, we set the epididymal tissue of 5 mice as a bio- T. gondii infection cases, such as granular degeneration logical replicate, the experiment group and the control of vas deferens epithelial cells, luminal stenosis and group were repeated three times). Mice in the experi- interstitium infiltration with inflammatory cells [7]. mental group were inoculated with four cysts of T. gondii The density and motility rate of sperm in epididymis PRU strain diluted with normal saline to 0.5 ml through infected with T. gondii were significantly lower than the the intragastric administration route (we have previously control group; however, the rate of sperm deformity studied the optimal number of PRU strains infections per was increased [8, 9]. mice, which tried to minimize the case damage caused Recently, various -omics technologies, including tran- by T. gondii infection in mice). Meanwhile, the control scriptomics, proteomics and metabolomics, have been groups were given the same amount of normal saline developed [10]. Transcriptomic analysis is one of the only. The male mice were sacrificed at 35 days post-infec- most common high-throughput techniques, which iden- tion. Under sterile conditions, the epididymides were tify the types and copy numbers of mRNAs, while the harvested. Under the microscope, the peripheral adipose cells are living in a functional state [11]. Genetic stud- tissue and blood vessels of the harvested epididymides ies have shown that mRNA act as a “bridge” for genetic were carefully removed. The processed epididymides information transmission between DNA and protein. were subjected to quick freezing by storing them in liquid Hence, it is a valuable source to identify the expression nitrogen at – 80 °C for subsequent analysis. of all genes with a specific time and space in the cell. Previous studies have investigated the association Transcriptome sequencing, data analysis and verification between T. gondii and male infertility using epidemiol- At present, transcriptome sequencing is a mature high- ogy, pathology and serology methods and techniques throughput second-generation sequencing method. A [12], detection of Toxoplasma DNA in semen [13] brief description of the experimental process is provided and observation of the tissue and cell damage [14]. To in Table 1; detailed methods of transcriptome sequenc- our knowledge, studies investigating the differentially ing, data analysis and verification can be found in Addi- expressed genes of host and T. gondii by transcriptome tional file 1: Text S1. sequencing of the male reproductive system are limited. The main objective of this study was to examine differen- Results tial gene expression by RNA sequencing (RNA-seq) tech- Pre‑experiment nology in order to identify key genes associated with T. Before the histological analysis, we conducted a pre- gondii (PRU strain) chronic infection of the epididymis in liminary study on the sperm quality of T. gondii infec- male Kunming mice. tion, including the statistics of sperm survival rate Table 1 Procedures for transcriptome sequencing, data analysis and validation Experimental procedure Experimental method or threshold considered Extraction of total RNA Trizol reagent (Invitrogen, CA, USA) Quality and purity detection of total RNA RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number > 7.0 cDNA library construction The cleaved mRNA fragments were reverse-transcribed to create the final cDNA library by the instruc‑ tions of the mRNA-Seq sample preparation kit (Illumina, San Diego, USA) Sample library construction Illumina Hiseq 2500 sequencing platform Transcript abundance estimation The RPKM of each gene were calculated by the MARS model in DEGseq program, when the RPKM greater than 1000, it is considered to be a highly expressed gene Threshold of differentially expressed genes FDR (false discovery rate) < 0.001 and |log2fold change| ≥ 1 Bioinformatics analysis GO and KEGG enrichment Verification of mRNA expression level Real-time fluorescence quantitative PCR Verification of protein expression level Western blot Zheng et al. Parasites Vectors (2019) 12:529 Page 3 of 11 and total sperm count. The sperm survival rate was and the number of cysts was counted. Cysts from each calculated according to the formula: Sperm survival mouse were counted three times and averaged. Statisti- rate = (Total sperm count - Dead sperm count)/Total cal results showed that 5 µl brain homogenate contained sperm count × 100%; three experiments were carried out three or more cysts. Therefore, we determined that all of the in each group. The results showed that the sperm survival experimental mice were infected with T. gondii. rate of the experimental group (32%) was significantly lower than that of the control group (74.67%). Quality analysis of RNA The sperm count per milliliter semen was calculated The purity of total RNA was high, and the ratios ­OD260/ according to the formula: Sperm count per milliliter OD280 and O ­ D260/OD230 were both higher than 2.0 semen = (5 medium square count/80) × 400 × dilution (Table 2). The RNA integrity number (RIN) was higher multiple × 10,000. We diluted sperm 10 times for micro- than 7, and the ratio of 28S/18S rRNA was higher than scopic examination. The experimental data are shown in 0.7. This indicated that the RNA had good integrity and Additional file 2: Figure S1; there were significant differ- met the requirements of the subsequent experiments. ences in total sperm count between the experimental group and the control group (P = 0.0058). The data showed that Sequencing data preprocessing T. gondii infection had a negative effect on sperm quality. The sample library constructed by Illumina Hiseq 2500 sequencing platform. We obtained 321,183,388 raw reads Identification of infection in mice (48.17G). After trimming, we obtained 314,632,244 clean The outward appearance changes of infected mice com- reads (47.21G). Among them, the amounts of sequencing prised mainly a stress reaction about a week after infection, data of each sample were more than 6G. In high through- which included depression and the fur is not smooth. Thirty put sequencing, each measurement of a basic group gives days after infection, 20 mice were selected for tail vein blood a corresponding value, which is a measure of the accu- collection and qPCR was used to verify the infection of mice racy (Table 3). (T. gondii ROP18 virulence gene was detected and using the β-actin as a reference); the result is shown in Additional Analysis of gene expression file 3: Figure S2. All of the 20 mice were infected. Fifteen RPKM/FPKM was used to measure the abundance of mice were randomly selected for dissection and epididymis gene expression in the transcriptome. We used FPKM to tissue was quickly removed. Meanwhile, the mouse brain estimate the abundance of known genes expresses in dif- was grinded and diluted with 100 µl saline solution; 5 µl of ferent samples. After the construction of new transcripts, brain homogenate was taken for microscopic examination 69,907 transcripts and 29,128 unigenes were obtained. Table 2 Quality analysis of RNA Sample Concentration (µg/µl) O.D. 260/280 O.D. 260/230 Amount (µg) rRNA RIN QC evaluation 28S/18S PRU-1 1.46 2.07 2.3 65.83 0.8 7 A PRU-2 1.16 2.09 2.25 52.24 1.8 7.2 A PRU-3 1.38 2.08 2.28 62.34 1.5 7.5 A Control-1 0.87 2.07 2.35 39.35 0.8 7 A Control-2 1.69 2.1 2.32 76.25 1.7 7.9 A Control-3 1.26 2.03 2.29 56.65 1.8 8.1 A Table 3 Pre-treatment results of data quality in the high throughput sequencing Sample Raw data reads Base Valid data reads Base Valid reads % Q20% Q30% GC% PRU-1 42,640,370 6.4G 41,484,632 6.22G 97.29 98.77 90.81 46.5 PRU-2 49,368,291 7.4G 48,326,910 7.25G 97.89 98.47 90.93 48.0 PRU-3 43,890,326 6.58G 42,897,451 6.44G 97.74 98.93 90.25 47.7 Control-1 62,208,188 9.33G 60,729,140 9.11G 97.62 98.86 91.00 47.5 Control-2 58,037,289 8.7G 57,291,719 8.6G 98.71 98.72 90.35 50.0 Control-3 65,038,924 9.76G 63,902,392 9.59G 98.25 99.04 91.37 46.5 Notes: Q20%, error rate is 1%, or the correct rate is 99%; Q30%, error rate is 0.1%, or the correct rate is 99.9% Zheng et al. Parasites Vectors (2019) 12:529 Page 4 of 11 The five-demographic data (distribution) of experimental and control groups are shown in Fig. 1. Analysis of DEGs Significant DEGs from the differential genes with P < 0.05, |log2 (fold change)| ≥ 1 and the FDR corrected P-value < 0.05 were selected. The overall distribution of DEGs was observed by drawing a volcano plot (Fig. 2). Compared with the control group, the number of up- and downregulated genes was 431 and 229, respectively. Par- tial DEGs are listed in Tables 4 and 5. Bioinformatics analysis for the DEGs GO database analysis was conducted on the DEGs, including biological processes (1746), cellular compo- nents (1275) and molecular functions (785); the distri- bution diagram of GO terms with significant changes Fig. 2 Distribution map of significant differentially expressed genes. Bright red dots indicate significantly upregulated genes, while blue dots indicate significantly downregulated genes in classification is shown in Fig. 3. The main categories in the biological processes include immune system pro- cess, response to stress and signal transduction. As for the cellular components, the GO terms with the most differentially expressed genes were cell, intracellular and organelle. The molecular functions were involved mainly in the protein binding followed by ion binding, and trans- membrane transporter activity. KEGG database analysis showed that the DEGs were enriched in 101 pathways. The most enriched pathways of DEGs are exhibited in Fig. 4. Markedly, cell adhesion (31), phagosome (27) and natural killer cell mediated cytotoxicity (17) were the major three enriched pathways (Fig. 4). However, the pathway directly related to male reproduction was not found. Verification results by qPCR analyzing and western blotting The eight genes related to reproduction were identified by qPCR (Table 6); these expression trends were coinci- dent with RNA-seq results (Fig. 5). The result of qPCR did not coincide with the RNA-seq perfectly; however, it is still possible to increase the reliability of these genes to a certain extent. DEGs including Piwil2, Spata18 and Tnfsf10 were selected for Western blot analysis. The results of the Western blot showed the same changing trend as the results of RNA-seq, although the specific amount of expression was different (Fig. 6). Fig. 1 Statistics of gene expression distribution. Infection group: the maximum, upper quartile, median, lower quartile and the minimum Discussion values are 92252.6, 1.84, 0.03, 0 and 0, respectively. Control group: We investigated the pathogenesis of T. gondii in the the maximum, upper quartile, median, lower quartile and minimum male reproductive tract of mice at the stage of sperm values are 107446, 1.7, 0.02, 0 and 0, respectively maturation using RNA-Seq technology. Additionally, Zheng et al. Parasites Vectors (2019) 12:529 Page 5 of 11 Table 4 Partial of upregulated DEGs in the high throughput sequencing data Symbol Name log2(FC) Function Iigp1 Interferon-inducible GTPase 1 6.5608 Required for disruption of the parasitophorous vacuole formed following T. gondii infection and subsequent killing of the parasite Klrb1c Killer cell lectin-like receptor subfamily B member 1C 6.17868 Plays a stimulatory role on natural killer (NK) cells cytotoxicity, activa‑ tion by cross-linking of the receptor induces ­Ca2+ mobilization and interferon-gamma production Tgtp1 T cell-specific GTPase 1 5.61362 Involved in innate cell-autonomous resistance to intracellular pathogens, such as T. gondii Tnfsf10 Tumor necrosis factor (ligand) superfamily, member 10 1.53097 Involved in male gonad development and regulation of the apoptotic process Spata18 Spermatogenesis associated 18 1.29567 A key regulator of mitochondrial quality that mediates the repairing or degradation of unhealthy mitochondria in response to mitochondrial damage Pgk2 Phosphoglycerate kinase 2 1.11099 Essential for sperm motility and male fertility but is not required for the completion of spermatogenesis Piwil2 Piwi-like RNA-mediated gene silencing 2 1.1907 Essential role in meiotic differentiation of spermatocytes and self-renewal of spermatogonial stem cells Table 5 Partial of downregulated DEGs in the high throughput sequencing data Symbol Name log2(FC) Function Lipf Gastric triacylglycerol lipase − 3.40022 Involved in lipid catabolic process and mitochondria when classification of cellular component Nrk Nik-related protein kinase − 3.41519 Involved in the TNF-alpha-induced signaling pathway Nwd1 NACHT and WD repeat domain containing 1 − 1.60721 Play a role in the control of androgen receptor (AR) protein steady-state levels Slc14a2 Solute carrier family 14 (urea transporter), member 2 − 1.11457 Mediates rapid transepithelial urea transport across the inner medullary collecting duct and plays a major role in the urinary concentrating mechanism Spink2 Serine protease inhibitor Kazal type 2 − 1.02159 Required for maintenance of normal spermatogenesis, involved in regulat‑ ing serine protease-dependent germ cell apoptosis we performed enrichment methodologies analysis to resistance to T. gondii. However, after T. gondii infec- screen the DEGs that might be related to the reproduc- tion, Tgtp1 and Tgtp2 had low levels of gene expression tive functions. Results were confirmed by using qPCR abundance. The average abundance of Tgtp1 was 13.7 for and Western blot methods. Our findings showed that the the experimental group and 0.58 for the control group RNA-Seq is accurate. and the average abundances of Tgtp 2 were 15.39 and The analysis of DEGs suggested that some genes were 0.31, respectively. Tgtp1 and Tgtp2 are mainly expressed interrelated with the infection of T. gondii, such as Iigp1 in the thymus and lymph nodes, predominantly T-cells (interferon-inducible GTPase 1), Tgtp1 (T-cell-specific and IFNG-stimulated macrophages [17]. In other words, guanine nucleotide triphosphate-binding protein 1) these genes are tissue-specific. After T. gondii infection, and Tgtp2. Iigp1 found in the parasitophorous vacuoles the immune system was stimulated, and the expression (PV) plays a role in the destruction of PV and death of level of these genes was greatly increased. the parasite, when the Iigp1 gene is overexpressed [15]. We found that Iigp1 was in regulation of autophagy in Glycolytic pathway and sperm motility the GO classification. Hence, it is possible that Iigp1 is There are several enriched pathways in our data analysis. involved in autophagy, although the specific mechanism Glycolysis was thought to be the oldest and most primi- is not clear. During infection of the type II of T. gondii, tive way of biological energy harvesting. We focused Tgtp1 and Tgtp2 were recruited to the PV membrane, PV on DEGs that affect glycolysis such as phosphoglycer- vesiculation, rupture and digestion of the parasite within ate kinase 2 (Pgk2), phosphoglycerate mutase (Pgam) the cytosol [15, 16]. In our study, the Iigp1, Tgtp1 and and lactate dehydrogenase C (LDHC). Phosphoglycerate Tgtp2 were significantly upregulated by 94-fold, 48-fold kinase is the first regulation factor of ATP generated in and 23-fold, respectively. This could be the result of host the glycolysis pathway. In the reproductive system, the Zheng et al. Parasites Vectors (2019) 12:529 Page 6 of 11 Fig. 3 GO analysis of the significant DEGs Fig. 4 The most enriched pathways of DEGs examination of sperm in elderly and young male patients gluconeogenesis. Moreover, Pgam2 is related to the with asthenospermia showed that the level of Pgk2 was fibrous sheath of the main segment in the sperm flagel- reduced. Pgk2, which is related to the quality of sperm, lum [20]. The fibrous sheath on the sperm flagellum is is mainly expressed in able-bodied spermatids [18]. Pgk2 the source of ATP and an essential structure of sperm deficiency leads to a decrease in sperm motility and ATP motility [21]. Therefore, Pgam2 is the key determinant to levels [19]. Pgam is an important enzyme of glycolysis maintaining sperm motility and morphology. LDHC is an and gluconeogenesis pathway, catalyzes the 3-phospho- enzyme that controls the conversion of pyruvate to L-lac- glycerate (3-PGA) into 2-phosphoglycerate (2-PGA), tic acid, which plays a key role in the process of sper- which dominates the balance between glycolysis and matogenesis and capacitation by participating in cellular Zheng et al. Parasites Vectors (2019) 12:529 Page 7 of 11 Table 6 Design of primers used for qPCR analysis Gene ID Gene Forward primer Reverse primer Product length (bp) 57746 piwil2 GGC​AGA​GGC​C TT​GTG​T TT​AG CGT​T TT​GAA​GGA​GGC​CGG​AA 134 73472 Spata18 TTA​TCA​CGT​GTG​GCC​TGC​TC TTA​AGC​TCC​TGC​TAC​GGC​AC 125 22035 Tnfsf10 ACG​C TT​CCA​AGA​TGG​TCT​CA ACA​GTC​CGT​ACT​CGG​CAT​C T 147 21822 Tgtp1 CGC​CAC​GTC​T TC​TCA​C TG​TC CAC​CAA​GTG​GAA​TGG​TGG​C T 130 60440 Iigp1 TCA​C TA​TGA​C TT​CCC​CGT​CCT​ TGC​T TC​AGA​AAT​TGC​CGC​T T 132 18663 Pgk2 GCC​TGT​GCC​AAC​CCA​GAT​AA ACA​GTG​ATG​C TT​GGA​AGG​C T 145 319555 Nwd1 CTC​T TT​GGA​CCA​CCA​GGC​AT AGG​C TC​AGC​T TT​GAT​GTC​CC 125 69982 Spink2 GCA​TGC​CCT​AGG​AAC​C TC​AA ATA​TGG​C TA​CCG​TCC​TCC​C T 101 11461 β-actin AGA​GAA​GCT​GTG​C TA​TGT​TGCT​ GGA​ACC​GCT​CGT​TGC​CAA​TA 128 Fig. 5 Results of qPCR and compared with RNA-seq energy metabolism [22]. LDHC was found in the human energy sources. Mukai & Okuno [24] noticed that when spermatozoa and spermatogenic cells and was increased the oxidative phosphorylation was blocked by rotenone gradually with meiosis. LDHC in mature sperm is mainly of mitochondrial respiratory chain inhibitors, the sperm located in the fibrous sheath of the main segment of the keeps moving. Therefore, glycolysis is not only an impor- tail of sperm, which is involved in the regulation of sperm tant source of energy for sperm but also plays a role in motility. In addition, LDHC is also participates in the sperm capacitation [25] and acrosome reaction [26, 27]. homeostasis of ATP. Deficiency/destruction of germline- specific LDHC leads to infertility, including a reduction Mitochondrial damage and apoptosis in sperm ATP content and reduced sperm motility [22, Spermatogenesis associated 18 (Spata18) is a key regu- 23]. Conclusively, the enzymes are closely linked with lator of mitochondrial quality that mediates repair or sperm activity and morphology. degradation of unhealthy mitochondria [28]. There is no In the present study, the DEGs of glycolysis indicated pathway for Spata18, but previous research has shown that T. gondii infection might also result in dysregulation that it is a monitor of cell differentiation process, which of those enzymes in humans, thus promoting a negative is involved in the maturation of spermatids into sperma- influence on the quality of male sperm. Normally, the tozoa [29]. energy production efficiency of oxidative phosphoryla- In our study, the expression of Tnfsf10 gene was upreg- tion is much higher than that of glycolysis, but glycolytic ulated to 2.89-fold in the pathway of cytokine-cytokine pathways have their particular importance in sperm receptor interaction, apoptosis and natural killer cell Zheng et al. Parasites Vectors (2019) 12:529 Page 8 of 11 calmodulin-dependent protein kinase [34]. This is con- sistent with the finding of Chen et al. [35] who stated ­ a2+ dependent calpain and/or ­Ca2+ independ- that the C ent cathepsins participate in apoptosis-like PCD and necrosis-like PCD. Trail gene-deficient (Trail2/2) mice were significantly reduced by 54% compared to the wild type mice, which explains the negative effects on sperm maturation in the reproductive system [30]. A high con- centration of soluble Tnfsf10 was detected in the semi- nal plasma, which revealed a variable expression of Trail receptors in the sperm cellular fraction among different participants [36]. Those results suggest that Tnfsf10 may play an important role in sperm maturation. The cytotoxicity of natural killer (NK) cells is moti- vated by the activating cell receptors. It can switch on the cascade reaction of downstream signal transduction. Fig. 6 Results of Western blot compared with RNA-seq. Lane 1: In our study, the Src family kinase (Fcgr4) was related to control group; Lane 2: infection group; from top to bottom, represent the cytotoxic pathway which is mediated by NK cells. the β-actin internal reference, spata18, tnfsf10 and piwil2 protein The Fcgr4 also could active the mitogen activated protein kinase (MAPK) pathway, which could produce the cyto- toxicity and secret proinflammatory cytokines (such as mediated cytotoxicity. Tnfsf10 (Trail) is a member of IFN-γ, TNF-α and GMCS). The upregulation of piwi and the tumor necrosis factor (TNF) superfamily, and is an Itgb2 genes mediates the target cell apoptosis by Tnfsf10, important signal molecule to maintain the stability of which is caused by perforin and granzyme. The Gzmb germ cells and the functional spermatogenesis in tes- (Granzyme B) gene is responsible for aspartate-specific tis [30]. Apoptosis, or programmed cell death (PCD), cysteine proteases (DNA fragmentation, low synthesis is a biological process controlled by genes and plays a of ploy and destruction of nuclear membrane integrity) vital role in maintaining the morphology of normal tis- which cause apoptosis. sues and organs. So far, the mitochondrial pathway is From these pathways, we consider that Tnfsf10 is considered to be the classical pathway of apoptotic sig- directly or indirectly connected with the male repro- nal pathways. Mitochondria control the life and death ductive system. Chronic infection of T. gondii gives rise of cells in aerobic environments, which is the main to dysregulation genes in these pathways, which in turn place of ATP production, and the major regulatory site have a negative consequence on the integrity and motil- of cell apoptosis. Mitochondrial permeability transition ity of sperm. At the stage where spermatids mature into pores (MPTP) exist in the inner and outer membranes spermatozoa, mitochondria formed the mitochondrial of mitochondria, and the permeability transition plays a sheath by gathering around the tail and wrapping around vital role in cell apoptosis [31]. The pathway of cytokine- the shaft spirally. Finally, it can provide the energy for cytokine receptor interaction shows Tnfsf10 as a cytokine rotational motion of the tail in the way of making full use and interacts with the cytokine receptor Tnfsf10b. In the of energy in plasma drops. The fertility was influenced by apoptosis pathway, Tnfsf10b (2.3-fold upregulation) as a various channels to create the apoptosis of germ cells. death receptor, interacts with Tnfsf10 causing production of the death inducing signaling complex (DISC), which Testis‑specific genes in the epididymis initiates the caspaseprotease family (aspartate-specific Our data analysis revealed that many DEGs were cysteine proteases) and activates apoptosis through DNA upregulated in the epididymis; however, previous stud- fragmentation, low synthesis of ploy, and destruction of ies have shown that they are testicular-specific genes, nuclear membrane integrity [32]. At the same time, the such as Piwil2 and RGS22 (regulator of G-protein sign- translocation and permeabilization of mitochondrial aling 22) genes. Piwil2 is a member of the Argonaute membrane could induce apoptosis through Bax/Bak [33]. family and PIWI (the P-element-induced wimpy tes- MPTP opening resulting from exogenous injury factors tis) subfamily. PIWI family proteins are only expressed (such as ­Ca2+ overload) would lead to necrosis and apop- in animal gonad tissues and have tissue specificity, tosis of cells. Some scholars have found that the syner- these are closely related to the maintenance of germ gism of melittin with Trail could increase the number stem cells and spermatogenesis [37]. The Piwil2 gene, of apoptosis in hepatoma cell through activating C ­ a2+/ expressed in tumor stem cells and germ cells, plays an Zheng et al. Parasites Vectors (2019) 12:529 Page 9 of 11 Table 7 The testis-specific gene was highly expressed in the epididymis Symbol log2 (FC) Tissue specificity Function Piwil2 1.1907 Expressed in adult testis, specifically in spermatocytes and in Plays an essential role in meiotic differentiation of spermatocytes spermatogonia and self-renewal of spermatogonial stem cells Rgs22 2.18187 Expressed in testis, including in Leydig cells and spermatogenic Negative regulation of signal transduction cells from the spermatogonia to spermatid stages Shcbp1l 1.78449 Testis-specific. Expressed in pachytene spermatocytes and Testis-specific spindle-associated factor that plays a role in elongating spermatids inside the seminiferous tubules spermatogenesis Spz1 1.08852 Expressed specifically in the testis (Sertoli and Leydig cells) and Play an important role in the regulation of cell proliferation and epididymis differentiation during spermatogenesis Spata18 1.29567 In testis, expressed primarily in spermatids Key regulator of mitochondrial quality that mediates the repair‑ ing or degradation of unhealthy mitochondria in response to mitochondrial damage important role in the self-renewal of stem cells, embryo information and a comprehensive database for future formation and transcriptional regulation [38]. Similarly, studies of the interaction between T. gondii infection Rgs22 is a specific gene in the male testis, which can and the male reproductive system. be expressed simultaneously in a variety of epithelial tumors. In testis, Rgs22 protein is associated with sper- Supplementary information matogenesis, which can be expressed in spermatogenic Supplementary information accompanies this paper at https​://doi. cells involved in the spermatogonia differentiation into org/10.1186/s1307​1-019-3783-2. sperm cells and Sertoli cells. Hu et al. [39] found that the expression of Rgs22 was downregulated in patients Additional file 1: Text S1. Detailed methods of transcriptome sequenc‑ ing, data analysis and verification. with azoospermia. Proteins are located in spermato- Additional file 2: Figure S1. Effect of T. gondii infection on total sperm genic cells and Leydig cells, which interact with the count. guanine-nucleotide-binding proteins [39]. Thus, we Additional file 3: Figure S2. Identification of infection in experimental consider that Rgs22 may be related to the meiotic stage mice. of spermatogenesis. In summary, Piwil2 and Rgs22 genes are testis-spe- Abbreviations cific genes, mainly expressed in spermatogenic cells and PRU: Prugniaud; DEGs: differentially expressed genes; FDR: false discovery rate; play a key role in the spermatogenesis process. Other FC: fold change; GO: Gene Ontology; AsAb: anti-sperm antibodies; RNA-seq: RNA sequencing; Gb: gigabases; FPKM: fragment per kilobase of exon model genes that were identified (see Table 7) are testis-spe- per million mapped reads; SAM: sequence alignment/map format; GTF: gene cific and highly expressed in the epididymis. One pos- transfer format; UCSC: University of California Santa Cruz; FRP: horseradish per‑ sible reason for the latter observation is the invasion of oxidase; DAB: diaminobenzidine; ECL: enhanced chemiluminescence; KEGG: Kyoto Encyclopedia of Genes and Genomes; MGI: mouse genome informatics; T. gondii, which contributes to the destruction of the RIN: RNA integrity number; PV: parasitophorous vacuoles. blood-testis barrier of the testis, and disruption of the spermatogenesis homeostasis [40]. Furthermore, sper- Acknowledgements Not applicable. matogenic cells in the seminiferous tubules were disor- dered, resulting in the increase of testis-specific gene Authors’ contributions expression in the epididymis. However, the epididymis YXZ contributed to the data analysis and the writing of the manuscript. XXZ contributed to the drafting of the manuscript. JAH and YSM contributed to has its tissue-specific microenvironment [41, 42] which the revising of the manuscript. WYH, GFL, YPW and XZ contributed to the data is different from that in the testis. The microenviron- collection and the laboratory work. YXZ and XXZ contributed to the animal ment in the epididymis is not appropriate to regulate experiment. ZGY and XML contributed to the conception of the idea and design. All authors read and approved the final manuscript. the cells in the testis and therefore, the specific mecha- nism is unclear and needs further investigation. Funding This work was supported, in part, by grants from the National Key Research and Development Programme of China (2017YFD0501305), National Natural Science Foundation of China (31972707), Major Scientific Research Pro‑ Conclusions jects (Characteristic Innovation) of Guangdong Province (2017KTSCX018), We sequenced the reproductive system of male mice the Key Research and Development Programme of Guangdong Prov‑ ince (2019B020218004), the Guangzhou Zoo Project (h2016141), the chronically infected with T. gondii, which provides a Natural Science Foundation of Guangdong Province (2019A1515011534, new direction for research into male sterility caused 2016A030313396), and Huizhou Science and Technology Projects by Toxoplasma infection. This work provides valuable (2015B040009001). Zheng et al. Parasites Vectors (2019) 12:529 Page 10 of 11 Availability of data and materials 13. Teixeira W, Tozato M, Pierucci JC, Vital GP, Cruz AC, Lopes W, et al. Investi‑ We have uploaded the transcriptome data to the Sequence Read Archive gation of Toxoplasma gondii in semen, testicle and epididymis tissues of (SRA) database, submission: SUB5863608, accession ID: PRJNA552423 primo-infected cats (Felis catus). Vet Parasitol. 2017;238:90–3. 14. Colosi HA, Jalali-Zadeh B, Colosi IA, Simon LM, Costache CA. Influence of Ethics approval and consent to participate Toxoplasma gondii infection on male fertility: a pilot study on immuno‑ This study was carried out in accordance with the principles of the Basel competent human volunteers. Iran J Parasitol. 2015;10:402–9. Declaration and recommendations of the People’s Republic of China, the 15. Martens S, Parvanova I, Zerrahn J, Griffiths G, Schell G, Reichmann G, et al. Animal Ethics Committee of South China Agricultural University. 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