Spermatogoniaapoptosis Induction as a Possible Mechanism of Toxoplasma Gondii Induced Male Infertility
Military Medicine Department, American Military University (AMU)
Received Date: 03/05/2020; Published Date: 26/05/2020
*Corresponding author: Sorush Niknamian, Board Member of Weston A Price Foundation (WAPF), Washington, USA
Cite this article: Sorush Niknamian, Spermatogoniaapoptosis Induction as a Possible Mechanism of Toxoplasma Gondii Induced Male Infertility. Op Acc J Bio Sci & Res 1(3)-2020.
Objectives: The protozoan Toxoplasma gondii as an intracellular protozoan is widely prevalent in humans and animals. Infection generally occurs through consuming food or drink contaminated with oocysts and tissue cysts from undercooked meat. This parasiteinfects different parts of men even from semen but there are little information about the effect of toxoplasmosis on male reproductive system.In this study, the effect of RH strain T. gondiitachyzoites on type Bspermatogonia(GC-1) cells was investigated.
Methods: Fresh tachyzoites taken of infected balb/c mice, GC-1 cells were infected with increasing concentrations of tachyzoites of T. gondii, then apoptotic cells identified and quantified by flow cytometry. The genes associated with the apoptosis were evaluated by RT2 Profiler PCR Array.
Results: PCR array analysis of 84 apoptosis-related genes demonstrated that 12 genes were upregulated at least 4-fold and that 1 genes were downregulated at least 2-fold in the T. gondii infection group compared with levels in the control group. The number of genes whose expression had increased during the period of infection with T. gondii was significantly higher than those whose expressions had decreased (18 versus 1) and Tnfrsf11b has the highest rate of gene expression.
Conclusions: T. gondii induce the in vitro apoptosis of GC-1 cells. This effect shows a trend of concentration-dependent increase, so that with increase in the ratio of parasite burden to spermatogonial cells, in addition to increase in the number of genes whose expression has changed, the fold of these changes has increased as well.
Keywords: Toxoplasma gondii; spermatogonia; apoptosis; In vitro
Infertility is a health problem that affects couples worldwide, regardless of their ethnicity, society and culture or economic status [1-3]. Infectious agents are able to compromise the reproductive functions in both sexes. Intriguingly, infection agents such as bacteria, fungi, viruses and parasites are involved in male reproductive failure and account for approximately 15% of the male infertility [4,5]. Toxoplasma gondii is an obligate intracellular protozoan parasite which infects all warm-blooded animals. Approximately one-third of human population has been exposed to this parasite [6,7].Toxoplasmosis is initiated firstly through ingestion of the parasite and then proliferation of tachyzoites within the host targeted cells. The parasite is usually tropism to brain, testes, and eyes in this phase as known acute phase [8,9]. The high prevalence of T. gondiiin human being and also considerable tropism of the parasite to reproductive organs such as testis postulate the potential roles of T. gondii in infertility pathogenesis.
There are increasing of evidences from human and animal models infertile that support the capability relationship between Toxoplasma and infertility [10-15]. An infection with T. gondii in infertile young couples is noticeably more than fertile couples [11,12]. Additionally, improving evidence reveals that T. gondii infection can impair testicular performance and also may be correlated with male infertility [12,16,17]. Nevertheless mechanism through which T. gondiiadjusts reproductive systems is not recognized yet . Programmed cell death (PCD), also known as apoptosis, is essential for homeostasis of spermatogenesis in mammals [18,19]. Apoptosis as a type of cellular death which regulated through genetic modifications leads to a series of cellular, morphological and biochemical alterations resulting in the cell death . Two different mechanisms are defined for apoptosis induction; intrinsic (mitochondrial) and extrinsic pathway . Although apoptosis is necessary in physiological conditions to regulate and fine-tune organelle function and architecture, it also can be induced or impeded during pathological conditions such as infections, inflammations and cancer [22-25].
Based on the vital roles of apoptosis in spermatogenesis, some studies have investigated the rate of apoptosis in infertile man and shown interestingly the higher rate of apoptosis in the individuals [18,26-28]. Moreover, they have also reported the percentage of apoptotic sperm is more abundant in ejaculated semen samples from the infertile men. Therefore, it seems the apoptosis can be accounted as one of the etiological molecular pathway involved in men infertility [29,30]. Intriguingly, T. gondii has a fascinating dual involvement in host cell apoptosis. Some previous studies demonstrated that T. gondiiinfected cells acquire the relatively resistance to some apoptotic stimuli [31-34]. On the other hand, it has been determined that T. gondii infection can induce apoptosis [35-39].The controversial findings about modulation of apoptosis following T. gondii infections can indicate the complexity of the toxoplasmosis-associated apoptosis which needs further investigations to build a clear picture in the issue. In the recent times profound negative effects of Toxoplasma infection on female reproductive performs however a few researches on the impacts of T. gondii on male reproductive system were executed [40-42]. Given the potential roles of toxoplasmosis-associated apoptosis in men infertility and the poorly undershoot of the underlying mechanisms, we aimed firstly to investigate the apoptosis induction rate with T. gondii in spermatic cells and then check the genes-related microarray to determine the underlying molecular mechanisms.
Materials and Methods
Animal care procedures in this study were performed according to International Guidelines on the Use of Laboratory Animals and ethical guidelines of the Animal Care Committee of Jundishapur University of medical sciences, Ahavz, Iran. The Jundishapur University of Medical sciences’s Ethics Committee also approved the procedures that were used in this study (code: IR.AJUMS.REC.1395.117). The animals’ health condition was checked via the experiments by a health surveillance program according to Federation of European Laboratory Animal Science Associations (FELASA) guidelines. All efforts were made to minimize suffering.
Experimental Design; Animal and Tachyzoites
Twelve balb/c mice strain 6-8weeks old, weighing 20–22 g were used in the present study. The experimental animals were obtained from the experimental Animal Unit of Razi vaccine and serum research institute Iran. Mice were acclimatized to the laboratory conditions for 7 days prior to the initiation of experimental treatments. The experimental animals were housed in standard plastic cages and maintained under controlled laboratory conditions of humidity (55%), temperature (22-23 oC) and 12:12 h light: dark cycle. Mice were fed ad libitum on normal commercial chow and had free access to water.
The RH virulent strains of T. gondii were grown and maintained by routine intraperitoneal passage in Balb/c mice that they were obtained from Department of Parasitology and Mycology Tehran University of Medical Sciences. In order to using fresh tachyzoites for exposuring with spermatogoniacells, firstly the fresh tachyzoites (2×104) were injected by ip to first mice, this mice was kept in a separate cage for 4 days until appearing peritonitis and general weakness signs, on the this day of observation of thus signs the animals were euthanazied humanly with ip injection of ketamine 10% (60 mg/kg) and xylazine 5% (10 mg/kg) according to standardized protocols supplied by the JundishapurMedical Science Ethics Committee, then peritoneal fluid containing fresh tachyzoites was extracted from their peritoneal fluid in sterile conditions. the peritoneal fluids from infected mice were collected and washed twice(at 1,000×g for 15 min) in sterile phosphate-buffered saline (PBS; pH 7.2) and maintained by serial passage in other mice (2×104 tachyzoites), This cycle was applied up to eleventh mice. The same procedure was followed to prepare tachyzoites for further infection experiments in Type B Spermatogonia Germ Cells (GC-1).
Cell Culture and Treatment with T. Gondii Infection
GC-1 cell line obtained from the Department of Cell Bank (Pasteur Institute of Iran) were used in this study. After preparing the cell line and confirmation of non-contamination of the medium, cell viability was measured using trypan blue 0.4% and then the Cells were routinely counted manually with a hemocytometer. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA), supplemented with 10% (v/v) fetal bovine serum (Gibco, USA), 100 U/ml penicillin (Sigma-Aldrich, USA) and 100 μg/ml streptomycin (Sigma-Aldrich, USA). The cells were incubated at 37°C in 5% CO2. The medium was replaced every 2 days. When the cell monolayer reached 70-80% confluence, the cells were detached with a solution of 0.05% trypsin-EDTA and reseeded. Suspension obtained from Peritoneum infected mice after centrifugation and washing used in the interaction assays. GC-1 cells were infected with T. gondiitachyzoites (parasite to cell with several ratio). 3 [4-dimethylthiazol-2-yl] -2-5-diphenyl tetrazolium bromide (MTT) assay were used to determine IC50 value. The equal ratios of cells (104 cells) were seeded in a 96-well tissue culture plate. The cells were treated with increasing concentrations of tachyzoites of T. gondii and MTT (Sigma, USA) assay was performed.
Detection of Apoptosis
3×105 GC-1 per well were seeded in a six-well tissue culture dish. Designated wells were infected with ratio 1x and 2x freshly passaged RH strain parasites for 18 hours. According to the instructions, Infected and noninfected cells were stained with annexin V using the Annexin V/PI kit (eBioscience™ Annexin V Apoptosis Detection Kit FITC) and apoptotic cells identified and quantified by flow cytometry. Briefly, cells were washed in PBS and incubated with 10 X binding buffer, propidium iodide (PI), annexin V-FITC, and dH2O (total 100 μL) for 15 min in the dark. Thenstained cells were analyzed using a Faces Calibur flow cytometer (BD Biosciences, USA).
Evaluation of Genes associated with Apoptosis process by RT2 Profiler™ PCR Array
Total RNA was isolated from target cells according to RNeasy Mini Kit (QIAGEN,Cat No. 74104) and the yield and quality of RNA were assessed by spectrophotometer at 260 nm (Thermo Scientific NanoDrop™ One/OneCMicrovolume UV Spectrophotometer, USA). Real-time PCR reactions were analyzed in total RNA using the Mouse Apoptosis RT2 Profiler™ PCR Array (QIAGEN, Cat No.PAMM-012ZA-24) according to manufacturer's protocol. Briefly, cDNA was prepared from 500 ng total RNA using RT2 PCR array first strand kit (QIAGEN, Cat No. 330401). C DNA was diluted by adding RNase-free water. The PCR was carried out using a LightCycler® 480 apparatus (Roche Applied Science). For one 96-well-plate of the PCR array, 2700 μl of PCR master mix (containing 1350μl 2× RT2 SYBR Green Mastermix, 102 μl of cDNA synthesis reaction and 1248 RNase-free water) were prepared, and aliquots of 25 μl were added to each well. The relative gene expression was determined by Quant Studio 3, 96-well (Applied Bios stems™, USA) real-time detection system software using an adaptive baseline to determine the threshold cycle (CT). The array includes the TNF ligands and their receptors; members of the BCL-2, TRAF, IAP, death domain, CARD, caspase, death effectors domain, and CIDE families; as well as genes involved in the p53 and DNA damage pathways.
The data were analyzed by the ΔΔCT method according to the manufacturer’s manual. Quality control was performed using genomic DNA and reverse transcription and positive PCR controls. The data were normalized to the housekeeping genes. Changes in gene expression were represented as fold increase/decrease. Calculate the fold-change for each gene from group test to group control as 2^-ΔΔct. Genes were considered to be upregulated or downregulated if changes in expression levels were ≥ 1.0-fold or ≤ 1.0-fold, respectively. qPCR for candidate gene. To validate the whole genome microarray data, qRT-PCR was performed, therefore total RNA was extracted from two group cells (control and treat-2x) using Super RNA extraction Kit for (yektatajhiz, Iran) and reverse transcription of the RNA was carried out using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s instructions. cDNA is utilized as a template for subsequent qPCR amplification using primers specific for candidate genes.The qPCR reaction included an initial activation step at 94˚C for 5 min, followed by 35 cycles at 94˚C for 30 secs, annealing at 60˚C for 30 secs, extension at 72˚C for 30 sec and a final extension at 72˚C for 7 min.qPCR was performed using 5x HOT FIREPol ®EvaGreen® qPCR Mix Plus the realtime PCR kit (Solis BioDyne,Estonia) and Rotor-Gene Q Real-Time PCR System (qiagen, USA).Raw data were obtained from Rotor-Gene Q Real-Time PCR System Software, exported in rdml format and imported to LinRegPCR to calculate the PCR efﬁciency. Ct values generated in each experiment were used as an indicator to obtain fold change in the expression of target genes normalized to Gapdh. The relative expression levels in terms of fold change were calculated by 2-ΔΔCt method using Gene software (MultiD Analyses AB, Sweden). The primers used for cDNA amplification are showed in Table 1.
Table 1: Primer sequences of candidate gene.
Gene’s expression was analyzed with RT2 PCR array data analysis version 3.4. Differences between groups were assessed by one-way analysis of variance (ANOVA). Differences were considered statistically significant at a P < 0.05.
Discussion and Results
Viability and Parasite entry process into CG-1 cell line Immediately after cell infecting with T. gondiitachyzoites, parasite entrywas checked at different times with light microscope, the process of attachment and entry took no longer than 30 minutes (Figure 1).
A: entry process of T. gondiitachyzoitesto Gc-1 cell line; A: Before entry; after entry (100x); C: after entry in 1x group; D: after entry in 2x group.
Figure1: Parasite entry to cell line in treated groups.
Investigation of Apoptosis in the Cells Exposed to T. gondii
MTT assays were performed to investigate the ratio of T. gondii to infection GC-1 cell line after exposure for 18h. The calculated IC50 value after 18h incubation of GC-1 cell lines with T. gondiitachyzoites was ratio 2:1 parasite / cell. Apoptosis was investigated by flow cytometry after annexin V/ PI double staining. Apoptosis rate in treated-2x group (29.6%) was higher significant compared treated-1x (18.7%) control group 16.5% (P<0.05) but there was not significant difference between treated-1x (18.7%) control group 16.5% (P>0.05) (Figure 2).
Figure 2: Effects of T. gondii infection on spermatogonia apoptosis levels in vitro.
Expression Changes of Apoptosis- Related Genes
To identify the potential effect of T.gondiitachyzoites on the cellular pathways of apoptosis- related genes of GC-1 cell line, differences in the mRNA levels of selected genes were examined using a custom RT2 Profiler PCR array by comparing different concentrations of tachyzoites-treated cells with control (untreated) cells. Our results showed that group II (2x-treated) had a greater effect on the apoptosis-related genes expression than the group I (1x-treated) compared control group. In both group, we observed a significant down and up-regulation of some genes. Four genes were more than 2-fold down-regulated in group I (1x-treated) compared to untreated group, but in group II (2x-treated), we showed the expression of a gene was decreased compare control group. In both treated groups, some genes showed increased expression. Specially, in group II (2x-treated), twelve genes were up-regulated more than 4-fold after treatment, with Tnfrsf11b presenting the most significant change (higher than 15-fold) (Table 2 and Figure 3). On the other hand, comparison between the two treated groups (I & II) showed that the expression of 14 genes in group II was significantly increased compared to group I (Table 2).
Table 2: Fold changes of apoptosis- related genes expression in group I (1x-treated), group II (2x-treated) and control group of GC-1 cell line.
Validation for Candidate Gene
Figure 3: Cluster analysis of the down and up-regulated apoptosis-related genes in GC-1cell line cells treated with different Concentrations of T.gondiitachyzoites (1x and 2x) compared with control group.
In order to confirm the accuracy of the results, qRT-PCR was carried out to validate the 10 genes. The results revealed that 8 of the 10 differently expressed genes were consistent with the data from microarray analysis. The fold changes of those 10 gene expressions, as determined by whole genome microarray and qRT- PCR, are presented in Figure 4 Whether T. gondii induces apoptosis or not is one of the questions that have long attracted the attention of the researchers around the world. Various reports have expressed contradictory results in this regard [31-39]. Undoubtedly, unfolding the attractive and dual role of T. gondii in inducing or controlling apoptosis in the host cell as well as its mechanism would increase our understanding on the interaction, with the host, of a significantly successful intracellular parasite.
Figure 4: Concordance in the expression of the apoptosis genes between microarray and qRT-PCR.
By secretion of virulence factors from its specialized organs, T. gondii affects the modulation of gene expression in host cells [52,53]. In general, some studies have mentioned the induction of high levels of apoptosis in different cell types, including splenocytes, T-cells in peyer's patches andfibroblasts following the contamination of mice with T. gondii. [39, 54, 55]. However, some other studies have shown that this parasite has no role in inducing apoptosis in cells, such as macrophages, neutrophils, lymphocytes B, placenta, spleen, and the brain; even in some cases, they have shown this protozoan to inhibit apoptosis with various mechanisms [31-56-60]. After Toxoplasma entry into the body and proliferation of tachyzoites during the acute phase, the parasite could affect various organs, including the reproductive system . In recent years, various studies have shown the negative effects of Toxoplasma on the reproductive system of humans and animals, especially females [61-64]. Moreover, few studies have indicated this parasite being a factor in infertility of men . To answer the question of whether T. gondii causes apoptosis or not, while assessing the apoptosis due to Toxoplasma in Spermatogonia cells, this study has examined changes in the expression of 84 genes involved in apoptosis.
In Spermatogonia cells, apoptosis usually takes place to prevent the excessive production of germ cells and to eliminate the damaged germ cells; however, infectious agents, smoking, radiation, pesticides, and so on also induce pathological apoptosis in these cells [18,19,65]. In this study, we showed that adjoining RH strain tachyzoites of T. gondii with B-type Spermatogonia cells with a ratio of 2 to 1 significantly induces apoptosisc .ompared to the control group. However, in lower ratios, no significant differences were observe between the two groups in terms of the incidence of apoptosis, which may be indicative of dose-dependent nature of apoptosis induced by T. gondii. Strains of this parasite with different virulent might be one of the causes of the discrepancy between previous studies regarding the onset of apoptosis from T. gondi . Overall, toxoplasma strains are divided into three groups (I, II and III) based on their virulence to mice, each of which has different strategies for stimulating or destroying the host immune response [66,67]. In a study, Gavrilescu et al. examined the role of virulent of various strains of T.gondii on the host immune response. They showed that the strain with highvirulent (RH), compared to the ones with less virulent (ME49), expands and multiplies faster in mice and is fatal. In addition, infecting the mice with a highly virulent strain increases nitric oxide and IFN-γ in serum and peritoneal fluid, whereas this does not occur in the case of strains with low acuity .
It has been shown that acute or chronic nature of infections affect the pathogenesis of toxoplasmosis too. Channon and Kasper have reported that the contamination of human monocytes with T. gondiiin the acute phase results in the release of soluble factors  The factors derived could lead to apoptosis induction and leading to reduction in immune system strength. Whether acute-phase induction of apoptosis is indicative of parasite strategy to escape the immune response is not known yet. The evidence indicates that non-contaminated cells suffer T. gondii under the influence of NO and other soluble factors released from infected cells. In acute infections, non-contaminated host cells could act as spectators, and excessive production of Th1 cytokines could greatly contribute to pathogenesis . Some other studies have shown that during acute toxoplasmosis, IFN-γ could result in apoptosis and suppression of the immune system [54,55,70]. In vitro conditions of previous studies could be one of the reasons for dual T. gondii's behavior in inducing or inhibiting apoptosis . The duration of infecting a target cell by parasite is another issue to be considered. T. gondii may in regulated to proliferation and surviving itself, play an opposite role in apoptosis at different times.
Nishikawa et al. have compared in vitro and in vivo methods. They have shown no significant differences in in vitro between two strains with high and low virulent in induction of apoptosis in cells, whereas in vivo method, this difference is significant in induction of apoptosis by the higher virulent strain . In vivo, due to the presence of different cell types in the environment and their interaction with each other, the situation is more complicated. In a study by Nash et al., human fibroblasts have been used to maintain the T. gondii tachyzoites . but in the present study, newly acquired tachyzoitesperitoneal fluid from Balb-c rats was used for testing. These different conditions could justify the contradictory result obtained in two studies.
Among the key issues to be considered in previous studies is the target cell being different in these studies. For instance, in a study, Young Hwang et al. showed that infection caused by T. gondii prevented the occurrence of apoptosis in human leukemia cell line (THP-1) . However, the study by Nishikawa et al. showed that T. gondii induces apoptosis in mice fibroblasts (BALB / 3T3 clone A31 fibroblasts) . As the biological conditions of these cells are different, it is quite normal that the responses of these cells vary in the presence of T. gondii as well. Two known pathways result in apoptotic cell-death. One of them is through the transfusion of receptors with extracellular intermediates (TNF-α / TNFR I; Fas / FasL) . and the other is through mitochondrial oxidation [75,76]. It has been reported that a number of factors, like TNF-α, Fas, NF-κB and p53 are involved in the regulation of apoptosis pathways. There is also evidence that T. gondii can significantly modulate host cell-related transcription factors .
In the present study, the comparison between 2X and control groups indicated that T. gondii led to increase in the expression of several pro and anti-apoptotic genes and only reduced the expression of one anti-apoptotic gene. The highest increase in expression (15.28) was related to Tnfrsf11b. In addition to Tnfrsf11b, Fasl, Cidea, Cd70, Naip1, Bcl2a1a, Cd40, Cd40lg, Trp73, Il10, Tnfsf10 and Nme5 genes also had an increased expression of more than 4 folds. The expression of Pro apoptotic genes induces apoptosis in three different pathways, including TNF-α family (Tnfrsf11b, Cd70, Cd40 and Tnfsf10), p53 pathway (Trp73), and the CIDE (Cidea) family apoptosis, whereas increasing the expression of anti-apoptotic genes involving TNF-α (Cd40lg), IAP (Naip1), NME / NM23 (Nme5), Bcl2 (Bcl2a1a) and Il10 families is involved in inhibiting apoptosis. The induction of apoptosis in the present study might be justified as changes in the expression of genes that having a positive effects on the pathway of apoptosis greater than those that have a negative effect. Moreover, the number of genes whose expression had increased during the period of infection with T. gondii was significantly higher than those whose expressions had decreased (18 versus 1) and this was indicative of that induction of gene expression might be the commonest response of Type B spermatogonia cells to infection. As mentioned, induction of apoptosis in Type B spermatogonia cells by T. gondii is dose-dependent, so that with increase in the ratio of parasite burden to Spermatogonia cells, in addition to increase in the number of genes whose expression has changed, the fold of these changes has increased as well.
In recent years, a great deal of progress has been made concerning the role of T. gondii in apoptosis of host cell. T. gondii apoptosis clearly plays an important role in the pathogenesis of the disease. What distinguishes this study from other ones is the simultaneous examination of 84 genes related to apoptosis. However, previous studies on the effect of apoptotic T. gondii on different cells have been conducted with a small number of genes involved in apoptosis. Obviously, T. gondii induces apoptosis in spermatogonial cells directly or indirectly (releasing soluble factors from infected cells). Many factors, including parasite virulent, duration of infection, contaminant dose, and target cell type could affect this process. As molecular mechanisms of apoptosis pathway by toxoplasma have not been completely specified yet, the present study's examination of a great number of genes involved in apoptosis pathway could pave the ground for future studies.
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