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Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology logoLink to Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology
. 2020 Nov 10;45(2):351–358. doi: 10.1007/s12639-020-01305-6

Toxoplasma gondii induced sperm DNA damage on the experimentally infected rats

Monir Taherimoghaddam 1, Maryam Bahmanzadeh 2,3, Amir Hossein Maghsood 1, Mohammad Fallah 1, Leili Tapak 4,5, Faeze Foroughi-Parvar 1,✉
PMCID: PMC8254694  PMID: 34295033

Abstract

Toxoplasma gondii, as an obligate protozoan parasite, can infect a wide variety of animals as well as human. As some studies have shown, toxoplasmosis decreases the fertility potency in different hosts, so there is a necessity for studies to determine the effects of T. gondii on reproductive system. Therefore, this project was aimed to investigate the effect of toxoplasmosis on the male reproductive system and sperm DNA integrity. In this experimental study, 80 Wistar male rats were divided into two groups as follows: infected group (inoculated by T.gondii tachyzoites) and control group [injected by Phosphate-buffered saline (PBS)]. Afterward, data were collected in every 10 days interval. The detailed description of the sperm parameters were recorded, and then, chromatin integrity of the epididymal sperm was analyzed using Aniline blue (AB), Acridine orange (AO), Chromomycin A3 (CMA3), and Toluidine blue (TB) staining. Sperm parameters (motility, viability, count, and normal sperm) significantly decreased in the infected rats. Sperm stained by AO staining showed a higher percentage in the infected rats compared to the control group on day 70 (P = 0.03). The mean percentages of AB stained sperm on days 30 (P = 0.01) and 50 (P = 0.02) were higher than the healthy group. Also, the significant rising of the stained sperm was observed in the infected group on day 20 (P = 0.01). Sperm stained with TB in the infected group has significantly increased on days 30 to 60 [day 30 (P = 0.001), 40 (P < 0.001), 50 (P = 0.014), and 60 (P = 0.001)]. T. gondii infection leads to the diminished fertility parameters as well as the damaged DNA sperm. The parasite could temporarily interfere with the male reproductive system.

Keywords: Toxoplasma gondii, Male rats, Infertility, Sperm, DNA

Introduction

Toxoplasma gondii is an obligate protozoan parasite that can infect a wide variety of animals as intermediate hosts (Dubey 2016). One third of human population are infected by T. gondii, but in pregnant women and immunocompromised groups, it is considered as a real threat (Tenter et al. 2000). Therefore, several studies have notably focused on the effects of T. gondii infection on female reproductive system and immunocmparative patients (Villena et al. 2010; Toporovski et al. 2012; Wong et al. 2018). Also, Toxoplasmosis impacts on male reproductive system were studied in few projects, for example; in several studies, T. gondii tachyzoites were isolated from animals and human semen (Dubey and Sharma 1980; Moura et al. 2007). It is suggested that, T. gondii through hypothalamic-hypophyseal axis alterations leads to damage on testis or the secondary hypogonadism (Martinez-Garcia et al. 1996). Moreover, studies detected a reduction of fertility in Toxoplasma infected male rats; such as the decreased sperm motility and viability, as well as an increase in abnormal sperm morphology (Abdoli et al. 2012; Dvorakova-Hortova et al. 2014). Pathological changes in testes, epididymis, vas deferens, and prostate of experimentally Toxoplasma infected mice were reported, while parasites were found in seminiferous tubules and sertoli cells cytoplasm as well as testicular macrophages (Shen 2001; Yang et al. 2005; Lopes et al. 2013). In addition, some studies showed that, sperm chromatin and DNA might be damaged under various exogenous and endogenous stress conditions during development (Tarozzi et al. 2007). Damage of DNA, directly or during apoptosis, induces mutation to the prevention of DNA production (Bahrke and Yesalis 2004). In addition, involvement of Toxoplasma infected cells in apoptosis was shown in previous studies and the increased mortality rate of the host cells were also proven (Gavrilescu and Denkers 2001; Payne et al. 2003). Studies also revealed that, T. gondii infection may lead to the sperm chromatin dysfunction through oxidative stress (Al-Kennany 2007; Nayak et al. 2019). Growing evidence implied that, oxidative stress play an important role in the infection induced apoptosis, and besides, excessive reactive oxygen species (ROS) are associated with DNA damage (genomic and mitochondrial) (Xu et al. 2012). Given that further studies should be done to indicate the effect of T. gondii on the male reproductive system and sperm chromatin, this project was designed to investigate if toxoplasmosis can damage sperm chromatin and male reproductive system.

Materials and methods

Animals

Eighty male Wistar rats were provided from animal house of Hamadan University of Medical Sciences. Animals were then divided into two groups. Virulent RH strain tachyzoites (gifted from Tehran University of Medical Sciences), which were obtained by serial intraperitoneal passage every 3–5 days in mice, were used for this experimental model (Foroghi Parvar et al. 2008). T.gondii tachyzoites (107) were inoculated via intraperitoneal injection to 40 rats as a test group. Control healthy rats were injected by PBS with the same volume. Animals were then monitored from day 10 to 80 under optimum optical (12 h light/dark) and thermal condition (at 22–25 °C). The research was performed in terms of the principles and ethical considerations of working with laboratory animals as confirmed by the ethics committee of Hamadan University of Medical Sciences (Ethics committee code: REC. 1396.125.IR.UMSHA).

Sperm analysis

Animals were euthanized by ketamine and xylazine. Then, the caudal part of their left epididymis was removed and finely minced into warm Ham`s F10 medium. Subsequently, spermatozoa were released after 10 to 15 min incubation at 37 °C and 5% CO2 in medium suspension (Bahmanzadeh et al. 2008). Afterward, the droplet of suspension was placed on cell counter chamber slide and sperm heads were counted after the cells settlement for 5 min at 200× magnification. The obtained data were expressed as million per milliliter. The percentage of normal sperm were counted regarding the abnormal sperm characteristics such as: headless, hook-less, double-headed, broken tail, coiled tail, and double-tailed using light microscopy at 200× magnification. Also, aliquots of sperm suspension were assessed for motile sperm using light microscopy. Viable spermatozoa were calculated based on eosin 2% penetration in the disrupted membrane in non-viable and dead spermatozoa (Organization 2010).

Sperm nuclear chromatin

Acridine orange (AO) staining

AO is a metachromatic fluorescence probe used for determination of the susceptibility of the sperm nuclear DNA to in situ acid-induced denaturation. Briefly, air-dried smears were fixed in Carnoy’s solution overnight (methanol:glacial acetic acid, 3:1). Afterward, each sample was stained for 10 min in a freshly prepared AO (0.19 mg/mL, Sigma-Aldrich) and in citrate phosphate buffer (McIlvain buffer, pH 4) for 5 min. The smears were then evaluated on the same day using Olympus fluorescence microscope (Zeiss) with a 460-nm filter. Accordingly, the duration of illumination was limited to 40 s per field. Native double-stranded DNA was distinguished from the denatured single-stranded DNA using green fluorescent versus red fluorescent in 100 spermatozoa (Pourentezari et al. 2014; Shokri et al. 2014).

Aniline blue (AB) staining

AB selectively stained lysine-rich histones. Air dried sperm smears were then fixed in 3% buffered glutaraldehyde in a 0.2 M phosphate buffer (pH 7.2) for 30 min at room temperature. Afterward, each smear was stained by 5% aniline blue solution (Sigma Co., St. Louis, MO) that was acidified to approximately pH 3.5 with acetic acid for 5 min, then they were washed with tap water and allowed to be completely air dried. At least, 100 sperm were evaluated using light microscopy. Also, dark blue stained spermatozoa were considered as abnormal, while those showing the unstained and pale blue stained were regarded as normal (Dadoune et al. 1988).

Toluidine blue (TB) staining

Briefly, air-dried spermatozoa smears were fixed in a freshly prepared 96% ethanol-acetone (1:1) for 30 min at 4 °C, and they were then hydrolyzed in 0.1 N HCl for 5 min at 4 °C. Subsequently, the slides were rinsed 3 times in distilled water for 2 min, and finally stained with 0.05% TB (Merck, Darmstadt, Germany) in 50% citrate phosphate (McIlvain buffer, pH: 3.5) for 10 min at room temperature. For performing the light microscopic assessment using 100× magnification, the chromatin quality of the spermatozoa was determined in terms of the metachromatic staining of sperm heads in light blue (TB−) and dark blue (TB+) (Jee et al. 2011; Bahmanzadeh et al. 2016).

Chromomycin A3 (CMA3) staining

CMA3 is fluorochrome specific for guanosine cytosine-rich sequences, which was used for the estimation of the degree of protamination of sperm chromatin in this study. Moreover, sperm smears were firstly dried and then fixed in Carnoy’s solution (methanol/glacial acetic acid 3:1) for 5 min at 4 °C. Afterward, slides were treated by 100 µl of CMA3 solution (C2659, Sigma-Aldrich) (0.25 mg/ml in a McIlvain buffer: 7 mL citric acid 0.1 M + 32.9 mL Na2HPO4 7H2O 0.2 M, pH 7.0, containing 10 mM MgCl2) for 20 min. After staining in darkness, the slides were washed in buffer and then mounted with buffered glycerol (1:1). Each sample was examined by Olympus fluorescence microscope (Zeiss), with the appropriate filters (460 to 470 nm) and 100× eyepiece magnifications. Notably, in CMA3 staining, bright yellow-stained Chromomycin-reacted spermatozoa (CMA3+) are considered as abnormal forms and yellowish green-stained non-reacted spermatozoa (CMA3−) are considered as normal forms (Bahmanzadeh et al. 2016).

Statistical analysis

All statistical analyses were performed using spss V.16. (IBM Corp., Armonk, NY.USA). Variables were analyzed by Mann–Whitney and Kruskal–Wallis tests. All the obtained data were expressed as mean ± standard deviation (SD). The statistical level of significance was set at P < 0.05.

Results

Sperm parameters

This study investigated the effect of Toxoplasma infection on DNA integrity as well as the sperm parameters. It was indicated that, T. gondii can significantly reduce sperm parameters. Normal sperm have decreased over the infection as reported as a significant reduction on days 20 and 70 (P = 0.008 for day 20 and P = 0.009 for day 70, respectively). At the same time, as sperm counts significantly diminished on days 10 to 70 except the day 30 (P < 0.05), their motility has also decreased on days 20 to 70 (P < 0.01). Moreover, the percentage of viable sperm significantly decreased on days 20, 30, and 60 (P < 0.02). This investigation indicates that, toxoplasmic rats have significantly higher death rate in sperm as well as atypical sperm (Fig. 1).

Fig. 1.

Fig. 1

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Comparison of sperm analysis results in days 10 to 80 of control and infected rats. Sperm viability a, Sperm count b, Sperm viability c and sperm total motility d. *P < 0.05

Sperm nuclear chromatin

The mean percentage of sperm stained by AO staining showed that, the number of the sperm stained was significantly higher in the infected group compared to the control group on day 70 (P = 0.03). AB sperm chromatin examination revealed that, the mean percentage of the stained sperm in the infected group has increased during the studied days compared to the control group. In addition, statistical analysis showed that, the mean percentages of the stained sperm were significantly higher on days 30 (P = 0.01) and 50 (P = 0.02) compared to the healthy group. Abnormal sperm have also increased in the infected rats compared to the control ones via CMA3 chromatin staining. A significant rising of the stained sperm was observed in the infected group on day 20 (P = 0.01) (Fig. 2).

Fig. 2.

Fig. 2

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Evaluation of the sperm nuclear chromatin integrity. a Acridine orange (AO), Orange-red-stained spermatozoa were considered as AO positive while, b green stained spermatozoa were considered to be AO negative (non-denatured DNA). c Aniline blue (AB), dark blue stained spermatozoa were considered abnormal. d Unstained or pale blue-stained spermatozoa were considered normal. e Toluidine blue (TB) dark blue-stained spermatozoa were considered abnormal. f Unstained or pale blue-stained spermatozoa were considered normal. g Chromomycin A3 (CMA3), bright yellow stained spermatozoa were considered as CMA3 positive or protamine deficient. h While green stained spermatozoa were considered to be CMA3 negative, with a normal amount of protamine

Mean percentage of the sperm stained with TB in the infected group significantly increased on days 30 to 60 [day 30 (P = 0.001), 40 (P < 0.001), 50 (P = 0.014), and 60 (P = 0.001) (Fig. 3).

Fig. 3.

Fig. 3

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The percentage of abnormal sperm chromatin. Results of Acridine orange a, Aniline blue b, Chromomycin A3 c and Toluidine blue d staining in T. gondii infected and control groups. *P < 0.05, #P < 0.001

Discussion

In the present study, the percentage of abnormal sperm, (sperm with no head or tail, and double-headed or fragmented sperm) increased along with the progression of Toxoplasma infection in the test group. Statistical analysis revealed that, abnormal sperm morphology significantly increased on days 20 and 70. The mean percentage of motile sperm of the infected group decreased on days 20 to 70 after being infected. In other words, the number of active sperm decreased as the toxoplasmosis progressed. In a study by Terpsidis et al., the mean percentage of sperm motility decreased on days 10 to 60 except the day 30 after the infection in the Toxoplasma infected rats (Terpsidis et al. 2009). In the present study, sperm count significantly declined on days 10 to 70 (except the day 30) in the infected group. Moreover, the percentage of live spermatozoa has significantly decreased on days 20 to 70 (except the day 40) in the infected group. Based on the above descriptions, proportion of normal sperm, rate of motility, and the total sperm count followed a similar decline trend. Also, since the parameters related to sperm are considered as the important criteria for fertility determination, it can be concluded that reduction in number of mobile spermatozoids and increase in the number of abnormal ones are the result of the effects of T. gondii on spermatogenesis. In a similar study by Abdoli et al. (2012), the mean percentage of abnormal sperm and other fertility factors showed a significant fall in the infected group. Terpsidis et al. (2009) reported an enhanced mean percentage of abnormal sperm as well as dead sperm in T. gondii infected rats during their study. Accordingly, in the current study, the percentage of the damaged sperm were significantly higher than natural spermatozoa in days 20 to 70 by TB, CMA3, AB, and AO staining (Acridine orange on day 70, Aniline blue on days 30 and 50, Chromomycinon day 20, and Toluidine Blue on days 30 to 60). Some studies reported that, if more than 30% of sperm are stained by AB, fertility rate will be decreased (Dadoune et al. 1988). In the current work, the maximum percentage of the stained sperm by AB reached 26% (day 70). Also, studies insisted that, if sperm parameters were not limited in the normal range, the percentage of the abnormal chromatin sperm would more likely increase. The results of this study also confirm that, there were significant changes of morphological factors (number and sperm's motility and viability) in Toxoplasma infected rats from days 30 to 70 (Dadoune et al. 1988). CMA3 staining indicates a protamine deficiency in the sperm nucleus chromatin. Moreover, a statistical significant relationship was suggested between the protamine deficiency and fertilization rate (Aoki et al. 2006). Aukey et al. demonstrated that sperm quality was inversely correlated with protamine deficiency (Carrel and Liu 2001; Aoki et al. 2005). Sperms that are stained in this way are mostly associated with the morphological abnormalities; as both morphological changes and protamine deficiency have independently affected the fertilization process (Nasr-Esfahani et al. 2001). In the current study, regarding the significant increase of the damaged sperm detected by CMA3 staining on day 20, the increased abnormal sperm morphology were recorded on days 20 and 70, which was in accordance with each other just on day 20, not on day 70. This finding may support the idea that, CMA3 staining, as a sensitive method, indicated damage to chromatin at the earliest stages prior to morphological changes (Bartoov et al. 2002). Aniline blue staining is also used to evaluate sperm chromatin stability. Histones of sperm, which were not replaced by protamine during the spermiogenesis, have a high amount of lysine amino acid and are stained in the blue and signed as impairment (Dadoune et al. 1988). Studies showed that there was a direct association between the ratio of intact sperm chromatin and the rate of in vitro fertilization (Kikuchi et al. 1976). Also, Reactive oxygen species (ROS) are half-life chemicals derived from oxygen in the metabolic pathways of all aerobic cells. Free radicals are highly reactive due to having unpaired electrons, and are also capable of attacking biological macromolecules such as amino acids or proteins, carbohydrates, lipids, and nucleic acids to compensate for their lack of oxygen, which ultimately lead to death by severe damage to cell structure and function (Sikka et al. 1995). In this regard, similar studies have suggested the effect of silver on sperm chromatin via oxidative stress establishment. Free radicals cause oxidative stress and also damage DNA and then destroy it (Seed et al. 1996). Reactive oxygen species (ROS) can also cause DNA fragmentation at the early stages of chromatin formation (Shrilatha 2007). Germ cell apoptosis and reduction in sperm production are resulted from excessive ROS may account for the male fertility deterioration (Agarwal et al. 2006). Increased apoptotic germ cells in seminiferous tubules, mainly lead to round spermatid and elongated spermatid and subsequently in chromatin abnormalities in the tests (Simşek et al. 1998).

Based on the current results, T. gondii can induce DNA damage. However, the extent of this degradation did not result in infertility and inability of sperm to fertilization. Also, production of antioxidants, as a result of parasite tendency to develop cysts in different tissues during infection, caused oxidative stress in tissue as well as in testis, and consequently, destroyed sperm chromatin (Sharma et al. 2018). It is documented in several research studies that diseases such as varicocele lead to reactive oxygen species (ROS) generation, peroxidation of sperm plasma membrane, nuclear DNA damage. The frequency of sperm cells with impaired chromatin is higher in infertile individuals with varicocele compared to other infertile men. Studies showed spermatozoa with abnormal DNA and immature chromatin caused failure in fertile sperm production (Twigg et al. 1998; Sakkas et al. 1999; Talebi et al. 2008).

Recent studies highlighted that, fertilization has been affected by toxoplasmosis, for example; it was described that, genital system had been damaged, and hypogonadism was reported due to T. gondii infection (Suresh Babu et al. 2007; Barreto et al. 2008); however, none of them focused on the effects of T. gondii on the sperm DNA quality. Other evidence has revealed a large number of T. gondii tachyzoites in seminal fluid of infertile patients as well as anti-sperm (Bohring et al. 2001). In accordance with the present study, Abdoli and Trepsidis separately detected the decreased number and motility of sperm and impaired sperm morphology in Toxoplasma infected rats (Terpsidis et al. 2009; Abdoli et al. 2012).

Conclusion

The most remarkable result to emerge from the data is that, T. gondii infection reduced the fertility parameters. Since most of the parameters were close to the mean of the control group at day 80 of this study, it could be concluded that, the effect of this parasite was on transient fertility factors, and the fertility conditions would be returned to the normal level in a case that the disease was controlled due to the immune system. Another interesting result of this study showed that DNA sperm has been damaged by T. gondii. Although further studies are needed to determine the mechanisms of T. gondii sperm chromatin degradation, it is clear that it can affect the quality of fertility.

Acknowledgements

This paper was a part of research, supported financially by Endometrium and Endometriosis Research Center, Hamadan University of Medical Sciences, Hamadan, Iran. We would like to show our gratitude to Ms. Ghadiri for sharing experiences during the course of this project. We are also immensely grateful to reviewers for their precious insights.

Author contributions

All authors have participated in conception and design, or analysis and interpretation of the project; drafting the article or revising it critically for important intellectual content; and approval of the final version.

Funding

This research was supported financially by Endometrium and Endometriosis Research Center, Hamadan University of Medical Sciences, Hamadan, Iran (Project No: 9602261247).

Compliance with ethical standards

Conflicts of interest

The authors have no conflicts of interest.

Ethical approval

The research was performed in terms of the principles and ethical considerations of working with laboratory animals as confirmed by the ethics committee of Hamadan University of Medical Sciences (Ethics committee code: REC. 1396.125.IR.UMSHA).

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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