<i>Toxoplasma gondii</i> Decreases the Reproductive Fitness in Mice

<div><p><i>Toxoplasma gondii</i> is a common protozoan parasite that infects warm-blooded animals throughout the world, including mice and humans. During infection, both, the parasite and the host, utilize various mechanisms to maximize their own reproductive success. Mice and humans are both the intermediate hosts for <i>Toxoplasma gondii</i>, which forms specialized vacuoles containing reproductive cysts in the formers’ tissue. As half of the human population is infected, developing a disease called toxoplasmosis, along with an ever-growing number of couples suffering with idiopathic infertility, it is therefore surprising that there is a lack of research on how <i>Toxoplasma gondii</i> can alter reproductive parameters. In this study, a detailed histometric screening of the testicular function along with the levels of the pituitary luteinizing hormone (LH) were analysed in infected mice. Data on relative testis and epididymis weight, and sperm count were also collected. Based on the results obtained, the level of LH in the urine of <i>Toxoplasma gondii</i> infected mice was lower compared to the control. In direct correlation with the hormone level, testicular function and sperm production was also significantly lower in <i>Toxoplasma gondii</i> positive group using sperm count and histometric analysis as a marker. Not only were the number of leptotene primary spermatocytes and spermatids lowered, but the number of Sertoli cells and the tubule diameter were elevated. In parallel, a pilot epigenetic study on global testicular methylation, and specific methylation of Crem, Creb1 and Hspa1genes essential for successfully ongoing spermatogenesis was performed. Global methylation was elevated in <i>Toxoplasma</i> infected mice, and differences in the DNA methylation of selected genes were detected between the <i>Toxoplasma</i> positive and control group. These findings demonstrate a direct relation between <i>Toxoplasma gondii</i> infection and the decrease of male reproductive fitness in mice, which may contribute to an increase of idiopathic infertility in humans.</p></div

Graphs showing a level of correlation between individual parameters within Toxo⁺ and Toxo⁻ group.

<p>Sertoli cells/Leptotene primary spermatocytes (A), Sertoli cells/spermatids (B) Leptotene primary Spermatocytes/Spermatides (C) The statistically significant coefficient of determination R² is indicated by an asterix*.</p

The LH level comparison in urine of Toxo⁺ and Toxo⁻ at Day 0 (the day prior) and Day 30 (at the end) of the experiment.

<p>The middle line indicates the arithmetic mean, the box extends from the 25th to 75th percentiles and the whiskers indicate the minimum and maximum of the measurement. The broken line indicates no significant difference between the Toxo⁺ and Toxo⁻ group at the beginning of the experiment. (*p value≤0.05).</p

Quantitative analysis of CpG methylation of the Crem gene promoter in Toxo⁺ and Toxo⁻ group.

<p>A. An exemplary pyrogram showing the methylation level in each CpG between the representative Toxo⁺ and Toxo⁻ samples. B. Summary of methylation in Toxo⁺ and Toxo⁻ groups. The middle line indicates the arithmetic mean, the whiskers indicate the standard deviations and the points indicate individual measurements (% of methylation of the appropriate CpG position). The red color indicates the significant difference (*p value≤0.05).</p

Histometric analysis of testicular cells.

<p>The number of Sertoli cells (A), Leptotene primary Spermatocytes (B), Spermatids (C) in Toxo⁺ and Toxo⁻ group. The middle line indicates the arithmetic mean, the box extends from the 25th to 75th percentiles and the whiskers indicate the minimum and maximum of the measurement. (***p≤0.001).</p

Quantitative analysis of CpG methylation of Hspa1 gene promoter in Toxo⁺ and Toxo⁻ group.

<p>A. An exemplary pyrogram showing the methylation level in each analysed CpG between the representative Toxo⁺ and Toxo⁻ samples. B. Summary of methylation in Toxo⁺ and Toxo⁻ groups. The middle line indicates the arithmetic mean, the whiskers indicate the standard deviations and the points indicate individual measurements (% of methylation of the appropriate CpG position). The red color indicates the significant difference (*p value≤0.05).</p

A The tubular diameter comparison between Toxo⁺ and Toxo⁻ group.

<p>The middle line indicates the arithmetic mean, the box extends from the 25th to 75th percentilesand the whiskers indicate the minimum and maximum of the measurement. (***p≤0.001). <b>B</b> The differences in the Index 250 between Toxo⁺ and Toxo⁻ group. The middle line indicates the arithmetic mean, the box extends from the 25th to 75th percentiles and the whiskers indicate the minimum and maximum of the measurement. (***p≤0.001).</p

The sperm concentration comparison between Toxo⁺ and Toxo⁻ group.

<p>The middle line indicates the arithmetic mean, the box extends from the 25th to 75th percentiles and the whiskers indicate the minimum and maximum of the measurement. (***p≤0.001).</p

Cross sections of <i>tubuli seminiferi</i> from mice testes infected with <i>Toxoplasma gondii.</i>

<p>No dramatic effect on the histological architecture could be observed in the testis of infected mice compared to controls. Magnifications 20x (A) and 40x (B) and control samples 20x (C) and 40x (D).</p

Effect of <i>Toxoplasma gondii</i> on testicular global DNA methylation.

<p>The middle line indicates the arithmetic mean, the whiskers indicate standard deviation and points indicate individual measurements. (*p≤0.05).</p