Bacteria-Mediated Anomalous Rho GTPase Activation Alters Sperm Structure and Provokes Premature Capacitation Events: A Possible Mechanism of Infertility

Abstract

Male infertility is often linked to sperm quality issues; however, the mechanisms behind these alterations remain unclear in certain contexts. This study investigates the impact of anomalous Rho GTPase activation-a process triggered by bacterial toxins-on human sperm structure and function. Human spermatozoa were exposed in vitro to a Rho GTPase activator derived from Escherichia coli under both capacitating and non-capacitating conditions. The results showed increased RhoA GTPase activity in non-capacitating conditions, without affecting viability or mitochondrial membrane potential. However, progressive motility decreased across both conditions, while non-progressive motility and acrosome reaction rates increased. Additionally, intracellular calcium levels rose exclusively in non-capacitating conditions. Structural analysis revealed an increase in abnormal sperm morphology, particularly vacuoles in the sperm head. These findings highlight that anomalous Rho GTPase activation disrupts essential processes like motility and capacitation, which are crucial for successful fertilization. This study provides novel insights into how bacterial infections may induce sperm damage, proposing that Rho GTPase activity could serve as a biomarker for evaluating sperm quality in cases of infertility linked to urogenital infections. Understanding these mechanisms may improve diagnostic and therapeutic approaches for male infertility associated with bacterial pathogens. Human spermatozoa were exposed in vitro to a Rho GTPase activator derived from Escherichia coli under both capacitating and non-capacitating conditions.

Keywords: Escherichia coli; Rho GTPases; acrosome reaction; cytotoxic necrotizing factor-1; human spermatozoa; sperm morphology.

Figure 1

Rho Activator II effect on RhoA activity and sperm motility. Two experimental conditions were examined: a capacitation condition and a non-capacitation condition. Spermatozoa were incubated with 0.25 μg/mL and 1 μg/mL Rho Activator II for 2 h at 37 °C. The figure represents four independent samples, each with technical duplicates plus the standard deviation bar. (A) RhoA GTPases activity was determined by immunoassay, where the absorbance at 490 nm is proportional to RhoA activity. (B) Results obtained from progressive sperm motility. (C) Results obtained from non-progressive sperm motility. Abbreviations: UC, untreated control group without Rho Activator II; *, p < 0.05 compared to the UC.

Figure 2

Rho Activator II effect on acrosome reaction and intracellular calcium content of spermatozoa. The figure represents four independent samples, each with technical duplicates plus the standard deviation bar. (A) Sperm AR measure after being incubated with 0.25 μg/mL and 1 μg/mL of Rho Activator II for 2 h at 37 °C in capacitation and non-capacitation conditions. (B) The sperm ARi was determined after 3 h of incubation in HTF with 5% BSA and then exposed to Rho Activator II in the presence or absence of progesterone. A negative control of sperm in the same initial medium and a positive control, to which only progesterone was added, were used. The ARi was calculated by subtracting AR spermatozoa of the negative control from experimental groups and expressed as a percentage in relation to AR spermatozoa from the positive control. (C) Percentage of spermatozoa with high calcium content (HCC), which were also incubated with 0.25 μg/mL and 1 μg/mL of Rho Activator II for 2 h at 37 °C in capacitated and non-capacitated conditions. (D) Representative image of spermatozoa staining with PSA-FITC under fluorescence microscopy (1000×) of the UC in the capacitation condition. (E) Representative image of spermatozoa staining with PSA-FITC under fluorescence microscopy (1000×) of the UC in the non-capacitation condition. The red line at the bottom of pictures D and E represents a 10 μm scale bar. Abbreviations: AR, acrosome reaction; HTF, human tubal fluid; BSA, bovine serum albumin; UC, untreated control without Rho Activator II; neg ctrl, negative control; Post ctrl, positive control; *, p < 0.05 compared to their UC group; and **, p < 0.05 for all groups with Rho Activator II compared to the negative control.

Figure 3

Rho Activator II effect on sperm morphology. Two experimental conditions were examined: a capacitation condition and a non-capacitation condition. Spermatozoa were incubated with 0.25 μg/mL and 1 μg/mL Rho Activator II for 2 h at 37 °C. (A) Percentage of spermatozoa with abnormal morphology. The figure represents four independent samples, each with technical duplicates plus the standard deviation bar. (B) Images of commonly observed sperm morphological abnormalities that would be associated with polymerized actin changes: 1. vacuolated head; 2. broken neck; 3. bent neck; 4. z-shaped tail; 5. coiled tail; 6. cut tail; and 7. bent tail. (C) Representative group of spermatozoa in the absence of Rho Activator II. It highlights the presence of vacuoles considered normal (nv). (D) Representative group of spermatozoa in the presence of Rho Activator II. The increased presence of morphological abnormalities such as pathological vacuoles (pv), coiled tail (ct), and bent tail (bt) compared to spermatozoa in (C) is highlighted. The blue line at the bottom of each picture represents a 10 μm scale bar. Abbreviations: UC, untreated group without Rho Activator II; *, p < 0.05 compared to their UC group.