Toll-like Receptor 2 is Involved in Calcium Influx and Acrosome Reaction to Facilitate Sperm Penetration to Oocytes During in vitro Fertilization in Cattle

Abstract

Cumulus cells of ovulated cumulus-oocyte complexes (COCs) express Toll-like receptor 2 (TLR2), pathogen recognition receptors, to recognize and react to sperm signals during fertilization. Sperm also express TLR2, but its contribution to the sperm-oocytes crosstalk is still unclear. Here, we adapted the in vitro fertilization (IVF) model to characterize the potential relevance of sperm TLR2 in sperm-oocytes interactions during fertilization in bovine. The IVF results showed that the ligation of sperm TLR2 with its specific antagonist/agonist resulted in down/up-regulation of the cleavage and blastocyst rates either in COCs or cumulus-free oocytes, but not in zona pellucida (ZP)-free oocytes. The computer-assisted sperm analysis (CASA) system revealed that sperm motility parameters were not affected in TLR2 antagonist/agonist-treated sperm. However, fluorescence imaging of sperm-ZP interactions revealed that the blockage or activation of the TLR2 system in sperm reduced or enhanced both binding and penetration abilities of sperm to ZP compared to control, respectively. Flow cytometrical analysis of acrosome reaction (AR) demonstrated that the TLR2 system adjusted the occurrence of AR in ZP-attached sperm, suggesting that sperm TLR2 plays physiological impacts on the sperm-oocyte crosstalk via regulating ZP-triggered AR in sperm. Given that calcium (Ca²⁺) influx is a pre-requisite step for the induction of AR, we investigated the impact of the TLR2 system on the ionophore A23187-induced Ca²⁺ influx into sperm. Notably, the exposure of sperm to TLR2 antagonist/agonist reduced/increased the intracellular Ca²⁺ level in sperm. Together, these findings shed new light that the TLR2 system is involved in sperm AR induction which enables sperm to penetrate and fertilize oocytes during the fertilization, at least in vitro, in cows. This suggests that sperm possibly developed a quite flexible sensing mechanism simultaneously against pathogens as well as COCs toward fertilization with the same TLR2 of the innate immune system.

Keywords: acrosome reaction; bovine; in vitro fertilization; intracellular Ca2+ influx; sperm; toll-like receptor 2.

FIGURE 1

Schematic representation of the study experimental design. Initially, to investigate the potential contribution of sperm TLR2 in sperm-oocyte interaction during in vitro fertilization, TLR2 antagonist (CU-CPT22; 100 µM) or TLR2 agonist (Pam3Cys; 100 ng ml⁻¹) was used for the blockage or activation of TLR2 in frozen-thawed bull sperm for 30 min, respectively. Sperm were then washed, adjusted to a concentration of 0.5 × 10⁶ sperm ml⁻¹, and co-cultured with matured intact COCs, cumulus-free (zona intact), or zona-free oocytes for 6 h. Cleavage rate and blastocyst rate were evaluated at 42 and 168 h post-insemination respectively. Moreover, the ability of TLR2 antagonist/agonist treated sperm to bind or penetrate ZP and the subsequent induction of AR was evaluated after 1 and 3 h of the co-culture using fluorescence microscopy and flow cytometry, respectively. Based on the results, more focus was given to the direct impact of blockage/activation of sperm TLR2 on sperm motility parameters, AR, and intracellular Ca²⁺ influx using CASA analysis, flow cytometry, and fluorescence microscopy, respectively.

FIGURE 2

Blockage/activation of sperm TLR2 suppressed/enhanced the cleavage and blastocyst rates in COCs. Sperm were pre-treated by (A) TLR2 antagonist (100 µM) or (B) TLR2 agonist (100 ng ml⁻¹) for 30 min, washed, and co-cultured with cumulus-free(zona-intact) and zona-free oocytes. Percentage of fertilized oocytes observed after 42 h post insemination and blastocyst on Day 7 (Day 0 = day of fertilization). Data reported as means ± S.E.M. Different superscript asterisks denote a significant difference (p < 0.05). The number of presumptive zygotes for each treatment group (from three independent experiments) is specified above each Figure.

FIGURE 3

Blockage/activation of sperm TLR2 suppressed/enhanced the cleavage rate in cumulus-free oocytes, but not in zona-free oocytes. Sperm were pre-treated by (A) TLR2 antagonist (100 µM) or (B) TLR2 agonist (100 ng ml⁻¹) for 30 min, washed, and co-cultured with cumulus-free (zona-intact) and zona-free oocytes. Percentage of fertilized oocytes observed after 42 h post insemination. Data reported as means ± S.E.M. Different superscript asterisks denote a significant difference (p < 0.05). The number of presumptive zygotes for each treatment group (from three independent experiments) is specified above each Figure.

FIGURE 4

TLR2 pathway mediates sperm-ZP binding and sperm-ZP penetration abilities. Sperm were pre-treated by TLR2 antagonist (100 µM) or TLR2 agonist (100 ng ml⁻¹) for 30 min, washed, and co-cultured with cumulus-free oocytes (n = 15–20) for 1 and 3 h. Sperm-oocyte complexes were washed to remove ZP-loosely attached sperm while only ZP-strongly attached sperm were counted as sperm-ZP binding ratio after staining with Hoechst 33342 and observed at ×200 magnification. Sperm with heads embedded in the ZP or perivitelline space were counted as sperm-ZP penetration ratio (A) Representative image of zona-binding observed in control and TLR2 antagonist treated group and the average number of sperm bound to the ZP at 1 and 3 h was counted (B) Representative image of zona-binding observed in control and TLR2 agonist treated group and the average number of sperm bound to the ZP at 1 and 3 h was counted (C) Representative image of zona-penetration observed in control and TLR2 antagonist treated group and the average number of sperm penetrated to the ZP at 1 and 3 h was counted (D) Representative image of zona-penetration observed in control and TLR2 agonist treated group and the average number of sperm penetration to the ZP at 1 and 3 h was counted. Data reported as means ± S.E.M of three independent experiments. Different superscript asterisks denote a significant difference (p < 0.05). Scale bar = 50 μm.

FIGURE 5

TLR2 system impacts the ZP-induced acrosome reaction in sperm under IVF conditions. Sperm were pre-treated by TLR2 antagonist (100 µM) or TLR2 agonist (100 ng ml⁻¹) for 30 min, washed, and co-cultured with cumulus-free oocytes for 1 and 3 h. Then, ZP-attached sperm were collected and incubated with 25 μg ml⁻¹ fluorescein peanut agglutinin FITC-conjugate (PNA-FITC) for 8 min at 38.5°C in dark. The percent of acrosome-reacted sperm was analyzed by flow cytometry (A) TLR2 antagonist reduces the ZP-induced acrosome reaction in sperm under IVF conditions (B) TLR2 agonist increases the ZP-induced acrosome reaction in sperm under IVF conditions. Data reported as means ± S.E.M of five independent experiments. Different superscript asterisks denote a significant difference (p < 0.05).

FIGURE 6

Effect of TLR2 antagonist and agonist treatment on sperm acrosome reaction (AR) triggered by A23187. Swim-up sperm were pre-treated by TLR2 antagonist (100 µM) or TLR2 agonist (100 ng ml⁻¹) for 30 min, washed, and triggered with or without 1 µM A23187 for 60 min. Then sperm were incubated with fluorescein peanut agglutinin FITC-conjugate (PNA-FITC; for detection of induction of acrosome reaction (AR)) and propidium iodide (PI; for detection of plasma membrane damage (PMD)) for 8 min at 38.5°C in dark. The percent of live and acrosome-reacted sperm was analyzed by fluorescence microscopy and flow cytometry. Untreated sperm were kept as a negative control (A) Representative dot plot diagram; a: Control; c: 100 μM TLR2 antagonist; b: Control +1 μM A23187; d: 100 μM TLR2 antagonist +1 μM A23187 and (C) Analysis of PNA-FITC and PI staining of bovine sperm in different treatment groups by flow cytometry (B) Representative dot plot diagram; a: Control; c: 100 ng ml⁻¹ TLR2 agonist; b: Control +1 μM A23187; d: 100 ng ml⁻¹ TLR2 agonist +1 μM A23187 and (D) Analysis of PNA-FITC and PI staining of bovine sperm in different treatment groups by flow cytometry (E) Representative images of TLR2 antagonist-treated sperm or (F) TLR2 agonist-treated sperm stained with FITC-PNA and PI viewed with fluorescence microscope (200×). a: live intact acrosome, b: dead intact acrosome, c: dead reacted acrosome, d: live reacted acrosome. Data reported as means ± S.E.M of five independent experiments. Asterisks denote a significant variance **** (p < 0.0001) between the different groups compared to control.

FIGURE 7

TLR2 system manipulates the intracellular calcium uptake by bovine sperm. Swim-up sperm were pre-treated by TLR2 antagonist (100 µM) or TLR2 agonist (100 ng ml⁻¹) for 30 min, washed, and loaded with 5 µM Fluo-4 AM for 40 min at 38.5°C in the dark before adding calcium ionophore A23187 (1 µM as a final concentration). The sperm fluorescence was measured by the fluorescence microscope. Images were captured every 5 min for a total of 1 h, with A23187 added after the initial five readings (every 1 min). The images were analyzed using BZ-X800 Analyzer and each sperm head was selected as the region of interest. Data were normalized using the following equation (F/F0)-1. where F0 is the average of the first five readings before the addition of A23187 and F is the fluorescence intensity obtained at each time point (A) Single-cell imaging of Fluo-4 fluorescence in bovine sperm in response to treatment: Control (a–c) and 100 µM TLR2 antagonist (d–f) (B) Single-cell imaging of Fluo-4 fluorescence in bovine sperm in response to treatment: Control (a–c) and 100 ng ml⁻¹ TLR2 agonist (d–f). Photos were taken before treatment (a, d), at 5 min (b, e), and 30 min (c, f) (Left) Measurement for individual sperm in each treatment for 60 min incubation. Original magnification ×200(a–f). Scale bar = 20 μm (C) TLR2 antagonist decreases the average intracellular calcium uptake by bovine sperm in response to treatment with A23187 (D) TLR2 agonist increases the average intracellular calcium uptake by bovine sperm in response to treatment with A23187. Data reported as means ± S.E.M of five independent experiments. Asterisks denote a significant variance * (p < 0.05); ** (p < 0.01); *** (p < 0.001); **** (p < 0.0001) between the treatment group compared to control.