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.
Highlights
Fertilization failure is one of the main reasons for infertility which has been attributed to either sperm or oocyte factors (Kashir et al, 2010)
The results showed that the blockage of sperm with Toll-like receptor 2 (TLR2) antagonist induced a substantial decline in the cleavage and blastocyst rates (Figure 2A)
To further explore the impact of sperm TLR2 on sperm crosstalk with cumulus cells and/or zona pellucida (ZP), TLR2 antagonist/agonist treated sperm were co-cultured with matured cumulus-free or zona-free oocytes for 6 h, and cleavage rate was analyzed
Summary
Fertilization failure is one of the main reasons for infertility which has been attributed to either sperm or oocyte factors (Kashir et al, 2010). Sperm capacitation is a prerequisite step to successful fertilization (Austin, 1951; Chang, 1951) that involves a series of biochemical transformations, including changes in sperm metabolism, intracellular pH, intracellular cyclic adenosine monophosphate (cAMP), and intracellular calcium concentration, all of which prepare sperm to undergo AR to penetrate the zona pellucida (ZP) and fertilize oocytes (Breitbart et al, 2005; Kwon et al, 2013). Despite investigations, it is not completely understood whether fertilizing spermatozoon initiates its AR during its voyage through the cumulus cells or when it binds to the ZP (Gadella, 2012). Only the acrosome-reacted sperm are existent in the perivitelline space and can fuse with the plasma membrane of the oocyte to effect fertilization (Austin, 1975; Saling et al, 1979; Toshimori, 1982; Inoue et al, 2005; Avella and Dean., 2011)
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