Inhibitors of zinc-dependent metalloproteases hinder sperm passage through the cumulus oophorus during porcine fertilization in vitro

Abstract

In this study, we report for the first time on a possible contribution of metalloproteases in sperm passage through the cumulus matrix in pigs. The presence of 20 mM 1,10-phenanthroline (1,10-PHEN), inhibitor of zinc-dependent metalloproteases, strongly inhibited the degree of sperm penetration in cumulus-intact (CI), but not in cumulus-free (CF), porcine oocytes during IVF. The inhibitory effect of 1,10-PHEN was due to the chelation of metal ions as a non-chelating analog (1,7-PHEN) did not affect IVF rates. Furthermore, incubation with 1,10-PHEN did not affect sperm binding to the zona pellucida nor sperm motility, membrane integrity, or acrosomal status. These findings led to the assumption that 1,10-PHEN interacts with a sperm-or cumulus-derived metalloprotease. Metalloproteases are key players in physiological processes involving degradation or remodeling of extracellular matrix. In vivo, their proteolytic activity is regulated by tissue inhibitors of metalloproteases (TIMP1-TIMP4). We tested the effect of TIMP3 on fertilization parameters after porcine IVF. Similar to 1,10-PHEN, TIMP3 inhibited total fertilization rate of CI but not CF oocytes and did not influence sperm quality parameters. Although the inhibitory effect was stronger in CI oocytes, TIMP3 also reduced the degree of sperm penetration in CF oocytes, suggesting the involvement of a metalloprotease in a subsequent step during fertilization. In conclusion, our results indicate the involvement of TIMP3-sensitive, zinc-dependent metalloprotease activity in sperm passage through the cumulus oophorus in pigs. The results should provide the basis for further biochemical research toward the localization and identification of the metalloprotease involved.

Oviductal estrogen receptor α signaling prevents protease-mediated embryo death

“…This idea is consistent with our findings that there were significant alterations in chemokines and their receptors expressed in the cKO oviduct and that there were alterations in PGs, which can modulate chemokine secretion ( Kalinski, 2012 ). Effects of altered oviductal PGs, chemokines, proteases, or PIs on the cumulus cells and/or extracellular matrix structure could also contribute to our observation of impaired fertilization in vitro ( Kim et al, 2008 ; Tamba et al, 2008 ; Beek et al, 2012 ).…”

Zinc ion flux during mammalian sperm capacitation

“…Matrix metalloproteinases are zinc-dependent enzymes known mainly for their ability to digest extracellar matrix 19 , with MMP-2 and MMP-9 being reported in human seminal plasma 20 . Inhibition of zinc-dependent metalloproteases hindered sperm passage through oocyte vestments during IVF 21 . Related to aforementioned protamine sperm chromatin stabilization, Zn-ion release from the nuclear zinc bridges described in human spermatozoa could facilitate sperm nucleus decondensation after fertilization, as a prelude to the formation of the zygotic paternal pronucleus 22 , 23 .…”

Porcine oocyte maturation <i>in vitro</i>: role of cAMP and oocyte-secreted factors – A practical approach

“…). This phenomenon indicates inferior matrix disassembly [84]. To explain this occurrence, the composition and degradation of the extracellular matrix of the COC should first be discussed in more detail.…”

“…To explain this occurrence, the composition and degradation of the extracellular matrix of the COC should first be discussed in more detail. Proteases are among the products secreted by cumulus cells [84]. Within the metalloproteases, a disintegrin and metalloproteinase with thrombospondin-like repeats-1 (ADAMTS-1), part of the ADAMTS family, plays a crucial role in the ovulation process [85].…”