Zona pellucida solubility and cortical granule complements in human oocytes following assisted reproductive techniques

Abstract

In this study the solubility to α-chymotrypsin of the zona pellucida (ZP) of human oocytes and polyploid embryos obtained during various clinical procedures of assisted fertilisation (IVF, ICSI, cyropreservation) was evaluated. The aim of the study was to determine whether changes in ZP solubility occur during such procedures and whether abnormal solubility could be likened to fertilisation failure. Correlation between ZP solubility and cortical granule (CG) density was also studied. The results showed that ZP solubility varied considerably among germinal vesicle or metaphase oocytes obtained from different subjects, but was essentially identical for the oocyte cohort obtained from individual women. On the basis of ZP solubility metaphase oocytes were subdivided into two classes: class I, average ZP dissolution time ± SE = 24.1 ± 0.9 min, n = 28; and class II, 46.7 ± 2.0 min, n = 13. Prolonged ZP dissolution times of metaphase oocytes were significantly correlated with a low in vitro fertilisation rate in sibling oocytes. The zonae of fertilised eggs (polyploid embryos) showed long solubilisation times (IVF: 45.3 ± 3.4 min, n = 18; ICSI: 48.9 ± 2.7 min, n = 19). ZP solubility of oocytes that failed to fertilise was intermediate between that of class I metaphase oocytes and embryos (unfertilised IVF: 33.0 ± 2.7 min, n = 13; unfertilised ICSI: 43.0 ± 2.4 min, n = 9). A moderate spontaneous ZP hardening occurred when metaphase oocytes were cultured for 24 h. Finally, cryopreservation of unfertilised oocytes caused hardening of their ZP, with dissolution times that were comparable to those found in fertilised eggs (49.5 ± 2.3 min, n = 10). In most cases, an inverse correlation was found between ZP dissolution time and CG density (longer solubilisation times corresponding to lower CG density). ZP hardening caused by cryopreservation, however, was not associated with a significant reduction in CG density in most of the oocytes examined.

Ultrastructural markers of quality are impaired in human metaphase II aged oocytes: a comparison between reproductive and in vitro aging

“…The above observation may exclude a defect in production (since inner density was unaffected), and so it can be likely attributable to a defective distribution accompanied by a slight degree of exocytosis (since ZP did not show noteworthy features, see below). The finding that only a moderate ZP hardening occurs in 24 h cultured human MII oocytes [63] supports this assumption.…”

What controls polyspermy in mammals, the oviduct or the oocyte?

“…The reported timescale for ZP resistance to proteolysis after fertilisation varies widely among species (Table 1). While in mice and rats ZP resistance is significantly higher after fertilisation or artificial activation (Gulyas & Yuan, 1985; Zhang et al , 1992; DeMeestere, Barlow & Leroy, 1997), there is no clear pattern in humans (Schiewe et al , 1995; Manna et al , 2001) and this increase does not take place in cattle (Iwamoto et al , 1999; Coy et al , 2005) or pigs (Coy et al , 2002; Kolbe & Holtz, 2005). Here we use the term “ZP hardening” when both resistance to proteolysis and a decrease in sperm binding and sperm penetration are implied, whereas the term “ZP resistance to proteolysis” will be used when there is no relationship with sperm penetration.…”

Prevention of zona hardening in non‐human primate oocytes cultured in protein‐free medium

“…Compared with other species, rhesus oocytes have similar digestion times. However, a recent report on enzymatic digestion of human oocytes shows much longer digestion times [21]. That study also measured digestion times for immature oocytes, but those were oocytes that failed to mature with the rest of the cohort of oocytes despite exposure to hCG in vivo .…”

“…Zona hardening has also been characterized in human oocytes and embryos [28), but as its study requires the destruction of oocytes, ethical concerns prevent detailed studies. The phenomenon is usually noted as a rationale for use of assisted reproduction technologies, such as intracytoplasmic sperm injection [8, 21].…”