Ultrastructural analysis of egg membrane abnormalities in post-ovulatory aged eggs

Abstract

Microscopic analyses of mammalian gamete ultrastructure have provided among the most seminal insights into fertilization. Here we present a summary of ultrastructural studies of mammalian fertilization, together with a review of the effects of post-ovulatory aging in eggs, and our own results using scanning electron microscopy to examine the effects of post-ovulatory aging on the egg membrane topography. Our previous work detected two abnormalities in egg membrane function in aged eggs: aged eggs appeared to be less able to support sperm-egg membrane interaction, thus rendering the eggs less fertilizable, and aged eggs appear to have a reduced ability to prevent polyspermy at the level of the egg membrane, i.e., to establish a membrane block to polyspermy. In the work presented here, we tested the suitability of both environmental (ESEM) and low-vacuum (LVSEM) modes of the FEI Quanta 200 Environmental Scanning Electron Microscope to study mammalian gametes. While ESEM mode was not sufficient under the conditions we used to observe fine cell surface details, the LVSEM mode proved to be an excellent way to view egg membrane topography. Unfertilized aged eggs have an abnormally distended amicrovillar region over the meiotic spindle, and fertilized aged eggs had three different abnormalities detected, from reduced sperm-induced membrane remodeling to abnormal sperm-induced membrane remodeling and membrane blebbing. Combining these insights of egg membrane function and topography with others in the field of post-ovulatory aging, we have expanded insights into why fertilization at increased times after ovulation is associated with poor reproductive outcomes.

MAPK3/1 (ERK1/2) and Myosin Light Chain Kinase in Mammalian Eggs Affect Myosin-II Function and Regulate the Metaphase II State in a Calcium- and Zinc-Dependent Manner1

“…Postovulatory aging is associated with unsuccessful fertilization and, in instances when aged eggs are fertilized, with poor reproductive outcomes, including pregnancy loss, smaller litter sizes, or in offspring with abnormalities [14,15,90,91]. Relevant to the data here, postovulatory aged eggs have reduced MAPK3/1 activity associated with a range of membrane and cortical abnormalities and the above-mentioned propensity to undergo parthenogenetic activation [92][93][94][95][96][97][98][99][100]. Thus, what we observe here with MAPK3/1 pathway inhibition and the associated reduction in myosin-II function is consistent with changes that occur with postovulatory aging, suggestive of a tie between reduced MAPK3/1 activity and several of the changes observed in aged eggs.…”

One‐step collagenase method for zona pellucida removal in unfertilized eggs: easy and gentle method for large‐scale preparation

“…Thus, the collagenase ZP removal method is a mild method with no observable harm to the oocytes that can simultaneously discriminate the spontaneously activated low quality oocytes from normal, young oocytes [16].…”

“…Eggs were collected in TYH-HEPES (Table 1) from oviducts at 13-15 h after hCG injection, which were referred to as young eggs. Eggs collected from oviducts at 26 h after hCG injection were referred to as post-ovulatory aged eggs [15][16][17]. Cumulus cells were removed by treatment with 5 min incubation in TYH-HEPES containing 0.05% hyaluronidase.…”

Prophase I Mouse Oocytes Are Deficient in the Ability to Respond to Fertilization by Decreasing Membrane Receptivity to Sperm and Establishing a Membrane Block to Polyspermy1

“…Our past work showed that fertilization cone formation was delayed in eggs fertilized by ICSI [54], of note because ICSI-fertilized eggs were deficient in the ability to establish a membrane block to polyspermy [54][55][56]. We therefore speculated that prophase I oocytes could have differences in sperm-induced membrane and cortical remodeling; we examined this by low-vacuum scanning electron microscopy [76]. Control fertilized metaphase II eggs showed clear signs of membrane remodeling, with the formation of an amicrovillar patch over the site of sperm incorporation (Fig.…”

“…Unfertilized and fertilized oocytes and eggs were prepared for scanning electron microscopy and viewed using an FEI Quanta 200 Environmental Scanning Electron Microscope with low-vacuum mode as previously described [76]. Micropipette aspiration to assess effective cortical tension was performed as previously described [77].…”