Zona Pellucida-free Eggs Research Articles

Multivariate analysis of male reproductive function in Inpp5b−/− mice reveals heterogeneity in defects in fertility, sperm–egg membrane interaction and proteolytic cleavage of sperm ADAMs

Past work indicated that sperm from mice deficient in the inositol polyphosphate 5-phosphatase Inpp5b have reduced ability to fertilize eggs in vitro and reduced epididymal proteolytic processing of the sperm protein A Disintegrin and A Metalloprotease 2 (ADAM2). On the basis of these data, our central working hypothesis was that reduced ADAM cleavage would correlate with reduced sperm-egg binding and fusion and in turn with reduced male fertility in Inpp5b(-/-) mice. Multiple endpoints of reproductive functions [mating trials, in vitro fertilization (IVF) assays and ADAM2 and ADAM3 cleavage] were investigated on a male-by-male basis, with pair-wise correlation analysis used to assess the relationships between these various parameters. Motile sperm from Inpp5b(-/-) mice showed significantly reduced fertilization of zona pellucida-free eggs due to reduced binding to the egg plasma membrane and subsequent fusion. Localization of a mouse sperm protein required for gamete fusion, IZUMO1, appears normal in Inpp5b-null sperm. To our surprise and differing from previous reports, we found that ADAM cleavage was only modestly impaired in numerous Inpp5b-null males and varied between individual animals. Performance in mating trials also differed from past reports. The pair-wise correlation analysis revealed that ADAM2 and ADAM3 cleavage was positively correlated, suggesting that processing of these proteins occurs by related/identical mechanisms, but otherwise, there were few correlations between the reproductive endpoints examined here. Nevertheless, this work provides detailed analysis of the Inpp5b(-/-) phenotype and also a blueprint for multivariate analysis to examine relationships between molecular characteristics and in vitro and in vivo physiological functions.

Mouse Sperm Lacking ADAM1b/ADAM2 Fertilin Can Fuse with the Egg Plasma Membrane and Effect Fertilization

Fertilin, a heterodimeric protein complex composed of alpha (ADAM1) and beta (ADAM2) subunits on the sperm surface, is believed to mediate adhesion and fusion between the sperm and egg plasma membranes. Here we have shown that mutant male mice lacking ADAM1b are fertile and that the loss of ADAM1b results in no significant defect in sperm functions such as migration from the uterus into oviduct, binding to egg zona pellucida, and fusion with zona pellucida-free eggs. ADAM1b-deficient epididymal sperm showed a severe reduction of ADAM2 on the cell surface, despite the normal presence of ADAM2 in testicular germ cells. The appearance of ADAM1b and ADAM2 on the sperm surface depended on formation and abundance of ADAM1b/ADAM2 fertilin in testicular germ cells. These results suggest that mouse ADAM1b/ADAM2 fertilin may play a crucial role not in the sperm/egg fusion but in the appearance of these two ADAMs on the sperm surface.

A novel aminophospholipid transporter exclusively expressed in spermatozoa is required for membrane lipid asymmetry and normal fertilization

Through the use of a functionally unbiased signal peptide trap screen, we have discovered an ATP-dependent aminophospholipid transporter that is exclusively expressed in the acrosomal region of spermatozoa; it is about 62% similar to the flippase, FIC1. We disrupted the transporter gene and found that the size of litters from male null mice was slightly smaller than found with wild-type males. Sperm morphology and motility were the same between null and wild-type littermates, but agents (merocyanine and annexin) that measure phospholipid packing or phosphatidylserine (PS) in the outer membrane leaflet showed that PS already existed in the outer leaflet of null spermatozoa before sperm capacitation. Fertilization rates were normal when null spermatozoa were added to zona pellucida-free eggs, but in the presence of the extracellular matrix, fewer transporter−/− spermatozoa bound tightly or penetrated the zona pellucida (ZP), and fewer underwent acrosome reactions. In vitro fertilization was compromised, especially at early time points or at low sperm concentrations after mixing null spermatozoa and eggs. Thus, a new aminophospholipid transporter expressed exclusively in spermatozoa is critical for normal phospholipid distribution in the bilayer, and for normal binding, penetration, and signaling by the zona pellucida.

Characterization of the Binding of Recombinant Mouse Sperm Fertilin β Subunit to Mouse Eggs: Evidence for Adhesive Activity via an Egg β1Integrin-Mediated Interaction

The sperm protein fertilin (also known as PH-30) is a candidate for mediating the interactions between sperm and egg plasma membranes. Fertilin is a heterodimer. The β subunit, which has a region with homology to the family of integrin ligands known as disintegrins, has been hypothesized to be involved in the binding of sperm to the egg surface. To investigate this hypothesis and determine what role fertilin β plays in fertilization, we have expressed the putative extracellular domain of mouse fertilin β in bacteria as a fusion protein with maltose-binding protein (hereafter referred to as recombinant fertilin β-EC) and used two assays to characterize its binding to mouse eggs. Immunocytochemistry was used to examine the localization of recombinant fertilin β-EC binding. A luminometric assay was also developed to quantify levels of binding of recombinant fertilin β-EC to single eggs. We find that recombinant fertilin β-EC binds to the region of the plasma membrane of the egg to which sperm bind, thus providing the first direct evidence that fertilin β has adhesive properties. Peptides corresponding to the disintegrin domain of fertilin β reduce its binding to eggs, suggesting that this domain is at least partially involved in the recognition of fertilin β by binding sites on the egg. Treatment of zona pellucida-free eggs with chymotrypsin reduces the ability of the eggs to support the binding of recombinant fertilin β-EC, implicating an egg surface protein as a binding site for recombinant fertilin β-EC. Binding of recombinant fertilin β-EC to eggs is also reduced in the absence of divalent cations and is supported by 2.0 mMCa2+, Mg2+, or Mn2+. Furthermore, eggs incubated in recombinant fertilin β-EC prior toin vitrofertilization show reduced levels of sperm binding. Finally, we have examined the possible role of integrins on eggs as receptors for fertilin β, since an anti-α6integrin subunit monoclonal antibody, GoH3, has been shown to inhibit sperm binding (E. A. C. Almeidaet al.(1995)Cell81, 1095–1104). We find that: (a) an increased amount of GoH3 epitope on the egg surface does not correlate with an increased ability of the eggs to bind sperm or recombinant fertilin β-EC; (b) the GoH3 antibody has virtually no inhibitory effect on recombinant fertilin β-EC binding; and (c) recombinant fertilin β-EC binding is reduced in the presence of anti-β1integrin antibodies. These results suggest that a β1-containing integrin participates in the binding of recombinant fertilin β-EC to mouse eggs.

Gene transfer and expression in mouse preimplantation embryos by recombinant adenovirus vector

Replication-defective recombinant adenovirus, Adex4SRLacZL, was used as a vector for transferring exogenous genes in mouse zona pellucida-free eggs at the pronuclear stage. The vector contained the E. coli LacZ reporter gene under the control of the SR alpha promoter (SV40 early promoter-fused HTLV-I LTR), and the expression of the reporter gene was examined during preimplantation development in culture. Histochemical staining of the embryos for beta-galactosidase activity showed that the exogenous LacZ gene as expressed in 98% of the embryos at the morula-blastocyst stages. As in the microinjection method, the exogenous genes could be pursued from the 2-cell stage. Neither apparent morphological changes nor cytotoxic effects were observed. Both the percentages of embryos expressing reporter genes and the rate of development to the blastocyst stage were higher in the adenovirus vector-treated embryos than in the microinjected ones. These results suggest that the adenovirus vector system is a useful tool in investigating the genetic control of early mammalian development.

Mouse sperm-egg plasma membrane interactions: analysis of roles of egg integrins and the mouse sperm homologue of PH-30 (fertilin) β

The guinea pig sperm protein, PH-30 (also known as fertilin), is postulated to participate in the interaction between the sperm and egg plasma membranes. The beta subunit of guinea pig PH-30 (gpPH-30 beta) contains a domain with homology to disintegrins, snake venom proteins that bind to integrins via an integrin-binding domain containing the tripeptide RGD. This raises the question of whether an egg integrin serves as a receptor for PH-30. Although mouse eggs express integrin subunits, their role in mouse fertilization is unresolved. Therefore, we examined fertilization for two different hallmarks of integrin function, namely, dependence of ligand binding on divalent cations and the ability to inhibit ligand binding with RGD peptides. We demonstrate that sperm binding to zona pellucida-free eggs is supported by Ca2+, Mg2+, or Mn2+. Ca2+ was necessary and sufficient for sperm-egg fusion, with 2.5 mM Ca2+ being the most effective concentration. In addition, fertilization could be partially inhibited with various RGD peptides, which caused a decrease in sperm-egg fusion by 30-58%. This partial inhibition of fusion with RGD peptides prompted the cloning of the mouse homologue of gpPH-30 beta (hereafter referred to as mPH-30 beta) to determine if it possessed the tripeptide RGD or a different amino acid sequence in its disintegrin domain. mPH-30 beta, which is expressed during meiotic and post-meiotic phases of spermatogenesis, shares significant similarities to gpPH-30 beta throughout the length of the molecule, from the signal sequence to the cytoplasmic tail. The full-length deduced amino acid sequence of mPH-30 beta. The disintegrin domain of mPH-30 beta has the tripeptide QDE (instead of RGD) in its cell recognition region. Peptides containing this QDE sequence decrease the binding and fusion of sperm with zona pellucida-free eggs by approximately 70%, suggesting that the disintegrin domain of mPH-30 beta participates in the interaction between sperm and egg membranes.

Initiation, Persistence, and Cessation of the Series of Intracellular Ca2+ Responses during Fertilization of Bovine Eggs.

A long-lasting series of transient rises in intracellular Ca2+ concentration ([Ca2+]i) were recorded with Ca2+ imaging during fertilization of bovine eggs matured in vivo or in vitro. The first Ca2+ response could be recorded in zona pellucida-free eggs within 1 min after sperm-egg contact, when a preceding slow [Ca2+]i rise reached 150-170 nM. The response had the peak of 565 ±32 nM (n=15; maximum 780 nM) and the duration of 4-6 min, being significantly larger and longer than any succeeding response (359 ± 12 nM, n=68; 2 min). The series of Ca2+ transients occurred at constant intervals of 19-20 min without attenuation in the magnitude for more than 9 h. Ca2+ transients became smaller progressively during pronuclear migration and nuclear envelope breakdown, and no response occurred in 2-cell embryo. In unfertilized eggs, injection of inositol 1, 4, 5-trisphophate (InsP3) induced a Ca2+ transient and the sulfhydryl reagent thimerosal caused repetitive Ca2+ transients. These responses were markedly inhibited or blocked by preinjection of a monoclonal antibody to the mouse InsP3 receptor. However, sperm-induced responses were not blocked in 2/3 of eggs examined. Another mechanism may be involved in addition to InsP3-induced Ca2+ release from intracelluar stores.

A cell surface block to polyspermy occurs in golden hamster eggs

We have examined the frequency and fate of supernumerary sperm in the perivitelline space (PVS) of in vitro fertilized hamster eggs to determine if there is a cell surface block to polyspermy. The zona pellucida block to polyspermy is very effective since only one sperm penetrated the zona pellucida in 72.8% of the 876 fertilized eggs examined. Of the polypenetrated eggs, 41.6% had a supernumerary sperm within the PVS. The proportion of polypenetrated eggs with PVS sperm did not change when the duration of coincubation was increased from 3 to 6 hr. PVS sperm were found in 67% of the inseminations. From these data we conclude that there is a cell surface block to polyspermy in the hamster. To investigate the mechanism of the cell surface block, we used the Hoechst-transfer technique ( R. Hinkley, B. Wright, and J. Lynn, 1986, Dev. Biol. 118, 148–154) to monitor sperm-egg fusion. We first demonstrated that dye transfer from zona pellucida-free eggs to sperm only occurred when fusion was possible, i.e., in the presence of calcium, and that dye was transferred to all fused sperm. When cumulus-free, zona-intact eggs were preloaded with Hoechst dye and viewed 3 hr postinsemination, three classes of eggs with supernumerary sperm in the PVS were observed: eggs with only Hoechst-positive sperm (62%), eggs with only Hoechst-negative sperm (27%), and eggs with both a Hoechst-positive and a Hoechst-negative sperm (11%). Because of the limited time resolution of the Hoechst-transfer technique, the cell surface block could operate by preventing sperm fusion (Hoechst-negative), by the failure of the eggs to incorporate fused sperm (Hoechst-positive), and/or by the “unfusing” of fused sperm (Hoechst-positive and Hoechst-negative). We are unable at this time to differentiate between these mechanisms.