Recovery of penetration ability in protease‐treated zona‐free mouse eggs occurs coincident with recovery of a cell surface 94 kD protein

Abstract

Previous studies have demonstrated that protease treatment of zona-free mouse eggs impairs sperm-egg interaction (Boldt et al.: Biol Reprod 39:19-27, 1988) and causes modification of a 94 kD egg plasma membrane protein (Boldt et al., Gamete Res 23:91-101, 1989). In this report, the ability of eggs to recover penetration ability following protease treatment was examined. Zona-free mouse eggs were isolated and treated with either trypsin or chymotrypsin (1 mg/ml, 20 min), then cultured for 0, 3, or 6 hr before insemination. Eggs cultured for 3 or 6 hr displayed significantly higher penetration levels than eggs inseminated immediately after protease treatment, indicating a recovery of penetration ability during the 3 or 6 hr incubation period. The recovery of penetration ability was not blocked by inclusion of cyclohexamide (50 micrograms/ml) during the 3 or 6 hr culture period, indicating that protein synthesis was not required for recovery of fusion ability. Cell surface radiolabeling studies with 125I revealed that a 94 kD cell surface protein was lost immediately following trypsin or chymotrypsin treatment but was found on the egg surface after the 3 or 6 hr recovery period. Recovery of the 94 kD egg surface protein occurred in the presence of cyclohexamide, and metabolic radiolabeling studies with 35S-methionine confirmed that synthesis of a 94 kD protein was blocked by cyclohexamide. These results suggest that the recovery of penetration ability after protease treatment of zona-free eggs is due to recovery of the 94 kD cell surface protein, providing further evidence for the involvement of the 94 kD protein in sperm-egg interaction.

Synchronous Sperm Penetration of Zona-Free Mouse Eggs in Vitro1

“…More recent studies seem to provide insight into the phenomenon from a molecular perspective. Cell-surface radiolabeling with 125I demonstrated that trypsin or chymotrypsin treatment was associated with loss or modification of a 94-kDa protein [14], and recovery of penetrating ability was coincident with recovery of the protein [15]. Although it is still unclear whether this protein molecule plays an integral role as a sperm receptor in sperm-egg fusion, the results seem to provide evidence that reconstruction of damaged surface molecules is needed for successful fertilization.…”

“…After 1 h preincubation of eggs, the fusion rate at 10 min was markedly improved. Kellom et al [15] suggested that a 94-kDa protein that was modified after protease treatment was not newly synthesized but was budding during egg preincubation. The molecule(s) damaged by acid treatment might be repaired in the same way, and it takes at least 2 h for eggs to be competent to fuse with sperm.…”

CD9 controls the formation of clusters that contain tetraspanins and the integrin α6β1, which are involved in human and mouse gamete fusion

“…ZP removal methods significantly alter oolemmal proteins and may reduce fertilization (Santella et al, 1992;Kinn and Allen, 1981;Kellom et al, 1992), a mechanical technique for ZP removal was performed using a pair of microdissection scissors under a binocular microscope (Ji et al, 1998). This technique is particularly gentle as no recovery time is necessary before insemination (Ji et al, 1998).…”

“…In vitro matured metaphase II were obtained by incubating germinal vesicle or metaphase I stage oocytes under a 5% CO 2 atmosphere at 37°C for 24 hours. ZP was removed with a chemical-and enzymatic-free technique, using a pair of microdissection scissors under a binocular microscope, in order to preserve the integrity of the surface proteins (Kellom et al, 1992).…”

Control of the surface expression of uvomorulin after activation of mouse oocytes

“…Their effects on early mouse embryos have not been investigated extensively; however, studies on 8-cell embryos have shown that, at this stage, embryos are sensitive to low concentrations of monensin, and that BFA and monensin appear to have qualitatively different effects on the distribution of newly synthesised gap junction protein connexin 43, suggesting different modes of action (De Sousa et al, 1993). However, a complete block on oocyte surface turnover does not occur, since unfertilised oocytes can recover rapidly from proteolytic removal of a putative sperm-binding protein by a process independent of protein synthesis (Kellom et al, 1992). The precise nature of the Golgi apparatus in maturing or Mil mammalian oocytes is not clear.…”