Development of a scanning electron microscopic technique for evaluation of oocytes and its utilization in the study of prolactin interaction with porcine zona pellucida

Since its commercial introduction in the 1960’s, the scanning electron microscope (SEM) has been used increasingly more by biologists studying microanatomical and macromolecular features of various cells and tissues. In this study, a technique for observation of oocytes by SEM was developed and utilized to demonstrate the presence of specific prolactin (PRL) binding entities on the zona pellucida. Details regarding the development of the SEM technique as well as results from the study of PRL interaction with the porcine zona pellucida are presented. Results from SEM evaluation of gold-labeled PRL treated oocytes suggest the presence of PRL binding entities on the ZP of porcine oocytes. Oocytes incubated with gold-labeled PRL equivalent to 920 ng/ml displayed an average of 55 ± 5 particles per field (x30,000). Control oocytes, treated with gold-labeled BSA, displayed 8 ± 1 particles per field. Oocytes treated with 920 ng/ml labeled PRL + 500 ug unlabeled PRL displayed an average of 15 ± 1 particles per field (maximum displacement). There was no further increase in gold-PRL displacement when oocytes were incubated with 920 ng/ml labeled PRL + 1.0 mg unlabeled PRL. The addition of unlabeled LH (500 ug) had no significant (p= .01) effect on the binding of gold-labeled PRL to the ZP (56 ± 4 particles per field). Scatchard analysis of the displacement curve was used to calculate an apparent K_D of 5.8 x 10^-6 for the PRL-binding entities. The role of PRL binding entities on the ZP is unclear but they possibly function in oocyte maturation or sperm-ZP interaction. The operational rules used to study and classify binding entities as receptors are addresed as well as future studies which will help prove that the binding entities reported are actually PRL receptors .