Characterization of an Ovarian Slice Model In Vitro to View Ovulation.

Abstract

Most studies of follicular maturation and ovulation in vitro utilize isolated follicles or whole ovaries through perfusion systems. Frequently, ovulation is established in vivo by the presence of an empty follicle. This study presents a method for real-time visualization of ovulation using an in vitro slice protocol that preserves the 3 dimensional structure of potentially key ovarian components. Ovulation is viewed from intact follicles within ovarian slices by either capturing daily images to examine progression at chosen time points or by using video microscopy to capture the continuity of events. Ovaries were collected from prepubertal (<5 wks) mice (C57 background), embedded in 8% agarose, cut at 200 µm and plated 3 slices per dish. Several different treatment combinations were investigated qualitatively, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), pregnant mare serum gonadotropin (PMSG), and PG600 (PMSG and hCG). For quantitative analysis, ovaries were treated with either no hormone, 5IU PG600, 5IU hCG, or 5IU PG600 followed 48 hours later by 5IU hCG. Results showed that dishes treated with both PG600 and hCG released approximately 22% of an average of 4.2 readily releasable oocytes per slice, while no oocytes were released without hCG. Ovulation was observed within 12 hours after the addition of hCG following PG600 pretreatment based on video microscopy or end point image comparisons. For other treatment groups, there was no evidence of stimulated oocyte release. Health and viability of the released oocytes was assessed by examining DNA labeled with 1mM Hoechst stain. Video microscopy indicated that one of the first steps in stimulated release is mobilization of the oocytes within the follicles within minutes. In summary, this study characterizes a novel method for studying the mechanisms of ovulation, the ability to examine and alter the progression of ovulation in a defined serum free media in vitro, and for the analysis of oocyte behavior within follicles. (platform)