Changes in the size, distribution and steroid content of rabbit ovarian follicles during early pseudopregnancy.

Abstract

The size and steroid content of ovarian follicles from the rabbit were examined during early pseudopregnancy. Diameters of all available follicles >0.8 mm were recorded upon dissection at Days 0, 2 and 6 after injection of an ovulatory dose of human chorionic gonadotropin (hCG). Each follicle was then analyzed for its content of estradiol, testosterone, and progesterone as an indica tion of its steroid biosynthetic capacity. The results of these measurements demonstrate a deple tion of large follicles (>1.6 mm) at 2 days after hCG injection with a subsequent repopulation of the ovaries with large follicles by Day 6. Additionally, the contents of all three steroids measured were shown to increase generally with increasing follicle size. The temporal aspects of the reap pearance of large steroidogenically active follicles at Day 6 of pseudopregnancy may be crucial to the survival of the developing corpora lutea. SIZE AND STEROID CONTENT OF RABBIT FOLLICLES 1041 was neutralized with 1 N HCI and then extracted 3 times with 4 volumes of freshly distilled diethyl ether. The aqueous phase was frozen in an acetone:dry ice bath, and the ether phase, containing the dissolved steroid, decanted. The ether was driven off in a nitrogen stream, and the dried extract reconstituted in 0.5 ml phosphate-gel buffer for assay of the contents of estradiol, testosterone and progesterone. The average recovery of the steroids was determined in randomly selected samples and was always greater than 95% of all three of the steroids. Steroids The followingsteroidswere purchasedfrom New England Nuclear Corp.: [2, 4, 6, 7@3 HI -estradiol-17@3 (sp act 91.3 Ci/mmol), [1, 2, 6, 7@3HI-testosterone (sp act 91.1 Ci/mmol), and [1, 2, 6, 7-3H]-progesterone (sp act 96 Ci/mmol). Purity of radioactive steroids was checked prior to use by either paper or column chromatography, with subsequent purification where indicated. All nonradioactive steriods were pur chased from Schwarz/Mann. Radioimmunoassays The steriod content of individual follicles was determined by a modification of the radioimmuno assay (RIA) procedure of Abraham (1975) generally used in this laboratory (Mills, 1975; Waterston and Mills, 1976; Mills and Osteen, 1977). However, due to the high specificity of the antibodies used, it was determined that direct assay was sufficient and the usual chromatographic separation of the steroids was not required. The antiserum to estradiol (E2 TGK) was a gift from Dr. D. C. Collins and Kristina Wright of Emory University, Atlanta, GA. This antiserum was raised against estradiol-6-(o-carboxymethyl) oxine-thyroglob ulin and was utilized at a dilution of 1:70,000. The lower limit of sensitivity of the estradiol RIA was 5 pg with an interassay coefficient of variation of 9.6%. The antiserum to testosterone utilized was R-181 (Radioassay Systems Laboratories, Inc., Carson, CA). It was raised against testosterone 19-carboxymethyl ether-BSA and was used at a dilution of 1:170,000. The lower limit of sensitivity of the testosterone assay was 10 pg with an interassay coefficient of variation of 11.3%. The antiserum to progesterone (D3) was prepared in rabbits in this laboratory against 4-preg nen-i 1-ol-3, 20-dione hemisuccinate-BSA. The D3 antibody was used at a final dilution of 1:24,500. The lower limit of sensitivity of the progesterone assay was 10 pg with an interassay coefficient of variation of 9.9%. Water blanks in all RIA procedures were below the sensitivity of the assays. Statistical Analysis Student's t test was used to analyze for statistical differences between groups. Follicle Populations days after hCG injection is presented in Fig. 1. The follicle distribution in ovaries of untreated does is marked by approximately equal num bers of small follicles (41% 1.6 mm). However, at 2 days after injection of an ovulatory dose of hCG, the large follicle population has been markedly depleted with only about 15% of the follicles present classified as large. By Day 6 of pseudo pregnancy the size distribution shows that a repopulation of the ovary with large follicles has occurred with more than 75% of the follicles in the large category. Calculations of