Evidence for androgen participation in induced ovulation in immature rats.

Rabbit antiserum to testosterone injected intravenously (iv) together with 10 IU human chorionic gonadotropin (hCG) markedly reduced the number of tubal ova in intact or hypophysectomized immature rats primed with 5 IU pregnant mare's serum gonadotropin. Normal rabbit serum (NRS) injected instead of the antiserum resulted in a normal complement of ovulated ova. Ten microgram of testosterone or 5alpha-dihydrotestosterone dissolved in NRS administered iv 1 h after simultaneous injection of hCG and antiserum to progesterone (anti-P) restored ovulation that would otherwise have been blocked by anti-P, with a comparable number of ova to control animals not given anti-P. The results indicate active participation of androgen in induced ovulation in immature rats.

Functional and structural relationships in steroidogenesis in vitro by human ovarian follicles during maturation and ovulation.

To investigate steroidogenic function of human follicles in the light of their structures, eight antral follicles of different sizes were mechanically isolated from ovaries of patients laparotomized in the follicular phase of the menstrual cycle with or without pretreatment with human menopausal gonadotropin (hMG). A portion of each follicle was taken for histology and slices of each follicle were incubated with [1-14C]-acetate. Incorporation into progestins, androgens, and estrogens was assessed by the reverse dilution technique with recrystallization to constant specific activity. A predominant incorporation into 17 beta-estradiol was observed in two maturing follicles, whereas a marked increase in incorporation into 17 beta-estradiol with a concomitant decrease in incorporation into androstenedione was verified in two other mature follicles. Remarkable enhancement in relative incorporation into C21 steroids was commonly noted in four preovulatory follicles. However, with the progress of preovulatory stages toward ovulation, as judged from structural changes of the follicles, actual incorporation into C19 and C18 steroids showed a moderate increase, followed by a drastic decrease around the time of ovulation. hMG injection induced similar relationships between the steroidogenic pattern and the follicle structure of different stages, although overall incorporation was considerably increased. We conclude that marked qualitative and quantitative changes in the steroidogenic function and accompanying corresponding changes in structure occurred over the period of follicular maturation and ovulation.

Covalent coupling of a steroid to microwell plates for use in a competitive enzyme-linked immunosorbent assay.

The covalent coupling of a model steroid, 17 alpha-hydroxypregnenolone, to the wells of the microtiter plate, CovaLink NH, for use in a competitive enzyme-linked immunosorbent assay is described. This plate has secondary amino groups bound to its surface. A carboxylated derivative of the steroid was coupled to the amino group to form an amide bond in a single step using a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (10 mM) as coupling reagent in the presence of N-hydroxysuccinimide (1 mM). After carrying out a competitive immune reaction, antibodies bound to immobilized steroids were estimated by means of a second antibody-enzyme conjugate. The non-specific background was reduced with blocking agents which did not interfere with the immune reaction between antibodies and the steroids coupled to the plastic surface. The following two procedures were effective for this purpose: pretreatment of wells with 0.01% Tween 20 solution followed by 0.5% bovine serum albumin in phosphate buffered saline, and addition of 0.01% Tween 20 to the assay buffer. With this method, the preparation of steroid-enzyme conjugates is unnecessary and optimization of conditions for ELISA procedures can be achieved in a simple manner.

Co-operation of progesterone and prostaglandins in ovulation induced by human chorionic gonadotrophin in immature rats primed with pregnant mare serum gonadotrophin.

Serial injections of a mixture of prostaglandin (PG) E2 and F2 alpha 0, 2, 4, and 6 h after simultaneous injection of human chorionic gonadotrophin (hCG) and indomethacin incompletely restored the ovulation that would have been blocked by indomethacin in immature rats treated with pregnant mare serum gonadotrophin followed by hCG. Serial injections of another mixture of PGE2 and PGF2 alpha 6, 8, 10 and 12 h after simultaneous injection of hCG and indomethacin similarly reversed, in part, the inhibitory effects of indomethacin on hCG-induced ovulation. In contrast, serial injections of the mixtures of PGE2 and PGF2 alpha 0, 2, 4, 6, 8, 10 and 12 h after simultaneous injection of hCG and indomethacin completely restored the indomethacin-blocked ovulation, suggesting that the prostaglandins mediate the action of hCG on ovulation both in the earlier and later stages of the preovulatory process. Six hours after simultaneous injection of hCG and indomethacin serial injections of a mixture of PGE2 and PGF2 alpha reproduced the acute and temporary increase in concentrations of progesterone and testosterone in plasma which would have been abolished by indomethacin. Progesterone given concurrently with hCG and indomethacin partially antagonized the inhibitory action of indomethacin on ovulation. Serial injections of a mixture of PGE2 and PGF2 alpha 6, 8, 10 and 12 h after concurrent administration of progesterone with hCG and indomethacin completely restored the indomethacin-blocked ovulation, suggesting that progesterone can substitute the action of prostaglandins injected serially in the first half of the preovulatory process. It was concluded that the co-operation of progesterone in the earlier stage and of prostaglandins in the later stage of the preovulatory interval is required to mediate the action of hCG on ovulation.

A progesterone-dependent step in ovulation induced by human chorionic gonadotrophin in immature rats primed with pregnant mare serum gonadotrophin.

In immature rats primed with pregnant mare serum gonadotrophin, antiserum to progesterone could prevent or reduce ovulation in response to injected human chorionic gonadotrophin (HCG). To be effective, antiserum treatment had to be within 6 h of gonadotrophin treatment; antiserum given 9 h after HCG was ineffective. Progesterone restored the antiserum blocked ovulation completely or incompletely when administered intravenously within 6 h of treatment with HCG. The first 6 h was shown to be a progesterone-dependent step in the ovulatory process in this experimental system.

Localization and distribution of unconjugated steroid hormones in normal placenta at term.

The localization and distribution of unconjugated oestrone, oestradiol-17 beta, oestriol and progesterone were studied in normal term placentae, using subcellular fractionation and indirect immunofluorescence techniques. Radioimmunoassay of these steroids in each subcellular fraction showed that they were detectable mainly in the cytosol fraction. The efficiency of fixatives for retaining steroids in placental tissue during immunohistochemical procedures was studied in order to validate the present experimental techniques. As compared with other fixatives, glutaraldehyde solution produced minimal leakage of steroid hormones from placental tissue. Using an indirect immunofluorescence technique oestrone, oestriol and progesterone were detectable in the cytoplasm of villous syncytiotrophoblast of fixed term placentae.

Chemiluminescence enzyme immunoassay of dehydroepiandrosterone and its sulfate using peroxidase as label.

We report a highly sensitive enzyme immunoassay for dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) using horseradish peroxidase as the label enzyme. Separation of free and bound DHEA-peroxidase conjugate was by insolubilized antibody, prepared by coupling purified IgG of goat anti-rabbit IgG serum with Sepharose 4B or a polystyrene tube. The enzyme activity was measured by the chemiluminescence reaction using luminol and hydrogen peroxide as substrate. The faint chemiluminescence was measured by a photon counter. The sensitivity was 25 pg/assay tube for DHEA and 100 pg/assay tube for DHEA-S. Upon comparison, results obtained by radioimmunoassay and this method showed good agreement ; r = 0.86 for free DHEA, r = 0.92 for acid-hydrolyzed DHEA-S and r = 0.91 for solvolyzed DHEA-S. The present method is applicable in the routine determination of DHEA and DHEA-S in biological fluid.

Studies on serum oestrogen and progesterone levels during the oestrous cycle and early pregnancy in mares.

Concentrations of progesterone and oestrogens were determined by radioimmunoassay in the peripheral blood of 22 Percheron and Breton breed mares from the 6th day of oestrus to the 150th day of pregnancy. Periodical variation patterns for the mean values of oestrone, oestradiol 17beta and total oestrogens in the cycling mares were found, with two peaks on the third day before and the 15th day after ovulation, and one depression on the 6th day of oestrus. In pregnant mares, the concentrations of oestrone and oestradiol 17beta increased rapidly (P less than 0.05) after Day 105 of gestation. Progesterone levels declined after Day 18 of the cycle in cycling mares, whereas they increased in the pregnant mares. Marked changes in the ratio of total oestrogen to progesterone concentrations were found in cycling mares, especially at oestrus, but in pregnant mares the ratio always remained below 1.00.

[Enzyme labeling methods and it's specificities].

Enzyme labeled antigen for use in ELISA of Hapten (steroids, prostanoid, carbohydrate, nucleic acid, peptide, herbicide, insecticide and antibiotic) have usually been prepared by condensation of carboxy group of hapten with amino groups of lysine residue in enzyme. The horseradish peroxidase (HRP) is best suitable as labeling enzyme, therefore it is small molecular, and substrate turnover is much higher compared to the other enzymes. The mixed anhydride and carbodiimide methods have mainly been used the preparation of hapten conjugate BSA, but not satisfactory for enzyme labeling. The N-hydroxysuccinimide ester (NHS: active ester) method is satisfactory with respect to reproducibility and sensitivity. The sensitivity is related to the bridging phenomenon: One of the disadvantages of the homologous labels is that the antibody shows an affinity not only for the Hapten but also for the bridge which connects the Hapten to carrier protein. We have developed a sensitive bridge heterologous EIA for progesterone (P) using geometrical isomers of P-3 (E/Z) (O-carboxymethyl) oxime-N-hydroxysuccinimide esters [ef Ab of P-3 (E) CMO-BSA/P-3 (Z) CMO-HRP]3). The sensitivity of heterologous proved to be higher than a homologous EIA or a conventional RIA. It seem like that a 1:1 steroid-enzyme conjugate is suitable for obtaining a high sensitivity. The avidin-biotin (AB) system provides great versatility, since by conjugation with an appropriate label, the AB assay can be used with any chosen detector. Furthermore, IgG can be labels with biotin without significantly influencing their immunological activity.(ABSTRACT TRUNCATED AT 250 WORDS)