Ovarian Function in the Preovulatory Rabbit.2

Transcription

1 BOLOGY OF REPRODUCTON 17, (1977) Ovarian Function in the Preovulatory Rabbit.2 CHUNG H. WU, LUS BLASCO, GEORGE L. FLCKNGER and GEORGE MKHAL Endocrine Section, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 1914 ABSTRACT Ovarian secretion of hormones was determined at hourly intervals for a period of 9 h following the administration of human chorionic gonadotropin (hcg) to estrous rabbits. Blood flow to the ovary was measured with radio-labeled microspheres immediately before peripheral and ovarian vein blood samples were obtained. The ovarian vein plasma concentration and secretion rates of estradiol-173 (E2) and testosterone (T) rose sharply at the 2 h period. By 4 h, when ovarian blood flow reached its maximum, the secretion rate of E2 and T reached a second peak. Secretion rates of esirone (E1), androstenedione (A), progesterone (P) and 2o-dihydroprogesterone (2a-OH-P) showed only a single peak at the 4 h period. There was no significant change in the peripheral blood levels of E1, E2 and A and there was a small rise in T between the second and fourth hours; in contrast, an increase in P noted in the first hour was sustained up to the eighth hour, while 2th-OH-P was elevated throughout with peaks at 2, 5 and 9 h. n spite of the maximum peripheral levels of hcg in the first hour, peak hcg removal rate by the ovary occurred an hour later. hcg seems to stimulate ovarian steroidogenesis, which is further augmented by increased ovarian uptake of hcg. The data also suggests that the significant but brief rise in E2 and T production serves an intraovarian rather than a systemic function. NTRODUCT Although the production of ovarian hormones is known to change during the preovulatory period, the rote of steroids in follicular maturation and ovulation is not clearly defined. n the rabbit, follicular growth and eventual ovulation can be induced by coitus or by treatment with a tuteinizing gonadotropin. Within a few hours these stimuli caused an increased ovarian secretion of progestins (Hilhard et al., 1971), androgens (Hilliard et al., 1974) and estrogens (Hilliard et at., 1971). Pharmacologic agents which inhibit this increased steroid synthesis by the ovary have also been shown to block ovulation (pner and Greep, 1971). The initial rise in steroid secretion is followed by a decline in hormone production to nadir levels just prior to ovulation (Hilliard et al., 1971). This period of Accepted April 26, Received February 1, Supported in part by National nstitutes of Health, Grant No. HD Reprint requests: Department of Ob-Gyn, Hospital of the University of Pennsylvania, 34 Spruce Street, Philadelphia, Pennsylvania ON reduced hormone production has been attributed to a refractoriness of the follicle to further gonadotropin stimulation (LeMaire and Marsh, 1975). n order to establish more precise relationships between the ovarian hormones during the preovulatory period, we have measured the peripheral and ovarian vein plasma concentrations of E1, E2, P, 2a-OH-P, A and T at hourly intervals after injection of HCG to estrous rabbits. Determination of ovarian blood flow with radio-labeled microspheres in the same animals allowed us to calculate hormone secretion rates. MATERALS AND METHODS New Zealand White rabbits were individually housed for 3 weeks before the intravenous injection of 75 U of hcg. Ovarian blood flow was measured by using radiolabeled microspheres (15 to technique as previously described (Blasco et al., 1973). mmediately after the measurement of blood flow and while the animals were still under phenobarbital (3 mglxg iv) anesthesia, the abdominal cavity was opened and the venous branches from the proximal end of the oviduct were ligated. Both ovarian veins were cannulated with a heparinized polyethylene tube (Adams ntramedic, P.E. 9). Blood was collected in heparinized test tubes for ten minutes. Peripheral arterial blood was obtained simultaneously from the catheter which was placed in 34

2 PREOVJLATORV RABBT OVARY 35 the femorat artery for blood flow study. Plasma was separated and frozen at -2#{176}Cuntil assay. Plasma steroid concentrations were determined by radioimmunoassays (RA). For the measurement of E1 and E2, diethylether extracts of aliquots from the plasma samples were subjected to LH-2 column chromatography and RA as previously reported (Wu and Lundy, 1971). charcoal-treated rabbit plasma gave a background of less than 2 pg/mi for both E1 and E3. Another aliquot of plasma, extracted with ether was chromatographed on Sephadex LH-2 columns with benzene:methanol (97:3, v:v) in order to separate P and 2a-Ol-P. The P and 2Ga-OH-P fractions were subjected to RA as previously reported (DeVilla et al., 1972; Wu et at., 1974). Charcoaltreated rabbit plasma had a background of less than 5 pg/m for P and 2n-OH-P. Similar sensitivity, accuracy and inter- and intra-assay errors for progestins and estrogens were observed when rabbit plasma was used in place of human plasma in these assays. The ether extract of a third aliquot of plasma was chromatographed on Sephadex LH-2 columns in benzene:methanol (95:5, v:v) to separate A and T. The antibodies (Ab) against A and T (A-Ab 862R and T-Ab 25a) were generously supplied by Dr. G. Niswender. The cross-reactivity of these antibodies to other steroids is shown in Table 1. Significant crossreaction of T-Ab with dihydrotestosterone and androstandiol was noted. n the A-RA, a 1/4, TABLE 1. Cross reaction of steroids with androstenedione-antibody and testosterone-antibody. Steroid % Cross r eaction A-Ab. T-Ab. Androstenedione 1 <.1 Testosterone 1. 1 Dihydrotestosterone Androstene-33,17f3diol <.1 1 5o-Androstan-3,173diol < Androstan-3o,ol-1 7-one <.1 <.1 Dehydroepiandrosterone <.1 <.1 Progesterone.3 <.1 Scs-Dihydroprogesterone <.1 <.1 2o-Dihydroprogesterone <.1 <.1 2t3-Dihydroprogesterone <.1 <.1 1 7n-Hydroxy-progesterone.2 <.1 Estrone <.1 <.1 Estradiol-17f3 <.1 <.1 Estriol <.1 <.1 Corticosterone <.1 <.1 Deoxycorticosterone <.1 <.1 Cortisol <.1 <.1 cross reaction = X/V X 1, X = ng of androstenedione (A) or testosterone (T) required to displace 5% of A-3 H or T-3 H bound to the respective antibody, Y = ng of steroid required to displace 5% of A-3 H or T-3 H bound to the same antibody. dilution of A-Ab and 5,-6, CPM 3H-A were used in each tube. The T-RA used a 1/1, dilution of T-Ab and 7,-8, CPM 3H-T. All the reagents were prepared in.1 M phosphate buffer (PH:7.) with.1 percent (wt/vol) gelatin. The free and bound fraction was separated by dextran-coated charcoal (charcoal 125 mg/1 ml for A-RA and 25 mg/loo ml for T-RA with 25 mg/i ml of dextran (T-7) in both RAJ. The sensitivity for A and T was 5 and 1 pg/tube, respectively. The inter- and intra-assay errors were 1 percent and 7 percent for A-RA and 12 percent and 8 percent for T-RA. The charcoal-treated rabbit serum showed a background of less than 2 pg/mi and 3 pg/m for A and T, respectively. The data are presented without subtracting this background. Plasma hcg concentrations was measured by a modification of the procedure for RA of human luteinizing hormone (LH) (Midgley, 1966). An aliquot (.5 ml) of plasma was used for asasy. n sample tubes, no additional normal rabbit serum was added while in the tubes of standard curve, normal rabbit serum was added to reach a final concentration of 5 percent normal rabbit serum. Thus, its concentration was equal to that in sample tubes. Because of higher rabbit serum concentration, a sufficient second antibody (anti-rabbit-i-globulin produced in sheep) was used to adequately precipitate the rabbit globulin in this RA system. The data was expressed as mu of second RP-HMG. Radioimmunoassays were performed in duplicate with each plasma sample. n animals where bilateral ovarian vein blood was obtained, the hormone concentration did not differ significantly between ovaries; thus, mean values were calculated for that animal. Student s t test was used to analyze for statistical differences between the groups. RESULTS n a pilot study, steroid concentrations of ovarian vein blood did not show significant differences between animals with or without microsphere injection. Thus, it appears that local capillary thrombi produced by microspheres do not influence ovarian steroid secretion. As previously reported (Blasco et at., 1975), ovarian blood flow reached its maximum level 4 h after the administration of hcg (Fig. 1). Prior to this sharp rise in blood flow, the ovarian vein plasma levels and secretion rates of E2 and T increased significantly 2 h after treatment (Figs. 2, 3). The ovarian vein concentrations of E2 and T declined steadily after their early rise, but the secretion of both hormones was again increased at 4 h in association with the high ovarian blood flow. n contrast to E2, the secretion and ovarian vein plasma level of E, increased only during the time of maximum blood flow (Fig. 2). Secretion of A also reached its highest level at 4 h, but the ovarian vein

3 36 WU ET AL. HCG 1 5V HOUR AFTER HCG NJECTON FG. 1. Mean (± S.E.) values of hourly ovarian blood flow of the eserous rabbits following V injection of 75 lu hcg. Number in parenthesis is the number of animals in each group. : P<.1 and t: P<.5 when compared with the value of estrous rabbit (Time-). plasma concentration of this steroid did not change significantly throughout the study (Fig. 3). These fluctuations in ovarian secretion of androgens and estrogens were associated with only minimal changes in the peripheral levels of the hormones. Following administration of hcg, the ovarian vein plasma concentrations of P and 2a- OH-P gradually rose to maximum levels at 4 h and at this time, a sharp increase in the secretion rate of the progestins was seen (Fig. 4). The ovarian vein level of P and 2-OH-P :: - () - 1) - FG. 3. The androgen concentration (Mean ± S.E.) androgen secretion rate of the preovulatory rabbit ovary. : P<.1 and t: P<.5 when compared with 8 4CG 8 z do. > 8 zooo A / 6 #{149} #{149}\ h SO. we ( J 1o. i. 5 1 )/ t U. HOUR AFTER HCG FG. 2. The estrogen concentration (Mean ± S.E.) estrogen secretion rate of the preovulatory rabbit ovary. : P<.1 and t: P<.5 when compared with HOUR AFTER HCG FG. 4.. The progestin concentration (Mean ± S.E.) progestin secretion rate of the preovulatory rabbit ovary. : P<. and t: P<o.5 when compared with

4 PREOVULATORY RABBT OVARY (Fig. 5) and coincided with the highest secretion rate for E2 and T. -, E W ḏ J t a- fo 8 * titti *kl HOUR AFTER HCG NJECTON FG. 5. hcg concentrations (Mean ± S.E.) in the ovarian vein and peripheral plasma and the hcg removal rate of the preovulatory rabbit ovary. : P<.O1 and t: P<.5 when compared with the value of estrous rabbit. declined more gradually after their period of maximum secretion than did the levels seen for E2 and T. n contrast to the androgens and estrogens, the changes in secretion rate of P and 2a-OH-P were reflected by similar patterns for the peripheral plasma concentrations. Throughout the study, the level of 2a-OH-P remained approximately 1-3 fold greater than that of P. The highest concentration of hcg in the peripheral blood was seen at the initial one hour time period, after which it declined rapidly throughout the remainder of the study (Fig. 5). The high peripheral levels of hcg resulted in only a small increase in ovarian vein blood levels of gonadotropin. Retention of hcg by the ovary, as calculated from the difference between peripheral and ovarian vein levels times the blood flow, was greatest at the 2 h period DSCUSSON Simultaneous measurement of ovarian blood E flow and hormones in the ovarian venous effluent during the preovulatory period provides an assessment of changing patterns of steroidogenesis. The maximum ovarian vein blood levels of E2 and T but not progestins, which were found 2 h after treatment with hcg, suggest a preference for the formation of C-19 and C-18 steroids at this early time. Since the higher levels of E2 and T were not accompanied by similar increases in the concentrations of E1 and A, it would appear that 1713-oh steroid dehydrogenase favors the formation of the more biologically active 17-hydroxylated steroids. Hilhiard and coworkers (1974) have shown that E2 is produced primarily by the rabbit follicle, while T appears to be synthesized by both the follicle and interstitial tissue. Armstrong and Papkoff (1976) have demonstrated in studies with rat ovaries that T may be an immediate precursor which is supplied to the follicle for estrogen formation. The simultaneous increase in ovarian secretion of T and E2, but not other ovarian hormones in our study, suggests a similar relationship during the early phase of the preovulatory period in the rabbit. The concentration of P and 2a-OH-P increases significantly above the levels found during estrus one hour following administration of hcg. The secretion rate of both progestins continues to rise, reaching a maximum at the 4 h period. The maximum secretion of all ovarian hormones at 4 h may be attributed to increased availability of steroid precursors as a result of the sharp rise in ovarian blood flow at this time. The increased secretion of progestins is associated with a significant increase in the peripheral plasma concentrations of P and 2a-OH-P. n contrast, the rises in T and E2 secretion following hcg are accompanied by only minimal changes in the peripheral plasma levels of these hormones. n addition, the decline of ovarian steroid secretion 4 h after hcg was reflected primarily in T and E2 and not P and 2a-OH-P. These findings suggest that E2 and T may have a limited intra-ovarian role, probably associated with ovulation rather than a systemic effect on a target organ. The highest peripheral blood levels of hcg

5 38 WU ET AL were seen at the first hour after administration of the tropic hormone. Thereafter, the levels declined approximately 85 percent during the ensuing 3 h. Removal of hcg by the ovary did not correspond to the peripheral levels of the hormone or to the changes in blood flow to the ovary. The maximum retention of hcg which occurred 2 h following treatment corresponds to the peak secretion of pituitary LH following coitus in rabbits (Goodman and Neill, 1976). Consequently, we were not surprised to find that ovarian steroid production reported herein agrees closely with that which occurs shortly after mating. t would appear that hcg initiates changes in the ovary which facilitate a more significant uptake of the tropic hormone at a hater time. Although Mills and McPherson (1974) were unable to demonstrate changes in binding of hcg by isolated follicles throughout the preovulatory period, our in vivo data on ovarian uptake of the gonadotropin would suggest that the increased retention at 2 h may represent an increase in cellular binding sites. The decreased retention of hcg at hater times of the preovulatory period, which was associated with a decline in steroid hormone secretion, may reflect either a loss of gonadotropin receptor or a saturation of receptor sites with hcg. ACKNOWLEDGMENT Materials for radioimmunoassay of LH were generously provided by the National Pituitary Agency and Endocrinology Study Section, National nstitutes of Health. Technical assistance of Mrs. Judith Wang and Mr. Robert Krill is gratefully acknowledged. REFERENCES Armstrong, D. T. and Papkoff, H. (1976). Stimulation of aromatization of exogenous and endogenous androgens in ovaries of hypophysectomized rats in vivo by follicle stimulating hormone. Endocrinology 99, Blasco, L, Wu, C. H., Flickinger, C. L, Wheeler, J. and Mikhail, G. (1973). Blood flow to the reproductive organs of the eserous rabbit. Gynec. nvest. 4, Blasco, L., Wu, C. H., Flickinger, C. L, Pearlmutter, D. and Mikhail, G. (1975). Cardiac output and genital blood flow distribution during the preovulatory period in the rabbit. Biol. Reprod. 13, DeVilla, G.., Jr., Roberts, K., Wiest, W. F., Mikhaih, C. and Flickinger, G. L (1972). A specific radioimmunoassay of plasma progesterone. J. Chin. Endocrinol. Metab. 35, Goodman, A. L and Nell, J. D. (1976). Ovarian regulation of postcoital gonadotropin release in the rabbit: reexamination of a functional role for 2a-dihydroprogesterone. Endocrinology 99, Hilliard, J. and Eaton, L. W., Jr. (1971). Estradiol-17a, progesterone and 2(-hydroxypregn-4-en-3-one in rabbit ovarian venous plasma.. From mating through implantation. Endocrinology 89, Hilliard, J., Scaramuzzi, R. J., Pang, C. N., Penardi, R. and Sawyer, C. H. (1974). Testosterone secretion by rabbit ovary in moo. Endocrinology 94, LeMaire, W. J. and Marsh, J. M. (1975). nterrelationships between prostaglandins, cyclic AMP and steroids in ovulation. J. Reprod. Fertil. Suppl. 22, pner, H. and Creep, R.. (1971). nhibition of steroid synthesis at various sites in the biosynthetic pathway in relation to induced ovulation. Endocrinology 88, Midgley, A. R., Jr. (1966). Radioimmunoassay: a method for human chroionic gonadotropin and human luteinizing hormone. Endocrinology 79, Mills, R. M. and McPherson, J. C.,. (1974). The binding of 3 -HCG to mature follicles isolated from the rabbit ovary. Proc. Soc. Exp. Biol. Med. 145, Wu, C. H. and Lundy, L. E. (1971). Radioimmunoassay of plasma estrogens. Steroids 18, Wu, C. H., Prazak, L, Flickinger, C. L and Mikhail, C. (1974). Plasma 2(ks-hydroxypregn-4-en-3-one in the normal menstrual cycle. J. Chin. Endocrinol. Metab. 39,