Relation Between Levels of Circulating Ovarian Steroids and Pituitary Gonadotropin Content During the Menstrual Cycle of the Rhesus Monkey

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1 BIOLOGY OF REPRODTIO 6, (198) Relation Between Levels of irculating Ovarian Steroids and Pituitary Gonadotropin ontent During the Menstrual ycle of the Rhesus Monkey J. HOTHKISS, D. J. DIERSHKE,3 W. R. BTLER,4 G. R. FRITZ and E. KOBIL Department of Physiology niversity of Pittsburgh School of Medicine Pittsburgh, Pennsylvania 1561 ABSTRAT Anterior pituitary glands were removed from 7 intact cycling rhesus monkeys sacrificed in the early (Day ), mid (Days 6-9) and late (Days 11-1) follicular phase, and in the early and late luteal phase (3-5 and 1-15 days after the midcycle luteinizing hormone (LH) surge). Assignment of cycle stage was confirmed by the pattern of circulating steroid and gonadotropin levels seen in the blood samples taken daily throughout the cycle. The anterior pituitary glands were weighed, stored at -3#{176} and assayed for LH and follicle-stimulating hormone (FSH) content by specific radioimmunoassays. Serum estradiol levels and pituitary LH and FSH contents rose simultaneously during the follicular phase. After the preovulatory gonadotropin surge, pituitary LH content was low and invariant. Pituitary FSH content reached a nadir in the early luteal phase and tended to rise in the late luteal phase. Multiple correlation analyses revealed that there is a positive correlation between rising levels of estradiol in the circulation and pituitary LH (p=o.oo3) and FSH (p=o.l7) content, and that there is a significant negative correlation between circulating progesterone levels and pituitary FSH content (p=o.). Pituitary LH content is less strongly related to circulating progesterone levels. There was no significant difference in the wet weights of the anterior pituitary glands during the five phases of the menstrual cycle studied. ITRODTIO The role of estrogen and progesterone in the control of pituitary gonadotropin secretion in the rhesus monkey has been well documented (Karsch et al., 1973a; Knobil, 1974; 198). These ovarian hormones may also regulate pituitary gonadotropin content because bioassayable LH and FSH in the pituitary rise during the follicular phase and fall during the luteal phase of the menstrual cycle in the rhesus Accepted October 1, Received July 7, A preliminary report of these findings has appeared in abstract form (Proc. Endocrine Society, 198, Abst. 61); this work was supported by grants HDO3968 and RR98 from the IH, SPHS and by a grant from the Ford Foundation. requests and correspondence: Dept. of Physiology, niversity of Pittsburgh, School of Medicine, Pittsburgh, PA Present address: Regional Primate Research enter, niversity of Wisconsin, Madison, Wisconsin Present address: Department of Animal Science, ornell niversity, Ithaca, ew York monkey (Simpson et al., 1956) and human (Bischoff et al., 1969). Since neither of these studies includes data on serum steroid levels, the correlation between ovarian function and pituitary gonadotropin content is presumptive. A rigorous evaluation of the relations among circulating estrogen and progesterone levels and pituitary LH and FSH content became possible during the course of other studies (Butler et al., 1975; Inskeep et al., 198) in which anterior pituitary tissue became available from rhesus monkeys whose reproductive histories and serum steroid and gonadotropic hormone levels were documented until the time of sacrifice. MATERIALS AD METHODS Twenty-seven rhesus monkeys (Macaca mulatta) with regular ovulatory menstrual cycles were bled daily by femoral venepuncture from Day 1 of the menstrual cycle (first day of vaginal bleeding) until sacrifice during the early or late luteal phase (3-5 and 1-15 days after the preovulatory gonadotropin surge) or during the early (Day ), mid (Days 6-9) or late (Days 11-1) follicular phase of the subsequent cycle. The animals were exsanguinated under sodium pentobarbital anesthesia (embutal, 3 mg/kg, i.v.) and the anterior pituitary glands were removed, weighed, and frozen in dry ice. 41 Downloaded from on 9 February 18

2 4 HOTHKISS ET AL. Each anterior pituitary gland was homogenized at #{176}in 1.5 ml. M phosphate buffer in. M ai I (ph 6.85) in an all-glass tissue grinder and centrifuged I (5, X g, 5 mm)to remove particulate matter. u I. The FSH and LH content of duplicate serial dilutions I oonor- of each pituitary extract was measured by specific. - radioimmunoassays (Monroe et al., 197; Yamaji et al., 1973). Serum levels of FSH, LH, progesterone (Karsch et n I al., 1973b; Goodman et al., 1978) and total estrogens (Hotchkiss et al., 1971) were measured by radioim- munoassay in samples taken from Day 1 until sacrifice.. I s The same rhesus FSH and LH standards (RhFSH#{176}WDP-. I B Ii XI , potency=14 X IH-FSH-S1 by bioassay; s I - n RhLH=WDP-X47-B, potency=1.9 X IH-LH-S1 by bioassay) were used for the tissue and serum radioimmunoassays. For statistical purposes, the value of the detection limit of each assay was assigned to those samples whose hormone concentration fell below the o assay sensitivity. E Data from groups of animals sacrificed at different. E times of the cycle were tested by one-way analysis of.. -. variance followed by paired comparisons between I, groups where appropriate. Simple and multiple regression analyses were performed to relate pituitary FSH or LH content to circulating estrogen and/or progesterone levels as the independent variable(s).. o -, ci RESLTS bo E.. P. -H I Table 1 shows the serum steroid and go-,,u, e - * nadotropin levels, and the anterior pituitary weights and gonadotropin content, for each animal as well as for the grouped data (mean ± S. E.) for the five stages of the menstrual cycle (early, mid and late follicular phase, early and late luteal phase). Figure 1 depicts the hormone. Q O -I data from these five groups arrayed temporally. fl lfl. from just after the midcycle gonadotropin surge until just prior to the next (presumptive) gonadotropin surge. While pituitary FSH content rose steadily from its nadir in the early luteal phase to a peak just prior to the expected preovulatory gonado- tropin surge, pituitary LH content remained E #{149}#{149}#{149}#{149}#{149} low throughout the luteal phase and did not. rise significantly until the middle of the follicu- b lar phase (Days 6-9). irculating estrogen levels were variable during the luteal phase and rose significantly during the mid and late,c - follicular phase. Serum progesterone was - undetectable in 14 of 15 animals during the - o I u -t. follicular phase, and was elevated significantly only during the luteal phase. irculating gonadotropin levels remained constant during this truncated cycle which excludes data from animals sacrificed during the preovulatory - gonadotropin surge. Anterior pituitary gland,,, weight did not change during this period of study. Downloaded from on 9 February 18

3 STEROIDS AD RHESS PITITARY GOADOTROPI OTET II: O 4.- O O W..- ( O.- O I O Il fl O Os -. 1 fl - -,... #{176} H -H * -. * -. O lwio t -( fl O IfI O,-I O O - n * - WI - l I I.. WI -H -H O O OO. O O-. flo *..-WIOWI. O* WI * WI * WI 1 WI WI WI * * * I * WI O WI * * +4 -H WIWIO *. OWIO - *,-,WI 1,1** O WI.OWIO WI o ** ,-4 -.-,-I *, * * - fl. o * 4.- s - * O* O WI O*WIO * -.4 O WI,1.,I - fl 1- n WI 1 1 * *.1 #{149}#{149}* #{149}#{149}#{149} +4 #{149} #{149} #{149} S. *.. WI 4I. si. c, I,-,,(4,I,,-,* o : * #{149} -H +1 *.- WI * - * O. - ri*o...io.-, Ofl I. * WI -. * - O WI. WI. WI O *4 - -,1,-l,-l.-I it, #{149} *.- - (F,,, (F, * O OOO -***WIWI.-,, **WI Downloaded from on 9 February 18

4 44 HOTHKISS ET AL..1 L) LTEAL PHASE DAYS POST LH PEAK il-i: FOLLILAR PHASE DAYS POST MESES FIG. 1. Pituitary FSH and LH content (panel a), circulating FSH and LH levels (panel b), and circulating estrogen and progesterone levels (panel c), at five points during the menstrual cycle in the rhesus monkey. The bars around the mean values indicate the standard error (n)=number of animals per point. Statistical analyses of the relations between pituitary gonadotropin content and serum steroid levels were performed on data from the entire menstrual cycle (7 animals) and from the follicular and luteal phases separately (Table.). Data from animals sacrificed during menses (Day of the cycle) are included in both cycle phases. There is a significant positive correlation between serum estrogen levels and pituitary gonadotropin content during the follicular phase (for FSH, R=.54, p=o.o4, n=15; for LH, R=.7, p=o.oo3, n=15) which is not evident during the luteal phase. The significance of the relation between serum estrogen and pituitary gonadotropin content becomes less striking when data from the follicular and luteal phases are pooled (for FSH, R=.46, p=o.ol6, n=7; for LH, R=.57, p=o.oo, n=7). In contrast, there is a negative correlation between circulating progesterone levels and pituitary FSH content during luteal phase Downloaded from on 9 February 18

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6 46 HOTHKISS ET AL. (R=-O.53, p=o.o9, n=17) and during the entire cycle. A marginally significant negative relation between progesterone and pituitary L content becomes evident only when data from the entire cycle is tested (R=-O.39, p=o.o45, n=7); Multiple regression analyses (Table ) suggest that serum estrogen (E) and progesterone (P) levels taken together are more strongly related to pituitary gonadotropin content than if these steroids are considered separately (R for FSH: E alone=o.1, P alone=o.3, E + P O.47;R for L: E alone=o.3, P alone=o.15, E + P=O.4). DISSSIO The patterns of pituitary gonadotropin content described in this study are qualitatively similar to those seen by Simpson et a!. (1956) who observed a midcycle maximum followed by a later (Days 15 and of the cycle) nadir in bioassayable gonadotropin content in the rhesus monkey. A similar study using human autopsy material also showed a midcycle elevation in pituitary FSH and LH content (more striking in the case of LH than FSH), and in addition, a perimenstrual elevation in pituitary LH content (Bischoff et al., 1969). Dating of the menstrual cycle phase was done by ovarian and endometrial histology. Because of the uncertainties of menstrual cycle dating and the lack of concomitant serum steroid data, neither study provides compelling evidence that ovarian steroid secretion may regulate pituitary gonadotropin content. The present study suggests that circulating estrogens and progesterone both play significant, albeit quite different, roles in regulating pituitary gonadotropin content during the menstrual cycle of the rhesus monkey. Pituitary FSH and LH rose as serum estrogens rose, while only pituitary FSH content appeared to rise in response to a decline in serum progesterone. However, since only 47% of the pituitary FSH rise and 4% of the pituitary rise can be attributed to these steroid changes (see multiple regression analysis R, Table ), there must be other factors which influence pituitary gonadotropin content as well. An action for gonadotropin-releasing hormone (GnRI-I) in this regard is suggested by the following evidence. 1) Little or no GnRH appears to be secreted by the hypothalamus in prepubertal rhesus monkeys as castration is followed by a long delay in the expected rise of gonadotropin secretion (Dierschke et al., 1974a). Pituitary LH content in such animals is generally less than 15% of that seen in the normal cycling animals in this study (Dierschke et al., 1974b). ) Ovariectomized adult monkeys whose endogenous GnRH secretion was abolished by lesions of the medial basal hypothalamus for -1 months prior to sacrifice have only 5-% of the pituitary gonadotropin content (W. D. Peckham, unpublished) of the unlesioned ovariectomized animal (Attardi et al., 198). 3) The administration of GnRH induces an increase in pituitary GnRH receptors in the rat (layton and att, 1981; Frager et al., 1981) and an increase in receptor number, in this species at least, is directly related to pituitary LH content (han et al., 1981). While pituitary FSH and L contents appear to change in parallel in this study (especially during the follicular phase), it is possible that each gonadotropin may in fact be regulated independently. For instance, while estradiol levels and pituitary LH content are low in both the immature female monkey (Dierschke et al., 1974b) and in the lactating monkey (Weiss et al., 1976), pituitary FSH content in these animals approaches that found in normal cycling animals. Estrogens, in addition to altering pituitary gonadotropin content, also alter the secretory dynamics of pituitary gonadotropins. Rises in circulating estrogens are responsible for both the negative feedback suppression of FSH and LH secretion and for the abrupt preovulatory gonadotropin surge in the rhesus monkey (Knobil, 1974; 198) by actions exerted directly on the pituitary gland, since both the inhibitory and stimulatory effects of estrogens can be seen in the absence of changes in GnRH infusion parameters in ovariectomized rhesus monkeys whose endogenous GnRH secretion has been terminated by medial basal hypothalamic lesions (akai et al., 1979). In fact, estradiol alone, in the absence of continued GnRH support, can elicit gonadotropin secretion in the suitably prepared rhesus monkey (Wildt et al., 1981a). In contrast to these striking effects of estrogen, progesterone by itself has no effect on LH secretion (Yamaji et al., 197) or on total bioassayable gonadotropin content of the pituitary gland in ovariectomized monkeys (Salhanick et al., 195). In the presence of estrogens, however, progesterone may facilitate or inhibit gonadotropin secretion (Helmond Downloaded from on 9 February 18

7 STEROIDS AD RHESS PITITARY GOADOTROPI OTET 47 et a!., 198; 1981; Resko et al., 1981). It has been argued that the inhibitory action of progesterone is exerted at the hypothalamic level while the facilitatory effect (i.e., the temporal advancement of the estradiol benzoate induced gonadotropin surge) may be exerted directly on the pituitary gland (Wildt et al., 1981b). The relation, if any, between the diverse effects of progesterone on gonadotropin secretion and the action of progesterone on pituitary FSH content described in this paper is obscure. In contrast to our findings that circulating estrogens are positively correlated with pituitary gonadotropin accumulation in the intact rhesus monkey, chronic high levels of estradiol cause a depletion of radioimmunoassayable (W. D. Peckham, unpublished) and bioassayable gonadotropin content (Salhanick et al., 195) in rhesus monkeys and in postmenopausal women (Rowlands and Sharpey-Schafer, 194). The mechanisms whereby the ovarian steroids influence pituitary gonadotropin content remain to be elucidated. AKOWLEDGMETS The authors are indebted to the staff of the Primate Research Laboratory of the niversity of Pittsburgh for expert animal care, to. Stehle and M. Forston for technical assistance, and to Dr. Floyd Taylor for statistical help. REFEREES Attardi, B., Hotchkiss, J. and Knobil, E. (198). Monkey pituitary oestrogen receptors and the biphasic action of oestradiol on gonadotropin secretion. ature 85:5-54. Bischoff, K., Bettendorf, G. and Stegner, H. E. (1969). FSH- und L- Gehalt in menschlichen Hypophysen wahrend des ovariellen yclus. Arch. Gynaekol. 8: Butler, W. R., Hotchkiss, J. and Knobil, E. (1975). Functional luteolysis in the rhesus monkey: Ovarian estrogen and progesterone during the luteal phase of the menstrual cycle. Endocrinology 96: han,., layton, It.., Knox, G. and art, K. J. (1981). Ontogeny of pituitary GnRH receptors in the rat. Endocrinology 18: layton, R.. and art, K. J. (1981). Regulation of pituitary gonadotropin-releasing hormone receptoes by gonadal hormones. Endocrinology 18: Dierschke, D. J., Karsch, F. J., Weick, R. F., Weiss, G., Hotchkiss, J. and Knobil, E. (1974a). Hypothalamic-pituitary regulation of puberty: feedback control of gonadotropin secretion in the rhesus monkey. In: ontrol of Onset of Puberty (M. M. Grumbach, G. D. Grave and F. E. Mayer, eds.). John Wiley & Sons, Inc., ew York, pp Dierschke, D. J., Weiss, G. and Knobil, E. (1974b). Sexual maturation in the female rhesus monkey and the development of estrogen-induced gonadotropic hormone release. Endocrinology 94: Frager, M. S., Pieper, D. R., Tonetta, S. A., Duncan, J. A. and Marshall, J.. (1981). Pituitary gonadotropin-releasing hormone receptors. Effects of castration, steroid replacement, and the role of gonadotropin-releasing hormone in modulating receptors in the rat. J. lin. Invest. 67: Goodman, R. L. (1978). A quantitative analysis of the physiologic role of estradiol and progesterone in the control of tonic and surge secretion of luteinizing hormone in the rat. Endocrinology 1: Helmond, F. A., Simons, P. A. and Hem, P. R. (198). The effects of progesterone on estrogen-induced luteinizing hormone and follicle-stimulating hormone release in the female rhesus monkey. Endocrinology 17: lelmond, F. A., Simons, P. A. and Hem, P. R. (1981). Strength and duration characteristics of the facilitatory and inhibitory effects of progesterone on the estrogen-induced gonadotropin surge in the female rhesus monkey. Endocrinology 18: Hotchkiss, J., Atkinson, L. E. and Knobil, E. (1971). Time course of serum estrogen and Iuteinizing hormone (LH) concentrations during the menstrual cycle of the rhesus monkey. Endocrinology 89: Inskeep, E. K., Wilson, L. Jr., Butler, W. R., Dierschke, D. J., Fritz, G. R. and Knobil, E. (198). Endometrial secretion of prostaglandins during the ovarian cycle and early pregnancy. In: The Endometrium (F. A. Kimball, ed.). Proc. 8th Brook Lodge Workshop on Problems in Reproductive Physiology. Spectrum Publications, ew York, pp Karsch, F. J., Weick, R. F., Hotchkiss, J., Dierschke, D. J. and Knobil, E. (1973a). An analysis of the negative feedback control of gonadotropin secretion utilizing chronic implantation of ovarian steroids in ovariectomized rhesus monkeys. Endocrinology 93: Karsch, F. J., Krey, L.., Weick, R. F., Dierschke, D. J. and Knobil, E. (1973b). Functional luteolysis in the rhesus monkey: The role of estrogen. Endocrinology 9: Knobil, E. (1974). On the control of gonadotropin secretion in the rhesus monkey. Recent Prog. Horm. Res. 3:1-36. Knobil, E. (198). The neuroendocrine control of the menstrual cycle. Recent Prog. Horm. Res. 36: Monroe, S. L., Peckham, W. D., eill, J. D. and Knobil, E. (197). A radioimmunoassay for rhesus monkey luteinizing hormone (RhLH). Endocrinology 86: akai, Y., Plant, T. M., Hess, D. L., Keogh, E. J. and Knobil, E. (1978). On the sites of the negative and positive feedback actions of estradiol in the control of gonadotropin secretion in the rhesus monkey. Endocrinology 1: Resko, J. A., Ellinwood, W. E. and Knobil, E. (1981). Differential effects of progesterone on secretion Downloaded from on 9 February 18

8 48 HOTH KISS ET AL. of gonadotropic hormones in the rhesus monkey. Am. J. Physiol. 4:E489-E49. Rowlands, I. W. and Sharpey-Schafer, E. P. (194). Effect of oestradiol benzoate on the amount of gonadotropin found in the pituitary gland and urine of post-menopausal women. Brit. Med. J. 1:5-7. Salhanick, H. A., l-iisaw, F. L. and Zarrow, M. X. (195). The action of estrogen and progesterone on the gonadotropmn content of the pituitary of the monkey (Macaca mulatta). J. lin. Endocrinol. Metab. 1:31-3. Simpson, M. E., an Wagenen, G. and arter, F. (1956). Hormone content of anterior pituitary of monkey (Macaca mulatta) with special reference of gonadotropins. Proc. Soc. Exp. Biol. Med. 91:6-11. Weiss, G., Butler, W. R., Dierschke, D. J. and Knobil, E. (1976). Influence of suckling on gonadotropin secretion in the postpartum rhesus monkey. Proc. Soc. Exp. Biol. Med. 153: Wildt, L., Hausler, A., Marshall,. and Knobil, E. (1981a). Estradiol as a gonadotropin releasing hormone in the rhesus monkey. Endocrinology 18: Wildt, L., Hutchison, J., Marshall, G. and Knobil, E. (1981b). The site of action of progesterone in the blockade of estradiol induced LH surges in the rhesus monkey. Proc. 6th Internat. ong. Endocrinology, Abst Yamaji, T., Dierschke, D. J., Bhattacharya, A.. and Knobil, E. (197). The negative feedback control by estradiol and progesterone of LH secretion in the ovariectomized rhesus monkey. Endocrinology 9: Yamaji, T., Peckham, W. D., Atkinson, L. E., Dierschke, D. J. and Knobil, E. (1973). Radioimmunoassay of rhesus monkey follicle-stimulating hormone (RhFSH). Endocrinology 9: Downloaded from on 9 February 18