Changes in rat ovaries of specific binding for LH, FSH and prolactin during the oestrous cycle and pregnancy

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1 Changes in rat ovaries of specific binding for LH, FSH and prolactin during the oestrous cycle and pregnancy K. W. Cheng Department ofphysiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E O W3, Canada Summary. In cyclic rats, the highest ovarian specific binding for LH was 6\m=.\0\m=+-\2\m=.\2% in pro-oestrus. During pregnancy, the specific binding of 125I-labelled bovine LH by rat ovaries increased gradually and reached a maximum of 24\m=.\1\m=+-\4\m=.\9%between Days 14 and 18 of gestation; a slight decrease in binding was observed at Day 20 of pregnancy. Ovarian specific binding for FSH was also highest in pro-oestrus (8\m=.\9\m=+-\2\m=.\1%), decreasing to about 50% in oestrus and metoestrus, but staying relatively constant during pregnancy. For prolactin, the specific binding in rat ovaries was highest (7\m=.\1\m=+-\ 1 \m=.\6%) in pro-oestrus, quite high in metoestrus and dioestrus and low in oestrus. Specific binding increased gradually only after Day 14 of pregnancy. Serum concentrations of rat LH, FSH and prolactin at different stages of the oestrous cycle and during pregnancy were determined by radioimmunoassays, and no obvious correlation was observed between levels of circulating hormones and the specific binding of these hormones in ovarian tissues. Affinity constants (Ka) for the hormones were very similar between ovaries from pro-oestrous rats and late-pregnant rats, being 0\m=.\31\m=x\109 M\p=n-\1 for LH, 0\m=.\65\m=x\1010M\p=n-\1for FSH,and 1\m=.\14\m=x\1010 M\p=n-\1for prolactin. Increases in specific binding for different hormones were due to increases of total binding sites in the ovary under different physiological states. Introduction Numerous studies have been reported on the specific uptake of radioiodinated gonadotrophic hormones by components of gonadal tissue (de Kretser, Catt, Burger & Smith, 1969; Catt, Dufau & Tsuruhara, 1972; Danzo, Midgley & Kleinsmith, 1972; Kammerman & Canfield, 1972; Gospodarowicz, 1973; Cheng, 1975a). Midgley (1971) showed that labelled or unlabelled HCG was taken up by luteinized cells in the ovaries of immature rats in which pseudopregnancy had been hormonally induced, and that HCG and prolactin were bound to components of luteinized rat ovarian tissues in vitro. The uptake of FSH by the ovary is enhanced by oestradiol treatment (Goldenberg, Vaitukaitis & Ross, 1972) and the amount of specific binding of protein hormones to the membrane-bound receptors is known to be dependent upon the sex and physiological state of the animal (Goldfine et ai, 1973). At present, however, little information is available on the specific uptake of gonadotrophic hormones by gonadal tissues in normal physiological states. The present report is of a study of the changes in the total receptor-binding sites for LH, FSH and prolactin in ovarian tissues of the rat during the oestrous cycle and pregnancy. Materials and Methods Animals. Mature cyclic rats and rats at known stages of pregnancy (Sprague-Dawley strain, g) were purchased from North American Breeding Laboratories, Manitoba, and were housed under controlled conditions of temperature (23 C), humidity (50-60%) and light (12 hr/24 hr). Oestrous cycles were monitored by daily vaginal smears, taken between and hours. Only

2 animals exhibiting at least three regular cycles were used in these studies. For timed pregnancy stages, males were placed with pro-oestrous females overnight and the day spermatozoa were found in the vaginal smear was designated as Day 1 of pregnancy. The animals were killed between and hours at particular stages of the oestrous cycle or pregnancy. Each animal was decapitated, and the trunk blood was drained into a 12-ml centrifuge tube. After centrifuging at 1000 g for 30 min at 4 C, the serum obtained was kept frozen at 20 C until assayed. The ovaries were dissected out, trimmed of fat and weighed immediately to the nearest 0-01 mg. The ovaries were stored at 70 C for no more than 3 weeks before determination of specific binding receptors for LH, FSH and prolactin. Hormones. Human FSH (LER-1577C), ovine prolactin (P-S10), and rat LH (LH-RP-1), FSH (FSH-RP-1) and prolactin (Prolactin-RP-1) were obtained through the Pituitary Distribution Program of NIAMDD, National Institutes of Health, Bethesda, U.S.A. Highly purified bovine LH (potency 20 = NIH-LH-S1) was a gift from Dr J. G. Pierce, UCLA, California, U.S.A. Radioiodination. Carrier-free Na125I was purchased from the New England Nuclear Corp., Boston, Massachusetts, U.S.A. Human FSH and ovine prolactin were radioiodinated with 1-0 mci l25i/5µg protein by the enzymatic method with lactoperoxidase (Calbiochem) as described previously (Cheng, 1975b). Bovine LH (5 µg) was iodinated with 0-5 mci 125I by the chloramine-t method (Hunter & Greenwood, 1962). Labelled rat LH, FSH and prolactin were prepared by the chloramine-t method with 1-0 mci 125I/2-0 µg protein. The radioiodinated hormones were separated from free 125I by gel filtration on Sephadex G-100 with 25 mm-tris-hcl buffer at ph 7-2. For determination, in duplicate or triplicate, of the specific activity of the labelled hormone, acetone was added to give a final concentration of 90% to 0-2 ml of the reaction mixture before gel filtration. The protein precipitated was washed 4 or 5 times with several volumes of acetone and then counted in an autogamma spectrometer. By knowing the amount of hormone present in the 0-2 ml aliquots (200 ng), the ct/min in the 90% acetone precipitate and the ct/min of 1 uci Na125I used for iodination, the specific activity of the labelled hormone was estimated and expressed in µci/µg hormone. The specific activities of 125I-labelled human FSH, ovine prolactin and bovine LH were \ici/\ig, \ic\l\ig and \ici/[ig, respectively. Preparation of ovarian homogenates. The ovaries from 3 rats at the same stage of the oestrous cycle or pregnancy were pooled and homogenized in 1-5 ml 25 mm-tris-hcl buffer, ph 7-2. The homogenate was layered on top of a glass-wool plug at the bottom of a polypropylene tube (12 72 mm; Falcon) with a hole pierced at the bottom, which in turn was situated inside a conical glass centrifuge tube ( mm, 12 ml ; Pyrex). After centrifuging for 30 min at 4 C at 2000g, the super natant in the glass centrifuge tube was discarded and the sedimentated particulate membrane fraction, contaminated by other cellular organelles was resuspended in 25 mm-tris-hcl buffer, ph 7-2, con taining 0-1 % bovine serum albumin and 10 mm-mgci2 to give a concentration of 150 mg wet weight of ovarian tissues/ml. The resuspended particles were again homogenized manually just before use in the specific binding studies. Specific binding of 125I-labelled bovine LH, human FSH and ovine prolactin. The hormones (each approx. 50,000 ct/min) were incubated separately with 01 ml of the resuspended ovarian particulate fraction ( mg/ml) in the absence or presence of excess unlabelled hormone (1 µg/tube) at a final volume of 0-5 ml mm-tris-hcl, ph 7-2, containing 0-1 % BSA and 10 mm-mgcl2. The incubation was carried out in mm glass tubes (Kimble Co. Ltd) in duplicate or triplicate at 23 C for 16 hr. This interval of overnight incubation was chosen for convenience in accordance with previous reports on receptor-binding studies of 125I-labelled human LH, human FSH and ovine prolactin receptor sites in partly purified plasma membranes from the rat testis and ovary (Catt et ai, 1972), bovine testis (Cheng, 1975a) and pregnant rabbit mammary gland (Shiu & Friesen, 1974), respectively. The reaction was terminated by adding 3-0 ml ice-cold 25 mm-tris-hcl buffer, containing 0-1 % BSA and 10 mm-mgcl2. Free and bound 125I-labelled hormones were separated by centrifugation for 30 min at 4 C at 2000 g. The supernatant was decanted, and the bound radioactive hormone in the pellet was counted in an Automatic gamma counter. Specific binding was expressed as (CB CN) 100/C-r where CB is the ct/min bound to the re-suspended ovarian particulate fraction, CN is the nonspecific bound ct/min in the presence of excess unlabelled hormone, and CT is the total ct/min

3 put into the tube (Cheng, 1975b). It has been demonstrated that the values of specific binding (%) expressed by this formula for calculations in radioligand-receptor assays are precise and reprodu cible, the within-assay variation being less than ± 10 % and the between-assay variation +15% (Cheng, 1975b). The specific binding of a labelled hormone to its receptor sites was demonstrated by specific displacement with the unlabelled preparation of that particular hormone; for example, the uptake of 125I-labelled bovine LH by ovarian tissues was inhibited only by unlabelled bovine LH and not at all by unlabelled human FSH or ovine prolactin. Radioimmunoassays. Radioimmunoassays for rat LH, FSH and prolactin were double-antibody systems using kits supplied by NIAMDD. The assays were carried out according to the standard procedure supplied with the kits, and results were expressed as ng NIAMDD rat standard (LH-RP-1, FSH-RP-1 or Prolactin-RP-l)/ml. LH Results The changes of ovarian binding activity for LH and serum concentrations of LH during the oestrous cycle and pregnancy are shown in Table 1. The highest values in the oestrous cycle were found at pro-oestrus, but in pregnancy the pattern of ovarian specific binding for LH increased and decreased in an inverse manner to that of serum LH. FSH The highest specific binding for FSH was again demonstrated in ovaries obtained from rats in pro-oestrus (Table 1), and serum levels were also highest at this stage of the cycle. During pregnancy, the specific binding for FSH stayed relatively constant, and the serum FSH concentrations varied only slightly. These findings suggest that FSH might not have a significant physiological role during pregnancy, although it is important for the maturation of follicles as indicated by the cyclic changes of specific binding during the oestrous cycle. Table 1. Mean + S.D.* serum concentrations of LH, FSH and prolactin and specific binding (%) of 125I-labeIled bovine LH, human FSH and ovine prolactin by ovarian tissues of rats at different stages of the oestrous cycle and during pregnancy (12 rats/group) Oestrous cycle Dioestrus Pro-oestrus Oestrus Metoestrus Pregnancy Day Serum level (ng/ml) 29-2 ± ± ± ± ± ± ± ± ± ± ± LH FSH Prolactin Specific bindingt (%) ± ± ± ± ± ± ± ± Serum level (ng/ml) ± ± ± ± ± ± ± Specific bindingt (%) ± ± ± ± ± ±0-7 Serum level (ng/ml) ± ± ± ± ± ± Specific bindingt (%) ± * Of 4 separate experiments with different preparations of radioiodinated hormones of different specific activities. t Expressed as a % of the total ct/min in the incubation mixture (see 'Materials and Methods').

4 Prolactin The serum concentrations of and specific binding for prolactin during the oestrous cycle were highest at pro-oestrus (Table 1). There was a slight increase in specific binding in late pregnancy. Degradation and binding studies The possibility of there being differences in the rate of degradation for hormones by ovaries at different physiological states was examined with ovaries obtained from rats in pro-oestrus or late pregnancy. Radioiodinated bovine LH, human FSH and ovine prolactin tracers were separately incubated with equal amounts of ovarian homogenates from both groups of rats for 3 hr at room temperature. After centrifugation, the supernatants of these incubation mixtures were examined for specific binding by re-incubating with fresh partly purified preparations of particulate receptors obtained from bovine corpus luteum (Gospodarowicz, 1973), bovine testis (Cheng, 1975a) and preg nant rabbit mammary gland (Shiu & Friesen, 1974), respectively, because these preparations have previously been shown to bind specifically these protein hormones. For controls, the respective tracers were incubated at room temperature for 3 hr in tris-hcl buffer, ph 7-2, in the absence of rat ovarian tissues. The structural integrity of the hormones used in the incubations was tested by crossreacting with excess amounts of the appropriate antisera. As shown in Table 2, these radioiodinated hormones showed no significant changes in specific binding after preincubation with ovarian homo genates, indicating that differences in specific binding for these hormones by ovarian homogenates at various physiological states (Table 1) are not due to different rates of degradation during incubation. Table 2. Degradation studies of 125I-labelled bovine LH, human FSH and ovine prolactin with ovarian homogenates from pro-oestrous and pregnant rats 125I-labelled tracer Bovine LH Human FSH Ovine prolactin Treatment (preincubation) Control Control Control Specific binding ( %) by partly purified particulate receptors Specific binding (%) by excess amounts of antiserum Table 3. Affinity constants (K ) and binding capacities (rv) of pro-oestrous and pregnant rat ovarian tissues for radioiodinated bovine LH, human FSH and ovine prolactin, reflecting corrections for the binding and degradation observed as a function of hormone concentration 125I-labelled tracer Source of membranes (1/mol) (fmol/loomg) Bovine LH Human FSH Sheep prolactin ' 0-63 IO The calculations are based on the data in Text-fig. 1 using the formula B/F= B/Kd, where 125I-labelled hormone specifically bound (fmol); F free = = 125I-labelled hormone (fmol); Kd= dissociation constant; and K= constant. The slope of the plot yields 1 Kd ( Ka) and the intercept on the abscissa yields the total 125I-labelled hormone binding capacity (fmol) for the amount of ovarian tissue used.

5 Scatchard plots (Scatchard, 1949) of the specific binding curves of LH, FSH and prolactin for ovaries from rats in pro-oestrus and at Day 18 of pregnancy are shown in Text-fig. 1, and the affinity constants (Ka) and binding capabilities in Table t- _i_l-l (labelled hormone (fmol) bound/100 mg ovarian tissue Text-fig. 1. Scatchard plot of specific binding of l25i-iabelled (a) bovine LH, (b) human FSH and (c) ovine prolactin by ovarian tissue homogenates from rats at pro-oestrus (O) or Day 18 of pregnancy ( ). The affinity constant (Ka) is the negative slope of the line, and the binding capacity (N) of the tissue is indicated by the intercept on the x-axis. A pool of ovaries from 18 rats was used. Tubes contained 01 ml ovarian homogenate (200 mg/ml) and different amounts of 125I-labelled hormones with and without unlabelled hormones. Tubes were incubated at 23 C for 16 hr. The calculation of the amount of bound I25I-labelled hormone (fmol) was based on the known specific activity of the radioiodinated hormone and an assumed mol. wt of 30,000 for LH and FSH, and 20,000 for prolactin.

6 Since these studies were carried out with 125I-labelled hormones, which would in general be contaminated with a small amount of non-iodinated hormones, it is possible that the behaviour of the radioiodinated hormones might not be identical to that of the native hormones. Nevertheless, the radioiodinated hormones used in these studies have been demonstrated to have identical elution volumes after gel filtration on Sephadex G-100 and to cross-react similarly with the appropriate antisera to the native hormones. Discussion These results show that during the oestrous cycle the rat ovary exhibits cyclic changes in its binding activities for LH, FSH and prolactin and that the number of binding sites for these functionally related hormones did not change in a parallel manner. The highest binding activity for all the gonado trophic hormones was found in pro-oestrus (Table 1). These findings are in good agreement with the results reported by Hunzicker-Dunn & Birnbaumer (1975) that LH- and FSH-stimulated adenylate cyclase activities in cyclic rat ovaries were highest in pro-oestrus and lowest in oestrus. Serum concen trations of LH, FSH and prolactin at different stages of the oestrous cycle (Table 1), which reflected the different physiological states of the ovary, were similar to the values reported by others (Daane & Parlow, 1971 ; Butcher, Collins & Fugo, 1974) in that there were surges of LH, FSH and prolactin during pro-oestrus. However, findings in the present report do not demonstrate any obvious correla tion between concentrations of circulating gonadotrophins and number of total binding sites in the ovarian tissues. The results from pregnant rats indicated that the ovarian binding sites for LH developed earlier than those for lactogenic activity, whilst FSH did not appear to be involved. Although the binding sites for and the concentrations of LH during pregnancy were inversely related, the pattern of increase of ovarian specific binding was nearly identical to that of serum progesterone concentrations during pregnancy (Morishige, Pepe & Rothchild, 1973), and was very similar to that of LH-stimulated adenylate cyclase activity in rat corpora lutea during pregnancy (Hunzicker-Dunn & Birnbaumer, 1975). The increases of ovarian uptake of LH and prolactin during pregnancy resulted from increases in the number of binding sites or the binding capacities of ovarian tissues, and the affinity constants (Ka) for all these gonadotrophins were similar for ovarian tissues in normal cyclic rats and pregnant rats (Text-fig. 1 and Table 2). No significant degradation of radioiodinated LH, FSH and prolactin was observed after incubation with ovarian homogenates; and no difference in degradation of these hormones was observed after incubation with homogenates of ovaries from cyclic or pregnant rats (Table 2). Membrane-bound radioiodinated human FSH (Cheng, 1975a) and ovine prolactin (Shiu & Friesen, 1974) have been demonstrated to retain their biological integrity after dissociating from their specific receptor-sites. Interactions of these hormones with their specific receptors do not therefore result in a significant alteration or degradation of the hormones themselves. The present findings indicate that changes in specific binding of hormones in target tissues occur in association with defined physiological states, which are in turn due to changes in the number of specific binding sites induced by another gonadotrophic hormone or ovarian steroids in the circula tion. Zeleznik, Midgley & Reichert (1974) demonstrated that administration of FSH in vivo acted on rat ovarian granulosa cells to induce or activate receptors for LH and HCG. Similarly, oestrogen has been reported to have stimulatory effects on specific binding for HCG and synthesis of progesterone by luteinized rat ovaries (Lee & Ryan, 1974). It is not known whether the appearance of available receptors is a process of unmasking, activation, or induction. The mechanism by which one hormone acts to stimulate the appearance of receptors for another hormone in target tissues is unknown. However, the exact relationship between serum levels of gonadotrophic hormones or oestrogenic steroids and the number of binding sites in the ovary under normal physiological conditions is believed to be worth further study. I am a scholar of the Medical Research Council of Canada. This work was supported by M.R.C. (Canada) Grant AM I am indebted to Miss Glenda Lagadi and Mrs Herminia Sy for expert technical assistance, Mrs Joanne Lough for typing the manuscript, and Mr Jeffery Harris for drawing the figure.

7 References Butcher, R.L., Collins, W.E. & Fugo, N.W. (1974) Plasma concentration of LH, FSH, prolactin, pro gesterone and estradiol-17ß throughout the 4-day estrous cycle of the rat. Endocrinology 94, Catt, K. J., Dufau, MX. & Tsuruhara, T. (1972) Radioligand-receptor assay of luteinizing hormone and chorionic gonadotropin. /. clin. Endocr. Metab. 34, Cheng, K.W. (1975a) Properties of follicle-stimulatinghormone receptor in cell membranes of bovine testis. Biochem. J. 149, Cheng, K.W. (1975b) A radioreceptor assay for folliclestimulating hormone. /. clin. Endocr. Metab. 41, Daane, T.A. & Parlow, A.F. (1971) Periovulatory patterns of rat serum follicle stimulating hormone and luteinizing hormone during the normal estrous cycle as revealed by radioimmunoassay: effects of pentobarbital. Endocrinology 88, Danzo, B.J., Midgley, A.R., Jr & Kleinsmith, L. J. 1972) Human chorionic gonadotropin binding to rat ovarian tissue in vitro. Proc. Soc. exp. Biol. Med. 139, de Kretser, D.M., Catt, K.J., Burger, H.G. & Smith, G.C. (1969) Radioautographic studies on the localization of I25I-labelled human luteinizing and growth hormone in immature rats. /. Endocr. 43, Goldenberg, R.L., Vaitukaitis, J.L. & Ross, G.T. (1972) Estrogen and follicle stimulation hormone interactions on follicle growth in rats. Endocrinology 90, Goldfine, I.D., Kahn, C.R., Neville, D.M., Jr, Roth, J., Garrison, M.M. & Bates, R. (1973) Decreased binding of insulin to its receptors in rats with hormone induced insulin resistance. Biochem. Biophys. Res. Commun. 53, Gospodarowicz, D. (1973) Properties of the luteinizing hormone receptors of isolated bovine corpus luteum plasma membranes. /. biol. Chem. 248, Hunter, W.M. & Greenwood, F.C. (1962) Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature, Lond. 194, 495^196. Hunzicker-Dunn, M. & Birnbaumer, L. (1975) Physiologic aspects of adenylate cyclase in follicles and corpora lutea ofrats. Program ofthe 57th Annual Meeting of the Endocrine Society, New York, p. 105, Abstr. No Kammerman, S. &CANFIELD, R.E. (1972) The inhibition of binding of iodinated human chorionic gonado tropin to mouse ovary in vitro. Endocrinology 90, Lee, C.Y. & Ryan, R.J. (1974) Estrogen stimulation of human chorionic gonadotropin binding by luteinized rat ovarian slices. Endocrinology 95, Midgley, A.R., Jr (1971) Gonadotropin binding to frozen sections of ovarian tissue. In Gonadotropins, pp Eds B. B. Saxena, C. G. Beling & H. M. Gandy. Wiley-Interscience, New York. Morishige, W.K., Pepe, G.J. & Rothchild, I. (1973) Serum luteinizing hormone, prolactin and pro gesterone levels during pregnancy in the rat. Endo crinology 92, Scatchard, G. (1949) Molecular interactions in protein solutions. Ann. N. Y. Acad. Sci. 51, Shiu, R.P.C. & Friesen, H.G. (1974) Properties of a prolactin receptor from the rabbit mammary gland. Biochem. J. 140, Zeleznik, A.J., Midgley, A.R., Jr & Reichert, L.E., Jr (1974) Granulosa cell maturation in the rat: increased binding of human chorionic gonadotropin following treatment with follicle-stimulating hor mone in vivo. Endocrinology 95, Received 20 February 1976