Pulsatile LH Release During Periods of Low Level LH Secretion in the Rat Estrous Cycle

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1 BIOLOGY OF REPRODUCTION 24, (1981) Pulsatile LH Release During Periods of Low Level LH Secretion in the Rat Estrous Cycle ROBERT V. GALLO Department of Physiology, University of California, San Francisco, School of Medicine, San Francisco, California ABSTRACT The present experiments were carried out to characterize LU release during periods of low level LH secretion in the rat 4-day estrous cycle. Unanesthetized animals with jugular cannulae were bled on the morning ( h) or afternoon ( h) of either estrus, diestrus 1 (i.e., metestrus), or diestrus 2, or on proestrus morning (75 l whole blood/6 mm). The percent coefficients of variation obtained for alterations in blood LH levels during the morning or afternoon of each of the four stages of the estrous cycle, or when morning and afternoon values for each cycle stage were combined, were all significantly greater than intra-assay variation, indicating that LH release was pulsatile during each stage of the cycle. No significant morning vs afternoon differences in mean blood LH levels, LH pulse amplitude, or pulse frequency existed in any cycle stage. As the cycle progressed, changes occurred in mean blood LH levels which were due to changes in the characteristics of pulsatile LI-I secretion on each day of the cycle. On estrus, blood LH levels are the lowest of the entire cycle due to the occurrence of the slowest frequency of pulsatile release in the cycle (1 pulse/2 h). On the next day, diestrus 1, blood LH levels are elevated, and are the highest of all the periods of low level LH release in the cycle. This increase in blood LH levels from estrus to diestrus 1 is due to a marked increase in LH pulse amplitude (15 vs 38 ng/ml) as well as a shortening of the LH interpulse interval to 60 mm. While this circhoral rhythm remained the same through diestrus 2 (55 mm) and proestrus morning (63 mm), the LH pulse amplitude decreased on these days to 20 and 16 ng/ml, respectively (compared with 38 ng/ml on diestrus 1), and therefore the mean blood level of LH on both days was significantly lower than on diestrus 1, although still higher than on estrus because of different pulse frequencies. Therefore, LH release is pulsatile during periods of low level LH secretion in the rat estrous cycle, and changes in the pulsatile characteristics of this release occur with different stages of the cycle. INTRODUCTION Studies on the neuroendocrine regulation of pulsatile LH secretion in the rat have used only the ovariectomized animal since removal of the ovaries allowed an elevation in blood LH levels and a pulsatile pattern to be readily discernible (for references see Gallo, 1980a). However, pulsatile LH release has been demonstrated for several other species with their gonads intact, including cattle (Katongole et al., 1971; Rahe et al., 1980), sheep (Foster et al., 1975; Baird, 1978), and humans (Midgley and Jaffe, 1971; Yen et al., 1972; Naftolin et al., 1973; Kapen et al., 1973; Santen and Bardin, 1973). Moreover, the pattern of pulsatile LH secretion during the human menstrual cycle (Yen et al., 1972; Santen and Bardin, 1973) and the estrous cycle of the cow (Rahe et al., 1980) or sheep (Foster et al., 1975; Baird, 1978) appears to depend on the steroid secretory patterns of each cycle. The aims of the present study then were twofold. The first was to determine if pulsatile LU secretion existed during the periods of low level LH release in the rat estrous cycle. If so, the second objective was to characterize it to determine whether any variations in LH secretory episodes occurred with different cycle stages and whether these could be correlated with the well known changes in ovarian steroid secretory patterns that occur during the rat 4-day estrous cycle (Kalra and Kalra, 1974; Butcher et al., 1974; Smith et al., 1975; Nequin etal., 1979). Accepted December 23, Received September 30, These studies were supported by NIH Grants HD05577 and AM MATERIALS AND METHODS Adult female Sprague-Dawley rats (Simonsen Labs., Gilroy, CA), weighing g, were main- 771

2 772 GALLO tamed on a 14L: 1OD schedule (lights on 0500 h) and fed Purina rat food and water ad libitum. Daily vaginal smears were taken up to and including the day of the experiment, and all rats showed two or more consecutive 4-day estrous cycles before that time. Bleeding and Experimental Groups The procedure for bleeding the unanesthetized, unrestrained rats in this laboratory has been reported (Drouva and Gallo, 1976). A cannula was inserted into or near the right atrium via the external jugular vein in each rat between h on the day preceding the use of the animal in an experiment. This procedure did not interfere with the stage of the estrous cycle, which was verified by a vaginal smear on the morning of the experimental day. Rats were bled on one of the four mornings ( h) of the cycle or in the afternoon ( h) of estrus, diestrus 1 (i.e., metestrus), or diestrus 2. Animals were injected with 350 U heparin 20 mm prior to being bled continuously through a peristaltic pump at a rate of 75 MI whole blood/6 mm for 3 h. Individual blood samples were collected in Hamilton microliter syringes and added directly to assay tubes kept on ice and containing phosphate-buffered saline + 0.1% gelatin. Hematocrits remained reasonably stable during these experiments (44.9 at onset, 40.5 at end; n 67). Radioimmunoassay Blood samples were analyzed for LH by a slight modification (Osland et al., 1975) of the ovine-ovine rat LH double antibody radioimmunoassay of Niswender et al. (1968). The minimum sensitivity of the assay in the present study was 0.5 ng. This was obtained by diluting the anti-ovine LH serum #15 by 1:160,000 and 48 h later adding 25,000-30,000 cpm of li25 I] -ovine LH to each assay tube. Second antibody was added 24 h later. Individual tubes used to determine the standard curve contained 75 p1 whole blood from hypophysectomized female rats to correct for observed differences in background counts in the presence or absence of whole blood lacking LH. Samples obtained from an individual rat were all assayed in a single assay, but samples from all animals were not measured in the same assay. The intra-assay CV determined at a mean LH level of 30 ± I ng/ml (n = 12) was 13.6%, and the interassay CV for a p001 of rat plasma containing 75 ± 3 ng LU/mI (n = 7) was 9.2%. LU values (ng/ml whole blood) are expressed in terms of the NIAMDD Rat LU RP-1, which has a biological potency equivalent to 0.03 X NIU-LH-S1. Values given in the text represent the mean ± SEM. Analysis of Data The percent coefficient of variation [CV; (SD mean) X 100] was determined for blood LH levels in individual rats and then for each group. To determine if the variation obtained in blood LH values indicated changes in the secretory pattern of LH rather than assay variation, this CV was compared (unpaired test) with the intra-assay variation determined at levels of LH comparable to those found during the different stages of the estrous cycle, i.e., ng/ml. These inrra-assay coefficients of variation were each based on determinations carried Out over 6 assays. Each determination consisted of 30 replicates of either 1.0, 1.4, 1.7, 2.0, or 2.5 ng NIAMDD Rat LH RP-1/75 p1 assay buffer, which corresponds to the 75 p1 samples taken from the rats. To this was added 75 p1 whole blood from hypophysectomized female rats to ensure that assay tubes used to determine intra-assay variation would be comparable in content to experimental rat samples used to determine the CV for rat mean blood LH levels. The intra-assay coefficients of variation differed within the range of the low mean blood LH levels found in this study. The intra-assay coefficients of variation for LH levels in the range of 9-14 ng/ml (i = 11.6 ± 0.5, n = 12), ng/mi (x = 20.6 ± 0.9, n = 15), and ng/ml (x = 29.8 ± 0.5, n = 12) were 22.4 ± 2.0, 17.1 ± 1.5, and 13.6 ± 1.6, respectively. Thus, if the mean LH level for a particular cycle stage was in one of these three LH ranges, the CV for rat blood LH levels in that stage of the cycle was compared with the intra-assay variation for this LH range. The CV and mean LU levels were also determined for all LH values composing the ascending (nadir to peak) as well as the descending phase of each potential LH pulse. A pulse was defined when this CV was greater than 1.5 times the intra-assay CV determined at a comparable mean level of LH. This definition was chosen since it takes into consideration a comparison of both the intra-assay coefficients of variation and the coefficients of variation for rat blood LU levels (see Results). Moreover, using such a procedure, the same definition was applicable to those LU pulses occurring in the rat during the plateau portion of the ovulatory LU surge on proestrus (Gallo, 1981). Thus, in the present study, for potential pulses having mean LH levels in the ranges 9-14, 15-25, and ng/mi, the minimum CV accepted for designation of a pulse was 34, 26, and 20, respectively. When it had been determined that LU release was pulsatile for a particular stage of the estrous cycle, this release was characterized by calculating mean blood LU levels, intervals between LU pulses, and the magnitude of increase in blood LH levels for individual pulses (pulse / blood LU; i.e., the difference between the nadir and ensuing peak in blood LU levels). Values for these three parameters were compared by one-way analysis of variance followed by Duncan s multiple range test. Morning and afternoon values for each parameter in one cycle stage were compared by the unpaired t test. RESULTS The percent coefficients of variation (mean ± SEM) obtained for alterations in blood LH levels during the morning and afternoon, respectively, were 38 ± 3 and 32 ± 3 on estrus, 61 ± 3 and 57 ± 6 on diestrus 1,43 ± 3 and 43 ± 6 on diestrus 2, and 32 ± 2 on proestrus morning. All morning and afternoon values, as well as the combined mean value for each stage of the cycle, were significantly greater than intra-assay variation (estrus afternoon, P<0.01; all others, P<0.001) indicating that LH release was pulsatile for each cycle stage.

3 ESTROUS CYCLE PULSATILE LU RELEASE 773 No significant morning vs afternoon differences in mean blood LI-I levels, LH pulse amplitude, or pulse frequency existed in any cycle stage. The combined mean values for each stage of the cycle are presented in Table 1. On estrus, blood LH levels are the lowest of the cycle due to the occurrence of the longest LU interpulse intervals of the cycle (119 ± 11 mm). On diestrus 1, blood LII levels are elevated and are the highest of all the periods of low level LH release in the cycle. This increase in blood LH levels from estrus to diestrus 1 is due to a marked increase in LU pulse amplitude (15.1 ± 2.0 vs 37.8 ± 2.8 ng/ml) as well as a shortening of the LH interpulse interval to 60 ± 7 mm. The LH interpulse interval was maintained through diestrus 2 (55 ± 5 mm) and proestrus morning (63 ± 11 mm). However, when compared with diestrus 1 the LH pulse amplitude decreased on both days, being 19.8 ± 1.3 ng/ml on diestrus 2 and 15.6 ± 1.2 ng/ml on proestrus morning, and therefore the blood level of LH on both days was significantly less than on diestrus 1, although still higher than on estrus because of different pulse frequencies. The increase in LH pulse amplitudes on diestrus 1 compared with other cycle stages can be seen by comparing Figs. 1 and 2. DISCUSSION The present study indicates that basal LU release in the intact rat, as in other species with their gonads intact, is pulsatile. Moreover, changes in the pattern of this pulsatile secretion are seen to occur at different stages of the estrous cycle. As the estrous cycle progresses, changes occur in mean blood LU levels which can be attributed to changes in the characteristics of pulsatile LI-I secretion. On estrus, the day following the ovulatory surge, blood LU levels are the lowest of the cycle due to the occurrence of the slowest frequency of pulsatile release in the cycle. On the next day, diestrus 1, in confirmation of previous reports (Naftolin et al., 1972; Kalra and Kalra, 1974; Goodman, 1978), blood LH levels are found to be elevated. Extending this finding, the present study indicates that a marked increase in LU pulse amplitude contributes to this rise. The height of these pulses is clearly the largest of the entire period of low level LU release in the rat cycle. In addition, mean blood LU levels are higher on diestrus 1 than on the preceding estrus since the LH interpulse interval is also shorter on diestrus 1, with LU pulses now occurring at hourly intervals. While this circhoral frequency remains the same through diestrus 2 and the morning of proestrus, the LU pulse amplitude decreases on these days, and therefore so does the mean blood level of LU. These changes in pulsatile LU secretion in the intact rat may well be related to changes in TABLE 1. Changes in the pulsatile characteristics of LU release during periods of low level LU secretion in the rat estrous cycle. Mean blood LH levelsc LU interuise Pulse blood LHC % CVb (ng/ml whole intervals0 (ng/ml whole Cycle stage na (mean ± SEM) blood ± SEM) (mm ± SEM) blood ± SEM) Estrus ± ± ± ± 2.0 Diestrus ± ± ± ± 2.8 Diestrus ± ± ± ± 1.3 Proestrus morning ± ± ± ± 1.2 anumber of rats/group. For estrus, diestrus 1, and diestrus 2 this includes 8-10 rats bled in either the morning or afternoon of each cycle stage. CV: All stages P<0.O01 vs intra-assay variation cmn blood LH levels: Diestrus 1: P<0.001 vs estrus; P<0.05 vs diestrus 2, proestrus morning. Estrus: P<O.O1 vs proestrus morning; P<0.05 vs diestrus 2. dlh interpulse intervals: Estrus: P<0.001 vs diestrus 1, diestrus 2, proestrus morning. epulse t blood LH: Diestrus 1: P<0.001 vs estrus, diestrus 2, proestrus morning.

4 774 GALLO Proestrus (AM) Diestrus 2 (PM) a.. 2O I -j 0 a Estrus (AM) \/#{149}-/ #{149}\ [ Diestrus 2 (PM) z E Diestrus 2 (AM) Time of day Time of day FIG. 1. Examples of pulsatile LU release on proestrus morning, estrus, and diestrus 2 of the rat estrous cycle. the secretory patterns of ovarian steroids that take place during the 4-day estrous cycle and have been elegantly described by others (Kalra and Kalra, 1974; Butcher et al., 1974; Smith et al., 1975; Nequin et al., 1979). In general, blood levels of estradiol and progesterone are low during estrus and early diestrus 1. Estradiol secretion begins to increase during diestrus 1. These elevated levels are maintained until diestrus 2 when a second increase in estradiol secretion begins, continuing until late morning of proestrus. Progesterone secretion from the Dlestrus 1 (AM) Dlestrus 1 (PM) #{149} f\\ Al \j \.J I\ a.. & a C z I60 C I I 1400 \J Time of day FIG. 2. Examples of pulsatile LU release on diestrus 1 (i.e., metestrus) of the rat estrous cycle.

5 ESTROUS CYCLE PULSATILE LH RELEASE 775 corpus luteum also begins on diestrus 1. Peak blood levels are reached early on the morning of diestrus 2, and soon after in the morning the corpus luteum regresses and progesterone secretion declines. Progesterone levels then remain low through diestrus 2 and early proestrus. Rats with 5-day estrous cycles have also been found to have increased mean blood LH levels on diestrus 1, i.e., metestrus (Goodman, 1978). In the latter study the increased LH secretion on diestrus 1 was suggested to be due to the ineffectiveness of the low blood levels of estradiol and progesterone on estrus and early diestrus 1 in suppressing LU secretion. Moreover, the effect of rising blood levels of progesterone late on diestrus 1 acting together with low levels of estradiol on diestrus 1 was suggested to eventually terminate the LU increase by diestrus 2. Despite differences in ovarian steroid secretion in 5-day vs 4-day cycles, in cycles of either duration blood levels of both ovarian steroids are low during estrus and early diestrus 1, and progesterone secretion increases on diestrus 1 (Smith et al., 1975; Goodman, 1978; Nequin et al., 1979). Estradiol may contribute more in rats with 4-day than 5-day estrous cycles to terminating the increase in LU secretion seen on diestrus 1 since, according to most studies, blood estradiol levels increase on diestrus 1 in the 4-day cycle (Kalra and Kalra, 1974; Butcher et al., 1974; Smith et al., 1975) and therefore earlier than in the 5-day estrous cycle (Goodman, 1978). Thus the increased LU pulse amplitude occurring in the late morning and early afternoon of diestrus 1, and the increased frequency of pulsatile release, may be due to an inadequate ovarian steroid negative feedback signal occurring during estrus and earlier on diestrus 1. Thereafter, the increase in progesterone and estradiol secretion occurring from diestrus 1 through early diestrus 2 may serve to terminate the heightened LU pulse amplitude seen on diestrus 1. Future studies in our laboratory will examine in depth the influence of both ovarian steroids on LU pulses on diestrus 1, and in the process test the above hypothesis. An increase in either LURU binding at the anterior pituitary (Park et al., 1976; Savoy- Moore et al., 1980; Clayton et al., 1980), in the sensitivity of the pituitary to LURU (Aiyer et al., 1974; Cooper et al., 1975; Greeley et al., 1975), or in the readily releasable pool of pituitary LU (Pickering and Fink, 1979) do not appear to be involved in contributing to the increased LU pulse amplitude on diestrus 1. While it has been suggested that an increase in portal blood levels of LURU does not occur on diestrus 1 (Sarkar et al., 1976), the necessity for a 30 mm collection technique in this study may have prevented detection of a small but significant increase in LURU release occurring in a much shorter time. Pulsatile LU release on diestrus 1 may be critical for normal estradiol and progesterone secretion. Although Uchida et al. (1969) suggested that the rat corpus luteum does not require gonadotropin stimulation on diestrus 1 to release progesterone, using LU injections others have suggested that LI-I can increase progesterone output on diestrus 1 (Barraclough et al., 1971; Buffler and Roser, 1974; Boehm et al., 1980). Divergent results have also been reported for estradio]. Injections of LU on diestrus 1 decreased estradiol secretion (Boehm et al., 1980), and LU added in vitro to ovaries of diestrous 1 rats had no effect on estradiol secretion (Chatterton et al., 1969). Uowever, pentobarbital given on diestrus 1 delayed vaginal cornification and decreased the appearance of uterine ballooning, presumably by decreasing LU and thus estradiol secretion since injection of hcg restored normal vaginal and uterine events (Okamoto et al., 1972; Dominguez and Smith, 1974). Further studies on the interactions between pulsatile LU release and estradiol and progesterone secretion are planned. During the luteal stage of the human menstrual cycle (Santen and Bardin, 1973) or the midluteal phase of the sheep or cow estrous cycle (Foster et al., 1975; Baird, 1978; Rahe et al., 1980), high amplitude LU pulses occur although with a slower frequency than at other times in the cycle. This has been suggested to be due to the effects of progesterone on the LU secretory mechanism (Foster et al., 1975; Baird, 1978; Rebar and Yen, 1979; Goodman and Karsch, 1980; Rahe et al., 1980). In contrast, in the rat estrous cycle the corpus luteum is comparatively short-lived, and while high amplitude pulses do occur on diestrus 1 the frequency of pulsatile release is more rapid than that on estrus and similar to that on diestrus 2 or the morning of proestrus. Further experiments are necessary to clarify the reason (s) for this difference. It is also possible now to compare pulsatile LU release in the rat in the presence or absence

6 776 GALLO of ovarian function. The magnitude and frequency of pulsatile LU activity increases in the ovariectomized rat when compared with pulsatile release during periods of low level LU secretion in the estrous cycle. LU interpulse intervals shorten from mm to mm, and there is a 5-13-fold increase in pulse amplitude (Arendash and Gallo, 1978; Gallo, 1980b). This may be contrasted with postmenopausal women wherein blood LU levels increase solely as a result of an increase in LU pulse amplitude (Yen et al., 1972). In conclusion, the release of LH in the rat is pulsatile both during periods of low level LU release, as indicated in this study, as well as during the proestrous LU surge (Gallo, 1981). Changes in the characteristics of this release occur with different stages of the cycle and may well cause and/or reflect well known changes in ovarian steroid secretory patterns that occur during the estrous cycle. The fact that LU is released in a pulsatile manner rather than continuously may well be of importance to ovarian mechanisms responding to this gonadotropic hormone on all days of the cycle since prolonged, steady hormonal input to a target cell often results in a loss of responsiveness to that signal while pulsatile input does not (Belchetz et al., 1978; Catt et al., 1979). ACKNOWLEDGMENTS I want to thank Cindy Voytek and Geoffrey Lewis for their skilled technical assistance in these experiments, Mark Brownfield and Antonella Bona Gallo for their valuable suggestions concerning the LH radioimmunoassay, Annette Lowe for figures, and Bonnie Iwamasa and Jeanne Connolly for typing the manuscript. Thanks also to Dr. G. D. Niswender for antiovine LU #15, Dr. U. Papkoff for highly purified ovine LU for iodination, Dr. J. Garcia for goat antiserum against rabbit gamma globulin, and Dr. A. F. Parlow and the NIAMDD for the rat LH used as a reference preparation. REFERENCES Aiyer, M. 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