Reduced Luteinizing Hormone Release by Synthetic Luteinizing Hormone-Releasing Hormone (LHRH) in Postpartum Lactating Rats K. H, LU,1 H. T. CHEN, L. GRANDISON, H. H. HUANG, AND J. MEITES2-3 Department of Physiology, Michigan State University, East Lansing, Michigan 48824 ABSTRACT. The ability of the pituitary to release LH in response to synthetic LHRH was tested in lactating female rats on days 7 and 17 post partum, and compared with that of normal cycling female rats pn diestrous day 2 (controls). Three consecutive injections of LHRH (100 ng/100 g BW, sc), each 50 min apart, were given to each rat and sequential bipod samples were collected at 25-min intervals by cardiac puncture under light ether anesthesia. In all 3 groups, the 2nd and 3rd injections of LHRH prpfluced much greater increases in serum concentratipns of LH than the 1st injection. However, this self-priming effect of LHRH on LH response was markedly attenuated in the postpartum lactating rats (PPL), compared with the normal cycling female rats on diestrous day 2. Three consecutive injections of LHRH produced significantly less LH release in PPL rats than in normal cycling female rats on diestrous day 2. Both day 7 and day 17

PPL rats released equally small amounts of LH in response to LHRH administration. The total amount of LH released by anterior pituitaries (APs) during a 5 h incubation in medium199, from day 7 or day 17 in PPL rats, was significantly less than that released by the APs from normal cycling female rats on diestrous day 2. APs from day 7 and day 17 PPL rats also released less LH in vitro in response to LHRH (50 ng) stimulation than APs from normal cycling female rats. When APs from normal cycling female rats on diestrous day 2 were incubated with LHRH, the increments in LH release were greater at the end of the 2nd and 3rd h than after the 1st h of incubation. However, such increments in LH release were relatively small when APs from day 7 or day 17 PPL rats were similarly incubated with LHRH. (Endocrinology 98: 1235, 1976)

T N POSTPARTUM rats, suckling by pups X stimulates pituitary prolactin release (1) and maintains lactation but greatly inhibits pituitary release of gonadotropins (2,3). Estrous cycles usually are not exhibited in female rats during postpartum lactation. Results, from a recent study in our laboratory showed that daily serum concentrations of both LH and FSH in postpartum rats were consistently low throughout the entire period of lactation (4). Pituitary release of gonadotropins returned to normal, and

estrous cycles resumed within a few days after the pups were removed on day 22 post partum. It was reported previously that postpartum lactating rats had less LHRH activity in the hypothalamus and less LH content in the pituitary than did non-lactating female rats (5), suggesting that suckling inhibits LHRH secretion by the hypothalamus and results in a reduction in pituitary LH secretion. Reduced gonadotropin responsiveness to synthetic LHRH has been reported in postpartum lactating ewes (6,7) and women (8). However, it was not clearly indicated whether decreased LH release following synthetic LHRH administration was due to decreased ability of the pituitary to release LH or to other in vivo influences on the pituitary. The present study was undertaken to compare the ability of the pituitaries of postpartum lactating rats (PPL) and normal cycling female rats on diestrous day 2 to release LH in response to synthetic LHRH both in vivo and in vitro.

Repeived August 11, 1975. ' Researph Associate during tenure of this work. Present address: Department of Reproductive Medipine, Schppl of Medicine, University of California, San Diegp, La Jolla, CA 92093. ? Aided in part by NIH research grants AM 04784 frpni the National Institute pf Arthritis, Metabplism, and Digestive Diseases, and CA 10771 from the National Cancer Institute. 3 Published with the approval of the Michigan Agricultural Experiment Station as Journal Article No. 7359.

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LU ET AL.

1236 Materials and Methods

Animals. Virgin female rats of the SpragueDawley strain were obtained from Spartan Research Animals, Inc., Haslett, MI. At approximately 3 months of age the rats were caged together with mature male rats of the same strain. After mating was confirmed by the presence of sperm in the vagina, the female rats were caged individually in transparent cages throughout pregnancy and postpartum lactation. In all experiments, the day following parturition was designated as day 1 post partum. Beginning on day 2 post partum the litters were uniformly adjusted to 8 pups per mother. The rats were housed in a temperature-controlled (25 ± 1 C) and artificially illuminated (fluorescent lights on from 0500 to 1900 h daily) animal room, and were fed a diet of Purina Rat Chow (Ralston Purina Co., St. Louis, Mo.) and tap water ad libitum. A group of 3 to 4-month-old, virgin, normal cycling female rats of the same strain were used for comparison with the PPL rats. Daily vaginal smears were taken from these rats and only animals exhibiting 2 consecutive 4-day estrous cycles were used. In vivo tests with synthetic LHRH. The ability of the pituitary to release LH in response to synthetic LHRH was tested in PPL rats on days 7 and 17 post partum, and in normal cycling female rats on diestrous day 2 (controls). LHRH (Abbott) was dissolved in 0.87% NaCl and a dose of 100 ng/lOOg BW in 0.1 ml of vehicle was injected SC. Three consecutive injections of LHRH, each 50 min apart, were given to each rat, and sequential blood samples of 0.3-0.4 ml each were collected by cardiac puncture under light ether anesthesia prior to the 1st injection and at 25 and 50 min after each LHRH injection. Blood samples were stored in a refrigerator at 4 C overnight before centrifuging at 4,000 x g for 15 min in a Sorvall refrigerated centrifuge. The serum was stored in a vial, capped with parafilm, and kept frozen at — 20 C until assayed for LH activity. During the LHRH tests all mother rats remained with their pups in individual cages. In vitro incubations of pituitary with and without synthetic LHRH. Anterior pituitaries (APs) from lactating rats on days 7 and 17 post partum and from normal cycling female rats on diestrous day 2 (controls) were used for in vitro incuba-

Endo . 1976 Vol 98 < No 5

tions. The rats were decapitated by guillotine at about 1000 h, and the APs were quickly removed, hemisected, and weighed. Each AP half was placed in a 5-ml culture tube (Kimble Products, Owen, IL) containing 2 ml of medium199 at a pH of 7.4. Medium-199 for the incubation was prepared by dissolving 1.10 g of medium-199 in powder form (Difco Labs., Detroit, MI) and 180 ing of NaHCO 3 (J. T. Baker Chemical Co., Phillisburg, NJ) in 100 ml of deionized distilled water. Incubations were carried out in a Dubnoff metabolic shaking incubator (Precision Scientific Co., Chicago, IL), 60 cycles/min, under constant gassing with 95% O 2 -5% CO2 at 37 ± 0.5 C. After 30 min pre-incubation, the medium was removed and replaced with 2.0 ml of fresh medium-199 containing 50 ng of synthetic LHRH (Abbott) or medium-199 alone. In a preliminary trial, we found that 50 ng of LHRH gave a near maximal LH response in this system. One AP half from each rat was incubated in medium-199 alone, while the other half was incubated with LHRH in medium-199 for5 h. Seven tubes were used for each treatment. During the course of incubation an aliquot of 200 )u,l of incubated medium was removed from each tube at hourly intervals, and an equal amount of fresh medium-199 containing 5.0 ng of LHRH or medium-199 alone was replaced. The 200 ix\ of incubated medium was immediately diluted to 1.0 ml with 0.1% gelatin in phosphate buffer saline (PBS), and stored. LH radioimmunoassay (RIA). LH concentrations in individual serum samples and in incubation medium were assayed by a double-antibody RIA using the ovine-ovine system (9). Since LH concentrations in the serum were expected to be high in samples collected after LHRH administration, each serum sample was diluted (1:10) with 0.1% gelatin in PBS before it was assayed by RIA. The 0.1% gelatin in PBS was used as the diluent in the LH RIA system. The incubation medium also was diluted with 0.1% gelatin in PBS to an appropriate concentration before assay. A small pilot assay of LH was first done before all samples were assayed. Concentrations of LH in the serum and in the incubation medium were expressed in terms of the reference standard, NIAMDD rat LH-RP-1. The significance of differences between the PPL rats and normal cycling female rats was determined by Student's t test.

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1237

LHRH IN POSTPARTUM LACTATING RATS

LHRH increased serum LH concentration from 11.2 ± 0.7 to 181 ± 26 ng/ml by 25 min. 1. Pituitary release of LH in response to Serum LH dropped slightly to 130 ± 18 consecutive injections of LHRH. ng/ml by the end of the next 25 min. In day 7 The average BW (±SE) of day 7 and day PPL rats LHRH produced an increase in 17 PPL rats and normal cycling female rats serum LH from 1.7 ± 0.2 to 164 ± 27 ng/ml on diestrous day 2 (controls) were 269 ± 16, by 25 min. Serum LH decreased to 80 ± 28 310 ± 7, and 263 ± 4 g, respectively. The ng/ml by 50 min. In day 17 PPL rats serum data in Figs. 1 and 1A show the serum con- LH increased from 2.5 ±0.2 to 111 ± 18 centrations of LH before and after each of ng/ml by 25 min, and decreased to 64 ± 14 the 3 consecutive injections of LHRH. In ng/ml by 50 min after a single injection of the controls (Fig. 1A), a single injection of LHRH. Thus, the increase in serum LH Results

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FIG. 1. Serum concentrations of LH before and after LHRH injections in lactating female rats on day 7 ( • • ) and day 17 • ) post partum and in (• normal cycling female rats on diestrous day 2 (A A). Three consecutive injections of LHRH (100 ng/100 g BW), each 50 min apart, were given to each rat as shown by the anows. Figure 1A shows the serum concentrations of LH before and after a single injection of LHRH. Each point on the graph represents the mean serum concentration of LH from 5 rats, and the vertical lines through each point represent the standard error (SE) of the mean.

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LU ET AL.

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Erido • 1976 Vol' 98 • No 5

injections of LHRH in these rats were not statistically different from each otheTj biit were significantly less than those in normal cycling female rats (P < 0.01). Thiis> each of the 3 consecutive injections of LHRH produced significantly less LH release in day 7 and 17 PPL rats than in normal female rats on diestrous day 2.

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in response to a single injection of LHRH in day 17 PPL rats was significantly less than that in normal cycling female rats (F < 0.05), whereas the increase in serum LH in day 7 PPL rats was not statistically different from that in normal cycling female rats on diestrous day 2. The data in Fig. 1 show the changes in serum concentrations of LH after the 2nd and 3rd injections of LHRH. In normal cycling female rats, serum LH increased from 130 ± 18 to 1,921 ± 100 ng/ml by 25 min after the 2nd injection of LHRH> and decreased slightly to 1,379 ± 66 ng/ml by the end of the next 25 min. Serum LH was further increased to 2,887 ± 131 ng/ml by 25 min after the 3rd injection of LHRH. The 2nd and 3rd injections of LHRH also produced large increases in serum LH in both day 7 and day 17 PPL rats. The increments in serum levels of LH after the 2nd and 3rd

2. Spontaneous release of LH by APs incubated in vitro. The wet weights of AP halves incubated in medium-199 alone were 5.02 ± 0.30, 4.72 ± 0.16, and 4.83 ± 0.14 mg for days 7 and 17 PPL rats, and for normal cycling female rats, respectively. These weights were similar to AP halves incubated with LHRH in medium-199. The data in Fig. 2 show that the APs from normal cycling female rats released 350 ± 85ngLH7 mg AP into the incubation medium, whereas the APs from days 7 and 17 PPL rats released only 151 ± 21 and 113 ± 19 ng LH/ mg AP, respectively, after 1 h incubation. During a 5 h incubation^ the total amounts of LH released by the APs from days 7 and 17 PPL rats were significantly less than those released by the APs from normal cycling female rats (P < 0.05). The total amount of LH released by the APs from day 17 PPL rats was only about 40-45% of that released by APs from normal cycling female rats on diestrous day 2. The amount of LH released by the APs from day 17 PPL rats was consistently lower than that from the APs of day 7 PPL rats during a 5 h incubation. 3. Pituitary release of LH in response to LHRH in vitro. The data in Fig. 3 show that the APs from normal cycling female rats released 850 ± 202 ng LH/mg AP, whereas the APs from day 7 PPL rats released only 493 ± 62 ng LH/mg AP after 1 h incubation with LHRH. Interestingly, during the first hour of incubation, the amount of LH released by the APs from day 17 PPL rats was close to that released by the APs of normal cycling female rats (679 ± 31 vs 850 ± 202 ng LH/mg AP). However, beginning at the second hour of incubation and continuing to 5 hj the amounts of LH released

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1239

LHRH IN POSTPARTUM LACTATING RATS by the APs from days 7 and 17 PPL rats were significantly less than those released by the APs of normal cycling female rats (P < 0.05). The total amount of LH released by the APs from day 7 PPL rats was only about 45-50% of that of the APs from normal cycling female rats. The amount of LH released by the APs from day 17 PPL rats was not statistically different from that of day 7 PPL rats. Since one half AP was incubated with LHRH in medium-199 while the other half was incubated in medium-199 alone, the difference in LH release between these 2 AP halves is believed to represent the net increase in LH release in response to LHRH stimulation. The data show that the net increase in LH release in response to LHRH by the APs from PPL rats was significantly less than that by the APs from normal cycling female rats on diestrous day 2 (P < 0.05). Discussion The present study demonstrates that the APs of PPL rats release less LH in vivo and in vitro in response to LHRH stimulation than the APs of normal cycling female rats during diestrus, when the response to LHRH was reported to be lower than at any other phase of the estrous cycle (10). PPL rats previously were reported to have significantly less LHRH in the hypothalamus and significantly less LH in the AP than non-lactating female rats (5), suggesting that the decreased LH release in PPL rats (4) may be due in part to diminished LHRH release from the hypothalamus and to a smaller pool of LH available for release in response to LHRH stimulation. Reduced LH release by LHRH administration has been demonstrated in lactating ewes (7) and lactating women (8), and also may be due mainly to a decreased pool of available LH in the AP. Pituitary LH on the day of parturition in ewes was reported to be only about 8% of that in non-lactating ewes, and injections of LHRH failed to promote a large release of LH until 40 days after parturition (7). Similarly, women during regular breast

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feeding showed less LH release in response to LHRH during the first 6 weeks post partum than women who began intermittent breast feeding after the 3rd postpartum week (8). The graded increases in LH release observed with consecutive injections of LHRH are in agreement with other recent reports (11,12). The 2nd and 3rd injections of LHRH produced much greater increases in serum concentrations of LH than the 1st injection of LHRH in both PPL and normal cycling female rats on diestrous day 2. Also, the difference in LH release between the PPL and normal cycling female rats after the 1st injection of LHRH was relatively small (Fig. 1A), whereas subsequent injections produced larger and more significant differences in LH release (Fig. 1). These observations are believed to indicate that the initial stimulation by LHRH increased the amount of releasable LH in the AP which then showed a greater LH response to subsequent LHRH administration (13). The in vitro increments in LH release by the APs

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LU ET AL.

from normal cycling female rats in response to constant stimulation by LHRH were greater by the 2nd and 3rd h than at the end of the 1st h of incubation, but these increments were relatively small when the APs from PPL rats were similarly incubated with LHRH in vitro. This again could be due mainly to a lower amount of releasable LH present in the AP of the PPL rats. Acknowledgments The authors wish to thank the Abbott Labs., N. Chicago, IL, for the gift of synthetic LHRH (A-41070). We also express our appreciation to Dr. L. E. Reichert, Jr., Emory University, Atlanta, Ga., for the generous supply of purified ovine LH (LER-1056-C2), and to Dr. G. D. Niswender of Colorado State University,

Fort Collins, CO, for the ovine LH antiserum (no. 15).

References 1. Amenomori, Y., C. L. Chen, and J. Meites, Endocrinology 86: 506, 1970.

2. Hammons, J.-A., M. Velasco, and I. Rothchild, Endocrinology 92: 206, 1973. 3. Ford, J. J., and R. M. Melampy, Endocrinology 93: 540, 1973. 4. Lu, K. H., H. T. Chen, H. H. Huang, L. Grandison, S. Marshall, and J. Meites, J E7idocrinol 68: 241, 1976. 5. Minaguchi, H., and J. Meites, Endocrinology 80: 603, 1967. 6. Jequier, A. M., C. Vanthuyne, and H. S. Jacobs, J Endocrinol 59: XIV, 1973. 7. Jenkin, C , and R. B. Heap, J Endocrinol 61: XII, 1974. 8. Tolis, C , H. Guyda, R. Pillorger, and H. G. Friesen, Endocrine Res Commun 1: 293, 1974. 9. Niswender, G. D., A. R. Midgley, Jr., S. E. Monroe, and L. E. Reichert, Jr., Proc Soc Exp Biol Med 128: 807, 1968. 10. Cooper, K. J., C. P. Fawcett, and S. M. McCann, Endocrinology 95: 1293, 1974.

11. Aiyer, M. S., S. A. Chiappa, and G. Fink, J Endocrinol 62: 573, 1974. 12. Castro-Vazquez, A., and S. M. McCann, Endocrinology 97: 13, 1975. 13. Lu, K. H., H. T. Chen, and J. Meites, Physiologist 18: 299, 1975.

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Reduced luteinizing hormone release by synthetic luteinizing hormone-releasing hormone (LHRH) in postpartum lactating rats.

Reduced Luteinizing Hormone Release by Synthetic Luteinizing Hormone-Releasing Hormone (LHRH) in Postpartum Lactating Rats K. H, LU,1 H. T. CHEN, L. G...
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