In vitro bioactive luteinizing hormone assay shows cyclical seasonal hormonal changes and response to luteinizing-hormone releasing hormone in the squirrel monkey (Saimiri boliviensis boliviensis).код для вставкиСкачать
American Journal of Primatology 14:167-175 (1988) In Vitro Bioactive Luteinizing Hormone Assay Shows Cyclical, Seasonal Hormonal Changes and Response to Luteinizing-Hormone Releasing Hormone in the Squirrel Monkey (Saimiri boliviensis boliviensis) RICHARD R. YEOMAN', SEZER AKSEL', JULIANNE M. HAZELTON', LAWRENCE E. WILLIAMSL.CHRISTIAN R. ABEE' Departments of 'Obstetrics an'd Gynecology and 'Comparative Medicine, University of South Alabama College of Medicine, Mobile, Alabama Studies on the reproductive mechanisms of the squirrel monkey have been hampered by inadequate measurements of luteinizing hormone (LH). The mouse interstitial cell bioassay, which measures testosterone production as the endpoint, was validated for use in the squirrel monkey by parallel responses of serum to LH standards and by in vivo responses to a n LHreleasing hormone (LHRH) analogue. The LH surge profile, as determined by daily blood sampling, was found to be of 1-2 days duration and comparable in amplitude to those of other primates. A 9.7day ovulatory cycle length was also calculated and was similar to previous estimates based on other hormonal and behavioral methods. A 150-fold decrease in basal LH was found in the nonbreeding season, as was a decreased LHRH response. This assay makes possible future studies on hypothalamic-ovarian mechanisms in this species. Key words: bioactive luteinizing hormone, estrous cycle, seasonal changes, releasing hormone challenge 1NTROI)UCTION Investigations into the reproductive cycle of the squirrel monkey have included attempts to improve the limited yield of offspring from a breeding colony [Lorenz et al, 1973; Kaplan, 19771, development of in vitro fertilization techniques [Dukelow, 1970; Dukelow, 19831, and studies of hormonally stimulated behavior [Wilson, 1977; Coe et al, 1981; Willams et al, 19861. Although the current market demand for use of this species in clinical studies [Langston et al, 1984; Pindak et al, 19851 has underscored the need to understand further and to optimize its reproductive mechanisms, relatively little is known. Until recently, gonadal steroids were the only hormones measurable in reproductive research. Measurements of gonadotropin levels have been limited to a preliminary report of a rise in luteinizing-hormone-like Received October 25, 1986; revision accepted September 20, 1987. Address reprint requests to Richard R. Yeoman, PhD, Department of Obstetrics-Gynecology, University of South Alabama, Room 336, Clinical Science Building, Mobile, AL 36688. 0 1988 Alan R. Liss, Inc. 168 / Yeoman et al. activity occurring at the time of the estradiol rise [Ghosh et al, 19821. This report used a radioimmunoassay with an antibody capable of measuring human luteinizing hormone (LH) but not previously validated in the squirrel monkey. The antibody for this assay is no longer available, and similar assays have failed t o show sensitivity to squirrel monkey LH in our hands. Irregularities with radioimmunoassay measurements also have been found in rhesus monkeys [Neill et al, 19771, suggesting that an in vitro bioassay is better suited for gonadotrophin measurements in certain primate species. The present study used the testosterone production of a mouse Leydig cell preparation to combine the sensitivity of a radioimmunoassay endpoint with the measurement of the biological activity of LH [Wickings et al, 1979; Van Damme et al, 19741. The bioassay was validated in the squirrel monkey by comparing dose responses to serum and standard LH preparations. Bioactive LH patterns were then quantitated in the breeding and nonbreeding seasons, and also after stimulation with an LH-releasing hormone analogue. MATERIALS AND METHODS Subjects were reproductively proven female squirrel monkeys (Saimiri boliviensis boliviensis) that were wild-caught and housed in laboratory breeding groups for several years. Breeding groups ranged from seven to nine females with one to two males per pen. Details of housing have been described previously [Diamond et al, 1984al. Animals were housed indoors but had limited access to the outdoors during the summer months. The building temperature was regulated at 22°C with a 15-h:9-h 1ight:dark schedule. Animals received a high-protein monkey chow and vitamin supplements daily. Animals were captured and manually restrained in the same sequence at each blood sampling time. One milliliter of blood was collected from the femoral vein within 3 minutes of capture. Serum was separated and stored frozen until assayed. The in vitro bioassay of LH is based upon the stimulation of testosterone biosynthesis by LH in dispersed mouse interstitial cells [Van Damme et al, 1974; Wickings et al, 19791. Four- to six-week-old male mice of the C3H inbred strain (Charles River, Wilmington, MA) were sacrificed with COa, and the testes were quickly removed on ice. Cells were physically dispersed in Medium 199 (Gibco, Grand Island, NY) containing 0.12 mM methyl isobutyl xanthene and 0.2% bovine serum albumin (Sigma, St. Louis). Cells were then gently agitated in a Dubnoff incubator for 1hour at 34°C. After centrifugation (150g x 10 min), resuspension of the pellet, and filtration (300 pm mesh), the solution was diluted to provide 2 x lo5 cells per 200 pl assay volume, as measured with hemocytometer. The NIH monkey pituitary standard, LER 1909-2,was used in a range of 0.5-10 ngltube. All serum samples were assayed in duplicate at 1.25, 2.5, and 5 pI per tube. The testosterone produced after a 4-hour incubation while agitated at 34°C was assayed as previously described [Wiebe et al, 19841, using antibody #250 from Dr. G.D. Niswender (Colorado State University, Fort Collins). The potency estimates for serum samples relative to the LH standard were calculated by parallel line analysis [Brownlee, 19651 adapted for computer by Dr. W.E. Bridson (Wisconsin Regional Primate Research Center, Madison). Sensitivity was 0.5 ng LER 1909-2/tube,and coefficients of variation were 9.6% within assays and 19.1% between assays. Series of multiple samples from individual animals were always assayed in a single assay to preserve the hormonal trends maximally. Parallelism was tested among LER 1909-2, the First International Standard for Bioassay and serial dilutions of squirrel monkey serum. Studies of cyclic LH patterns during the breeding season involved daily blood draws between 0900 and 1100 h for 19-to 20-day periods from late January to early BIOLH in the Squirrel Monkey / 169 March. Twenty-two animals were sampled in this aspect of the study. Samples for determination of nonbreeding LH levels were collected in a different group on an October morning in conjunction with the evaluation of pituitary responsiveness. Pituitary responsiveness was evaluated with an LH analogue (LHRH-A)(WY/ 40972, Wyeth, Philadelphia) that had been previously characterized [Corbin, 19821. Challenges in comparable groups during nonbreeding (n = 4) and breeding (n = 6) seasons involved administration of 0.1 pg LHRH-A in 0.2 ml saline IV preceded by baseline blood sampling and followed by further sampling at 20-minute intervals for 100 minutes. A second LHRH-A injection was given 60 minutes after the first injection in the nonbreeding season to evaluate a priming effect in the pituitary. Progesterone was also measured in breeding animals challenged to aid in cycle stage determination. The radioimmunoassay involved extraction and binding with antibody kindly provided by Dr. V. Mahesh (Medical College of Georgia, Augusta). Details have been reported previously [Diamond et al, 1984al. RESULTS Results from the mouse interstitial cell in vitro bioassay for LH showed that a serial dilution of squirrel monkey serum was linear and parallel to the monkey standard, LER 1909-2 (Fig. 1).Dilutions that contained as little as 1.25 pl serum were able to stimulate testosterone production above baseline. The First International Standard for Bioassay also stimulated testosterone production in a parallel manner at a potency of 35.6 pIU/ng LER 1909-2. Sera from cycling monkeys were analyzed for LH, and increases of greater than 2 SD of preceding day values were detected in normal cycles prior to pregnancy and after abortion. Incidental observations of matings corroborated the onset and duration of the annual breeding season. Animals suspected of being pregnant, as judged by sustained elevated bioactive levels, were not included in these analyses. The first day of LH rise was marked and consistently greater than the following day and thus was termed the peak. The data were normalized to the peak of the LH release and compared from 4 days before peak through 3 days after. The mean peak LH serum levels for the 17 LH surges encountered was 2,865.6 425.7 ng/ml (mean k SEMI, as compared to 487.4 & 31.9 ng/ml measured on the preceding day (Fig. 2). Thus, the levels rose dramatically to a peak, yet fell slowly so that values were still above baseline 24 hours later. In ten animals, two surges were detected in each animal during the 20-day blood draw, and a cycle period of 9.7 & 0.4 days was determined. Stimulation of an LH rise with LHRH-A in the nonbreeding season produced a highly significant (P < .Ol) elevation in serum LH at 20 minutes postinjection (Fig. 3). The response to a second injection at 60 minutes while the levels were still elevated was negligible, which is consistent with the effects of a long-acting agonist. This stimulation trial was done in October, before the breeding season, as suggested by the low basal levels of LH. When an equivalent LHRH-A injection was repeated during the breeding season in a comparable group (n = 6), a much greater response was observed, and the baseline levels were more than two orders of magnitude greater (Table I). The estrous cycle phase during breeding season testing was random, but an analysis of daily LH and progesterone levels in serum collected before the LHRH-A challenge showed that four of the six animals were tested 8 to 10 days after their last LH surge and had 219.5 & 15.5 ng progesterone per ml serum. The other two animals had an LH surge 2 days previously with 173.5 i-7.5 ng progesterone per ml. On the day of the challenge, preinjection LH levels were comparable in all animals tested (1,790.2 & 132.6 ng/ml); however, the early luteal animals responded with a larger increase in LH (1,100 100 ngiml) than did the late luteal * * 170 I Yeoman et al. 1000- 1 Y LER 1909-2 800 - 9 '7 c 2 600- C e 0 B I c. 01 2 400c3 0 200 - 0.5 OL 1 0.5 1 2 5 10 20 ng or pl or pIU 50 100 200 Fig. 1. Dose-response curves from the monkey pituitary extract LER 1909-2(NIH),female monkey serum, and 1st International Standard for Bioassay (MCR 70145). Days from LH surge Fig. 2. LH surge profile from 17 cycles normalized to the day of LH surge (mean f SEM). BIOLH in the Squirrel Monkey / 171 LHRH-A LHRH-A 0.1ug 0.1 ug 50 - I 1, 40 - B 3rn 30- 20 - 10 0 , 100 -20 0 20 40 60 Minutes After First LHRH-A 80 Fig. 3. LH responses from four individual animals administered 0.1 pg LHRH-A IV at time 0 and 60 minutes later during the nonbreeding season. TABLE I. Comparison of Bioactive LH Levels (nglml)at 0 and 20 Minutes After 0.1 pg LHRH-A Administration Intravenously in the Nonbreeding Season to One Group and in the Breeding Season to a Comparable Group t = O Nonbreeding (n = 4) Breeding + OBa 1,790.2 + 132.6 11.4 t = 20 Net increase 51.8 f 8.2 40.4 2,572.5 f 259.9 782.3 (n = 6) "Mean SEM. animals (598.5 91.4 ng/ml). All breeding season animals released much more LH when stimulated than did animals during the nonbreeding season. DISCUSSION In the study reported here, the in vitro LH bioassay with mouse interstitial cells was validated for use in the squirrel monkey by obtaining consistent parallel responses with recognized pituitary and urinary LH standards. The specificityof the assay was further established by stimulating the pituitary with LHRH-A and observing an appropriate rise in serum LH. Previous studies have shown no interfer- 172 I Yeoman et al. ence from serum levels of follicle-stimulatinghormone in the interstitial cell response [Van Damme et al, 19741. A particular advantage of this assay for use with squirrel monkeys, whose body weight is only 500-800 g, is the small sample volume (20 p1) needed to prepare the dilution curve. Application of this assay to serum samples collected during the breeding season showed short-duration LH surges occurring frequently in cyclic patterns. These daily samples, although limited in precision by the collection frequency, revealed that LH was markedly elevated for only 1 day and decreased to less than one-half the peak level on the 2nd day. This pattern contrasts with the 2- to 3-day marked elevation detected with radioimmunoassay in this species [Ghosh et al, 19821. This difference may represent a more dramatic cyclic pattern in biological LH activity than in immunological activity. A change in the bio- to immuno-activity ratio during the ovarian cycle has been reported previously in rhesus monkeys [Neill et al, 19771. Another difference in these studies was housing conditions, since our samples were taken from animals in breeding pens, whereas the previous study used females housed without males. The sample-collecting technique may be another source of difference, since our study collected 1 ml of blood per day with manual restraint, and the previous study collected 2 ml blood per day from Nebutal-anesthetized animals. In another New World species, the marmoset, marked LH elevations of 1day duration have been reported [Harlow et al, 19831, similar to elevations observed in certain Old World species, rhesus [Weick et al, 19731 and stumptail macaques [Wilks, 19771. The magnitude of the LH surge, as sampled with daily morning blood draws, was remarkable in that the change from the previous day was comparable percentagewise to other species and similar in concentration to human ovulatory LH levels measured in the same assay [Abuzeid et al, 19861. Other peptide hormones in the squirrel monkey such as growth hormone, thyroid-stimulating hormone, and adrenal corticotrophic hormone also have been found to be in concentrations comparable to other primates [Kaack et al, 1980; Brown et al, 1970; Coe et al, 19781. The conventional range of LH levels in squirrel monkeys is notable when considering the extremely high levels of estrogen and progesterone that have been measured in this species [Diamond et al, 1984a; Chrousos et al, 19821 and may relate t o a defective receptor mechanism [Diamond et al, 1984133. Observations of ovulatory cycle length also could be determined in some animals from the LH patterns, and our calculation of 9.7 days was comparable to previous estimates based on ovarian steroids, estrous behavior, and vaginal cytology [Diamond et al, 1984a; Wolf et al, 1977; Wilson, 1977; Castellanos & McCombs, 19681. A comparison of cycle lengths reveals that the squirrel monkey has the shortest ovulatory cycle of all monkeys reported as a result of its brief follicular phase [Pohl & Hotchkiss, 19831. The annual occurrence of a nonbreeding, noncyclic state in squirrel monkeys housed indoors is reflected in LH measurements. LH levels were 150-foldless at this time of the year than during the breeding season. Seasonal reproductive mechanisms likely involve dynamic interactions within the entire hypothalamic-pituitaryovarian axis, with the response of each component facilitated by prior stimulation. Thus, it is difficult to identify the cause of reduced hormonal activity during this nonbreeding season. In our colony of squirrel monkeys, serum levels of estrogen and progesterone are markedly reduced in the nonbreeding season [Diamond et al, 1984al. Other studies have shown that ovarian sensitivity to an ovulation-inducing regimen of exogenous gonadotropins decreases during the summer nonbreeding season [Harrison & Dukelow, 19731. This decreased sensitivity was explained as possibly due to a decreased endogenous gonadotropin background. The expected BIOLH in the Squirrel Monkey / 173 decrease in LH during the nonbreeding season was confirmed in the present study. The decrease in serum LH levels observed with the change from breeding to nonbreeding status is comparable to but much greater than the twofold decrease observed in nonbreeding rhesus monkeys [Walker et al, 19841. Although marmosets are New World monkeys, as are squirrel monkeys, they are not seasonal breeders [Hearn & Lunn, 19751. However, within a breeding group of marmosets, only the dominant male and female breed. These dominant animals have measurable levels of LH, while subordinate animals have undetectable levels [Abbott et al, 19811. Follicle-stimulating hormone is another pituitary gonadotropin with major importance in ovarian stimulation, yet to date it has not been measured in New World monkeys because of lack of an appropriate assay. Folliclestimulating hormone has been found to decrease significantly in rhesus monkeys during the nonbreeding season [Walker et al, 19841, suggesting that this hormone also has an involvement in seasonal reproductive mechanisms. The seasonal change in serum LH of the squirrel monkey may involve a diminished pituitary responsiveness to releasing hormones in the nonbreeding season. The present study has shown that equivalent amounts of LHRH-A induce much less LH release in the nonbreeding season than in the breeding season. This decrease in pituitary responsiveness may be due to markedly reduced background levels of estrogen, since estrogen has been shown to enhance gonadotroph responses t o luteinizing hormone-releasing hormone [Higuchi & Kawakami, 19821. A more important mechanism responsible for decreased serum LH in the nonbreeding season may be a decrease in the frequency of luteinizing hormone-releasing hormone pulses from the hypothalamus. Seasonally breeding sheep exhibit a decrease in the number of luteinizing hormone pulses during the nonbreeding season [Scaramuzzi & Baird, 19771, and LH pulses are highly associated with pulses of releasing hormone [Clarke & Cummins, 19821. Thus, the dramatic contrast in baseline LH levels observed between breeding and nonbreeding periods may suggest a hypothalamic-pituitary mechanism of seasonal reproductive change in this species. A similar conclusion has been reached concerning seasonality in other mammals [Clarke, 19811 and is nicely demonstrated in ovariectomized sheep, which have a seasonal increase in LH while receiving a constant level of estradiol treatment [Legan et al, 19771. CONCLUSIONS 1.The mouse interstitial cell in vitro bioassay for LH, which measures testosterone production as the endpoint, was validated for squirrel monkeys by parallel responses of serum and LH standards. Appropriate serum LH increases in animals in response to stimulation with an LHRH analogue were also observed. 2. The LH surge profile determined by daily blood sampling was found to be of 1-2 days duration and comparable in amplitude t o those of other primates. 3. A 9.7-day ovulatory cycle length was also calculated and was similar to previous estimates using other hormonal and behavioral methods. 4.A 100-folddecrease in basal LH was found in the nonbreeding season. 5. This assay makes possible future studies on hypothalamic-ovarian mechanisms in this species. ACKNOWLEDGMENTS These studies were supported by NIH grant RR01254. The authors wish to thank Dr. William E. Bridson of the Wisconsin Regional Primate Research Center, University of Wisconsin, and Dr. David R. Mann of Morehouse School of Medicine 174 I Yeoman et al. for technical guidance on this assay. We also wish to thank the Keproductive Endocrinology Laboratory staff, the Primate Research Laboratory staff, and Mr. Robert Ricker for their excellent technical assistance. The proficient secretarial assistance of Mrs. Mary Lou Russell and Mrs. Melissa Gillis is very much appreciated. We are indebted to Dr. G. Bialy, NIH, and Dr. P.L. Storring, WHOMBSB, for standards; to Dr. G. Niswender, Colorado State University, for testosterone antibody; to Dr. V. Mahesh, Medical College of Georgia, Augusta, for progesterone antibody; and to Dr. A . Corbin, Wyeth Laboratories, for the LHRH analogue. All experiments presented in this manuscript were performed in accordance with standards established by the Animal Welfare Act and by the document entitled “Guide for the Humane Care and Use of Laboratory Animals.” REFERENCES Abbott, D.H.; McNeilly, AS.; Lunn, A.F.; Hulme, M.J.; Burden, F.J. Inhibition of ovarian function in subordinate female marmoset monkeys (Callithrix jacchus Jac chus) JOURNAL O F REPRODUCTION AND FERTILITY 63:335-345,1981. Abuzeid, M.I.; Yeoman, R.R.; Aksel, S. The role of luteinizing hormone bioactivity in HMB-HCG induced cycles. 33rd Annual Meeting, SOCIETY FOR GYNECOLOGICAL INVESTIGATION, Abstract #134, 1986. Brown, G.M.; Grota, L.J.; Penny, D.P.; Reichlin, S. Pituitary adrenal function in the squirrel monkey. ENDOCRINOLOGY 86:519-529, 1970. 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