Serum Levels of FSH and LH During the Menstrual Cycle of the Chimpanzee

BOLOGY OF RPRODUCTO 4, -5 (97) Serum Levels of FSH and LH During the Menstrual Cycle of the Chimpanzee B.. HOWLAD, C. FAMA,3 AD T. M. BUTLR Departnzents of Oral Biology arid Physiology, The University of Manitoba, Winnipeg 3, Ca,,acla, and the 65 7st Aeroinedical Research Laboratory (AFSC), Hollonian Air Force Base, ew Mexico 8833 Received December 8, 97 The serum levels of FSH and LH were measured throughout the menstrual cycle of four chimpanzees by means of human gonadotropin radioimmunoassays. A midcycle LH peak was observed during each cycle. Two periods of elevated FSH levels were noted; one early in the cycle commencing near the time of onset of menstruation; the second at or near the time of the midcycle LH peak. n one animal that became pregnant, a second rise in the levels of LH, presumably due to cross reactivity in the assay by chorionic gonadotropin, occurred days after the first LH peak. The data suggest that the fluctuations in FSH and LH during the menstrual cycle and early pregnancy are similar in chimpanzees and women. Subhuman primates, particularly the chimpanzee, should provide excellent models for studying problems in human reproductive physiology. The advantages of investigating human-related problems in an experimental species with a similar reproductive cycle are obvious. Unfortunately there is limited information regarding urinary levels of gonadotropins in subhuman primates, and until recently no data on blood levels were available. McArthur and Perley (969) reported levels of general gonadotropin in the urine of the rhesus monkey, stumptail monkey, baboon, and chimpanzee. Only in the chimpanzee was it possible to use specific assay techniques for quantitating FSH and LH throughout the menstrual cycle. n the human, radioimmunoassay methods have made it possible to measure blood levels of FSH and LH (Jaffe and Midgley, 969). Recently this technique has The animals used in this study were handled in accordance with the Guide for Laboratory Animal Facilities and Care established by the ational Academy of Science-ational Research Council. Address reprint requests: Dr. B.. Howland, Department of Oral Biology, University of Manitoba, Winnipeg 3, Manitoba, Canada. M. R. C. Canada Scholar. been extended to the rhesus monkey (Kirton et a!., 97; Monroe ci a!., 97) and the baboon (Stevens ci a!., 97) making it possible to measure blood levels of LH in these species. Radioimmunoassay methods for estimating gonadotropins in other primate species have not been reported. The present report describes the changes in serum FSH and LH levels during the menstrual cycle in chimpanzees as measured by human gonadotropin radioimmunoassay methods. One animal became pregnant during the menstrual cycle under study providing the opportunity to determine changes in gonadotropin levels at the inception of pregnancy. MATRALS AD MTHODS xperimental A,,imals. Four adult female chimpanzees from the breeding colony at the 657 st Aeromedical Research Laboratory were studied. Menstrual cycle histories were available on all animals. Changes in sex skin swelling and periods of menstrual bleeding were recorded for each animal. Venous blood samples were collected daily during the midportion of the menstrual cycle and at 2-3-day intervals at other stages. The blood was allowed to clot and the serum was frozen at - 2C until assayed. Gonadotropi, Assays. All FSH values and the LH values of two cycles were measured using human

2 HOWLAD, FAMA, AD BUTLR TABL FFCT OF VOLUM OF CHMPAZ SRUM ASSAYD Volume of pooled serum assayed (ml) O FSH AD LH MASURD Hormone concentration Pool Pool 2 Pool 3 Pool 4 FSH LW LW LHb LW.5 32.6.4 2. 22. 4.. 32.4 9. 23.3 9. 4..5 - - 24. - -.2 32.5 9.9 22.2 8.5 5..3 3.9 3.3-9.5 4.5.4 28.3.5 - - -.5 28.4.8 - - - #{76}Concentration in g LR-97/l ml. Concentration in mlu 2nd RP-HMG/ml. gonadotropin radioimmunoassays previously described (Faiman and Ryan, l967a, b; Ryan and Faiman, 968). The human pituitary preparation LR- 97 was used as the assay standard. The LH values in the other two cycles were determined by a similar double-antibody method, using an anti-hcg serum and 25-labeled human LH (LR 822-2) as the trace (Howland, unpublished). For these assays the Second nternational Reference Preparation of Human Menopausal Gonadotropin (2nd RP-HMG) was employed as the assay standard. The antibody showed some cross reactivity with purified human FSH. However, when the same series of samples was assayed using this method and the above-mentioned specific method, the relative changes in LH from sample to sample were in close agreement. Most values represent the mean of duplicate determinations. RSULTS The chimpanzee serum behaved in the human gonadotropin radioimmunoassays in a manner identical with that of human serum. Dose-response curves that paralleled the human standards were obtained in each of the methods used. Reliable estimates of either FSH or LH concentration were obtained using volumes of serum ranging from.5 to.5 ml (Table ). The gonadotropin levels and sex skin changes are shown in Fig.. A midcycle elevation in serum LH concentrations lasting for -3 days was observed in each animal. The timing of the LH peak varied from day of the cycle in animal 247 to day 32 in animal 23. Two periods of elevated FSH values were noted, one early in the cycle with the rise usually beginning with the onset of menstruation and the second at or near the time of the LH peak. n three of the four cycles, the midcycle LH and FSH peaks were coincident. The cycle length of animal 247 was only 22 days or some 8 days shorter than usual. Animal 23 had a 43-day cycle which was about 6 days longer than usual. Animal 25 showed a normal cycle length and the cycle for animal 48 which previously had been about 3 days in length terminated in pregnancy. The length of the luteal phase of the cycle (LH peak to menses) was much less variable than the length of the entire cycle and ranged from 2-4 days. The levels of an LH-like hormone began to rise days following the LH peak in animal 48 and appeared to be still rising at the time of the last sampling 6 days later. Dilution of one of the samples during this second elevation in immunoreactive LH showed nonparallelism with the assay standard (Table 2). During this period FSH levels remained low. o subsequent menstruation occurred and the animal was later found to be pregnant. Fluctuations in sex skin swelling continued during early pregnancy; however, this is not unusual in the chimpanzee. There appeared to be no relationship between the onset of sex skin swelling and the time of the LH peak. n the three nonpregnant animals, sex skin regression began 4-7 days after the LH peak while in the pregnant animal the regression was delayed until days following the LH peak. DSCUSSO The cross reaction between chimpanzee gonadotropins and antibodies to human gonadotropins has enabled us to measure serum levels of both FSH and LH in the chimpanzee. Because chimpanzee hormones

CHMPAZ FSH AD LH 3 8 4( o 25 SH 2C S R _. 2i -J 5 #{49}2 i 5 o 23 C 4 C -J 2 k::5tt 7 2636 2 FG.. Serum LH and FSH concentrations and sex skin changes during the menstrual cycle in four adult chimpanzees. The horizontal bars indicate days of menstrual flow: S indicates the start of sex skin swelling; M indicates that sex skin swelling had reached a maximum; R indicates the day the sex skin started receding. were not available, it was necessary to use human standards. Although the degree of cross reactivity is unknown, the parallelism between chimpanzee serum and human standards, as well as the observation that serum gonadotropin levels are comparable to the human, suggest the possibility of a close antigenic similarity between chimpanzee and human gonadotropins. This study demonstrates for the first time that available human assays can be utilized to measure other primate pituitary gonadotropins. We believe that this is also the first report of serum FSH levels in subhuman primates. Previous studies have failed to show evidence of cross reactivity between monkey and human pituitary gonadotropins (eill eta!., 967; Faiman eta!., 967). These TABL 2 FFCT OF VOLUM OF PRGAT CHMPAZ Volume of serum assayed (ml) SRUM O LH MASURD LH concentration.5 22.4. 2.2.5 5..2 3.5 #{76}Concentration in zg LR-97/l ml. findings have prompted the development of a specific assay for rhesus monkey LH (Monroe ci a!., 97) and the use of antimonkey LH serum for developing an assay for baboon LH (Stevens ci a!., 97). Methods for FSH in these species are still not available. A thorough screening of available

4 HOWLAD, FAMA, AD BUTLR antibodies to human gonadotropins for possible cross reactivity with various primate gonadotropins may reveal other antisera which are suitable for assaying gonadotropins in the chimpanzee and other species. This may also apply to other pituitary hormones and, in fact, a radioimmunoassay system for human TSH has been applied to the measurement of circulating TSH levels in the rhesus monkey (Wherry ci a!., 97). The use of human standards allows direct comparison of human and chimpanzee hormone levels. Although these comparisons may not be entirely valid with respect to absolute levels, certainly the relative changes in gonadotropin levels can be compared. f the differences in standards are taken into account, the absolute levels and the cyclic changes in LH in chimpanzees are similar to those reported for women (Midgley and Jaffe, 966; Faiman and Ryan, 967c, Cargille ci a!., 969). Similarly, the levels and cyclic changes in FSH in the chimpanzee resemble those observed in women by Faiman and Ryan (l967c) and Cargille ci a!. (969). Further evidence of similarity between humans and chimpanzees with respect to LH secretion was presented by McArthur and Perley (969). They observed that the urinary LH excretion pattern, as well as the absolute levels of urinary LH in cycling chimpanzees resembled those in women. They also found maximal urinary FSH levels during the follicular phase of the cycle, which is consistent with our serum measurements, but were unable to detect a midcycle rise. The baboon, unlike the chimpanzee, has been found to have peak levels of LH in both serum and urine which are about one-third human levels (Stevens ci a!., 97). Also, the duration of the midcycle rise in serum LH concentrations was found to be shorter in the baboon (Stevens ci a!., 97) and the rhesus monkey (Kirton ci a!., 97) than that reported in the human (Jaffe and Midgley, 969), or observed in the chimpanzee in the present study. The rise in immunoreactive hormone days after the LH peak in animal 48 was presumed to be due to chorionic gonadotropin. This material cross-reacted in a nonidentical fashion in the LH assay (Table 2). The presence of chorionic gonadotropin in the urine of pregnant chimpanzees has been reported (Zuckerman, 935; Schultz and Snyder, 935). The timing of this rise in serum levels of this placental hormone is in close agreement with the two observations in women where serum HCG levels began to rise 9 days after the LH peak (Jaffe ci a!., 969; Parlow ci a!., 97). The FSH level remained low at the time the chimpanzee chorionic gonadotropin level was rising. n women, the rise in HCG in early pregnancy has been associated with a drop in serum FSH to very low or undetectable levels (Jaffe ci a!., 969; Parlow ci a!., 97). The visible changes in the degree of sex skin swelling in the female chimpanzee provide a convenient means of identifying the approximate stage of the menstrual cycle. n attempting to correlate the time of ovulation with sex skin changes, lder (938) observed that conception took place only in cases where coitus had occurred within the last 6 days of maximum sex skin swelling. n a later study (Young and Yerkes, 943), examination of chimpanzee ovaries at laparotomy indicated that ovulation usually occurred just prior to sex skin detumescence. n the three nonpregnant animals in this study, the ovulatory surge of LH occurred 4-7 days before sex skin swelling started to regress. n the pregnant animal, however, the peak occurred days prior to initial sex skin regression. A very constant time interval of - days between the first day of detumescence and the beginning of the next menstrual period was reported by lder and Yerkes (936). This was not observed in the animals of this study where the intervals ranged from 5- days. Young and Yerkes (943) found that a short postswelling phase was not indicative of ovulatory failure. This

CHMPAZ FSH AD LH 5 is consistent with our observations of apparently normal gonadotropin levels and LH peaks in all animals while the interval between first observable detumescence and menstrual bleeding was only 5 and 8 days in two of them. Our observations suggest that although the length of the luteal phase in this species is relatively constant, the timing of the onset of sex skin regression during this phase is variable and is of limited value in determining the time of ovulation or predicting the time of onset of the next menses. ACKOWLDGMTS This work was supported in part by a grant from the University of Manitoba Graduate Research Fund and by M. R. C. Canada Grant MA-2997. The human LH preparations, LR-97 and LR-822-2, were kindly supplied by Dr. L.. Reichert, mory University. The 2nd RP-HMG was a gift from the Medical Research Council, ational nstitute for Medical Research, Mill Hill, London. RFRCS CARGLL, C. M., Ross, G. T., AD YOSHM, T. (969). Daily variations in plasma follicle stimulating hormone, luteinizing hormone and progesterone in the normal menstrual cycle. J. Chin. ndocrinol. 29, 2-9. LDR, J. H. (938). The time of ovulation in chimpanzees. Yale J. Bin!. Med., 347-364. LDR, J. H., AD YRKS, R. M. (936). The sexual cycle of the chimpanzee. Anat. Rec. 67, 9-43. FAMA, C., AD RYA, R. J. (l967a). Radioimmunoassay for human follicle stimulating hormone. J. Chin. ndocrinol. 27, 444-447. FAMA, C., AD RYA, R. J. (l967b). Radioimmunoassay for human luteinizing hormone. Proc. Soc. xp. Biol. Med. 25, 3-33. FAMA, C., AD RYA, R. J. (l967c). Serum folliclestimulating hormone and luteinizing hormone concentrations during the menstrual cycle as determined by radioimmunoassays. J. Cliii. ndocrinob. 27, l7-7t6. FAMA, C., RYA, R. J., GRSL, J. G., AD R- CHRT, L.., JR. (967). Species specificity of FSH and LH as determined by radioimmunoassay. Proc. Soc. xp. Biol. Med. 25, 232-234. JAFF, R. B., L, P. A., AD MDGLY, A. R., JR. (969). Serum gonadotropins before, at the inception of, and following human pregnancy. J. Cliii. ndorinol. 29, 28-283. JAFF, R. B., AD MDGLY, A. R. (969). Current status of human gonadotropin radioimmunoassay. Obstet. Gynecob. Surv. 24, 2-23. KRTO, K. T., SWDR, G. G., MDGLY, A. R., JR., JAFF, R. B., AD FORBS. A. D. (97). Serum luteinizing hormone and progesterone concentration during the menstrual cycle of the rhesus monkey. J. Cliii. ndocrinol. 3, 5-. MCARTHUR, J. W., AD PRLY, R. (969). Urinary gonadotropin excretion by infrahuman primates. ndocriiiology 84, 58-53. MDOLY, A. R., JR., AD JAFF, R. B. (966). Human luteinizing hormone in serum during the menstrual cycle: determination by radioimmunoassay. J. Cliii. ndocri,iol. 26, 375-38. MORO, S.., PCKHAM, W. D., LL, J. D., AD KOBL,. (97). A radioimmunoassay for rhesus monkey luteinizing hormone (Rh LH). ndocrinology 86, 2-8. LL, J. D., PCKHAM, W. D., AD KBL,. (967). Apparent absence of immunological crossreactivity between human and simian gonadotropic hormones as determined by radioimmunoassay. ature Loiido,i 23, 4-5. PARLOW, A. F., DAA, T. A., AD DGAM, W. J. (97). On the concentration of radioimmunoassayable FSH circulating in blood throughout human pregnancy. J. Chin. ndocrinol. 3, 23-24. RYA, R. J., AD FAMA, C. (968). Radioimmunoassay of human follicle stimulating hormone: A comparison of several antisera. xcerpla Med. Fou,ich. hit. Co,igr. Ser. o. 6, 29-33. SCHULTZ, A. H., AD SYDR, F. F. (935). Observations on reproduction in the chimpanzee. Bulb. Jo/iiis Hopkins Hosp. 57, 93-25. STVS, V. C., SPARKS, S. J., AD POWLL, J.. (97). Levels of estrogens, progestogens and luteinizing hormone during the menstrual cycle of the baboon. ndocrinobogy 87, 658-666. WHRRY, F.., PGTO, L. L., BATS, R. W., GARRSO, M. M., HL, A. L., AD MASO, J. W. (97). mmunologic cross-reactivity between monkey and human thyroid stimulating hormone as determined by radioimmunoassay; a rapid procedure for purifying labeled 5-TSH. ndocrinology 86, 769-773. YOUG, W. C., AD YRKS, R. M. (943). Factors influencing the reproductive cycle in the chimpanzee; the period of adolescent sterility and related problems. ndocriiiobogy 33, 2-54. ZUCKRMA, S. (935). The Ascheim-Zondek diagnosis of pregnancy in the chimpanzee. Amer. J. Phi ysiob., 597-!.