Circulating and excreted hormones during the ovarian cycle in the cotton-top tamarin Saguinus oedipus.код для вставкиСкачать
American Journal of Primatology 31:55-65 (1993) Circulating and Excreted Hormones During the Ovarian Cycle in the Cotton-Top Tamarin, Saguinus oedipus TONI E. ZIEGLER, DANIEL J . WITTWER, AND CHARLES T. SNOWDON Department of Psychology and Wisconsin Regional Primate Research Center, University of Wisconsin, Madison Five parous, cycling cotton-top tamarin females were used to compare circulating and urinary profiles of the major reproductive hormones during the ovarian cycle. Blood samples were collected on unanesthetized, hand restrained females three times per week for 25 days and first morning void urine was collected daily for 30-40 days including the serum sampling period. Serum and urine samples were analyzed for estrone, estradiol, and bioactive LH. Additionally, serum was analyzed for progesterone and urine for estrone conjugates (ElG) and immunoreactive LH. Both serum and urinary hormonal profiles revealed ovarian cyclicity in four females; the fifth one stopped cycling during the study. All hormones were referenced to the urinary immunoreactive LH peak. Urinary estrone conjugates followed similar profiles to serum progesterone. Progesterone levels increased two- to threefold on the day of the urinary immunoreactive LH peak. Peak levels of circulating bioactive LH occurred on the same day or the day preceding the urinary bioactive and immunoreactive LH peak for all four cycling females. Serum estradiol levels rose during the follicular phase and peaked prior to or on the day of the urinary LH peak while urinary estradiol levels did not rise until the day of the urinary LH peak. Serum and urinary estrone levels did not rise until the day of the urinary LH peak and remained high throughout the luteal phase. The measurement of urinary LH corresponded well with serum LH, establishing urinary LH as an important hormone for monitoring the ovulation in the tamarin. The highest circulating estradiol concentrations were found in the follicular phase of the cycle, but estradiol did not appear in urine during the follicular phase of the cycle. Elevated estrone levels occur during the luteal phase following the progesterone pattern, and therefore elevated urinary concentrations of estrone and estrone conjugates may be indicative of luteal function. o 1993 Wiley-Liss, Inc. Key words: ovarian cycle, serum hormones, urine hormones, tamarins Received for publication October 5, 1992;revision accepted March 1, 1993. Address reprint requests to Dr. Toni E. Ziegler, Department of Psychology, 1202 W. Johnson St., University of Wisconsin, Madison, Wisconsin 53706. 0 1993 Wiley-Liss, Inc. 56 1 Ziegler et al. INTRODUCTION Reproductive events are best detected and timed while monitoring hormonal concentrations, especially in species of primates that do not exhibit external signs of ovulation and conception. Traditionally, reproductive events have been described through measurement of circulating hormones which allow for precise timing of ovulation and early detection of pregnancy. However, when monitoring the reproductive events in endangered, excitable, or very small animals, urinary assays for reproductive hormones have proven to be reliable and less stressful [Hodges et al., 1979; Shideler et al., 1983; Lasley et al., 1985; Dahl et al., 1987; Ziegler et al., 1987a,bl. While many primate species have been examined for reproductive function using urinary assays, with very few species have the patterns of urinary hormones been compared directly to circulating hormones to determine their effectiveness in timing ovulation and early detection of pregnancy. To date, this comparative information is only available for the human [Munro et al., 1991; Dahl et al., 1987; Beitins et al., 19801, the cynomolgus monkey [Mehta et al., 19861, the rhesus macaque [Monfort et al., 19871, the common marmoset [Eastman et al., 1984; Hodges et al., 19831, and the owl monkey [Bonney et al., 19791. Most of these studies were limited to a comparison of only one or two hormones. Most of our knowledge of the reproductive hormones in the cotton-top tamarin comes from measurements of urinary hormones [Brand, 1981; French et al., 1983; Ziegler et al., 1987a,bl. Both urinary estrone and estradiol show elevated levels following the LH surge, paralleling progesterone concentrations, without a preovulatory urinary estrogen rise. Since the major urinary estrogens show a luteal instead of follicular rise and only 5% of progesterone is excreted into the urine [Ziegler et al., 19891, a comparative study on circulating and urinary hormones is essential for the interpretation of the urinary hormonal patterns. The present study documents the patterns of hormone secretion in the peripheral circulation and compares the serum hormonal profiles to urinary metabolites. MATERIALS AND METHODS Subjects and Sampling Five cycling cotton-top tamarins ranging in age from 20 months to 9 years were used as subjects. All but one female (the youngest) had given birth to live infants. One female lived alone in a pair cage, three females were paired with a male and two of these females were hysterectomized with ovaries intact while the other one lived with a vasectomized male, and one female was the breeding female in a large family (five offspring in the cage) where the breeding male had died. Thus, all five females were unable to conceive during the study. Sizes of pair and family cages, husbandry, and diet have been reported [Snowdon et al., 1985; Ziegler et al., 1987bl. Blood and urine samples were collected from the tamarins for 25-30 days. Three times per week between 1300 and 1400, the females were caught from their cages and hand restrained while 0.5-0.9 ml of blood was collected by venipuncture of the femoral vein on the unanesthetized females. The collected blood was stored in serum separator tubes (Microtainer, Becton Dickinson, Rutherford, NJ) for 30 min, then centrifuged for 10 min, and the serum portion stored at -20°C until hormonal assay analyses. Once a week, an aliquot of the whole blood collected from each female was used to monitor hematocrit levels. Hematocrit levels decreased 15-18% over the four week bleeding period falling from a mean of 54 to a mean of 47. Urine was collected as first morning void daily between 0800 and 0900 when Serum and Urinary Hormones in Cotton-Top Tamarins / 57 TABLE I. Parallelism, Recovery of Extracted Steroid, Assay Sensitivities, and Precision for the Hormones Measured in the Serum and Urine of Cotton-Top Tamarins Sensitivity Hormone ~ Parallel Recoveryb pg/tubeb Serum' 80.55 2 0.95 0.21 rt 0.01 10.6 pg/ml 6.5 pg/ml 26 ng/ml 0.14 ng/ml 15.82 mIUf Assay precision" Urine' Intra Inter ~~~ estrone yes serum yes urine estradiol yes serum yes urine estrone no serum conjugates yes urine progesterone yes serum yes urine bioLH yes serum yes urine immunoLH yes urine 82.30 * 2.89 0.56 * 0.01 - 3.10 ? 0.91 87.70 ? 0.21 1.06 5 0.51 103.6 t 2.60 1.04 0.28 ng/mlTd 0.29 C 0.01 Flltube - * - 17.61 4.32' (3) 13.44 (7) ng/ml 3.9 5.43 (3) 11.78 (7) ng/ml 196 2.78 (3) 10.68 (7) ng/ml 2.92 (3) 3.53 (3) 23.07 mIU 8.0 bum1 3.36 (3) 13.72 (4) 2.66 (3) 8.88 (3) "Expressed as coefficient of variation. 'Values are mean s.e.m. 'Based on dilutions used for assay. dT = testosterone. "Numberin parentheses = n. 'mIU = milli-international units of purified human chorionic gonadotropin. * lights were turned on in the rooms. A collection container was held directly underneath the female of interest while she urinated. A detailed description of the collection technique has been described [Ziegler et al., 1987a,bl. Collected urine was centrifuged and stored at -20°C until hormonal analyses were performed. Ovarian cycling that is not followed by conception rarely occurs in paired cotton-top tamarins. We therefore assessed all previous complete ovarian cycles that have been monitored in our colony by measuring daily urinary estrone conjugates and LH. These were analyzed for ovarian cycle length and for length of the follicular and luteal phase of the cycle. The seven females who had shown ovulatory cycles were between 1 and 10 years of age and represented all aspects of reproductive condition (i.e., nulliparous to multiparous, paired or living alone after a mate had been removed). Assays Both serum and urine samples were analyzed for estrone, estradiol, and bioactive LH. Additionally, serum was analyzed for progesterone and urine for immunoreactive LH and estrone conjugates. The methods and validation of urinary hormones for these assays have been described [Ziegler et al., 1987a,bl but brief descriptions of the assays and the modifications for serum analyses are reported below. Table I gives information on parallelism, recoveries, sensitivity, and precision for the various assays. Serum samples (60-100 pl) analyzed for estrone and estradiol were measured by ether extraction, chromatography separation using LH-20 Sephadex columns and a dich1oromethane:methanol solvent system with individual RIA of each steroid. This method separates estrone and estradiol from each other as well as from other steroids. The estrone antiserum crossreacted < 0.1%with other steroids but the estradiol antisera crossreacted 5.8%with estrone, 0.3% with estriol, and 0.82% 58 / Ziegler et al. for testosterone. Serial dilutions of a serum pool with the addition of 50 pgI100 p1 of estrone and 75 pg/lOO p1 of estradiol were parallel to the standard curve and gave recoveries of each added steroid at 80.6% and 82.3%, respectively. Urine samples were assayed at volumes between 0.1 and 1pl using a 1:lOO dilution of the samples with an additional dilution of 1:25 for estrone after chromatographic separation. To measure total estrone and estradiol, urine was hydrolyzed overnight with 25 p1 beta-glucuronidase (Type H2, containing sulphatase activity as well, Sigma Chemical Co., St. Louis, MO) a t 37°C prior to column chromatography. Serum and urine samples were run in the same RIA for each female. The intra- and interassay coefficient of variations are reported in Table I. Measurement of estrone conjugates (ElGI in the urine was accomplished by adding 0.01-0.1 p1 of sample and direct assay with estrone-3-glucuronide antibody and trace. Validation and description of this assay has been reported [Ziegler et al., 1987bl. Serum samples were not analyzed for E1G since serial dilutions of a serum pool were not parallel to the standard curve. Serum samples were analyzed for progesterone concentration using 10 p1 of serum. Samples were extracted with 5 ml of petroleum ether to separate progestins from other steroids, dried under air, and measured by RIA using a progesterone antiserum a t 1:2,800 in 100 p1 (HH, P-1, Holly Hill Biologicals, Inc., Hillsboro, OR) and [1,2,6,7-3Hl-progesterone a t 7,500 cpm in 100 pl (P4:New England Nuclear, Boston, MA) as ligand. The antiserum crossreacted 14.4%with 5a-pregnane-3,20dione, 1.6%with pregnenolone, 0.6% with 5P-pregnane-3,20-dione, 0.2% with 17ahydroxyprogesterone, 0.2% with 20P-dihydroprogesterone,and 0.2% with 20adihydroprogesterone. Progesterone (Sigma) was used as standards in the range from 5-250 pg/ml. Five serial dilutions of pooled tamarin serum were parallel to the standard curve. Bioactive LH (bioLH) was measured by the use of the mouse interstitial cell bioassay using the methods already reported [Ziegler et al., 1987al. Serum and urine samples were added as 5 p1 of sample diluted with 95 pl of tissue culture medium 199 with 0.2% BSA. Pooled tamarin serum with the addition of serial dilutions of human chorionic gonadotropin (0.1,0.25,0.5 mlU) were parallel to the standard curve and yielded an accuracy of 103.6 ? 2.6%. Immunoreactive LH was measured in urine using a heterologous LH RIA which measures LH and CG in the tamarin. Methods and validations have been reported [Ziegler et al., 1987al. All urinary hormonal concentrations were expressed as milligrams of creatinine to compensate for variability in fluid intake. The creatinine assay was performed according to the methods in Tietz [19761. RESULTS Four of the five tamarins displayed hormonal cyclicity during the blood and urine sampling. The fifth female ceased cycling and all her hormone levels dropped to basal levels during the sampling period. Ovarian cycle lengths as defined by successive urinary immunoreactive LH peaks ranged from 15-24 days for the four females with a mean level of 20 & 2.5 SEM. LH peaks were defined as two- to fivefold increases in basal LH for 1-2 days before declining to near basal levels. Levels of hormones were determined for the four cycling females and adjusted for day from the urinary LH peak. Figure 1A presents the serum LH and progesterone profiles and Figure 1B presents the corresponding urinary LH and E1G profiles in one representative female. Highest elevations of LH in both serum and urine occurred on the same day in two of the females, while highest elevations in serum LH may have occurred the day before the urinary LH peak in the two females Serum and Urinary Hormones in Cotton-Top Tamarins / 59 A - 15 -E 60 E SO- . 2 -E - - Ol -10 = n I A 40 - 30 - 5 L al E 2al u) -5 . - ' I ' ' ' ' 1 10 I , I ' , ' -10-9 - 8 - 7 - 6 - 5 - 4 - 3 - 2 - 1 . . 0 ' 1 1 ' ' 2 1 . . 4 3 ) 8 I 5 6 . ' 7 I 8 I ' 9 . 1- 0 10 Days from urinary LH peak B 30 6 6 . E m PI E 1 20 C 2 0 5 F W P 0 .-C 10 L f 0 I . , . , . , . , . , . , . I . , - 1 0 - 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2 - 1 . , 0 . , 1 . , 2 . , 3 . , 4 . , 5 P 0 .c 5 . ,.,.,.,.,0 6 7 8 9 10 Days from urinary LH peak Fig. 1. Serum and urinary hormonal profiles for a representative female cotton-top tamarin during the ovarian cycle. A: Circulating levels of serum bioactive LH (bioLH, expressed in mIU/human chorionic gonadotropin reference preparation) and progesterone (P). B Urinary levels of immunoreactive LH and estrone conjugates (ElG). All graphs are normalized to the day of the urinary LH peak and urinary hormones are expressed per milligram of creatinine (Cr). where a matched serum sample was not obtained on the day of highest urinary LH levels. Serum progesterone and urinary E1G showed similar profiles of basal levels of hormone prior to the LH peak, increasing the day before or the day of the LH peak and remaining elevated following the presumed ovulation. Thus, ovulation in 60 / Ziegler et al. 100 t 80 cn E . 2 E I 60 50 F m .-C - L 40 0 . , . , . , . , . , . ) . 1 . r . , . - 1 0 - 9 - 6 - 7 - 6 - 5 - 4 - 3 - 2 - 1 a 20 0 1 2 3 4 5 6 7 8 9 10 Days from urinary LH peak Fig. 2. Serum and urinary bioactive LH levels (bioLH, expressed &s mIUlml human chorionic gonadotropin reference preparation) from a representative female cotton-top tamarin during the ovarian cycle. Data are normalized to the day of the urinary LH peak. the tamarin may best be reflected by a serum LH peak coincident with increased progesterone and a urinary LH peak coincident with increased E1G. The follicular phase for the females was described by basal levels of serum progesterone (< 5 ng/ml) and urinary E1G (< 10 pg/mgCr) for 5 days prior to the urinary and serum LH peak. Based on the data revealing serum progesterone and urinary E1G patterns to be similar, we examined eleven females for ovarian cycle length by assessing urinary LH and E1G. There was a mean cycle length of 21.36 f 1.05 SEM. days (range 15-24 days). Follicular and luteal phases of the cycle were identified by basal E1G for the follicular phase and elevated E1G for the luteal phase. The follicular phase was an average of 5 2 0.45 days with a coefficient of variation (c.v.) of 8.94%. The luteal phase was found to be a mean of 16.36 f 3.64 days with a C.V. of 22.25%indicating that the majority of variance in cycle length between females was due to the variation in length of the luteal phase. Serum and urinary bioactive LH profiles are shown in Figure 2 for an individual female. Although serum samples were not collected daily, the highest concentrations of the hormone appeared to coincide in serum and urine or else the serum LH peak preceded the urine peak by a day. Serum and urinary bioactive LH concentrations were highly correlated for all females, r = 0.83, n = 43, P < 0.01. Comparisons of serum estrogens to urinary estrogens are shown in Figure 3A,B for one representative female. All females showed comparable profiles. Serum estradiol levels were elevated during the follicular and ovulatory phases, but urinary estradiol did not increase until the day of the LH peak. Neither serum nor urinary estrone concentrations showed follicular increases, but they were both elevated on the day of the LH peak and throughout the luteal phase of the cycle. Even though serum and urinary estrone followed similar patterns, t b b viere not Serum and Urinary Hormones in Cotton-TopTamarins I 61 A 500T+ I'800 serumE2 -E -E . . 0 0 n P (Y W -10 - 5 0 5 10 15 B t . E" 0 C hl w Q) .-c L a Fig. 3. Serum and urinary levels of estrone (El)and estradiol (E2)in one representative cotton-top tamarin female. A: Serum estradiol and serum estrone are measuredas days from urinary LH peak. B: Urinary estradiol and urinary estrone measured as days from urinary LH peak. Urinary estrogens were expressed per milligram of creatinine (Cr). highly correlated for any of the four females. The ratio of estrone:estradiol was higher in the urine by a mean factor of 20.5 times than in serum, indicating a shift to increased estrone secretion in the urine. The female who stopped cycling during the serum collection time had elevated 62 I Ziegler et al. levels of serum progesterone, estradiol, estrone, and urinary E1G for the first 10 days of collection. Levels then dropped and remained low for the next 15 days. After the blood collection stopped, E1G levels remained low for approximately 10 days and then the female resumed cycling. DISCUSSION The measurement of circulating hormones documented the ovarian cycle in the cotton-top tamarin. The patterns of the reproductive hormones indicated normal ovarian function and resembled the hormonal profiles of other New World primates. Although it was impossible to collect daily samples from these small primates, the increase in follicular estradiol and elevated LH was detected in all four cycling females. The follicular phase of the cycle was short, lasting only 5 days with the luteal phase varying considerably. Although the present data are limited, it appears that cotton-top tamarins differ from humans, great apes, and Old World monkeys where follicular and luteal phases are usually of comparable lengths [Knobil & Hotchkiss, 19881. Data from the common marmoset have indicated a similar profile to the cotton-top tamarin where the follicular phase is shorter than the luteal phase (mean of 8 days follicular phase: mean of 21 days luteal phase) [Harlow et al., 1983, 19841. Serum patterns in the cotton-top tamarin were similar to those reported in the common marmoset with a few exceptions. Both the common marmoset and the cotton-top tamarin show follicular increases in estradiol with peak concentrations occurring the day before or the day of the LH peak and elevated estrone levels occurring during the luteal phase of the cycle. However, the onset of increased circulating progesterone appears to occur when LH is elevated in the cotton-top tamarin, whereas the progesterone increase occurs 1-2 days following the LH peak in the common marmoset [Harlow et al., 19841. It is not possible to make direct comparisons between the present data on serum profiles with the data presented by Preslock et al. [19731 on serum estrogen and progestin profiles due to the non-specific assays that were used for that early study. However, estrogen levels did appear to precede progestin levels in the earlier study. The urinary profiles of immunoreactive LH and E1G paralleled the serum profiles of bioactive LH and progesterone. It is therefore feasible to define the ovulatory period in the cotton-top tamarin from the urinary LH peak and onset of elevated estrone. This comparison means that urinary hormones can be used to monitor ovarian function without the need of collecting serial blood samples in this small and excitable primate. With the ability to collect daily urine samples reliably, we can examine social and behavioral effects on fertility at times that require minimal disturbances to the female and her social group. This also enables us to collect long-term data that would be impossible with serial blood sampling. Even without daily blood sampling in the four cotton-top tamarins, it was possible to see that the serum and urine LH levels were elevated during the same time frame. It is possible that serum LH levels may peak the day before the urinary LH peak in some females, but the highly elevated serum LH on the same day as the urinary LH peak seen in two of the four females suggests that the serum LH peak may also occur on the same day. Human LH and FSH have a fairly rapid clearance rate through the hepatic system due to a lower sialic acid content and may be excreted rapidly into the urine [Ashwell & Morell, 19741. Previous investigations examining urinary estrogens in tamarins have all revealed similar profiles of urinary estrogens increasing a t the time of the ovulatory LH peak and remaining elevated during the luteal phase of the cycle [Ziegler et al., 1987a,b, 19891. Serum profiles from the present study indicated that the Serum and Urinary Hormones in Cotton-TopTamarins / 63 cotton-top tamarin, as in other primates, showed follicular increases in circulating estradiol that peak before or on the day of the LH peak. Only serum estrone showed the urinary pattern of elevated levels beginning a t the time of the LH peak and extending through the luteal phase. There appears to be either a delay in estradiol excretion into the urine or estradiol is converted to nonhydrolyzable forms since there are no increases in urinary estradiol until the time of the LH peak. It also appears that estradiol is partially metabolized to estrone in the urine since serum levels are comparable for the two estrogens while urine levels are considerably higher for estrone than estradiol. Ziegler et al. [19891reported that approximately half of injected radiolabelled estradiol was converted to estrone in the cotton-top tamarin and that estrone is excreted a t a slower rate than estradiol. Circulating estrone and urinary estrogens are following a progesterone-like pattern. This may suggest that some estrogens are produced by the corpus luteum. Elevated levels of estrogens are produced by the corpus luteum in many primates, including humans, gorillas, chimpanzees, and orangutans, but in lesser amounts in the Old World monkeys such as the rhesus, baboon, and cynomolgus monkeys [see Knobil & Hotchkiss, 19883.Both the common marmoset [Harlow et al., 19841 and the cotton-top tamarin display elevated circulating levels of estrone during the luteal phase of the ovarian cycle. An alternative explanation may be peripheral conversion of another luteal steroid to estrone. The active interstitial gland tissue found in tamarins of all ages after puberty may also contribute to the elevated estrone levels [Mansdotter et al., 1992;Tardif & Ziegler, 19921. One of the five females from which repeated serum samples were taken ceased ovulating and showed basal hormone levels. This female was the only one living in a large family cage where repeated capture took longer than 5 min and involved extended chasing of the tamarins before capture occurred. Since the entire group (six tamarins) was highly agitated, it is possible that the stress of the extended capture experience adversely affected ovarian cyclicity. The female did return to ovulatory cyclicity 10 days following the cessation of capture and bleeding. In summary, the ovarian cycle of four female cotton-top tamarins was evaluated by both serum and urinary hormones. Serum estradiol levels were elevated during the follicular phase of the cycle when progesterone levels were a t basal levels as is typical of primates. The pattern of urinary LH and E1G followed the same pattern of serum LH and progesterone in defining the periovulatory events. This allows us to continue to monitor reproductive functioning by using urinary measurements that can be collected daily with minimal intervention. CONCLUSIONS 1. Serum LH levels were at highest elevations the day before or the day of the urinary LH peak. 2. Urinary estrogens follow the same profile as serum progesterone and can be used to indicate the luteal phase in ovarian cycling cotton-top tamarins. 3. Serum and urine estradiol profiles differ during the ovarian cycle with serum estradiol indicating a preovulatory peak and urinary estradiol showing luteal elevations. ACKNOWLEDGMENTS We are extremely grateful to G.Scheffler and F.Wegner for technical advice concerning assays, P. Cofta, L. Converse, and T. Dreyfus for assisting in the capture and bleeding of tamarins, J. Scheffler and Dr. D. Houser for veterinary advice, and R. Roush and T. Porter for colony management. We also acknowl- 64 I Ziegler et al. edge Dr. John Mara of Hill Pet Products, Topeka, KS, for donation of Zu-Preem Marmoset Diet. This research was supported by grant MH 35,215 to C.T. Snowdon, RR 00167 to the Wisconsin Primate Research Center, and The University of Wisconsin Graduate School Research Committee. This is publication 32-049 of the Wisconsin Regional Primate Research Center. REFERENCES Ashwell, G.; Morell, A.G. The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. ADVANCED ENZYMOLOGY 41:99-105, 1974. Beitins, I.Z.; Shah, D.; OLaughlin, K.; Johnson, L.; Ostrea, T.R.; Van Wart, J.; MacArthur, J.W. The effects of fasting on serum and urinary gonadotropins in obese postmenopausal women. JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM 51:26-30, 1980. Bonney, R.C.; Dixson, A.F.; Fleming, D, D. Cyclic changes in the circulating and urinary levels of ovarian steroids in the adult female owl monkey (Aotus triuirgutus). JOURNAL OF REPRODUCTION AND FERTILITY 56~271-280,1979. Brand, H.M. Urinary oestrogen excretion in the female cotton-topped tamarin (Suguinus oedipus oedipus). JOURNAL OF REPRODUCTION AND FERTILITY 62:467473, 1981. Dahl, K.D.; Czekala, N.M.; Lim, P.; Hsueh, A.J.W. Monitoring the menstrual cycle of humans and lowland gorillas based on urinary profiles of bioactive follicle-stimulating hormone and steroid metabolites. JOURNAL CLINICAL ENDOCRINOLOGY 64:483-486, 1987. Eastman, S.A.K.; Makawiti, D.W.; Collins, W.P.; Hodges, J.K. Pattern of excretion of urinary steroid metabolites during the ovarian cycle and pregnancy in the marmoset monkey. JOURNAL OF ENDOCRINOLOGY 102:19-26, 1984. French, J.A.; Abbott, D.H.; Scheffler, G.; Robinson, J.A.; Goy, R.W. Cyclic excretion of urinary oestrogens in female tamarins (Suguinus oedipus). JOURNAL OF REPRODUCTION AND FERTILITY 68:177184,1983. Harlow, C.R.; Gems, S.; Hodges, J.K.; Hearn J.P. The relationship between plasma progesterone and the timing of ovulation and early embryonic development in the marmoset monkey (Cullithrix jucchus).JOURNAL OF ZOOLOGY 201:273-282, 1983. Harlow, C.R.; Hearn, J.P.;Hodges, J.K. Ovulation in the marmoset monkey: Endocrinology, prediction and detection. JOURNAL OF ENDOCRINOLOGY 103:17-24, 1984. Hodges, J.K.; Brand, H.; Henderson, C.; Kelly, R.W. Levels of circulating and urinary oestrogens during pregnancy in the marmoset monkey (Cullithrix jacchus). JOURNAL OF REPRODUCTION AND FERTILITY 67:73-82, 1983. Hodges, J.K.; Czekala, N.M.; Lasley, B.L. Estrogen and luteinizing hormone secretion in diverse primate species from simplified urinary analysis. JOURNAL OF MEDICAL PRIMATOLOGY 8:349-364, 1979. Knobil, E.; Hotchkiss, J. The menstrual cycle and its neuroendocrine control. Pp. 1971-1994 in THE PHYSIOLOGY OF REPRODUCTION, Vol. 2. E. Knobil, J.D. Neill, eds. New York, Raven Press, Ltd., 1988. Lasley, B.L.; Stabenfeldt, G.H.; Overstreet, J.W.; Hanson, F; Czekala, N.M.; Munro, C. Urinary hormone levels at the time of ovulation and implantation. FERTILITY AND STERILITY 43:861-867, 1985. Mansdotter, S.; Epple, G.; Kuderling, I. Agerelated changes in ovarian morphology of the South American tamarin Suguinus fuscicollis (Callitrichidae). JOURNAL OF ZOOLOGY, LONDON 227:239-255,1992. Mehta, R.R.; Jenco, J.M; Gaynor, L.V.; Chatterton Jr, R.T. Relationships between ovarian morphology, vaginal cytology, serum progesterone, and urinary immunoreactive pregnanediol during the menstrual cycle of the cynomolgus monkey. BIOLOGY OF REPRODUCTION 35~981-986,1986. Monfort, S.L.; Hess, D.L; Shideler, S.E.; Hendrickx, A.G.; Lasley, B.L. Comparison of serum estradiol to urinary estrone conjugates in the rhesus macaque (Macacu rnulutta). BIOLOGY OF REPRODUCTION 37~832-837,1987. Munro, C.J.; Stabenfeldt, G.H.; Cragun, J.R.; Addiego, L.A.; Overstreet, J.W.; Lasley, B.L. Relationship of serum estradiol and progesterone concentrations to the excretion profiles of the major urinary metabolites as measured by enzyme immunoassay and radioimmunoassay. CLINICAL CHEMISTRY 37:838-844. 1991. Preslock, J.P.; Hampton, S.H.; Hampton, J.K, Jr. Cyclic variations of serum progestins and immunoreactive estrogens in marmosets. ENDOCRINOLOGY 92:10961101,1973. Shideler, S.E.; Czekala, N.M.; Kasman, Serum and Urinary Hormones in Cotton-Top Tamarins / 65 L.B.; Lindburg, D.G.; Lasley, B.L. Monitoring ovulation and implantation in the liontailed macaque (Mucacu silenus) through urinary estrone conjugate evaluations. BIOLOGY OF REPRODUCTION 29:905911,1983. Snowdon, C.T.; Savage, A.; McConnell, P.B. A breeding colony of cotton-top tamarins (Suguinus oedipus). LABORATORY ANIMAL SCIENCE 35:477-480,1985. Tardif, S.D.; Ziegler, T.E. Peatures of female reproductive senescence in tamarins (Suguinus spp.), a New World primate. JOURNAL OF REPRODUCTION AND FERTILITY 94:411-421, 1992. Tietz, N.W. FUNDAMENTALS OF CLINICAL CHEMISTRY. Philadelphia, WB Saunders, 1976. Ziegler, T.E.; Bridson, W.E.; Snowdon, C.T.; Eman, S. Urinary gonadotropin and estro- gen excretion during the postpartum estrus, conception and pregnancy in the cotton-top tamarin (Suguinus oedipus oedipus). AMERICAN JOURNAL OF PRIMATOLOGY 12:127-140, 1987a. Ziegler, T.E.; Savage, A.; Scheffler, G.; Snowdon, C.T. The endocrinology of puberty and reproductive functioning in female cotton-top tamarins (Suguinus oedipus) under varying social conditions. BIOLOGY OF REPRODUCTION 37:618627, 198713. Ziegler, T.E.; Sholl, S.A.; Scheffler, G.; Haggerty, M.A.; Lasley, B.L. Excretion of estrone, estradiol, and progesterone in the urine and feces of the female cotton-top tamarin (Suguinus oedipus oedipus). AMERICAN JOURNAL OF PRIMATOLOGY 17:185-95 1989.