Annual Report 1997

Assay Services continue to provide modern, efficient and cost-effective endocrine measurements in a centralized facility for all scientists affiliated with the Primate Center and non-Center Principal Investigators requiring our specialized expertise. We maintain and execute numerous established assay procedures, sample preparation techniques and chromatographic separation procedures, while acquiring new technology and developing and adapting new methodology and techniques to meet the progressive research requirements of investigators. Our current major assay procedures include radioimmunoassays (RIAs; including our in-house iodination suite), enzymeimmunoassays (EIAs), electrochemiluminescence assays (ECLs), bioassays and automated, quantitative high performance liquid chromatography (HPLC). Our major sample preparation techniques include HPLC and celite chromatography. Our diversity in state-of-the-art techniques enables Assay Services to employ the most appropriate combination of procedures to solve the new, and increasingly more complex, analytical challenges from investigators. We also offer different levels of service support to meet the varied needs of investigators, so that assays can be performed, calculated and assessed either entirely by Assay Services personnel (Full service) or by Assay Services personnel working together with research personnel (Self service). Quality control assessment is maintained by Assay Services personnel for each hormone, in each medium, for each species. Assay Services operates as an integrated team and our progress and productivity are, for the most part, products of cohesive group effort.
Assay Services operates under the Radiation Users License, Biosafety Protocol and Animal Care and Use Protocols held and maintained by

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Productivity

In 1997, Assay Services maintained a high level of productivity, executing 109,562 biochemical measurements in order to determine hormone concentrations in 68,601 samples. This output is especially notable because sample preparation prior to assay can be lengthy, particularly when discrete isolation of hormones is required using celite or high performance liquid chromatography. This high level of hormone determination is expected to continue due to (1) the proliferation of funded requests for hormone assays from investigators within the Primate Center's Research Groups and from laboratories not affiliated with the Primate Center, (2) to increasing numbers of research personnel performing portions of hormone assays in Assay Services, and (3) the continuing collaborative research agreement between Dr. Joseph Kemnitz (Aging and Metabolic Diseases and Interim Director) and the National Institute for Aging for the regular determination of a variety of steroid and peptide hormone concentrations in serum samples from aged rhesus macaques at NIH.

Steroid hormones

Steroid hormone assays accounted for 49,386 determinations in 1997, including assays for androgens, estrogens, progestins (and their conjugated forms) and glucocorticoids. The most frequent determinations were made for cortisol (28%), progesterone (25%) and testosterone (23%), accounting for 74% of all steroid hormone determinations. This work also included research development and full validation of the following assays:

The vitamin D metabolite validations performed by Mr. Wegner were instrumental in enabling funding of an NIH grant submitted by Dr. Wendy Saltzman (Physiological Ethology) to utilize the common marmoset as a novel model for estrogen-depletion bone loss (Dr. Abbott and Dr. Neil Binkley (Aging and Metabolic Diseases) are Co-Investigators on this award). The fecal and urinary hormone validations performed by Messrs. Guenther Scheffler and

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High Performance Liquid Chromatography (HPLC)

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(1) detection and identification of androgen metabolites in the urine of male cotton-top tamarins: Several HPLC fractions showed crossreactivity with our testosterone antibody necessitating the use of celite column chromatography before EIA.

(2) using the HPLC system with UV detection to quantify multiple steroids simultaneously in each sample: Male rhesus monkey urine was selected for this pilot project since testosterone in this species is metabolized and excreted in the urine as many different steroids for which no antibodies are available for assay development. Radiolabelled studies of male rhesus monkey urine injected into the HPLC revealed that six particular steroids were metabolites of testosterone (estradiol, testosterone, epitestosterone, epiandrosterone, etiocholanolone and androsterone). Other urinary metabolites of testosterone in the male rhesus monkey, such as 5 Beta-androstane-3 Alpha,17 Beta-diol and 5 Alpha-androstane-3 Alpha,17 Beta-diol do not UV absorb and cannot be detected by our UV detection system. Reference preparations for all six steroids detected (ranging from 10 ng to 10 (g) produced regression coefficients of 0.96 to 1.00, indicating the usefulness of the HPLC technique as an accurate assay for the testosterone metabolites without the need for EIA or RIA assessment. We are currently in the process of completing the validation of this technique for quantitative assessment of testosterone concentrations in male rhesus urine. This technique for measuring multiple androgens simultaneously will also be used for androgen determination in male cotton-top tamarin urine and male muriqui feces.

(3) detection and separation of estradiol and pregnanediol in the feces of female blue monkeys: HPLC separation permitted assessment of EIA antibody crossreactivity.

(4) detection of glucocorticoids, estrogens and progestins in the feces of female howler monkeys: HPLC separation permitted identification of individual steroid hormones present in the feces of female howler monkeys.

(5) detection of androgen metabolites in the feces of male oribi: This permitted separation of androgen metabolites for assessment of EIA antibody crossreactivity.

(6) detection of androgen metabolites in male impala feces: This permitted assessment for EIA testosterone antibody crossreactivity.

(7) separation and identification of testosterone and cortisol in the feces of male Thompson's gazelle: This permitted assessment of EIA antibody cross-reactivity.

(8) separation and identification of glucocorticoids in the serum of both the Coho salmon and Rainbow trout: this enabled Dr. Terry Barry and his colleagues in the Aquaculture Program at the University of Wisconsin-Madison to progress with their research projects into understanding of the negative effects of stress on the farming of these fish species.

(9) detection of glucocorticoids and mineralocorticoids in the serum of domestic dogs: This project was carried out in collaboration with Dr. Paul Miller in the Department of Veterinary Sciences at the University of Wisconsin-Madison. Dogs with Sudden Acquired Retinal Degeneration (SARD) were suspected of having an altered secretion of adrenal hormones. Serum samples from both inflicted and healthy animals were subjected to HPLC separation and UV absorption identification. Results indicated almost identical patterns of adrenal hormones in both conditions with the exception of aldosterone which was unusually elevated in the healthy control dogs. This suggested that the initial hypothesis of disturbed adrenal function causing SARD was incorrect.

Peptide hormones

Peptide assays accounted for 60,176 (55%) of the total 109,562 determinations in 1997, including assays for a wide range of peptide hormones (including both biological and immunoreactive measurements) and peptide neurotransmitters. The most frequent determinations were for gonadotropin-releasing hormone (45%), luteinizing hormone (17%), insulin (18%) and neuropeptide-Y (5%), accounting for 85% of all peptide determinations. This work included research, development and full validation of the following assays:

Web Site Development for Assay Services at
http://www.primate.wisc.edu/assay

Dr. Abbott introduced this new initiative in 1997.

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Significant increases in training in assay techniques in 1997

Mr. Wegner attended a week long "Assay Development Course" at IGEN, International in Gaithersburg, MD in February, 1997 where he learned many important procedures for the new electrochemiluminescence (ECL) machine purchased the previous fall. This was a "hands-on" course where specific reagents were purified, labeled and optimized for use. General maintenance of the machine was explained, as well as the computer software package.

Theoretical and practical training in assay methodology (commonly requiring 3-6 weeks per trainee) is provided by our highly experienced personnel to a continual influx of new staff, research students and visiting scientists. In 1997, the demand for training in Assay Services greatly exceeded the levels of previous years, and was given to six scientists, seven graduate students and seven technicians (a total of 20 trainees). This increased training requirement reflects the greater use of the diverse analytical techniques available in Assay Services by investigators and their staff. Training in assay techniques has now become a large component of Assay Services activities:

Scientists:
Dr. David Fernandez (Neurobiology) - radioimmunoassay of peptide hormones
Dr. Masaharu Mizuno (Neurobiology) - radioimmunoassay of peptide hormones
Dr. Pamela Tannenbaum (Physiological Ethology) - radioimmunoassay of peptide hormones
Dr. Maria Bernardete Cordiero de Sousa (University of Natal, Brazil: Physiological Ethology) - fecal steroid analysis.
Dr. Adrian Treves, (Dept. of Psychology, UW-Madison) - fecal steroid analysis.
Dr. Elke Zimmerman (Dept. of Zoology, University of Hanover, Germany) - consulting on collection and biological validation of cortisol and testosterone in the mouse lemur.

Graduate students:
Ms. Ricki Colman (Aging and Metabolic Diseases) - radioimmunoassay of peptide hormones
Ms. Suzanne Rust (Dept. of Anthropology, UW-Madison) - fecal steroid analysis.
Ms. Anita Ginther (Dept. of Psychology, UW-Madison) - urinary protein and steroid analysis.
Ms. Karen Pazol (Department of Anthropology, University of Pennsylvania) - fecal steroid analysis.
Mr. Kurt Klomberg (Dept. of Psychology, UW-Madison) - serum steroid analysis and celite chromatography.
Ms. Anne Carlson (Dept. of Zoology, UW-Madison) - fecal steroid analysis.
Ms. Cristina Santos (Dept. of Biology, Sao Paulo University, Brazil) - radioimmunoassay of peptide hormones

Technicians:
Ms. Justine Clotfelter, (Physiological Ethology) - fecal steroid analysis
Mr. Scott Baum (Aging and Metabolic Diseases) - serum steroid analysis.
Ms. Kellie Gari (Aging and Metabolic Diseases) - radioimmunoassay of peptide hormones
Ms. Pat Martin (Dept. of Psychology, UW-Madison) - serum steroid analysis.
Ms. Cecily Mui (Dept. of Wildlife Ecology, UW-Madison) - urine and fecal steroid analysis, urinary protein analysis and HPLC.
Ms. Mary Ann Abiado (Dept. of Food Science, UW-Madison) - HPLC.
Mr. Jeremy McBride (Dept. of Anthropology, UW-Madison) - fecal sample preparation.

Improved computerized data analysis and accounting

Computerized data reduction methods and statistical analyses continued to provide the necessary accuracy and efficiency for calculation of assay parameters and results. The calculated results are made readily available for investigators or for transmission to individual researcher's office computers. Messrs. Scheffler and Wegner continued to collaborate with Mr. DuBois (Data Management, Operational Services) to improve the ease and efficiency of data entry and to obtain additional data summaries to permit more efficient monitoring of assay quality control. Summaries of assay determinations made by hormone, by investigator, and by funding source are automatically generated each month by the Primate Center's Sun SPARCstation 2 computer for all of Assay Services' personnel. This computerized summary of Assay Services output permits routine monitoring of demands on the Service, research and development, and monthly billing of investigators (including details of assays requested or performed).

Neurobiology service unit to measure neurotransmitters by HPLC

In our most recent Base Grant renewal, Assay Services had proposed and was approved for establishing an HPLC system fitted with an electrochemical detector to measure neurotransmitter concentrations in a variety of media and tissues. Following meetings with Drs. Abbott, Ziegler and Terasawa (Neurobiology) to discuss re-organization of units within the Primate Center, Dr. Kemnitz decided to form a Neurobiology Service Unit to perform this task. Development of techniques to measure a variety of neurotransmitters will now switch to Dr. Terasawa in Neurobiology.

Primate Center publications that utilized Assay Services in 1997

(1) Kasuya, E., M. Mizuno, G. Watanabe, and E. Terasawa. 1997. Effects of an antisense deoxyoligo-nucleotide for neuropeptide Y mRNA on in vivo LHRH release in ovariectomized female rhesus monkeys. Regulatory Peptide (in press).
(2) Abbott, D.H., D.A. Dumesic, J.R. Eisner, R.J. Colman, and J.W. Kemnitz. 1998. Insights into the development of PCOS from studies of prenatally androgenized female rhesus monkeys. Trends in Endocrinology and Metabolism 9(2): 62-67.
(3) Kemnitz, J., K.A. Holston, and R.J. Colman. 1997. Nutrition, aging and reproduction in rhesus monkeys. Nutrition and Reproduction. LSU Press. New Orleans, LA. (In press.)
(4) Ziegler, T.E., and C.T. Snowdon. 1997. Role of prolactin in paternal care in a monogamous New World primate, Saguinus oedipus. Annals of the New York Academy of Sciences 807: 599-601.
(5) Ziegler, T. 1997. Hormones associated with non-maternal infant care: A review of mammalian and avian studies. Folia Primatologica. (In press).
(6) Ye, F., K. Imamura, N. Imanishi, L. Rhodes, and H. Uno. Effects of Topical Antiandrogen and 5 Alpha-reductase Inhibitors on Sebaceous Glands in Male Fuzzy Rats. Skin Pharmacology.
(7) Abbott, D.H., W. Saltzman, N.J. Schultz-Darken, and P.L. Tannenbaum. 1997. Adaptations to subordinate status in female marmoset monkeys. Comparative Biochemistry Physiology C. (In press.)
(8) Carlson, A.A.., T. E. Ziegler, and C.T Snowdon. Ovarian function of pygmy marmoset daughters in motherless and intact families. American Journal of Primatology (in press).
(9) Porter, T.A., Snowdon, C.T. 1997. Female reproductive status and male pairmate behavior in cotton-top tamarins. Annals of the New York Academy of Sciences 807: 556-558.
(10) Ziegler, T.E., G. Scheffler, A.A. Carlson. 1997. Methods and use of fecal steroid analyses for monitoring reproductive functioning in marmosets and tamarins. A Primatologia No Brasil. 5 (in press).
(11) Strier, K.B. and Ziegler, T.E. 1997. Behavioral and endocrine characteristics of the reproductive cycle in wild muriqui monkeys, Brachyteles arachnoides. American Journal of Primatology 42:299-316.
(12) Ziegler, T.E., Santos, C.V., Pissinatti, A. and Strier, K.B. 1997. Steroid excretion during the ovarian cycle in captive and wild muriquis, Brachyteles arachnoides. American Journal of Primatology; 42:311-321.
(13) Abbott, D.H., Saltzman, W., Schultz-Darken, N.J. and Smith, T.E. 1997. Specific neuroendocrine mechanisms not involving generalized stress mediate social regulation of female reproduction in cooperatively breeding marmoset monkeys. Annals of the New York Academy of Sciences 807:219-238.
(14) Saltzman, W., Schultz-Darken, N. and Abbott, D.H. 1997. Familial influences on ovulatory function in common marmosets (Callithrix jacchus). Am. J. Primatol. 41:159-177.
(15) Terasawa, E., and C.L. Nyberg. 1997. LHRH pulse generation in the monkey: in vivo and in vitro studies. In: Neural control of reproduction: physiology and behavior, K. Maeda, H. Tsukamura, and E. Yokoyama, eds., pp. 57-70. JSS Press, Tokyo.
(16) Saltzman, W., J.M. Severin, N.J. Schultz-Darken, and D.H. Abbott. 1997. Behavioral and social correlates of escape from suppression of ovulation in female common marmosets housed with the natal family. American Journal of Primatology. 41(1):1-21.
(17) Dumesic, D.A., Abbott, D.H., Eisner, J.R. and Goy, R.W. 1997. Prenatal exposure of female rhesus monkeys to testosterone propionate increases serum luteinizing hormone levels in adulthood. Fertility and Sterility. 67(1):155-163.
(18) Abbott, D.H., Dumesic, D.A., Eisner, J.E., Kemnitz, J.W. and Goy, R.W. 1997. The Prenatally Androgenized female rhesus monkey as a model for polycystic ovarian syndrome. In: Androgen Excess Disorders in Women. R. Azziz, J.E. Nestler, and D. Dewailly (eds.), Raven-Lippencott Press (in press).
(19) Saltzman, W., Schultz-Darken, N.J., Severin, J.M. and Abbott., D.H. 1997. Escape from Social Suppression of Sexual Behavior and of Ovulation in Female Common Marmosets, in The Integrative Neurobiology of Affiliation, Annals of the NY Academy of Sciences. 807: 567-570.
(20) Coe, C.L., G.R. Lubach, J.W. Karaszewski, and W.B. Ershler. 1996. Prenatal endocrine activation alters postnatal cellular immunity in infant monkeys. Brain, Behavior, and Immunity. 10. 221-234.
(21) Uno, H. 1997. Age-related pathology and biosenescent markers in captive rhesus macaques. Journal of the American Aging Association. 20:1-13.
(22) Obana, N., Chang, C. and Uno, H. Inhibition of hair growth by testosterone in the presence of dermal papilla cells from the frontal bald scalp of the postpubertal stumptailed macaque. Endocrinology 138:356-361,
(23) Ye, F., Imamura, K., Imanishi, N., Rhodes, L., and Uno, H. Effects of topical antiandrogen and 5 a reductase inhibitors on sebaceous glands in male fuzzy rats. Skin Pharmacology (in press)
(24) Uno, H., Ye, F., Imamura, K., Obana, N., and Bonfils, A. Dose-dependent and long term effects of RU58841 (androgen receptor blocker) on hair growth in the bald stumptailed macaque. J. Invest. Dermatol. 108: 651, 1997
(25) Obana, N., Allen-Hoffmann, L., Pan, H., and Uno, H. The role of dermal papilla cells in macaques alopecia in testosterone-dependent inhibition of human keratinocytes. J. Invest. Dermatol. 108: 653, 1997
(26) Uno, H., M, Franklin., G. Kramer., S. Shelton, and N. Kalin. Gender differences in brain volume and size of corpus callosum and amygdala of rhesus monkey measured from MRI images. Neuroscience Abst. 23: 863, 1997
(27) Marshall, V.S., J. Kalishman, J.A. Thomson. 1997. Nonsurgical embryo transfer in the common marmoset monkey. Journal of Medical Primatology 26: 241-247.
(28) Ramsey, Jon J., R.J. Colman, A.G. Swick, and J.W. Kemnitz. 1997. Energy expenditure, body composition, and glucose metabolism in lean and obese rhesus monkeys treated with ephedrine and caffeine. American Journal of Clinical Nutrition (in press).
(29) Sousa, M.B. and Ziegler, T.E. 1998. Diurnal variation on the excretion patterns of fecal steroids in common marmoset, Callithrix jacchus, females. Am. J. Primatol. (in press).
(30) Kapke, C.A., Arcese P., Ziegler T.E. and Scheffler G.R. 1998. Identification and measurement of estradiol and progesterone metabolites in the feces of white-tailed deer (Odocoileus virginanus). Journal of Zoo and Wildlife Medicine. (in press).
(31) Coe, C. L., F. Hou, and A. S. Clarke. 1996. Fluoxetine treatment alters leukocyte trafficking in the intrathecal compartment of the young primate. Biological Psychiatry. 40:361-367.
(32) Kalin, N.H., S.E. Shelton, M. Rickman, R.J. Davidson. 1997. Individual differences in freezing and cortisol in infant and mother rhesus monkeys. Behav Neurosci (in press).
(33) Kalin, N.H., C. Larson, S.E. Shelton, R.J. Davidson. 1997. Asymmetric frontal brain activity, cortisol, and behavior associated with fearful temperaments in rhesus monkeys. Behav Neurosci (in press).
(34) Kalin, N.H., S.E. Shelton. 1997. The ontogeny of separation and threat-induced defensive behaviors in rhesus monkeys during the first year of life. Am J Primatology (in press).
(35) Kalin NH, Shelton SE. 1998. The regulation of defensive behaviors in rhesus monkeys: implications for understanding anxiety disorders. Wisconsin Symposium on Emotion Volume 1.

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