Detection of urinary gonadotropins in callitrichid monkeys with a sensitive immunoassay based upan a unique monoclonal antibody.

A radioimmunological method for measuring urinary luteinizing hormone (LH) and chorionic gonadotropin (CG) excretion in the family Callitrichidae is described. The method uses a monoclonal antibody that will be available in virtually unlimited quantity. Several polyclonal antisera that have been useful for the detection of callitrichid gonadotropins are near depletion. The monoclonal antibody-based RIA provided similar results when compared with the mouse Leydig cell bioassay for LH and a previously validated polyclonal antibody-based RIA. When the monoclonal antibody is used for immunodetection of Saguinus oedipus LH by non-reducing SDS-PAGE, a single entity is recognized that corresponds with the molecular weight range of bioactive LH and appears to be in the normal range for LH in nonhuman primates. LH and CG were detected by the monoclonal antibody-based RIA in urine from representatives of species from all genera of callitrichids. Hormonal profiles of daily urine samples revealed the detection of the preovulatory LH surge by both RIA methods in the cotton-top tamarin (Saguinus Oedipus) and pygmy marmoset (Cebuella pygmea) and the increase CG due to pregnancy in both species. Serial dilutions of midcycle and pregnancy urine from Saguinus, Callithrix, Leontopithecus, and Cebuella exhibited parallelism when compared with our in-house reference standard of rhesus monkey CG. Cultured Saguinus oedipus pituitary cells showed an increased release of LH when challenged by gonadotropin releasing hormone (GnRH) providing further support that the monoclonal antibody-based RIA measures LH in this species. © 1993 Wiley-Liss, Inc.

Biochemical and biological properties of urinary follicle-stimulating hormone (FSH) from the rhesus monkey (Macaca mulatta).

Follicle-stimulating hormone (FSH) is routinely used for the induction of superovulation in women. Homologous gonadotropin preparations that could be used for reproductive studies in macaques would have valuable research applications. Accordingly, we set out to characterize the physical and biological characteristics of urinary FSH (UFSH) in the ovariectomized rhesus monkey. In urine from 7 monkeys, concentrations of bioactive FSH ranged from 16 to 57 µg/1, relative to cynFSH-RPI (NIDDK). UFSH was contrasted to pituitary FSH (PFSH) by non-reducing SDS-polyacrylamide gel electrophoresis (PAGE), native disc PAGE, and FPLC chromatofocusing. The apparent molecular weights of UFSH and PFSH are similar (approximately 35 kD); however, UFSH is more negatively charged and demonstrates a lower overall isoelectric (pl) range than PFSH. The bioactivity of UFSH was assessed by the stimulation of aromatase activity in cultured Sertoli cells and by induction of follicular maturation in hamsters. Two fractions of pituitary FSH, which differed in isoelectric properties, were obtained by chromatofocusing. The in vivo biological activity of FSH-A (acidic, pl 3.8-4.6) and UFSH (pl 3.5-4.5) were similar, but greater than FSH-B (basic, pl 4.6-5.5). These results support the hypothesis that heavily sialylated, low pl FSH expresses high in vivo bioactivity. This may reflect the well-known effect of sialic acid in prolonging the circulating half-life of glycoproteins. Thus, the quality and quantity of FSH present in ovariectomized rhesus monkey urine indicates that this may be a useful source for the preparation of enriched hormone preparations. © 1992 Wiley-Liss, Inc.

The secretion of bioactive and immunoreactive follicle-stimulating hormone (FSH) and luteinizing hormone (LH) throughout the menstrual cycle of the rhesus monkey (Macaca mulatta).

The present study provides the first evaluation of related changes in serum levels of bioactive FSH (Bio FSH) and immunoreactive FSH (iFSH), and concurrent dynamics of LH and FSH bioactivity throughout the menstrual cycle of the rhesus monkey. Mean concentrations of Bio FSH were elevated on days 0 and 1 (n = 7; P < 0.05; day 0 = preovulatory LH surge). Data from individual animals revealed that an average (± SEM) of 1.43 ± 0.29 and 2.71 ± 0.61 discrete surges of Bio FSH occurred in each monkey's follicular and luteal phase, respectively. Analysis of the collective data indicated that periods of increased Bio FSH secretory activity spanned days -1 to 1 and 8 to 10 (P < 0.025). Increases in serum Bio FSH and iFSH concentrations were not precisely correlated on a daily basis (38.9%), although 72.2% of the peaks of Bio FSH and iFSH surges occurred within a day of one another. Similarly, only 36.1% of the Bio FSH surges were accompanied by elevations in bioactive LH (Bio LH). A significant rise in Bio LH, but not Bio FSH, occurred on day -1 (P < 0.01). Concentrations of Bio LH, but not Bio FSH, were elevated in the early luteal phase (P < 0.01). The bioactivity/immunoactivity ratios (Bio/I) of LH and FSH were maximal on the day of the preovulatory surge (P < 0.01). On day -1, LH Bio/I significantly increased (P < 0.05), but no change in FSH Bio/I was detected. The Bio/I of LH, but not FSH, remained elevated in the early luteal phase. In summary: the relative increase in Bio FSH exceeds iFSH during the preovulatory surge. Surges of Bio FSH occur during the follicular and luteal phases which potentially could support follicle selection/maturation. Divergencies between circulating LH and FSH biopotency may reflect a differential regulation of secretion and/or biosynthesis of these hormones. The prolonged early luteal elevation of LH Bio/I is consistent with the idea of a functional role of elevated LH biopotency in the maintenance of the corpus luteum.

Detection of nonhuman primate gonadotropins in polyacrylamide gels: An alternative to the western blot.

The lack of appropriate antibodies restricts the use of western blots in studies of nonhuman primate gonadotropins. We now present the evaluation of an alternative method that can be applied in situations in which antibody is limiting or nonexistent. The use of nonreducing sodium dodecyl sulfate­polyacrylamide gel electrophoresis (SDS­PAGE) sample buffer (Swack et al., 1987) retains a significant degree of α­ß subunit association, as determined by the western blot analysis of human and rhesus luteinizing hormone (LH; 33 and 36 kD, respectively). This observation was confirmed when eluates of gel slices were analyzed by specific gonadotropin immuno­ and bioassays. The molecular weight of rhesus LH in pituitary protein was found to be 36 kD by bioassay and immunoassay. Similarly, the molecular weight of rhesus follicle­stimulating hormone (FSH) did not differ between assay systems (36 kD). The analysis of chorionic gonadotropin (CG)/LH activity in gel eluates of pregnant rhesus and tamarin urinary protein revealed major activity at 43 kD, with lesser bands between 34 and 39 kD. Guanidine­induced dissociation of gonadotropin subunits in pregnant rhesus urinary protein resulted in the disappearance of dimer at 43 and 36 kD and the appearance of 25 and 16 kD subunit peaks. This alternative to the western blot is not limited to SDS­PAGE. Native gel electrophoresis (no SDS) showed that rhesus FSH possesses a greater negative charge than rheus LH (Rf = 0.49 vs. 0.35). Isoelectric focusing PAGE resolved distinct isoforms of rhesus LH and FSH (pl range 5.2­7.2 and 4.0­5.8, respectively). The electrophoretic analysis of nonhuman primate gonadotropins in impure protein samples will provide fundamental data on the comparative biochemistry of these hormones. Applications will be found in biomedical and comparative studies.

Determination of bioactive FSH in rhesus monkeys (Macaca mulatta).

Studies of the reproductive biology of nonhuman primates are commonly hindered by the lack of homologous, sensitive immunoassay systems for gonadotropins. The recent development of a bioassay based upon folliclestimulating hormone (FSH)­stimulated estradiol production by rat Sertoli cells (Padmanabhan et al.: Endocrinology 121:1089­1098, 1987) offers a novel opportunity to expand our knowledge of the physiological importance and regulation of this important reproductive hormone in these species. Accordingly, we have established the utility of the rat Sertoli cell FSH bioassay to study the reproductive biology of rhesus monkeys. Analysis of medium from cultured rhesus monkey pituitary cells demonstrates the ability of this assay system to specifically and sensitively detect macaque FSH. Serial dilutions of unextracted urinary samples stimulate estradiol in parallel with the reference standard. The pattern of bioactive FSH in daily urine samples throughout the menstrual cycle resembles that normally observed in serum, displaying a clear preov alatory gonadotropin surge. In attempts to analyze rhesus monkey serum, severe interference with cellular estradiol production was found with extremely low serum sample volumes. In order to circumvent this problem, a serum extraction procedure utilizing metaphosphoric acid was developed which permits the accurate determination of bioactive FSH in serum. The pattern of serum bioactive FSH secretion throughout the menstrual cycle is in general agreement with that observed by FSH RIA, although immunoreactive FSH values are consistently higher. The ability to detect bioactive FSH in macaques will significantly enhance our research capabilities with nonhuman primate species which share many physiological similarities with man. Important applications also may be found in studies on the reproductive biology in endangered species of primates.