Recognition by recombinant autoimmune thyroid disease-derived Fab fragments of a dominant conformational epitope on human thyroid peroxidase.

To characterize the nature of thyroid peroxidase (TPO) autoantibodies present in the sera of patients with autoimmune thyroid disease, we cloned three IgG1/kappa Fab fragments which bind 125I-TPO. This was accomplished by the molecular cloning and expression in bacteria of IgG gene fragments from B cells infiltrating the thyroid of a patient with Graves' disease. The three Fab fragments (SP2, SP4, and SP5) are coded for by a common heavy chain (VH1, D, JH3) and three related, but different, light chains (VK1, JK2). The SP Fab fragments bind specifically to TPO with high affinities (6 x 10(-11)-2 x 10(-10) M) comparable to those of serum TPO autoantibodies. TPO autoantibodies represented by the SP Fab fragments are present in all 11 patients studied, constitute a high proportion (36-72%) of serum TPO autoantibodies in individual patients and interact with a conformational epitope on TPO.

Human organ-specific autoimmune disease. Molecular cloning and expression of an autoantibody gene repertoire for a major autoantigen reveals an antigenic immunodominant region and restricted immunoglobulin gene usage in the target organ.

The most common organ-specific autoimmune disease in humans involves the thyroid. Autoantibodies against thyroid peroxidase (TPO) are present in the sera of virtually all patients with active disease. We report the molecular cloning of the genes for 30 high-affinity, IgG-class human autoantibodies to TPO from thyroid-infiltrating B cells. Analysis of the putative germline genes used for the TPO human autoantibodies suggests the use of only five different H and L chain combinations involving four H chains and three L chains. In addition, the same combination of H and L chains was found in multiple patients. The F(ab) proteins expressed by these genes define two major, closely associated domains (A and B) in an immunodominant region on TPO. These A and B domains contain the binding sites of approximately 80% of IgG-class TPO autoantibodies in the sera of patients with autoimmune thyroid disease. The present information permits analysis, not previously possible, of the relationship between autoantibody H and L chain genes and the antigenic domains on an autoantigen. Our data, obtained using target organ-derived autoantibodies, indicate that there is restriction in H and L chain usage in relation to the interaction with specific antigenic domains in human, organ-specific autoimmune disease.

The effect of cessation of growth on protein synthesis in a mutant of Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase.

A temperature-sensitive mutant of Chinese hamster ovary cells with an altered leucyl-tRNA synthetase fails to grow and to incorporate amino acids into protein properly at or near the non-permissive temperature. This mutant was used to determine whether cessation of growth at the elevated temperature affected elongation factor EF-1, since the activity of EF-1 is markedly lower in non-growing cells in stationary phase than in rapidly-growing cells in exponential phase. Cell-free extracts prepared from cells maintained at 39 degrees C for 24 h showed a marked decrease in the ability to translate natural mRNAs, compared to cells incubated at 34 degrees C. However, the ability to translate poly(U), which requires elongation factor EF-1 (and EF-2), was not affected. Analyses of activities involved in the initiation of protein synthesis and in the activation of amino acids revealed that, with the exception of leucyl-tRNA synthetase, the rest of the components required for translation also appeared to be relatively stable even after 24 h at the elevated temperature. The effects of elevated temperature on cell-free extracts were also investigated. The results were similar to those obtained with intact cells; that is, except for leucyl-tRNA synthetase which was rapidly inactivated in vitro at 39 degrees C, other aminoacyl-tRNA synthetases and translational components involved in chain initiation and elongation were relatively stable. Thus, no change in EF-1 activity was detected as a result of arrested cell growth, an inherent lability of the elongation factor, or metabolic degradation as a consequence of a rapid turnover rate in the absence of protein synthesis.

Kinetics for changes in enzyme synthesis and mRNA content and hormones required for induction of 6-phosphogluconate dehydrogenase in hepatocytes.

Rats fasted for 2 days were refed a 60% glucose diet for varying periods of time in order to follow the kinetics for changes in 6-phosphogluconate dehydrogenase synthesis and mRNA content. Hepatocytes isolated from control or induced rats were incubated with actinomycin D and the rate of decline in 6-phosphogluconate dehydrogenase mRNA was determined by translating RNA in a nuclease-treated reticulocyte lysate. The half-life for 6-phosphogluconate dehydrogenase mRNA under both of these conditions was about 2 h. Thus, increases in transcription or the processing of nuclear RNA may increase 6-phosphogluconate dehydrogenase mRNA during the dietary induction of this enzyme. Hepatocytes prepared from fasted rats were cultured with 5% serum and various hormones and energy sources. If hepatocytes were isolated from thyroidectomized rats and cultured in serum from a thyroidectomized calf, the 4-fold induction of 6-phosphogluconate dehydrogenase was primarily dependent upon added insulin. In the presence of optimal insulin concentrations (10(-7) M) triiodothyronine slightly stimulated 6-phosphogluconate dehydrogenase induction. The gut hormones somatostatin and secretin had no effect on 6-phosphogluconate dehydrogenase induction in cultured hepatocytes. Hepatocytes cultured in carbohydrate-free medium and 5% serum required added insulin for maximal induction. 8-Br-cGMP did not significantly affect 6-phosphogluconate dehydrogenase induction in hepatocytes either in the presence or absence of added insulin. Dibutyryl cAMP did not alter the time course or extent of 6-phosphogluconate dehydrogenase induction in cultured hepatocytes. We have concluded that under these conditions insulin is a potent signal regulating the levels of 6-phosphogluconate dehydrogenase mRNA and that this induction is not mediated by cyclic nucleotides.

The rhesus monkey corpus luteum is dependent on pituitary gonadotropin secretion throughout the luteal phase of the menstrual cycle.

Experiments were conducted in rhesus monkeys to determine whether the corpus luteum of the menstrual cycle requires pituitary gonadotropin for progesterone production and normal functional lifespan. Eight adult females were rendered anovulatory by placement of radiofrequency lesions in the arcuate region of the medial basal hypothalamus. Endogenous gonadotropin secretion and ovulatory cycles were reestablished by chronic pulsatile infusion of GnRH. Control luteal phases exhibited typical plasma progesterone patterns and ranged from 14-17 days in length. In experimental cycles, endogenous gonadotropin secretion was interrupted during the luteal phase by stopping the infusion of GnRH. When the GnRH infusion was stopped in the early luteal phase (3 days after the preovulatory estradiol peak; day 3), plasma LH fell to undetectable levels within 90 min. Plasma progesterone concentrations (1.5 +/- 0.4 ng/ml) declined to undetectable levels (less than 0.2 ng/ml) by the afternoon of day 5 (P less than 0.05). Premature menses occurred 2-5 days later. When the GnRH infusion was stopped in the midluteal phase (day 8), plasma LH fell below the limits of detectability within 150 min. Circulating progesterone (4.5 +/- 1.0 ng/ml) declined to undetectable levels by the afternoon of the following day. Premature menses occurred 3 days after the fall in plasma LH, 11 days after the preovulatory estradiol peak. Plasma LH and progesterone remained undetectable as long as exogenous GnRH was withheld (18 days), and progesterone did not reappear until the next GnRH-induced ovulatory cycle. These results demonstrate that the normal functional lifespan of the primate corpus luteum requires the presence of circulating pituitary gonadotropin during both the early (developmental) and middle (fully functional) stages of the nonfertile luteal phase.

The corpus luteum of the primate menstrual cycle is capable of recovering from a transient withdrawal of pituitary gonadotropin support.

To further our understanding of the role of pituitary gonadotropin secretion in the control of corpus luteum function during the primate menstrual cycle, we have used an experimental model which enables us to directly control pituitary gonadotropin secretion throughout the luteal phase. Specifically, we have asked whether cessation of progesterone secretion, or functional luteolysis, resulting from a 3-day withdrawal of gonadotropin support, culminates in an irreversible loss of luteal responsiveness to further gonadotropic stimulation; and do the effects of gonadotropin deprivation vary with the age of the corpus luteum? Endogenous gonadotropin secretion was abolished in seven adult rhesus monkeys by placing radiofrequency lesions in the arcuate nucleus region of the medial basal hypothalamus. Endogenous gonadotropin secretion and ovulatory menstrual cycles were then restored by chronic pulsatile infusion of GnRH (1 pulse/h). Control luteal phases supported by this GnRH regimen exhibited typical plasma progesterone patterns and ranged from 14-17 days in length. In experimental cycles, endogenous gonadotropin secretion was interrupted for a 3-day period during the early (days 2-5), mid (days 8-11), or late (days 13-16) stages of the luteal phase. During the GnRH deprivation period, bioassayable and immunoreactive serum LH was undetectable. The disappearance of circulating LH was followed by a rapid fall in plasma progesterone levels regardless of the stage of the luteal phase. The restoration of gonadotropin secretion resulted in a resumption of progesterone secretion when the gonadotropin deprivation period was imposed during the early or midluteal phase. In each instance, the resumption of progesterone secretion continued for a period of time which effectively completed the typical 14- to 17-day functional lifespan of the corpus luteum of the menstrual cycle. Thus, the luteal phase was neither shortened nor lengthened by a 3-day interruption of luteal function resulting from withdrawal of gonadotropic support. When gonadotropin secretion was interrupted during the late luteal phase (days 13-16), restoration of gonadotropin secretion on day 16 did not result in resumption of progesterone secretion. Our findings confirm our earlier demonstration that progesterone secretion during the luteal phase of the non-fertile menstrual cycle is dependent on pituitary gonadotropic support.(ABSTRACT TRUNCATED AT 400 WORDS)

The relationship between the rat of testosterone infusion and gonadotropin secretion.

Prepubertal and young adult male rats were castrated and continuous intravenous infusions of testosterone were administered for 2-3 days. Blood samples were obtained at various intervals and serum concentrations of FSH and LH were determined. In 43-day-old males a dose of approximately 57 mug/day was required to suppress serum LH to levels observed in intact controls, and FSH was also suppressed at this dose. Within one day after termination of infusions serum LH and FSH concentration had returned to (or exceeded) the levels observed in castrates infused with vehicle only. This was true even after a very large dose of testosterone had been administered (918 mug/day). In 59-day-old males serum LH was suppressed somewhat by 40 mug testosterone/day and further by 200 mug/day. FSH did not appear to be suppressed as readily in these rats as compared to the younger animals. Further experiments in which castrated rats were injected with testosterone or testosterone propionate revealed marked differences in the magnitude and time course of the action of these compounds upon LH release. It is suggested that the rate of metabolism of these steroids is important in limiting their effects, especially when administered by single daily injections. Furthermore, it appears that "recovery" of the hypothalamic-pituitary system from exposure to high concentrations of testosterone is rapid following clearance of the steroid from the blood.

Regulation of serum gonadotropins by photoperiod and testicular hormone in the Syrian hamster.

Male hamsters were maintained on long (14L:10D) or short (10L:14D) photoperiods. The serum concentrations of LH and FSH were reduced in the animals kept on the short photoperiod, and these animals had atrophied testes and sex accessories. Serum gonadotropin concentrations increased following castration in both long and short photoperiods, but gonadotropin secretion was inhibited by much smaller doses of testosterone in the males maintained on the short photoperiod as compared to males kept on a long photoperiod. The levels of testosterone required to suppress serum gonadotropin levels in castrated animals corresponded reasonably well with the serum androgen levels observed in intact males, suggesting that serum androgen is a major regulator of gonadotropin secretion in the male hamster.

Estradiol as a gonadotropin releasing hormone in the rhesus monkey.

The temporal relationship between cessation of GnRH delivery to the pituitary gland and the loss of responsiveness to the stimulatory action of estradiol (E2) was examined in 4 ovariectomized rhesus monkeys whose endogenous GnRH production had been abolished by hypothalamic lesions. Gonadotropin secretion was re-established by the intermittent administration of GnRH. The GnRH-replacement regimen was then discontinued and estradiol benzoate (EB) injected 24, 48, 72 and 96 hours later. Unambiguous gonadotropin discharges were induced when EB was administered 24 or 48 hours after discontinuation of GnRH replacement. We conclude that E2 can initiate gonadotropin discharges in the absence of circulating GnRH. E2 may, therefore, be viewed as a gonadotropin releasing hormone.

Interference with the gonadotropin-suppressing actions of estradiol in macaques overrides the selection of a single preovulatory follicle.

We examined the role of the gonadotropin-suppressing effects of estradiol on the maturation of a single ovulatory follicle in cynomolgus monkeys (Macaca fascicularis) by administering ovine antiestradiol antibodies during the mid through late follicular phase of the menstrual cycle. In each of three control animals, when the ovary containing the maturing follicle was removed during the late follicular phase, histological examination of the remaining ovary 10 days later revealed the presence of a single large maturing follicle. In contrast, in three experimental animals, when estradiol antibodies were infused from days 5 through 10 after unilateral ovariectomy, serum FSH and LH concentrations were elevated above those of control animals, and histological examination of ovaries 10 days after unilateral ovariectomy revealed the presence of two large maturing follicles in the remaining ovary of two animals and four large maturing follicles in the remaining ovary of the third animal. The ability of follicles recruited during passive immunization with estradiol antibodies to respond to exogenous gonadotropin was studied. In three control animals, the maturing follicle was destroyed on day 10 of the follicular phase, and 3 days later, each animal received an ovulatory dose of human CG. None of these control animals produced progesterone. In three experimental animals a continuous infusion of estradiol antibodies was initiated on day 5 of the follicular phase, and the largest antral follicle was destroyed on day 10. Three days thereafter the antibody infusions were terminated and each animal received an ovulatory dose of human CG. Each of these animals produced progesterone despite the destruction of the largest follicle 3 days earlier. These observations demonstrate that estradiol is the principal ovarian modulator of gonadotropin secretion during the follicular phase of the cycle and that interference with the gonadotropin-suppressing actions of estradiol results in continued recruitment and maturation of secondary follicles in the presence of a dominant follicle.

Effects of different gonadotropin pulse frequencies on corpus luteum function during the menstrual cycle of rhesus monkeys.

In the nonfertile menstrual cycle, the frequency of episodic LH secretion declines from approximately 1 pulse/h in the early luteal phase to 1 pulse/4-8 h in the mid- to late luteal phase, but the relevance of this phenomenon to the initiation of functional luteal regression is not completely understood. We investigated whether a reduction in LH pulse frequency causes a decline in luteal progesterone production by experimentally reducing LH pulse frequency during the early luteal phase, and measured the effects on the subsequent plasma progesterone pattern and the onset of luteal regression. Rhesus monkeys were rendered anovulatory by placing radiofrequency lesions in the arcuate region of the medial basal hypothalamus or surgically transecting the hypothalamic-pituitary stalk. Endogenous gonadotropin secretion and ovulatory menstrual cycles were restored by pulsatile infusion of synthetic GnRH at a frequency of 1 pulse/h. Commencing on days 3-6 of the luteal phase, GnRH frequency was changed to either 1 pulse/8 h (four animals) or 1 pulse/24 h (four animals), or maintained at the standard 1 pulse/h frequency (four animals). Luteal phases of 13- to 17-day duration were observed in all animals kept on the 1 pulse/h frequency and in three of four animals in which the frequency was changed to 1 pulse/8 h on day 3 of the luteal phase. Daily midluteal phase (days 5-10) plasma progesterone levels observed in response to the 1 pulse/h and 1 pulse/8 h infusion regimens were similar (mean +/- SE, 4.1 +/- 0.4 vs. 3.2 +/- 0.4 ng/ml; P greater than 0.1). In contrast, short luteal phases were observed in all animals after the LH pulse frequency was reduced to 1 pulse/24 h. Comparison of plasma LH responses to a representative GnRH pulse of each GnRH infusion regimen revealed that the maximal LH levels attained in response to 1 pulse/8 h (47.5 +/- 11.5 ng/ml) were significantly greater (P less than 0.05) than the maximal LH levels attained in response to 1 pulse/h (30.5 +/- 3.2 ng/ml) or 1 pulse/24 h (27.2 +/- 5.0 ng/ml). Progesterone levels remained elevated for 140-200 min after the LH pulse resulting from the 1 pulse/8 h infusion regimen. In response to the 1 pulse/24 h infusion regimen, plasma progesterone levels remained elevated for 60 min after the LH pulse.(ABSTRACT TRUNCATED AT 400 WORDS)

Estrogen induces premature luteal regression in rhesus monkeys during spontaneous menstrual cycles, but not in cycles driven by exogenous gonadotropin-releasing hormone.

Administration of exogenous estradiol during the mid- to late luteal phase of the menstrual cycle results in premature regression of the corpus luteum. The present study was initiated to identify the site of action of estrogen as well as to determine why administration of estrogen during the early luteal phase of the menstrual cycle does not result in luteolysis. Based upon extant literature, we hypothesized that estrogen and progesterone synergize to promote premature luteal regression. We tested this hypothesis in intact, spontaneously cycling rhesus monkeys by inserting estradiol, progesterone, or estrogen plus progesterone capsules on days 2 through 6 of the luteal phase. Insertion of estrogen or progesterone capsules alone did not advance luteolysis compared with the effect of control empty implants (n = 3). In contrast, insertion of estrogen plus progesterone implants on days 2 through 6 of the luteal phase resulted in a significant lowering of serum progesterone concentrations, and menses was advanced 5-6 days compared with control cycles. On the basis of these findings in spontaneously cycling monkeys, we speculated that estrogen treatment causes luteal regression only in the presence of a progesterone-mediated decrease in LH pulse frequency. To test this hypothesis, we used rhesus monkeys whose endogenous gonadotropin secretion was abolished by either placement of radiofrequency lesions in the mediobasal hypothalamus or transection of the hypothalamic-pituitary stalk. Ovulatory menstrual cycles were restored by pulsatile administration of exogenous synthetic GnRH. Insertion of estradiol capsules during the luteal phase into animals whose gonadotropin pulse frequency was set at either one pulse per h or one pulse per 8 h failed to cause premature luteal regression (n = 4). These findings indicate that whereas estrogen promotes luteal regression in intact, spontaneous cycling rhesus monkeys, it does not do so in animals whose gonadotropin secretion is controlled by exogenous GnRH. On the basis of these observations, we conclude that the hypothalamus is a major site of action of estrogen in the initiation of luteal regression in macaques.

Hypophysiotropic signal frequency and the functioning of the pituitary-ovarian system in the rhesus monkey.

In adult rhesus monkeys bearing hypothalamic lesions that abolished secretions of LH and FSH, normal circulating levels of these hormones and consequent follicular development culminating in ovulation were reestablished by the administration of a GnRH pulse once every hour (control frequency). We examined the effects of slowing the frequency of these pulses on ovarian follicular development, as assessed by circulating concentrations of estradiol. A reduction in frequency to one pulse every 90 min supported follicular development in most instances, albeit with a diminished incidence of ovulation. One pulse of GnRH every 2 h resulted in anovulatory follicular cycles with lower peak concentrations of estradiol than those achieved on the control frequency. Follicular development was absent when GnRH pulses were delivered once every 3 h. When mean concentrations of gonadotropins were determined during periods of low circulating estradiol levels, plasma FSH concentrations in monkeys receiving GnRH pulses at the slow rates did not differ from control, whereas LH concentrations were significantly reduced. It can be concluded that small reductions in the frequency of GnRH stimulation have profound effects on the quality of follicular development, even when FSH concentrations are maintained at normal levels.

On the site of action of progesterone in the blockade of the estradiol-induced gonadotropin discharge in the rhesus monkey.

The site of action of progesterone (P) in the blockade of estradiol-induced gonadotropin discharges was examined in rhesus monkeys bearing hypothalamic lesions which abolish endogenous GnRH production. Normal ovulatory menstrual cycles were re-established in these animals by the pulsatile, hourly administration of GnRH. In the follicular phase of these induced menstrual cycles, P-containing Silastic capsules were implanted sc yielding luteal phase plasma P concentrations which normally block estradiol-induced gonadotropin surges. P failed to block estradiol-induced surges in lesioned animals on GnRH replacement. In such animals, however, P advanced the initiation of these surges. While estradiol acts on the pituitary to cause gonadotropin discharges, P appears to block this effect by acting on the central nervous system. On the other hand, the results also suggest that the site of facilitatory action of P on gonadotropin release is at the level of the pituitary gland.

Frequency and amplitude of gonadotropin-releasing hormone stimulation and gonadotropin secretion in the rhesus monkey.

In adult ovariectomized rhesus monkeys bearing hypothalamic lesions which reduced circulating LH and FSH to undetectable levels, sustained elevated gonadotropin concentrations were reestablished by the intermittent administration of gonadotropin-releasing hormone (GnRH) at the rate of 1 microgram/min for 6 min once every hour. The effects of varying either the frequency or the amplitude of these GnRH pulses on gonadotropin secretion were examined in such animals. Increasing the frequency of GnRH administration from the physiological one pulse per h to two, three, or five pulses h while maintaining a constant infusion rate and pulse duration resulted in gradual declines in plasma gonadotropin concentrations. These declines were most profound at the highest frequencies and the consequence of reduced pituitary responses to individual GnRH pulses. Decreasing the frequency of GnRH pulses from one per h to one every 3 h led to variable declines in plasma LH levels, but circulating FSH invariably rose. Reducing the GnRH infusion rate from 1 to 0.1 mg/min while maintaining constant frequency and pulse duration resulted in abrupt declines in plasma LH and FSH to immeasurable levels, although pulsatile increments in circulating GnRH concentrations without a concomitant reduction in plasma LH concentrations, which remained unchanged. An infusion rate of 0.5 microgram/min resulted in unstable plasma LH and FSH levels. These results demonstrate that changes in the frequency or amplitude of hypophysiotropic stimulation have profound effects on plasma gonadotropin levels as well as on FSH to LH ratios in the circulation. The physiological implications of these observations are discussed.

Administration of human luteinizing hormone (hLH) to macaques after follicular development: further titration of LH surge requirements for ovulatory changes in primate follicles.

After stimulation of multiple follicular development, endogenous LH surges elicited by GnRH or GnRH agonist were of insufficient duration (4-14 h) to evoke oocyte maturation and luteinization in this species. In this study, periovulatory LH surge requirements were further titrated using hLH as the ovulatory stimulus. Beginning at menses, rhesus monkeys were treated with human gonadotropins for 9 days to stimulate follicular growth. To induce ovulatory maturation on day 10, animals received: 1) hCG (1000 IU, im; n = 8); 2) highly-purified, urinary hLH (2542 IU, im; n = 4); or 3) hLH (2542 IU, im) followed by three injections of hLH (200 IU, im) at 8-h intervals (0800, 1600, 2400 h) daily during the luteal phase until menses (n = 3). Oocytes and luteinizing granulosa cells were obtained via follicle aspiration 27 h after the initial hLH or hCG injection. Estradiol and progesterone levels were measured in daily serum samples by RIA. Bioactive LH levels were determined at selected intervals within 36 h of the hLH ovulatory stimulus. Nuclear maturity of oocytes was evaluated as an indicator for reinitiation of meiosis. Luteinizing granulosa cells were processed for indirect immunocytochemistry using a monoclonal antibody to human progesterone receptor. In vitro progesterone production by luteinizing granulosa cells over 24 h was also assessed in the absence and presence of hCG. In all groups, serum estradiol rose to similar peak levels on day 10. After hLH, bioactive LH levels peaked (1262 +/- 79 ng/mL; mean +/- SEM) by 2-6 h, declined thereafter but remained above surge levels (100 ng/mL) for 18-24 h. Within 24 h of hLH injection, serum progesterone increased to 13 +/- 3 nmol/L, but returned to baseline in 1-6 days. In contrast, higher levels of progesterone were observed after hCG (114 +/- 51 nmol/L) and during luteal phase treatment with hLH (137 +/- 25 nmol/L) and the luteal phase was longer (11.5 +/- 0.4 and 14.3 +/- 0.7 days, respectively). Of the total cohort of oocytes aspirated, the proportion of oocytes resuming meiotic maturation (metaphase I plus metaphase II) was similar after hCG (76%) and hLH (74%). However, the proportion of oocytes maturing to metaphase II tended to be less (P = 0.08) after hLH (13%) than hCG (22%). Fertilization rates were similar between the two groups. Progesterone receptor was detected in nuclei of luteinizing granulosa cells from all animals receiving hCG, but only in some given hLH.(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulation of primate luteal function by recombinant human chorionic gonadotropin and modulation of steroid, but not relaxin, production by an inhibitor of 3 beta-hydroxysteroid dehydrogenase during simulated early pregnancy.

CG produced by fetal tissues extends the functional lifespan of the primate corpus luteum during early pregnancy. Previous studies showed that urinary hCG administered to monkeys to simulate the rising CG levels associated with early pregnancy enhanced both progesterone (P) and relaxin (RLX) production by the corpus luteum. The current study was designed: 1) to compare the ability of recombinant (r) and urinary (u) hCG to stimulate luteal function, and 2) to assess the role of P in the regulation of luteal RLX secretion during simulated early pregnancy by concomitant administration of hCG and the 3 beta-hydroxysteroid dehydrogenase inhibitor trilostane to reduce P production. Rhesus monkeys received injections of either r-hCG or u-hCG (Ares Serono) in increasing doses (15-2880 IU/dose, twice daily) for 9 days beginning on day 9 of the luteal phase (n = 5/group). An additional group (n = 4) received r-hCG as described above, with concomitant oral administration of trilostane (500 mg/dose twice daily; Sanofi Winthrop). Daily serum samples were assayed for hCG by immunoradiometric assay, steroid hormones by RIA, and RLX by enzyme-linked immunosorbent assay. Serum hCG levels typically were not different between the r-HCG and u-hCG groups during or after treatment. Concentrations of hCG peaked 1 day after the final injection in monkeys receiving r-hCG (mean +/- SEM. 2759 +/- 120 mIU/mL) and u-hCG (2120 +/- 60 mIU/mL) and dropped below 5 mIU/mL by 10 days after the final treatment in all groups. Both r-hCG and u-hCG stimulated luteal P and RLX production. Progesterone levels rose rapidly after the initiation of hCG treatment and peaked in animals receiving r-hCG (14.4 +/- 2.8 ng/mL) and u-hCG (11.9 +/- 1.4 ng/mL) 4 days after initial administration. RLX levels peaked in the r-hCG (400 +/- pg/mL) and u-hCG (323 +/- 85 pg/mL) groups within 4 days of the final hCG treatment. Trilostane with r-hCG reduced P concentrations to very low levels (< 0.5 ng/mL; P < 0.01) within 1 day of administration compared to those in animals receiving r-hCG only and maintained these low levels for the entire treatment interval. Nevertheless, trilostane administration did not alter luteal RLX production, with serum levels peaking at 377 +/- 76 pg/mL. These data indicate that r-hCG and u-hCG were equally efficacious in stimulating the steroidogenic and peptidergic activities of the corpus luteum during simulated early pregnancy. In addition, P deprivation during r-hCG administration did not alter circulating RLX levels, suggesting that P is not a major regulator of RLX production by the primate corpus luteum during early pregnancy.

Initiation of periovulatory events in primate follicles using recombinant and native human luteinizing hormone to mimic the midcycle gonadotropin surge.

The amplitude and duration of the midcycle LH surge required for periovulatory changes in the primate follicle are incompletely defined. We reported that short (4- to 14-h) LH surges were insufficient to induce periovulatory events after multiple follicular development in macaques. In contrast, an 18- to 24-h LH surge induced oocyte maturation plus granulosa cell luteinization, but did not support corpus luteum function. In this study, the periovulatory changes following LH surges of 48 h elicited using pituitary (pit) or recombinant (r) human (h) LH were compared to those after 24-h LH surge durations or after urinary hCG (u-hCG) treatment. Beginning at menses, rhesus monkeys were treated with human gonadotropins for 9 days to stimulate follicular growth. On day 10, animals (n = 3-5/group) received 1) a single injection of u-hCG [79 +/- 3 micrograms RP-1 equivalents (equiv), im], 2) two injections of pit-hLH (91 +/- 4 micrograms RP-1 equiv, im), 3) one injection of r-hLH (21 +/- 1 micrograms RP-1 equiv, im), or 4) two injections of r-hLH (21 +/- 1 micrograms RP-1 equiv). Oocytes and granulosa cells were obtained via follicle aspiration 27 h after the initial LH or hCG injection. In all groups, serum estradiol rose to similar peak levels by day 10. Circulating LH-like bioactivity was elevated for more than 48 h after u-hCG. Peak serum LH bioactivities were proportional to the administered LH doses, as determined in the in vitro bioassay. Two injections of either r-hLH or pit-hLH elicited surge levels (> 100 ng/mL) of bioactive LH for 36-48 h, whereas one injection sustained surge levels for only 18-24 h. The proportions of oocytes resuming meiosis (68-76%) were similar in all groups. Immunocytochemical staining for progesterone receptor and in vitro progesterone production by granulosa cells in all LH-treated groups were comparable to those of cells form the hCG-treated group. Peak levels of progesterone in the luteal phase were comparable in monkeys treated with two doses of pit-hLH and r-hLH (18.5 +/- 10.4 vs. 8.1 +/- 1.5 ng/mL) and approached that in u-hCG treated monkeys (39.5 +/- 18.0 ng/mL). However, progesterone levels in animals treated once with r-hLH (3.4 +/- 1.5 ng/mL) were less (P < 0.05) than those in u-hCG-treated monkeys.(ABSTRACT TRUNCATED AT 400 WORDS)

Suppression of ovarian estradiol secretion by a single injection of antide in cynomolgus monkeys during the early follicular phase: immediate, sustained, and reversible actions.

We examined the effects of the GnRH antagonist antide on ovarian estrogen secretion after a single administration in intact cycling cynomolgus monkeys (n = 5/group) during the early follicular phase. Antide treatment on menstrual cycle day 2 resulted in a dose-dependent increase in menstrual cycle lengths (mean +/- SEM) to 38 +/- 3, 49 +/- 8, and 96 +/- 15 days for 3.0, 10.0, and 30.0 mg/kg antide, respectively, in association with inhibition of folliculogenesis and suppression of estradiol concentrations to therapeutic levels. Subsequent resumption of apparently normal ovulatory menstrual cycles occurred in all 15 individuals. In addition, all four monkeys from the group treated with 30 mg/kg antide that were available for subsequent matings became pregnant and had normal babies. Thus, no irreversible consequences or adverse effects of antide on reproductive function in these primates was observed. No allergic or other adverse reactions were found locally or systemically in these primates, even at the highest dose of antide. To the extent that this primate model is a paradigm for clinical therapeutics, a single treatment of antide (30 mg/kg) provides sustained inhibition of ovarian estradiol secretion for about 2 months, thus demonstrating the feasibility of using antide for clinical management of steroid-dependent conditions.

A mouse monoclonal antibody to a thyrotropin receptor ectodomain variant provides insight into the exquisite antigenic conformational requirement, epitopes and in vivo concentration of human autoantibodies.

We used the secreted TSH receptor (TSHR) ectodomain variant TSHR-289 (truncated at amino acid residue 289 with a 6-histidine tail) to investigate properties of TSHR autoantibodies in Graves' disease. Sequential concanavalin A and Ni-chelate chromatography extracted milligram quantities of TSHR-289 (approximately 20-40% purity) from the culture medium. Nanogram quantities of this material neutralized the TSH binding inhibitory activity in all 15 Graves' sera studied. We generated a mouse monoclonal antibody (mAb), 3BD10, to partially purified TSHR-289. Screening of a TSHR complementary DNA fragment expression library localized the 3BD10 epitope to 27 amino acids at the N-terminus of the TSHR, a cysteine-rich segment predicted to be highly conformational. 3BD10 preferentially recognized native, as opposed to reduced and denatured, TSHR-289, but did not interact with the TSH holoreceptor on the cell surface. Moreover, mAb 3BD10 could extract from culture medium TSHR-289 nonreactive with autoantibodies, but not the lesser amount (approximately 25%) of TSHR-289 molecules capable of neutralizing autoantibodies. Although the active form of TSHR-289 in culture medium was stable at ambient temperature, stability was reduced at 37 C, explaining the mixture of active and inactive molecules in medium harvested from cell cultures. In conclusion, studies involving a TSHR ectodomain variant indicate the exquisite conformational requirements of TSHR autoantibodies. Even under "native" conditions, only a minority of molecules in highly potent TSHR-289 preparations neutralize patients' autoantibodies. Therefore, Graves' disease is likely to be caused by even lower concentrations of autoantibodies than previously thought. Finally, reciprocally exclusive binding to TSHR-289 by human autoantibodies and a mouse mAb with a defined epitope suggests that the extreme N-terminus of the TSHR is important for autoantibody recognition.