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Implantation of the mammalian embryo into the wall of the uterus is regulated by a timely interplay of the ovarian hormones, estrogen and progesterone. These hormones orchestrate a set of modifications in the uterine endometrium that transforms it from a nonreceptive to a receptive phase allowing the implantation of the developing blastocyst. The molecular and cellular mechanisms underlying this complex process, however, remain largely unknown. To investigate the endocrine basis of uterine receptivity, we employed a gene expression screen technique to identify factors whose expressions are modulated in the rat uterus in response to estrogen and progesterone at the onset of implantation. Here we report that the expression of calcitonin, a peptide hormone involved in calcium homeostasis, is markedly enhanced in the uterus during pregnancy. By Northern blot analysis, we show that the synthesis of calcitonin messenger RNA is induced at the time of implantation. Immunocytochemistry with calcitonin antibody demonstrates further that the peptide is localized in the glandular epithelial cells of the uterus. The antiprogestin drug RU486, which is known to block implantation, abolishes calcitonin expression, suggesting a regulatory role for progesterone in this process. Consistent with this observation, progesterone significantly stimulates calcitonin messenger RNA and protein synthesis in the uteri of ovariectomized animals. Our study, therefore, identifies calcitonin as a stage- and cell-specific marker of progesterone action in the uterus during pregnancy. Estrogen exhibits no significant effect on calcitonin expression when administered alone to ovariectomized animals. However, a low dose of estrogen synergizes with progesterone, and a high dose antagonizes progesterone-mediated gene induction. Both estrogen and progesterone, therefore, modulate calcitonin gene expression in the uterus. The stage-specific regulation of calcitonin is apparently determined by the relative concentrations and the sequences of appearance of these two hormones and possibly other as yet unknown regulatory factors during pregnancy. We propose that calcitonin, a known regulator of calcium levels in the bone and kidney, may play an important regulatory role in the uterus of pregnant animals during the early events leading to implantation of the embryo.

In an attempt to correlate serum LH isoform distribution with its bio/immuno ratio, follicular phase blood samples from four normally cycling women and four patients with polycystic ovarian disease (PCOD) were studied. Chromatofocusing of peripheral serum samples across a pH gradient of 9.5-4.5 yielded a broad area of LH immunoreactivity comprising several peaks in the pH range of 7.2-9.0, and six other major peaks at pH values of 9.4, 6.8, 6.4, 5.1, 4.7 and less than 4.0. In addition, the bioactive and immunoreactive levels of LH were measured in the unfractionated serum samples. In three out of four PCOD patients and one healthy woman, the majority of LH species (approximately 70%) were distributed at a pI value greater than 7.0. All of them had a high bio/immuno ratio (greater than 2.0) and an elevated bioactive level of serum LH (greater than 40 IU/l). Conversely, fewer alkaline pI isoforms of LH were found in the other three normal women and one PCOD patient, who had low levels of the bio/immuno ratio of LH (less than 2.0) and bioactive LH (less than 40 IU/l). A significant (P less than 0.05) direct correlation was observed between the bio/immuno ratio of serum LH and the proportion of alkaline LH eluted. In conclusion, as demonstrated previously in the pituitary, the bio/immuno ratio of serum LH also correlates well with the charge distribution of LH isoforms. The results indicate that an altered isoform distribution with more basic LH forms is associated with a high biological activity of serum LH.

The purpose of this study was to re-evaluate the quantitative and qualitative responses of LH to exogenous and endogenous GnRH stimulation in normally cycling women and in polycystic ovarian disease (PCOD). Responses of serum LH to GnRH (100 micrograms i.v.) and the opioid antagonist naloxone (10 mg i.v.) were determined in healthy women in the early (n = 5) and late (n = 4) follicular phase, and in patients with PCOD (n = 20). Serum bioactive (B) LH was determined by a mouse interstitial cell in vitro bioassay, and immunoreactive LH by a conventional RIA (I-LH) and a novel sensitive (0.05 IU/l) and specific immunofluorimetric assay (F-LH). The B/I (2.4 +/- 0.1) and B/F (2.7 +/- 0.6) ratios in basal serum samples of the PCOD patients were significantly higher (p less than 0.05) than the corresponding ratios (1.6-1.8 and 1.8-2.0) of the control women. GnRH stimulated the secretion of I-LH (2-4-fold), F-LH and B-LH (3-5-fold each) in all groups studied. There were no apparent changes of the B/I and B/F ratios in normal women during early follicular phase or in patients with PCOD. However, the normal women during late follicular phase displayed a significant (p less than 0.05) increase in the B/I ratio, albeit no change was found in the B/F ratio. During naloxone-induced endogenous GnRH responses, the control women during late follicular phase showed a 3-6-fold increase in B-LH, I-LH and F-LH, with unchanged B/I and B/F ratios.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum bioactive and immunoreactive LH and FSH were measured in clinical conditions with increased or decreased gonadotropin secretion. Gonadotropin immunoreactivity was measured using a conventional RIA (I) and an ultrasensitive immunofluorometric method (F). Bioactive (B) LH was assessed by the mouse interstitial cells in vitro bioassay, and B-FSH using the immature rat granulosa cell assay. Acute GnRH stimulation of adult men (n = 6) increased LH levels measured by the different methods 4.3- to 5.3-fold. The B/I ratio of LH increased from 2.34 +/- 0.21 to 3.71 +/- 0.36 (mean +/- SEM) at 120 min (P less than 0.05), but no change was found in the B/F ratio. After ovariectomy of premenopausal women (n = 6), the LH levels increased in 1 week 4- to 6-fold, the B/I ratio from 1.85 +/- 0.22 to 2.59 +/- 0.24, and the B/F ratio from 1.78 +/- 0.22 to 2.90 +/- 0.30 (P less than 0.05 for both). In addition, the LH levels were measured during GnRH agonist treatment of ovarian carcinoma (n = 8), endometriosis (n = 8), and prostatic carcinoma after orchiectomy (n = 8). In the two former groups, serum B-LH decreased in 1 month to undetectable levels (less than 0.5 IU/L), and in the prostate cancer patients to 1.2 (0.8-1.9) IU/L (log mean and range of +/- SEM). The concomitant decline of I-LH was to 1.5-1.9 IU/L in the agonist-treated female patients, and that of F-LH to 0.10-0.15 IU/L; in the prostate cancer patients, respectively, these values were 7-8 and 0.3-0.7 IU/L. The B/I and B/F ratios during the agonist treatments could only be calculated in the prostate cancer patients (in the others, B-LH became undetectable). The B/I ratio decreased from 2.34 +/- 0.5 to 0.14 +/- 0.03 (P less than 0.01), but no suppression was found in the B/F ratio from a pretreatment value of 3.6 +/- 0.8. B-, I-, and F-FSH levels were measured in the GnRH agonist-treated orchiectomized prostate cancer patients. The pretreatment level of B-FSH was 154 (137-175), that of I-FSH was 38.0 (34.4-42.0), and that of F-FSH was 39.8 (35.3-44.9) IU/L. The B/I ratio of FSH was 3.76 +/- 0.49, and the B/F ratio was 3.53 +/- 0.59. The mean B-FSH level decreased during treatment by 87-93.5%, that of I-FSH by 98%, and that of F-FSH by 91.5% (P less than 0.01 for all).(ABSTRACT TRUNCATED AT 400 WORDS)

Publisher Summary The C cells of the thyroid gland are known as the major site of synthesis of calcitonin. This polypeptide hormone is released in response to hypercalcemia and mediates calcium homeostasis in both bone and kidney. This chapter presents a study that identifies the uterus as a novel site of calcitonin gene expression during preimplantation stages of pregnancy. The calcitonin synthesized in uterus acts in a paracrine or autocrine fashion to regulate calcium levels in certain uterine cells. Calcium controls many cellular processes, including muscle contraction, nerve conduction, and numerous enzymatic reactions. In the uterus, calcitonin regulates processes such as myocontraction and, therefore, plays a crucial role in embryonic implantation and maintenance of pregnancy. The antiprogestin RU486, which effectively blocks implantation and terminates pregnancy, also inhibits calcitonin expression. RU486-induced termination of pregnancy in preimplantation stage animals results from down regulation of certain genes, including calcitonin, which are primarily regulated by progesterone and may be crucial for implantation.

The purpose of this study was to find out whether the ratio of bioactive (B)/immunoreactive (I) LH varies during pulsatile LH secretion in healthy men when I-LH is determined using novel immunometric methods with improved sensitivity and specificity. Blood samples were collected for LH measurements from eight healthy men (aged 20-26 yr) at 20-min intervals over a period of 10 h. LH was measured in all samples using two novel sensitive and specific immunometric methods [Delfia, Wallac (sensitivity, 0.04 IU/L); and Amerlite, Amersham (sensitivity, 0.1 IU/L)] and for comparison with a conventional RIA. In addition, the plasma samples representing the LH secretion peaks and the preceding interpulse nadirs (two to five pairs of samples per subject; n = 23) were measured for B-LH using the mouse interstitial cell in vitro bioassay. The three immunoassay methods demonstrated the well known pulsatile mode of I-LH secretion in all subjects. A good correlation of B- and immunometric I-LH levels was observed (bioassay vs. Delfia, r = 0.89), but independent of the immunometric method used, no difference could be demonstrated between the B/I ratios of the mean basal and peak levels of LH (1.72 +/- 0.19 and 1.63 +/- 0.19 with Delfia, and 2.18 +/- 0.32 and 2.10 +/- 0.26 with Amerlite, respectively). Only when I-LH was measured by RIA was there a significant (P < 0.001) 2-fold increase in the B/I ratio in the secretion pulses. In conclusion, the previously documented increase in the B/I ratio of LH during the endogenous secretion pulses could not be documented when I-LH was measured, instead of RIA, using the novel immunometric methods. This finding further emphasizes the need for reassessment of the B/I ratio changes in LH in various physiological and clinical conditions.

Plasma bioactive (B) and immunoreactive (I) FSH and LH were measured every 10 min for 8 h in the same postmenopausal women in a three-phase study: 1) during normal pulsatile gonadotropin secretion (basal study; n = 8), 2) 8 h after a single injection of a GnRH antagonist (5 mg Nal-Glu, sc; n = 5), and 3) 21 days after a GnRH agonist injection (D-Trp6, 3.75 mg depot preparation, im; n = 7). I-FSH and I-LH were measured by monoclonal antibody immunoradiometric assays. B-FSH and B-LH were measured in selected samples with the immature rat granulosa cell and mouse interstitial cell assays, respectively. Significant pulsatility of B- and I-FSH and LH was demonstrated in the basal samples, but only the B/I ratio of LH was slightly elevated within the secretion peaks. After GnRH antagonist treatment, I-FSH decreased from a mean pretreatment level of 55.7 +/- 7.8 IU/L by 26% (P less than 0.001), and B-FSH from 313.8 +/- 61.9 IU/L by 44% (P less than 0.01). The B/I ratio decreased from 6.4 +/- 1.7 to 4.5 +/- 1.0 (P less than 0.05). After agonist treatment, the I- and B-FSH levels decreased by 92% and 83% (P less than 0.0001), respectively, but the B/I ratio increased to 17.3 +/- 4.7 (P less than 0.05). The concentrations of I- and B-LH decreased by 75% and 80%, respectively (P less than 0.001), after antagonist treatment. After agonist treatment, I-LH decreased by 92%, and B-LH by 93% (P less than 0.0001). No changes in the B/I ratios of LH were found after either treatment. In conclusion, no changes were found in the quality of circulating LH during the treatments, whereas the antagonist treatment decreased and the agonist treatment increased the B/I ratio of FSH. These findings provide further evidence that the qualitative responses of FSH and LH to treatment with the same GnRH analog are different, and that the suppressive mechanisms of GnRH antagonist and agonist action on gonadotropin secretion are different.

An investigation was undertaken to characterize an immunological LH variant in a 31-yr-old healthy woman whose serum LH was either poorly or not at all recognized by two monoclonal antibodies. The two antibodies recognize epitopes present on the intact LH dimer, but not on the free subunits. It was found that the immunologically aberrant LH of the subject was bioactive, as evidenced by an in vitro bioassay for LH. Nothing in the personal history of the subject or in the results from a number of hormone analyses revealed any endocrine abnormalities. In a GnRH stimulation test, the increase in immunoreactive LH using two reference immunometric assays for LH was less than 10% of the mean response of five control subjects. In relative terms, the maximal increase in LH in the subject was only 60-100%, in contrast to 340-560% for the control subjects. The bio/immuno ratio of the LH in the subject was high and was further increased in the GnRH stimulation test. A low proportion of acid LH isoforms in basal and stimulated samples from the subject was in agreement with the high bio/immuno ratio. Gel filtration studies showed the presence of molecular species of apparently lower molecular size than the intact LH, but different from the free beta-subunit. The results suggest the presence of fragments of the alpha-beta-dimer where at least part of the beta-subunit has been lost. A pedigree analysis involving the parents, siblings, and children of the subject strongly suggests a genetic origin of the LH variant described with an autosomal mode of inheritance. This report on an immunological variant of LH illustrates the potential dangers of using monoclonal based immunoassays where a protein hormone with fully maintained biopotency may be partially or totally missed due to the monospecificity of the immunoreagent. This possibility should be kept in mind when inappropriately low levels of gonadotropins are detected in diagnostic routine.

We compared five two-site immunometric assays (including three commercially available kits) for measuring lutropin (LH) in serum. Four of the assays involved monoclonal antibodies directed against the alpha-beta dimer, intact LH; these assays measured significantly lower concentrations of LH in 19 (out of 83) samples than did a commercial method not involving such antibodies. In five serum samples, two of the intact LH assays failed to detect any significant immunoreactivity above the detection limit. Findings of normal in vitro LH bioactivity in these samples did not confirm the low immunoreactivity of the intact LH assays. The inability of these assays to detect bioactive LH creates confusion in daily, routine testing as well as in research monitoring of bio/immuno ratios. The data presented here confirm our previous findings (Clin Chem 1991;37:333-40) and emphasize the need to avoid the use of monoclonal antibodies specific for the intact LH dimer.

Objective To investigate the interactions of hyperinsulinemia and inappropriate gonadotropin secretion in women with polycystic ovarian disease (PCOD). Design Comparative study of endocrinologic parameters in subjects with PCOD. Setting Open patient clinic of reproductive endocrinology at University Central Hospital of Turku, Finland. Patients Fourteen nonobese and 10 obese patients with PCOD. Seven healthy women for reference data collection. Normal thyroid function, serum prolactin concentration, normal diurnal cortisol variation, euglycemia in all subjects. Main Outcome Measures Serum concentrations of insulin, testosterone, androstenedione, dehydroepiandrosterone sulfate, sex hormone-binding globulin, immunoreactive luteinizing hormone (LH), bioactive LH, and follicle-stimulating hormone (FSH). Results The concentration of insulin was higher and that of bioactive LH was lower in obese than in nonobese PCOD women in whom the levels were also above the upper reference value. There was a negative correlation between insulin and bioactive LH levels (r = —0.57). Bioactive LH correlated inversely with the body mass index (BMI) (r = —0.50). After eliminating the effect of the BMI, the correlation between bioactive LH and insulin was no longer significant (r = —0.37). The bioactive LH and immunoreactive LH/FSH ratio correlated significantly (r = 0.68). Conclusions These data demonstrate that hyperandrogenic women can be divided into two subgroups: those with insulin resistance, normal or minimally elevated LH, and markedly elevated insulin levels; and those with elevated LH levels, no insulin resistance, and normal insulin concentrations. Obesity is associated with the former, and high bioactive LH levels with the latter subgroup.

The present study aimed at investigating the nature and causes of non-parallelism in testosterone responses to serial dilutions of peripheral serum and standard LH preparations in the mouse Leydig cell in vitro bioassay of LH. Immunoadsorption with monoclonal antibody to the β-subunit of LH was used to obtain LH-free serum; the procedure removed more than 98% of the immunoassayable LH. When a constant amount of the LH-free serum was added to standard dilutions, the bioassay dose-response curves to serum dilutions and standards became parallel, i.e. the well-known source of error of this assay system was eliminated. When standard curves prepared in medium and LH-free serum (final concentration 10%) were compared, no effect of the serum was found on basal cAMP and testosterone production. However, the LH-stimulated testosterone and cAMP production were suppressed by serum by a rather constant factor of 40%. Mild heating (60°C, 15 min) or treatment with dextran-coated charcoal, but not ether extraction, was able to eliminate the inhibitory activity of the LH-free serum. Binding studies demonstrated that [125I]hCG interaction with mouse Leydig cell homogenates was inhibited by LH-free serum in a fashion indicative of reduced LH receptor number, but not of reduced binding affinity. In conclusion, these data show that human serum contains LH inhibitor(s) which affect the LH-receptor interaction and LH stimulated testosterone production in mouse Leydig cell in vitro. The effect is marked in serum concentration over 1.5% and it shows only minor variation between individual sera. This source of error can be effectively removed from the LH in vitro bioassay by using LH-free serum for preparation of dilutions of LH standards.