The role of AIP and CDKN1B/p27Kip1 in endocrine neoplasia

Identification of genes predisposing to tumor syndromes has raised general awareness of tumorigenesis. Genetic testing of tumor susceptibility genes aids the recognition of individuals at increased risk of tumors. Identification of novel predisposing genes enables further studies concerning the classification of potential associated tumors and the definition of target patient group. Pituitary adenomas are common, benign neoplasms accounting for approximately 15% of all intracranial tumors. Accurate incidence estimation is challenging since a great portion of these adenomas are small and asymptomatic. Clinically relevant adenomas, that cause symptoms due to the expansion of the cell mass or the over-secretion of normally produced hormones, occur in approximately one of 1 000 individuals. Although the majority of pituitary adenomas are sporadic, a minority occur as components of familial syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 syndrome is caused by germ-line mutations in the MEN1 gene, whereas most of the CNC patients carry the mutated protein kinase A (PKA) regulatory subunit-1-α (PRKAR1A) gene. Recently, other conditions predisposing to endocrine tumors have been identified: Pituitary Adenoma Predisposition (PAP) and MEN type 4 (MEN4). PAP was originally identified in a genetically homogeneous Finnish population. In a population based cohort from Northern Finland, aryl hydrocarbon receptor-interacting protein (AIP) gene mutations were found in 16% of all patients diagnosed with growth hormone (GH) producing pituitary adenoma, and in 40% of the subset of patients who were diagnosed under the age of 35 years. Since AIP mutations were originally described in a defined, homogeneous population from Northern Finland, it was relevant to study whether mutations also occur in more heterogeneous populations. In patient cohorts with different ethnic origins and variable clinical phenotypes, germ-line AIP mutations were detectable at low frequencies (range 0.8-7.4%). AIP mutation-positive patients were often diagnosed with a GH-producing adenoma at a young age, and usually had no family history of endocrine tumors. The low frequency of AIP mutations in randomly selected patients, and the lack of any family history of pituitary adenomas create a challenge for the identification of PAP patients. Our preliminary study suggests that AIP immunohistochemistry may serve as a pre-screening tool to distinguish between the AIP mutation-negative and the mutation-positive tumors. Tumors of various endocrine glands are components of MEN1 and CNC syndromes. Somatic MEN1 and PRKAR1A mutations in sporadic pituitary adenomas are rare, but occur in some of the other tumors related to these syndromes. The role of AIP mutations in endocrine neoplasia was studied and our results indicated that somatic AIP mutations are rare or non-existent in sporadic tumors of endocrine glands (0 of 111). Furthermore, germ-line AIP mutations in prolactin producing adenomas (2 of 9) confirmed the role of this pituitary tumor type in the PAP phenotype. Thyroid disorders are common in the general population, and the majority of them are sporadic. Interestingly, it has been suggested that thyroid disorders might be more common in PAP families. For this reason we studied germ-line AIP mutations in 93 index cases from familial non-medullary thyroid cancer (NMTC) families. The underlying gene or genes for familial NMTC have not been identified yet. None of the patients had any potentially pathogenic AIP mutation. This suggests that AIP is unlikely to play a role in familial NMTCs. A novel multiple endocrine syndrome was originally described in rats with phenotypic features of human MEN type 1 and 2. Germ-line mutations of cyclin-dependent kinase inhibitor 1B (CDKN1B also known as p27Kip1) gene were reported later in these rats and a germ-line mutation was also identified in one human family with MEN1-like phenotype (later named MEN4). To confirm the importance of this gene’s mutations in humans, we performed a mutation screening in MEN-like patients and in patients with pituitary adenoma. Our results indicate that CDKN1B/p27Kip1 mutations appear in a small portion of MEN1-like patients (one of 36), and that such mutations are rare or non-existent in both familial (0 of 19) and sporadic pituitary adenoma patients (0 of 50). In conclusion, this work strengthens the tumor susceptibility role of AIP and CDKN1B/p27Kip1 in endocrine neoplasia. Clarifying the PAP phenotype facilitates the identification of potential AIP mutation carriers. Genetic counseling can be offered to the relatives and follow-up of the mutation carriers can be organized, hence an earlier diagnosis is feasible.

Follicle stimulating hormone secretion and dominant follicle growth during treatment of Bos indicus heifers with intra-vaginal progesterone releasing devices, oestradiol benzoate, equine chorionic gonadotrophin and prostaglandin F-2 alpha

The aim of this study was to investigate the effects on follicle stimulating hormone (FSH) secretion and dominant follicle (OF) growth, of treatment of Bos indicus heifers with different combinations of intra-vaginal progesterone releasing devices (IPRD), oestradiol benzoate (ODB), PGF(2 alpha), and eCG. Two-year-old Brahman (BN; n=30) and Brahman-cross (BNX; n=34) heifers were randomly allocated to three IPRD-treatments: (i) standard-dose IPRD [CM 1.56 g; 1.56 g progesterone (P-4); n = 17]; (ii) half-dose IPRD (CM 0.78 g; 0.78 g p(4); n=15); (iii) half-dose IPRD + 300 IU eCG at IPRD removal (CM 0.78 g+G; n=14); and, (iv) non-IPRD control (2 x PGF(2 alpha); n=18) 500 mu g cloprostenol on Days -16 and -2. IPRD-treated heifers received 250 mu g PGF(2 alpha) at IPRD insertion (Day 10) and IPRD removal (Day -2) and 1 mg ODB on Day -10 and Day -1. Follicular dynamics were monitored daily by trans-rectal ultrasonography from Day -10 to Day 1. Blood samples for determination of P-4 were collected daily and samples for FSH determination were collected at 12 h intervals from Day -9 to Day -2. A significant surge in concentrations of FSH was observed in the 2 x PGF(2 alpha), treatment 12 h prior and 48 h after follicular wave emergence, but not in the IPRD-treated heifers. Estimated mean concentrations of total plasma P-4 during the 8 days of IPRD insertion was greater (P<0.001) in the CM 1.56 g P-4 treated heifers compared to the CM 0.78 g P-4 treated heifers (18.38 ng/ml compared with 11.09 ng/ml, respectively). A treatment by genotype interaction (P=0.036) was observed in the mean plasma P4 concentration in heifers with no CL during IPRD insertion, whereby BN heifers in the CM 1.56 g treatment had greater plasma P-4 than the BNX heifers on Days-9, -7, -6, -5, and -4. However, there was no genotype effect in the CM 0.78 g +/- G or the 2 x PGF(2 alpha) treatment. Treatment had no effect on the DF growth from either day of wave emergence (P=0.378) or day of IPRD removal (P=0.780) to ovulation. This study demonstrates that FSH secretion in B. indicus heifers treated with a combination of IPRD's and ODB to synchronise ovulation was suppressed during the period of IPRD insertion but no significant effect on growth of the DF was observed. (C) 2013 Elsevier B.V. All rights reserved.

Studies on follicular atresia: role of tropic hormone and steroids in regulating cathepsin-D activity of preantral follicles of the immature rat

The ovary of the immature female rat is comprised of primary and medium-sized preantral follicles. Upon stimulation with FSH or PMSG, the cathepsin-D activity, a representative lysosomal enzyme of granulosa cells, is reduced by 50% (P < 0.01). 17β-Estradiol at the doses tried was unable to mimic this effect. Blockade of steroidogenesis with cyanoketone also had no effect on the cathepsin-D activity of isolated granulosa cells. Dihydrotestosterone (DHT), however, at a dose of 1 mg/rat was able to inhibit PMSG's tropic action. It brought about an increase in cathepsin-D activity and reduction in steroidogenic activity of isolated granulosa cells. The atretogenic activity of DHT could be relieved by supplementation with exogenous FSH. DHT was observed to significantly reduce (P < 0.01) endogenous FSH and LH levels within 12–18 h of its injection suggesting that its atretic effect was due to its action at the pituitary rather than the gonad. In addition to the above the ability of 15 IU of PMSG to reduce cathepsin-D activity of granulosa cells was also significantly reduced (P < 0.01) if endogenous FSH was neutralized by a specific FSH antiserum. The present study suggests that as far as small and medium-sized primary and preantral follicles are concerned, FSH lack is the essential signal for onset of atresia.

Effect of antiserum to luteinizing hormone (LHAS) on the physiology of the epididymis and accessory glands in the albino rat

Rabbit antiserum specific to ovine luteinizing hormone free of contaminating antibodies to nonspecific proteins and FSH was administered to adult, intact rats at a dose of 0.1 and 0.2 ml/day for five days. LHAS had no effect on the weights of the epididymis but decreased their secretory activity to castrate level. Administration of 0.2 ml of LHAS or castration resulted in a marked and comparable reduction in the weights and secretory activity of the accessory glands. LHAS, even at a lower dose (0.1 ml/day), caused a significant reduction in the content of sialic acid in the vas deferons and Cowper's glands. These results are discussed in relation to the factors that regulate the functions of the epididymis and accessory glands.

Comparative mparative Effects of Antiserum to Luteinizing Hormone on Pseudopregnancy and Pregnancy Induced in the Same Rat

The comparative role of luteinizing hormone (LH) in maintaining pregnancy and histamine-induced decidualization in the rat was studied with the help of a new system, wherein the above two states could be brought about simultaneously in the same animal, but in different uterine horns. Specific and well-characterized LH antiserum, administered daily, both during the pre-trauma (days 1-4) and post-trauma (days 5-8) periods, resulted in the termination of pregnancy and inhibition of decidualization. This antiserum effect could be reversed by suitable steroid therapy. Results suggest that the antiserum blockade of ovarian steroidogenesis continued even after cessation of its treatment. Early pregnancy and decidualization seem directly comparable in that both are dependent upon LH to stimulate the ovarian synthesis of much-needed progesterone and estrogen.

Cadmium stress alters the redox reaction and hormone balance in oilseed rape (Brassica napus L.) leaves

In order to understand the physiological response of oilseed rape (Brassica napus L.) leaves to cadmium (Cd) stress and exploit the physiological mechanisms involved in Cd tolerance, macro-mineral and chlorophyll concentrations, reactive oxygen species (ROS) accumulation, activities of enzymatic antioxidants, nonenzymatic compounds metabolism, endogenous hormonal changes, and balance in leaves of oilseed rape exposed to 0, 100, or 200 μM CdSO4 were investigated. The results showed that under Cd exposure, Cd concentrations in the leaves continually increased while macro-minerals and chlorophyll concentrations decreased significantly. Meanwhile, with increased Cd stress, superoxide anion (O 2 • − ) production rate and hydrogen peroxide (H2O2) concentrations in the leaves increased significantly, which caused malondialdehyde (MDA) accumulation and oxidative stress. For scavenging excess accumulated ROS and alleviating oxidative injury in the leaves, the activity of enzymatic antioxidants, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), was increased significantly at certain stress levels. However, with increased Cd stress, the antioxidant enzyme activities all showed a trend towards reduction. The nonenzymatic antioxidative compounds, such as proline and total soluble sugars, accumulated continuously with increased Cd stress to play a long-term role in scavenging ROS. In addition, ABA levels also increased continuously with Cd stress while ZR decreased and the ABA/ZR ratio increased, which might also be providing a protective role against Cd toxicity.

Studies on Rat Ovarian Receptors for Lutropin (Luteinizing Hormone): Applicability of radioimmunoassay to measure lutropin bound to receptors.

Measurement of receptor-bound unlabelled physiologically active lutropin (luteinizing hormone, LH) was possible by a modified radioimmunoassay. The conventional radioimmunoassayconducted at 4°C was inadequate, whereas the modified assay performed at 37'C could measure receptor-bound lutropin. The radioimmunoassay at 37'C takes only 36h for completion compared with 5-7 days at 4°C. The sensitivity and range of dose-response curves are, however, unaltered. The validity of the technique was established by a number of criteria.

Termination of pregnancy in macaques (Macaca radiata) using monkey antiserum to ovine luteinizing hormone

The ability of a monkey antiserum to ovine LH to interrupt gestation in monkeys has been established. The antiserum has been shown to neutralize monkey pituitary LH by a number of criteria. The significant increase in serum progesterone level on day 23 of the cycle shown by mated monkeys has been used as an index of pregnancy. Injection of LH antiserum during the first week of missed menses (day 29–31 of cycle or day 18–20 of gestation) causes significant reduction in serum levels of progesterone followed by onset of bleeding which is interpreted as the termination of gestation. The same dose of non-immune serum given to monkeys during the same period does not have any deleterious effect on the progress of pregnancy. The antiserum-treated animals after the termination of gestation, resume cyclicity. Injection of antiserum after day 25 of gestation does not bring about termination of pregnancy. It is suggested that by using antisera raised in humans to ovine LH, this method may be developed as a fertility control measure in humans.

Dynamics of Circulating Concentrations of Gonadotropins and Ovarian Hormones Throughout the Menstrual Cycle in the Bonnet Monkey: Role of Inhibin A in the Regulation of Follicle-Stimulating Hormone Secretion

In higher primates, increased circulating follicle-stimulating hormone (FSH) levels seen during late menstrual cycle and during menstruation has been suggested to be necessary for initiation of follicular growth, recruitment of follicles and eventually culminating in ovulation of a single follicle. With a view to establish the dynamics of circulating FSH secretion with that of inhibin A (INH A) and progesterone (P-4)secretions during the menstrual cycle, blood was collected daily from bonnet monkeys beginning day 1 of the menstrual cycle up to 35 days. Serum INH A levels were low during early follicular phase, increased significantly coinciding with the mid cycle luteinizing hormone (LH) surge to reach maximal levels during the mid luteal phase before declining at the late luteal phase, essentially paralleling the pattern Of P-4 secretion seen throughout the luteal phase. Circulating FSH levels were low during early and mid luteal phases, but progressively increased during the late luteal phase and remained high for few days after the onset of menses. In another experiment, lutectomy performed during the mid luteal phase resulted in significant decrease in INH A concentration within 2 hr (58.3 +/- 2 vs. 27.3 +/- 3 pg/mL), and a 2- to 3-fold rise in circulating FSH levels by 24 hr (0.20 +/- 0.02 vs. 0.53 +/- 0.14 ng/mL) that remained high until 48 hr postlutectomy. Systemic administration of Cetrorelix (150 mu g/kg body weight), a gonadotropin releasing hormone receptor antagonist, at mid luteal phase in monkeys led to suppression of serum INH A and P-4 concentrations 24 hr post treatment, but circulating FSH levels did not change. Administration of exogenous LH, but not FSH, significantly increased INH A concentration. The results taken together suggest a tight coupling between LH and INH A secretion and that INH A is largely responsible for maintenance of low FSH concentration seen during the luteal phase. Am. J. Primatol. 71:817-824, 2009.

Effect Of Human Chorionic Gonadotrophin And Ovine Luteinizing Hormone On Rat Ovarian Macromolecular Metabolism

Administration of human chorionic gonadotrophin (HCG) or ovine LH to immature rats primed with pregnant mare serum gonadotrophin (PMSG) stimulated the rate of synthesis of polyadenylic acid (poly A)-rich RNA in the ovaries. The rate of total RNA synthesis was not affected significantly by hormone treatment, whereas protein synthesis was enhanced. The increase in the rate of synthesis of poly(A)-rich RNA in the ovaries could be inferred as induction of messenger RNA synthesis after the hormone treatment. The poly(A)-rich nature of the isolated RNA was established by oligo(dT)–cellulose chromatography, binding to Millipore filter disks and hydridization with [3H]polyuridylic acid. The level of cyclic AMP in the ovaries of such rats was also raised after administration of LH, the increase coincided with the increase in the rate of synthesis of poly(A)-rich RNA. The implications of these results are discussed in the light of the biochemical basis of luteinization and the action of LH.

Prolactin suppresses release of luteinising hormone during lactation in the monkey

STUDIES with rats have shown that during lactation there is an inhibition of luteinising hormone (LH)-dependent physiological events, such as implantation1, and a return to oestrus cyclicity2. This inhibition has been shown to occur only during the intense suckling phase and it has been correlated with the high levels of prolactin present in the circulation at this time. Although exogenous prolactin could substitute for the effects of intense suckling, it could do so only under the permissive influence of minimal suckling stimulus. We have shown that there is, in these conditions, a lowering of LH levels, and that this is due to interference by prolactin with the pituitary responsiveness to LH-releasing hormone (LHRH) (K. Muralidhar, R. M. and N. R. M., unpublished). Using the lactating monkey, we have now demonstrated a similar inhibitory effect of prolactin on pituitary responsiveness to LHRH, suggesting a mechanism by which amenorrhoeic conditions are maintained during lactation.