Elevated chorionic gonadotropic hormone in transgenic mice induces parthenogenetic activation and ovarian teratomas.

Both male and female reproductive functions are impacted by altered gonadotrophin secretion and action, which may also influence the development of endocrine tumours. To ascertain if chronic hypersecretion of human chorionic gonadotropin (hCG) contributes to the development of gonadal tumours, double transgenic (TG) mice that overexpress hCGα- and ß-subunits were analysed. By the age of two months, ovarian tumours with characteristics of teratomas developed with 100% penetrance. Teratomas were also seen in wild-type ovaries orthotopically transplanted into TG mice, demonstrating an endocrine/paracrine mechanism for the hCG-induced ovarian tumorigenesis. Both in vitro and in vivo experiments showed oocyte parthenogenetic activation in TG females. In addition, ovaries showed reduced ovulatory gene expression, inhibited ERK1/2 phosphorylation, and impaired cumulus cell expansion. Hence, persistently high endocrine hCG activity causes parthenogenetic activation and development of ovarian teratomas, along with altered follicle development and impaired ERK1/2 signalling, offering a novel mechanism associated with the molecular pathogenesis of ovarian teratomas.

Acromegaly caused by a GHRH-producing pancreatic neuroendocrine tumor: a rare manifestation of MEN1 syndrome.

SUMMARY: Multiple endocrine neoplasia type 1 NM_001370259.2(MEN1):c.466G>C(p.Gly156Arg) is characterized by tumors of various endocrine organs. We report on a rare, growth hormone-releasing hormone (GHRH)-releasing pancreatic tumor in a MEN1 patient with a long-term follow-up after surgery. A 22-year-old male with MEN1 syndrome, primary hyperparathyroidism and an acromegalic habitus was observed to have a pancreatic tumor on abdominal CT scanning, growth hormone (GH) and insulin-like growth factor 1 (IGF1) were elevated and plasma GHRH was exceptionally high. GHRH and GH were measured before the treatment and were followed during the study. During octreotide treatment, IGF1 normalized and the GH curve was near normal. After surgical treatment of primary hyperparathyroidism, a pancreatic tail tumor was enucleated. The tumor cells were positive for GHRH antibody staining. After the operation, acromegaly was cured as judged by laboratory tests. No reactivation of acromegaly has been seen during a 20-year follow-up. In conclusion, an ectopic GHRH-producing, pancreatic endocrine neoplasia may represent a rare manifestation of MEN1 syndrome. LEARNING POINTS: Clinical suspicion is in a key position in detecting acromegaly. Remember genetic disorders with young individuals having primary hyperparathyroidism. Consider multiple endocrine neoplasia type 1 syndrome when a person has several endocrine neoplasia. Acromegaly may be of ectopic origin with patients showing no abnormalities in radiological imaging of the pituitary gland.

A nationwide study on parathyroid carcinoma.

BACKGROUND: Parathyroid carcinoma (PC) is rare and diagnostically challenging. Reported outcomes are rather poor and the incidence might be increasing. MATERIAL AND METHODS: We performed a nationwide study on all cases (n= 32) diagnosed in 2000-2011 in Finland, and compared clinical and histopathological characteristics and outcome to atypical parathyroid (APA; n= 28) and parathyroid adenomas (PA; n= 72). The incidence in years 1955-1999 was compared to that in 2000-2013. RESULTS: Preoperatively, calcium and parathyroid hormone concentrations were higher in PC compared to APA and PA (1.76, 1.56 and 1.44 mmol/l, p < .001; and 989, 355 and 160 µmol/l, p < .001, respectively). Calcium was ≤1.77 mmol/l for all PAs. Hospitalization (44% vs. 22% and 3%, respectively, p = .01), renal (50% vs. 48% vs. 22%, respectively, p = .01) and bone (47% vs. 15% vs. 38%, respectively p = .002) manifestations were more common. PC and APA tumors were larger than PA (p < .001). Histopathological characteristics of PC compared to PA are increased mitotic activity (p= .001), chief cells (p = .003), diffuse growth pattern (p < .001), higher Ki67 (p< .001) and negative parafibromin (p < .001). One PC (1/18) and one APA (1/16) patient had a CDC73 mutation. After 6.7 (2-13.9) years of follow-up, 9.4% of PC had residual, 21% recurrent disease and 12.5% died of disease. Overall mortality did not differ between subgroups (p = .094). Recurrent PC was characterized by vascular invasion, lymph node metastases, high mitotic activity, necrosis and negative parafibromin. Incidence increased from 1.42 (range 0.52-2.14) to 7.14 (range 3.42-10.38)/10.000.000/years; (p < .001). CONCLUSIONS: PC associates with severe primary hyperparathyroidism and must be suspected if calcium ≥1.77 mmol/l. The prevalence of CDC73 germline mutations in PC and APA in Finland is 6%. PC has distinct histopathological characteristics and its incidence has increased over the past decades.

TSH elevations as the first laboratory evidence for pseudohypoparathyroidism type Ib (PHP-Ib).

Hypocalcemia and hyperphosphatemia because of resistance toward parathyroid hormone (PTH) in the proximal renal tubules are the most prominent abnormalities in patients affected by pseudohypoparathyroidism type Ib (PHP-Ib). In this rare disorder, which is caused by GNAS methylation changes, resistance can occur toward other hormones, such as thyroid-stimulating hormone (TSH), that mediate their actions through G protein-coupled receptors. However, these additional laboratory abnormalities are usually not recognized until PTH-resistant hypocalcemia becomes clinically apparent. We now describe four pediatric patients, first diagnosed with subclinical or overt hypothyroidism between the ages of 0.2 and 15 years, who developed overt PTH-resistance 3 to 20 years later. Although anti-thyroperoxidase (anti-TPO) antibodies provided a plausible explanation for hypothyroidism in one of these patients, this and two other patients revealed broad epigenetic GNAS abnormalities, which included loss of methylation (LOM) at exons AS, XL, and A/B, and gain of methylation at exon NESP55; ie, findings consistent with PHP-Ib. LOM at GNAS exon A/B alone led in the fourth patient to the identification of a maternally inherited 3-kb STX16 deletion, a well-established cause of autosomal dominant PHP-Ib. Although GNAS methylation changes were not detected in additional pediatric and adult patients with subclinical hypothyroidism (23 pediatric and 39 adult cases), hypothyroidism can obviously be the initial finding in PHP-Ib patients. One should therefore consider measuring PTH, along with calcium and phosphate, in patients with unexplained hypothyroidism for extended periods of time to avoid hypocalcemia and associated clinical complications.

The permissive role of prolactin as a regulator of luteinizing hormone action in the female mouse ovary and extragonadal tumorigenesis.

Transgenic female mice overexpressing the hCGß subunit (hCGß(+)) and producing elevated levels of luteinizing hormone (LH)/hCG bioactivity present as young adults with enhanced ovarian steroidogenesis, precocious puberty, and infertility. They subsequently develop pituitary prolactinomas, high circulating prolactin (PRL) levels, and marked mammary gland lobuloalveolar development followed by adenocarcinomas. None of these phenotypes appear in gonadectomized mice, indicating that the hCG-induced aberrations of ovarian function are responsible for the extragonadal phenotypes. PRL receptor-deficient (PRLR(-/-)) female mice are sterile, despite ovulating, due to a failure of embryo implantation, as a consequence of decreased ovarian LH receptor (Lhcgr) expression and inadequate corpus luteum formation and progesterone production. To study further the presumed permissive role of PRL in the maintenance of gonadal responsiveness to LH/hCG stimulation, we crossed the hCGß(+) and PRLR(-/-) mice. The double-mutant hCGß(+)/PRLR(-/-) females remained sterile with an ovarian phenotype similar to PRLR(-/-) mice, indicating that LH action, Lhcgr expression, and consequent luteinization are not possible without simultaneous PRL signaling. The high frequency of pituitary prolactinomas in PRLR(-/-) mice was not affected by transgenic hCGß expression. In contrast, none of the hCGß(+)/PRLR(-/-) females showed either mammary gland lobuloalveolar development or tumors, and the increased mammary gland Wnt-5b expression, possibly responsible for the tumorigenesis in hCGß(+) mice, was absent in double-mutant mice. Hence, high LH/hCG stimulation is unable to compensate for missing PRL signaling in the maintenance of luteal function. PRL thus appears to be a major permissive regulator of LH action in the ovary and of its secondary extragonadal effects.

Short-term pharmacological suppression of the hyperprolactinemia of infertile hCG-overproducing female mice persistently restores their fertility.

Female infertility is often associated with deregulation of hormonal networks, and hyperprolactinemia is one of the most common endocrine disorders of the hypothalamic-pituitary axis affecting the reproductive functions. We have shown previously that transgenic female mice overexpressing human chorionic gonadotropin ß-subunit (hCGß+ mice), and producing elevated levels of bioactive LH/hCG, exhibit increased production of testosterone and progesterone, are overweight and infertile, and develop hyperprolactinemia associated with pituitary lactotrope adenomas in adult age. In the present study, we analyzed the influence of the hyperprolactinemia of hCGß+ females on their reproductive phenotype by treating them with the dopamine agonists, bromocriptine and cabergoline. Long-term bromocriptine treatment of adult mice was effective in the control of obesity, pituitary growth, and disturbances in the hormone profile, demonstrating that hyperprolactinemia was the main cause of the hCGß+ female phenotype. Interestingly, short-term treatment (1 wk) with cabergoline applied on 5-wk-old mice corrected hyperprolactinemia, hyperandrogenism, and hyperprogesteronemia, prevented pituitary overgrowth, normalized gonadal function, and recovered fertility of adult hCGß+ females after hormone-induced and natural ovulation. The same cabergoline treatment in the short term applied on 3-month-old hCGß+ females failed to recover their reproductive function. Hence, we demonstrated that the short-term cabergoline treatment applied at a critical early stage of the phenotype progression effectively prevented the hyperprolactinemia-associated reproductive dysfunction of hCG-overproducing females.

Enhanced LH action in transgenic female mice expressing hCGbeta-subunit induces pituitary prolactinomas; the role of high progesterone levels.

The etiology of pituitary adenomas remains largely unknown, with the exception of involvement of estrogens in the formation of prolactinomas. We have examined the molecular pathogenesis of prolactin-producing pituitary adenomas in transgenic female mice expressing the human choriongonadotropin (hCG) beta-subunit. The LH/CG bioactivity is elevated in the mice, with consequent highly stimulated ovarian progesterone (P(4)) production, in the face of normal estrogen secretion. Curiously, despite normal estrogen levels, large prolactinomas developed in these mice, and we provide here several lines of evidence that the elevated P(4) levels are involved in the growth of these estrogen-dependent tumors. The antiprogestin mifepristone inhibited tumor growth, and combined postgonadectomy estradiol/P(4) treatment was more effective than estrogen alone in inducing tumor growth. Evidence for direct growth-promoting effect of P(4) was obtained from cultures of primary mouse pituitary cells and rat somatomammotroph GH3 cells. The mouse tumors and cultured cells revealed stimulation of the cyclin D1/cyclin-dependent kinase 4/retinoblastoma protein/transcription factor E2F1 pathway in the growth response to P(4). If extrapolated to humans, and given the importance of endogenous P(4) and synthetic progestins in female reproductive functions and their pharmacotherapy, it is relevant to revisit the potential role of these hormones in the origin and growth of prolactinomas.

Extragonadal LH/hCG action--not yet time to rewrite textbooks.

Gonadotropins are indispensable in both sexes in the regulation of gonadal sex steroid production and gametogenesis. In addition to their well-established classical actions, numerous recent publications have indicated the presence and function of luteinizing hormone/chorionic gonadotropin receptors (LH/hCG-R) in a variety of extragonadal tissues. However, the physiological significance of such effects has remained unclear. We have generated two genetically modified mouse models, one with excessive production of hCG and the other with targeted disruption of LH/hCG-R gene, and used them to address the functions of LH and hCG. Numerous gonadal and extragonadal phenotypes were found in the models with the two extremes of LH/hCG action. However, when the extragonadal effects were scrutinized in greater detail, they all appeared to arise through modification of gonadal function, either through enhanced or inhibited response to LH/hCG stimulation. Hence, further evidence is needed before the extragonadal LH/hCG-R expression can be considered functionally significant.

Phenotypic characterisation of mice with exaggerated and missing LH/hCG action.

In order to study the physiology and pathophysiology of gonadotrophin action, we have produced transgenic (TG) mice overexpressing human chorionic gonadotrophin (hCG) alpha and beta subunits (hCG+ mice) and knockout (KO) mice for the luteinising hormone receptor (LHR; LuRKO mice). The two extremes in LH function, i.e. strong LH/hCG stimulation and total blockade of this action, confirm numerous earlier concepts about LH function, but they also reveal new aspects about gonadal function during excessive LH production and in the absence of this trophic stimulus. The purpose of this review is to summarise the key findings on these two genetically modified mouse models.

Genetically modified mouse models in studies of luteinising hormone action.

Numerous genetically modified mouse models have recently been developed for the study of the pituitary-gonadal interactions. They include spontaneous or engineered knockouts (KO) of the gonadotrophin-releasing hormone (GnRH) and its receptor, the gonadotrophin common-alpha(Calpha), luteinising hormone (LH) beta and follicle-stimulating hormone (FSH) beta subunits, and the two gonadotrophin receptors (R), LHR and FSHR. In addition, there are also transgenic (TG) mice overexpressing gonadotrophin subunits and producing supraphysiological levels of these hormones. These models have offered relevant phenocopies for similar mutations in humans and to a great extent expanded our knowledge on normal and pathological functions of the hypothalamic-pituitary-gonadal (HPG) axis. The purpose of this article is to review some of our recent findings on two such mouse models, the LHR KO mouse (LuRKO), and the hCG overexpressing TG mouse (hCG+).

Fetal but not adult Leydig cells are susceptible to adenoma formation in response to persistently high hCG level: a study on hCG overexpressing transgenic mice.

We have previously demonstrated that male transgenic (TG) mice overexpressing human chorionic gonadotropin (hCG+) develop reproductive organ defects, but no tumors, in adult age. In this study, the effects of persistently elevated hCG were followed in TG males between day 5 postpartum and adulthood. Leydig cell (LC) adenomas were found in prepubertal mice, most prominently at the age of 10 days, but not in adult age. Serum testosterone concentrations were significantly increased in TG males at all ages studied. The phenotype of the prepubertal hCG+ males resembled that found in boys upon expression of constitutively activating luteinizing hormone (LH) receptor mutations. The temporal expression patterns of the fetal LC marker gene, thrombospondin 2, and those of adult LCs, hydroxysteroid dehydrogenase-6, delta5-3-beta and prostaglandin D synthase, were similar in wild-type and hCG+ males. Hence, the postnatal adenomas resemble functionally fetal LCs, and only these cells are susceptible to hCG-induced tumorigenesis. Our findings demonstrate a novel intriguing difference between the fetal and adult LC populations and provide further insight into the potential tumorigenic effects of gonadotropins.

Multiple sites of tumorigenesis in transgenic mice overproducing hCG.

We have produced transgenic (TG) mice expressing under the ubiquitin C promoter either the glycoprotein hormone common alpha-subunit (C(alpha)) or human chorionic gonadotropin (hCG) beta-subunit. C(alpha) overexpression alone had no phenotypic effect, but the hCG(beta) expressing females, presenting with moderately elevated levels of bioactive LH/hCG, due to dimerization of the TG hCG(beta) with endogenous C(alpha), developed multiple gonadal and extragonadal neoplasias. Crosses of the C(alpha) and hCG(beta) mice (hCG(alpha)beta) had >1000-fold elevated hCG levels, due to ubiquitous transgene expression, and presented with more aggressive tumour formation. The ovaries displayed initially strong luteinisation of all somatic cell types, leading to formation of luteomas, and subsequently to germ cell tumours (teratomas). The pituitary glands of TG females were massively enlarged, up to >100 mg, developing macroprolactinomas with very high prolactin (PRL) production. This endocrine response probably induced breast cancers in the mice. In contrast to the females, similar high levels of hCG in male mice had only marginal effects in adulthood, with slight Leydig cell hyperplasia and atrophy in the seminiferous epithelium. However, clear Leydig cell adenomas were observed in postnatal mice, apparently originating from fetal Leydig cells. In conclusion, these studies demonstrate marked tumorigenic effects of supraphysiological hCG levels in female mice, but clear resistance to similar changes in males. The extragonadal tumours were induced by hCG stimulated aberrant ovarian endocrine function, rather than by direct hCG action, because gonadectomy prevented all extragonadal phenotypes despite persistent hCG elevation. The phenotypes of the TG mice apparently represent exaggerated responses to hCG/LH and/or gonadal steroids. It remains to be explored to what extent they simulate respective responses in humans to pathophysiological elevation of the same hormones.

Elevated steroidogenesis, defective reproductive organs, and infertility in transgenic male mice overexpressing human chorionic gonadotropin.

We previously developed a transgenic (TG) mouse model that overexpresses the human chorionic gonadotropin (hCG) beta-subunit under the universal human ubiquitin C promoter, displaying in males a modest 3-fold increase in circulating levels of LH/hCG bioactivity. The males were fertile and presented with a mild reproductive phenotype. To achieve higher levels of hCG, a double TG model was generated by cross-breeding the hCG beta-expressing mice with another TG line harboring a ubiquitin C/common alpha-subunit fusion gene. The double-TG mice expressed excessive levels of dimeric hCG, with 2000-fold elevated circulating LH/hCG bioactivity. These male mice were infertile, primarily due to inability to copulate, and they showed enhanced testicular androgen production despite clear down-regulation of LH/hCG receptors. Their intratesticular inhibin B was unaltered, but serum FSH was markedly reduced. Apparently the chronic hCG hyperstimulation led to focal Leydig cell proliferation/hypertrophy at 6 months of age, but failed to promote testicular tumors. Even though full spermatogenesis occurred in most of the seminiferous tubules, progressive tubule degeneration was apparent as the males grew older. The prostate and seminal vesicles were enlarged by distension of glandular lumina. Functional urethral obstruction was indicated by distension and sperm accumulation in distal vas deferens as well as by dilated urinary bladder and enlarged kidneys. The abnormal function of accessory sex glands and/or lower urinary tract as a consequence of the disturbed sex hormone balance or direct action of hCG may be the main cause of infertility in this model. The present study provides in vivo evidence that exposure of male mice to chronically elevated levels of hCG severely affects their urogenital tract function at multiple sites and causes infertility, but, unlike in LH/hCG overexpressing female mice, it is not tumorigenic.