Unconventional Actions of Glycoprotein Hormone Subunits: A Comprehensive Review.

The glycoprotein hormones (GPH) are heterodimers composed of a common α subunit and a specific ß subunit. They act by activating specific leucine-rich repeat G protein-coupled receptors. However, individual subunits have been shown to elicit responses in cells devoid of the receptor for the dimeric hormones. The α subunit is involved in prolactin production from different tissues. The human chorionic gonadotropin ß subunit (ßhCG) plays determinant roles in placentation and in cancer development and metastasis. A truncated form of the thyrotropin (TSH) ß subunit is also reported to have biological effects. The GPH α- and ß subunits are derived from precursor genes (gpa and gpb, respectively), which are expressed in most invertebrate species and are still represented in vertebrates as GPH subunit paralogs (gpa2 and gpb5, respectively). No specific receptor has been found for the vertebrate GPA2 and GPB5 even if their heterodimeric form is able to activate the TSH receptor in mammals. Interestingly, GPA and GPB are phylogenetically and structurally related to cysteine-knot growth factors (CKGF) and particularly to a group of antagonists that act independently on any receptor. This review article summarizes the observed actions of individual GPH subunits and presents the current hypotheses of how these actions might be induced. New approaches are also proposed in light of the evolutionary relatedness with antagonists of the CKGF family of proteins.

Ontogeny of immunoreactive Lh and Fsh cells in relation to early ovarian differentiation and development in protogynous hermaphroditic ricefield eel Monopterus albus.

Luteinizing hormone (Lh) and follicle-stimulating hormone (Fsh) control many aspects of gonadal development and function in teleosts. In the present paper, the specific antisera against ricefield eel Lhb (Lh beta subunit), Fshb (Fsh beta subunit), and Cga (the common pituitary glycoprotein hormone alpha subunit) were generated, and the cellular localization, initial appearance, and subsequent development of gonadotrophs in relation to early ovarian differentiation and development in the ricefield eel, a protogynous sex-changing teleost, were examined with immunochemistry. Lhb- and Fshb-immunoreactive signals were identified in distinct pituitary cells that occupied primarily the peripheral regions of the adenohypophysis. During ontogeny, Lhb-immunoreactive signals were first detected in the pituitary around 40 days after hatching (dah) when the oogonia transitioned into early primary growth oocytes, and the intensity of immunoreactivity increased concomitantly with the growth of primary oocytes from 60 to 140 dah. During overwintering from 170 to 230 dah, Lhb-immunoreactive signals were significantly decreased when a large proportion of perinucleolus oocytes contained intense Balbiani bodies. In contrast, Fshb-immunoreactive signals were not detectable in the pituitary until around 230 dah (in the spring after hatching) and slightly increased from 285 dah when the late perinucleolus oocytes began to enter the secondary growth phase. Both Lhb- and Fshb-immunoreactive cells were increased when the early cortical alveoli oocytes emerged at 300 dah. The mRNA expression of lhb and fshb coincided with their immunoreactive signals. Taken together, these results suggest that only Lh is involved in primary oocyte growth in ricefield eels, but both Fsh and Lh are important for the secondary ooctye growth.

Mechanisms for pulsatile regulation of the gonadotropin subunit genes by GNRH1.

The frequency of gonadotropin-releasing hormone (GNRH1, or GnRH) pulses secreted from the hypothalamus determine the ratios of the gonadotropin subunit genes luteinizing hormone beta (Lhb), follicle-stimulating hormone beta (Fshb) and the common alpha-glycoprotein subunit gene (Cga) transcribed in the anterior pituitaries of mammals. Fshb is preferentially transcribed at slower GNRH1 pulse frequencies, whereas Lhb and Cga are preferentially transcribed at more rapid pulse frequencies. Producing the gonadotropins in the correct proportions is critical for normal fertility. Currently, there is no definitive explanation for how GNRH1 pulses differentially activate gonadotropin subunit gene transcription. Several pathways may contribute to this regulation. For example, GNRH1-regulated GNRH1-receptor concentrations may lead to variable signaling pathway activation. Several signaling pathways are activated by GnRH, including mitogen-activated protein kinase, protein kinase C, calcium influx, and calcium-calmodulin kinase, and these may be preferentially regulated under certain conditions. In addition, some signaling proteins feed back to downregulate their own levels. Other arms of gonadotroph signaling appear to be regulated by synthesis, modification, and degradation of either transcription factors or regulatory proteins. Finally, the dynamic binding of proteins to the chromatin, and how that might be regulated by chromatin-modifying proteins, is addressed. Oscillations in expression, modification, and chromatin binding of the proteins involved in gonadotropin gene expression are likely a link between GNRH1 pulsatility and differential gonadotropin transcription.

Absence of apparent circadian rhythms of gonadotropins and free alpha-subunit in postmenopausal women: evidence for distinct regulation relative to other hormonal rhythms.

Aging is associated with a decrease in gonadotropin levels in postmenopausal women (PMW) and is also associated with alterations in a number of circadian rhythms. The goals of this study were to determine the presence of circadian rhythms of gonadotropins and glycoprotein free alpha-subunit (FAS) in young and old PMW. Healthy, euthyroid PMW, ages 45 to 55 years (n = 11) and 70 to 80 years (n = 11), were admitted in the morning to start a 24-h constant routine of light, temperature, position, and activity. Subjects remained awake and semirecumbent for the duration of the study and were fed hourly snacks, and activity was monitored continuously. Blood was sampled every 5 min for two 8-h periods corresponding to the estimated acrophase and nadir of the temperature rhythm. Luteinizing hormone (LH) and FAS were measured in all samples and follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and cortisol in 20-min serum pools. Mean LH (p < 0.001), FSH (p < 0.002), and FAS (p < 0.002) were lower in older compared with younger PMW. Day/night differences in cortisol and TSH (p < 0.001) were present in all subjects. However, there were no day/night differences in LH in younger or older PMW or in FSH in younger or older PMW. There were no day/night differences in mean FAS in younger or older PMW or in FAS pulse frequency or amplitude. Thus, in controlled studies in which differences in cortisol and TSH were demonstrated, there were no day/night differences in LH, FSH, or FAS in PMW. These studies suggest that despite evidence of intact circadian rhythms of cortisol and TSH, gonadotropin secretion does not appear to follow a circadian pattern in PMW. Thus, the age-related decline in gonadotropin secretion in PMW is not associated with a dampening of circadian rhythmicity. The absence of day/night differences in FAS suggests that GnRH plays a more prominent role in FAS regulation than does thyrotropin-releasing hormone in PMW.

Targeted disruption of LH receptor gene revealed the importance of uterine LH signaling.

Numerous previous studies have demonstrated that LH and hCG can directly regulate several uterine functions. We investigated in the present study, whether uterine phenotype in LH receptor knockout animals resulted also from the absence of direct actions of LH in the uterus. The phenotype consisted of marked growth reduction of uterus, decreased thickness of endometrial and myometrial layers, number of endometrial glands, height of luminal epithelium and vascular space. Analysis of uterine gene expression by mouse genome U74Av2 Affymetrix genechips revealed a differential expression of 155 genes by more than 3-fold (range 3-53-fold) between null and wild-type animals. Of these, 89 genes decreased and 66 increased in uterus of null animals. Semi-quantitative RT-PCR confirmed the differential expression of several selected genes. The decreased genes can be clustered into 18 functional families and the increased into 15 functional families. Semi-quantitative RT-PCR, Western blotting and immunocytochemistry demonstrated a decreased expression of ERbeta, PR-A, PR-B and AR in uterus of null animals as compared with wild-type siblings. Twenty-one-day estradiol and progesterone replacement therapy did not normalize the decrease in the number of endometrial glands and several genes that either decreased or increased in expression. The partial success of therapy suggests that direct LH actions could be required to completely normalize the uterus. In summary, findings on the knockout model reaffirm that LH and hCG control uterine functions directly as well as indirectly through increasing ovarian synthesis of steroid hormones and both actions are required for normal uterine biology.

Fisiología de la pubertad femenina / Female puberty physiology

El objetivo del presente artículo es el estudio de la fisiología de la pubertad femenina, con descripción del concepto de pubertad, su cronología, las etapas del desarrollo puberal y los cambios hormonales que acontecen desde la lactancia a la niñez, en la prepubertad y en la pubertad

The aim of this review is the study of female puberty physiology with the description of the puberty concept, its chronology, the several stages of puberty development, and the hormonal changes in the different stages

Factors influencing response to ovarian stimulation.

Ovarian stimulation is an integral part of assisted reproduction treatments. Ovarian response to gonadotrophin treatment, besides other factors, determines the outcome of treatment, as the number and quality of oocytes retrieved are related to the chance of achieving a pregnancy. A number of factors have been identified that might predict ovarian response, such as age of the patient and antral follicle count. In addition, it has been shown that genetic factors such as the patient's FSH-receptor genotype also determine individual response to FSH treatment. Besides patient-related factors, the choice of drugs for ovarian stimulation plays a significant role. Until recently, biopotency of gonadotrophin preparations was tested by an in-vivo bioassay with an intrinsic variability up to 20%. Due to a superior manufacturing technique, follitropin alpha can now be filled by mass. This allows assessment of FSH with a precise SE-HPLC assay and variability of the FSH content between production lots has now been estimated at 1.6%. Results of recent studies indicate that treatment with follitropin alpha filled by mass results in consistent ovarian response, fewer treatment days and fewer cancelled cycles. This is an important step towards further minimizing drug-related variability of ovarian response to FSH treatment.

Overexpression of human chorionic gonadotropin causes multiple reproductive defects in transgenic mice.

Human CG is a pregnancy marker secreted by the placenta, and it utilizes the same receptors as does LH. Human CG is a heterodimer, and its subunits are expressed in tissues other than placenta. Similarly, LH/hCG receptors are also expressed in multiple tissues; however, the physiological significance of this expression is unknown. Free hCGbeta is efficiently secreted in vitro in transfected cells and is highly expressed in many human cancers; however, the biological effects of free hCGbeta in vivo are unknown. To study in vivo consequences of elevated levels of free hCGbeta and hCG dimer in both male and female reproductive physiology, we used mouse metallothionein 1 promoter to generate multiple lines of transgenic mice that overexpressed either one or both subunits of hCG. Although mice expressing the glycoprotein hormone alpha subunit are normal and fertile, both male and female transgenic mice overexpressing only the hormone-specific hCGbeta subunit are infertile. The hCGbeta subunit-expressing transgenic female mice progressively develop cystic ovaries, whereas the male transgenic mice are infertile but otherwise are not phenotypically discernible. In contrast, both the male and female transgenic mice coexpressing high levels of the hCG subunits (i.e., the hCG dimer) demonstrate multiple reproductive defects. The male transgenic mice have Leydig cell hyperplasia, very high levels of serum testosterone, reduced testis size, and dramatically enlarged seminal vesicles and are infertile and display overly aggressive behavior when caged with females. The female transgenic mice are also infertile, have elevated levels of serum estradiol, and progressively develop hemorrhagic and cystic ovaries with thecal layer enlargement and stromal cell proliferation and degenerating kidneys. These results suggest that the in vivo biological effects of ectopically expressed free hCGbeta subunit are distinct from those of the hCG dimer and are gender specific. These transgenic mice are useful models for studying the biology of free hCGbeta subunit, for further analyzing the gain of function effects of hCG during early Leydig cell development, and for studying the roles of hCG in ovarian and kidney pathophysiology and function.

Thyroid hormone-responsive pituitary hyperplasia independent of somatostatin receptor 2.

Mice homozygous for the targeted disruption of the glycoprotein hormone alpha-subunit (alphaGsu) display hypertrophy and hyperplasia of the anterior pituitary thyrotropes. Thyrotrope hyperplasia results in tumors in aged alphaGsu(-/-) mice. These adenomatous pituitaries can grow independently as intrascapular transplants in hypothyroid mice, suggesting that they have progressed beyond simple hyperplasia. We used magnetic resonance imaging to follow the growth and regression of thyrotrope adenomatous hyperplasia in response to thyroid hormone treatment and discovered that the tumors retain thyroid hormone responsiveness. Somatostatin (SMST) and its diverse receptors have been implicated in cell proliferation and tumorigenesis. To test the involvement of SMST receptor 2 (SMSTR2) in pituitary tumor progression and thyroid hormone responsiveness in alphaGsu(-/-) mutants, we generated Smstr2(-/-), alphaGsu(-/-) mice. Smstr2(-/-), alphaGsu(-/-) mice develop hyperplasia of thyrotropes, similar to alphaGsu(-/-) mutants, demonstrating that SMSTR2 is dispensable for the development of pituitary adenomatous hyperplasia. Thyrotrope hyperplasia in Smstr2(-/-), alphaGsu(-/-) mice regresses in response to T4 treatment, suggesting that SMSTR2 is not required in the T4 feedback loop regulating TSH secretion.

Glycoprotein hormone structure-function and analog design.

Human chorionic gonadotropin (hCG), luteinizing hormone, follicle-stimulating hormone (FSH), and thyrotropin (TSH) are hormones that share a common alpha subunit but differ in their beta subunits. Recombinant DNA techniques, valuable tools for structure-function analyses, provide an approach for designing therapeutic analogs. FSH is used clinically to stimulate the ovarian follicles for in vitro fertilization and to initiate follicular maturation in women with infertility problems. The CG beta subunit contains a carboxy-terminal extension (CTP) with four serine O-linked oligosaccharides, which is important for the long half-life of hCG. A clinical problem of FSH is its relatively short half-life in circulation. Fusing CTP to the FSH beta coding sequence increased the in vivo potency of the resulting FSH dimer over three-fold. Analogs of the other hormones containing CTP also increase their biologic half-life. Subunit assembly is vital to the function of these hormones. To address whether alpha and beta subunits can be synthesized as one chain and also maintain biological activity, a chimera comprised of the hCG beta subunit genetically fused to the alpha subunit was constructed. The resulting polypeptide was efficiently secreted and displayed an increased biologic activity in vitro and in vivo. Similarly, the single-chain form of FSH also retained in vivo activity. Since subunit dissociation inactivates the activity of the heterodimer, single-chain analogs should have longer biological half-lives. These analogs represent suitable substrates for engineering potent and stable agonists and antagonists.

Functional uncoupling between intracellular calcium dynamics and secretion in the alphaT3-1 gonadotropic cell line.

Gonadotropin releasing hormone (GnRH) stimulates both transcription and secretion of the alpha subunit of the gonadotropins in a Ca2+-dependent fashion. In this study, we examined the role of Ca2+ as the signal coupling agonist occupancy of GnRH receptors to hormone secretion using the gonadotropic cell line alphaT3-1. Treatment of alphaT3-1 cells for 60 min with GnRH (0.1-100 nM), veratridine (50 microM) or high K+ (56 mM) was completely ineffective in stimulating secretion. The lack of effect occurred in spite of a robust, specific, and dose-dependent biphasic [Ca2+]i response consisting of a rapid peak sensitive to thapsigargin (200 nM) followed by a smaller plateau sensitive to the extracellular application of EGTA (5 mM). On the other hand, treatment of alphaT3-1 cells with the Ca2+ ionophore ionomycin resulted in a significant dose-dependent stimulation of secretion and [Ca2+]i responses comparable to those elicited by GnRH. Binding assays revealed the presence of Ins(1,4,5)P3 receptors (Kd = 3.2 nM, Bmax = 50.5 fmol/mg protein) but not ryanodine receptors in alphaT3-1 cell membranes. Together, these results show a functional uncoupling between the [Ca2+]i response and secretion in this cell line, suggesting that the increase in [Ca2+]i triggered by GnRH and depolarization may be necessary but not sufficient to stimulate exocytosis.

Follicle-stimulating isohormones: regulation and biological significance.

Follicle-stimulating hormone (FSH) is a key hormone in the regulation of follicular development. Although the existence of FSH heterogeneity is well established, the physiological significance of this pleomorphism remains unknown. Observed changes in circulating FSH heterogeneity during critical reproductive events such as puberty and reproductive cyclicity suggest that different combinations of FSH isoforms reach the target sites during different physiological states to influence a variety of biological end points such as cellular growth, development, steroidogenesis and protein synthesis. Considering that these FSH isoforms have different physicochemical properties and potential to bind not only their cognate receptors but also structurally related, non-FSH receptors with various affinities, the regulatory implications of FSH heterogeneity in modulating the various FSH-induced functions are enormous. However, assigning functional significance to FSH heterogeneity has been hampered because of (1) difficulties associated with procurement of highly purified, naturally occurring, circulating FSH isoforms; (2) absence of reference standards that contain the entire repertoire of FSH isoforms present in biological fluids; and (3) specificity issues inherent to the detection systems used. If particular FSH isoforms do possess selective biological functions, specific combinations of FSH isoforms could be generated to regulate fertility in farm animals and humans.

Structure-function relationship of follicle-stimulating hormone and its receptor.

Follicle stimulating hormone (FSH) is one of the two pituitary gonadotrophins involved in the regulation of gonadal function. Structurally, this gonadotrophin is a heterodimer composed of two non-covalently associated subunits containing several heterogenous oligosaccharide residues which play an important role in both the in-vivo and in-vitro bioactivity of the hormone. Its cognate receptor, which belongs to the superfamily of the G protein-linked cell surface receptors, also displays a high degree of functional and molecular complexity. Studies employing monoclonal antibodies, synthetic peptides and/or site directed mutagenesis, have unveiled some of the multiple structural determinants involved in FSH and FSH receptor function and interaction. Despite their structural complexity, both molecules exhibit a high degree of plasticity and diversity that allows formation of distinct ligand-receptor complexes capable of selectively activating or deactivating a variety of signalling pathways. Knowledge and mapping of the structural determinants and functional epitopes for intra- and extracellular hormone action are of paramount importance not only for a better and more detailed understanding of the molecular basis of FSH action and FSH receptor function but also for the rational design of analogues with predicted properties and effects.

Análisis comparativo entre la TSH de tercera generación y la prueba de estimulación con THR en pacientes con hipertiroidismo, hipotiroidismo y sujetos normales

El análisis de la hormona tiroestimulante (TSH) de tercera generación permite la diferenciación de pacientes con hipertiroidismo real y pacientes con niveles suprimidos de TSH asociados con otras patologías, lo cual era imposible determinar con los análisis de primera y segunda generación

Evidence that the alpha-subunit influences the specificity of receptor binding of the equine gonadotrophins.

Horse LH/chorionic gonadotrophin (eLH/CG) exhibits, in addition to its normal LH activity, a high FSH activity in all other species tested. Donkey LH/CG (dkLH/CG) also exhibits FSH activity in other species, but about ten times less than the horse hormone. In order to understand the molecular basis of these dual gonadotrophic activities of eLH/CG and dkLH/CG better, we expressed, in COS-7 cells, hybrids between horse and donkey subunits, between horse or donkey alpha-subunit and human CG beta (hCG beta), and also between the porcine alpha-subunit and horse or donkey LH/CG beta. The resultant recombinant hybrid hormones were measured using specific FSH and LH in vitro bioassays which give an accurate measure of receptor binding specificity and activation. Results showed that it is the beta-subunit that determines the level of FSH activity, in agreement with the belief that it is the beta-subunit which determines the specificity of action of the gonadotrophins. However, donkey LH/CG beta combined with a porcine alpha-subunit exhibited no FSH activity although it showed full LH activity. Moreover, the hybrid between horse or donkey alpha-subunit and hCG beta also exhibited only LH activity. Thus, the low FSH activity of dkLH/CG requires an equine (donkey or horse) alpha-subunit combined with dkLH/CG beta. These results provide the first evidence that an alpha-subunit can influence the specificity of action of a gonadotrophic hormone.

Disulfide bonds 7-31 and 59-87 of the alpha-subunit play a different role in assembly of human chorionic gonadotropin and lutropin.

CG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit but differ in their hormone-specific beta-subunits. Both subunits have five and six disulfide bonds, respectively, which consists of cystine knot structure. We previously eliminated the disulfide bonds 7-31 and 59-87 in alpha-subunit without significantly affecting assembly with human CG beta-subunit. To study the role of these disulfide bonds in dimerization with other beta-subunits, the wild-type or mutated alpha gene was contransfected with the wild-type human LH beta or FSH beta gene into Chinese hamster ovary (CHO) cells or GH3 cells, and assembly was assessed by continuous labeling with [35S]methionine/cysteine, immunoprecipitation with anti-alpha or-beta serum, and SDS-PAGE. Our data revealed that disruption of either disulfide bond 7-31 or 59-87 in the alpha-subunit markedly reduced the dimer formation with LH beta-subunit in both CHO and GH3 cells, whereas it did not significantly affect the assembly of FSH. This suggests that the regions in the alpha-subunit recognized by beta-subunits for assembly are different among gonadotropins.

Glycoprotein hormone alpha-subunit functions synergistically with progesterone to stimulate differentiation of cultured human endometrial stromal cells to decidualized cells: a novel role for free alpha-subunit in reproduction.

Glycoprotein hormone-free alpha subunit is secreted by the pituitary throughout the menstrual cycle and by the placenta during pregnancy. We showed previously that free alpha subunit stimulated PRL secretion from term pregnancy decidual cells, suggesting a function for free alpha in pregnancy. However, no role has been ascribed to free alpha in the normal menstrual cycle. Using an in vitro model, we examined the role of alpha subunit in regulating human endometrial stromal cell differentiation (decidualization). PRL and insulin-like growth factor binding protein-1 (IGFBP-1), specific decidual secretory products, were used as markers of decidualization. We found that alpha subunit acted synergistically with progesterone (P) to induce more rapid decidualization with higher output (2- to 6-fold) of PRL and IGFBP-1, compared with P alone (P < 0.01). The effect of alpha was dose dependent, with stimulatory activity starting at 0.05 ng/ml and reaching maximal levels at 1-2 ng/ml. These levels correspond to serum concentrations of free alpha found during the luteal phase of the menstrual cycle when endometrial decidualization occurs in vivo. These findings demonstrate new biological activity for alpha subunit in the regulation of human endometrial decidualization and indicate that free alpha plays a role in human reproduction. Furthermore, demonstration of potential bioactivities of free alpha subunit has important implications for understanding normal endocrine function and various pathological conditions.

Effect of immunization against synthetic peptide sequences of the alpha N-subunit of bovine inhibin on ovulation rate, gonadotrophin concentrations and fertility in heifers.

The effects of immunizing cattle against either of two peptides from the amino terminal peptide (alpha N) of the alpha 43-subunit of bovine inhibin on ovulation rate, gonadotrophin concentration and fertility were investigated. Two peptide sequences from the alpha N-subunit of bovine inhibin (P1N, bI alpha-(8-20) and P2N, bI alpha-(153-167)) were synthesized and conjugated to human serum albumin (HSA). Hereford-cross heifers (n = 5 per group) were given an initial injection of 3 mg of one of the peptide conjugates, followed by three booster injections (1.5 mg) at intervals of 11 weeks. Control heifers (n = 5) were injected with HSA only. Blood samples were taken once a week to measure antibody titre and every hour at about the time of the first oestrus and during the mid-luteal phase after the second booster injection, to measure FSH and LH concentrations. Ovulation rate was measured by ultrasonography. Gonadotrophin concentrations were analysed for four periods relative to the peak (time = 0 h) of the preovulatory LH surge as follows: pre-surge: -16 to -5 h; surge: -4 to 4 h; post-surge: 5 to 16 h and a period of 12 h during the mid-luteal (days 10-12) phase. Antibodies that bound to the individual peptides were generated and the ovulation rate increased (P < 0.05) in immunized heifers. Control heifers had one ovulation at all ovulatory cycles monitored. In group P1N, one heifer had two ovulations at each of the six cycles monitored, while another heifer had two ovulations at one cycle.(ABSTRACT TRUNCATED AT 250 WORDS)