Cancer Treatment and Bone Health.

Considerable advances in oncology over recent decades have led to improved survival, while raising concerns about long-term consequences of anticancer treatments. In patients with breast or prostate malignancies, bone health is a major issue due to the high risk of bone metastases and the frequent prolonged use of hormone therapies that alter physiological bone turnover, leading to increased fracture risk. Thus, the onset of cancer treatment-induced bone loss (CTIBL) should be considered by clinicians and recent guidelines should be routinely applied to these patients. In particular, baseline and periodic follow-up evaluations of bone health parameters enable the identification of patients at high risk of osteoporosis and fractures, which can be prevented by the use of bone-targeting agents (BTAs), calcium and vitamin D supplementation and modifications of lifestyle. This review will focus upon the pathophysiology of breast and prostate cancer treatment-induced bone loss and the most recent evidence about effective preventive and therapeutic strategies.

Molecular mechanisms of enhancing porcine granulosa cell proliferation and function by treatment in vitro with anti-inhibin alpha subunit antibody.

BACKGROUND: This study was conducted to clarify the effect of the inhibiting action of inhibin on porcine granulosa cell proliferation and function, and to investigate the underlying intracellular regulatory molecular mechanisms. METHODS: Porcine granulosa cells were cultured in vitro, and were treated with an anti-inhibin alpha subunit antibody, with or without co-treatment of follicle-stimulating hormone (FSH) in the culture medium. RESULTS: Treatment with anti-inhibin alpha subunit antibody led to a significant increase in estradiol (E2) secretion and cell proliferation. Anti-inhibin alpha subunit antibody worked synergistically with FSH at low concentrations (25 microg/mL) to stimulate E2 secretion, but attenuated FSH action at high concentrations (50 microg/mL). Immunoneutralization of inhibin bioactivity increased FOXL2, Smad3, and PKA phosphorylation, and mRNA expression of the transcription factors CEBP and c-FOS. The expression of genes encoding gonadotropin receptors, FSHR and LHR, and of those involved in steroidogenesis, as well as IGFs and IGFBPs, the cell cycle progression factors cyclinD1 and cyclinD2, and the anti-apoptosis and anti-atresia factors Bcl2, TIMP, and ADAMTS were upregulated following anti-inhibin alpha-subunit treatment. Treatment with anti-inhibin alpha subunit down regulated expression of the pro-apoptotic gene encoding caspase3. Although expression of the pro-angiogenesis genes FN1, FGF2, and VEGF was upregulated, expression of the angiogenesis-inhibiting factor THBS1 was downregulated following anti-inhibin alpha subunit treatment. CONCLUSIONS: These results suggest that immunoneutralization of inhibin bioactivity, through augmentation of the activin and gonadotropin receptor signaling pathways and regulation of gene expression, permits the development of healthy and viable granulosa cells. These molecular mechanisms help to explain the enhanced ovarian follicular development observed following inhibin immunization in animal models.

What can tubular progenitor cultures teach us about kidney regeneration?

Cultures of stem or progenitor cells have made critical contributions to the comprehension of tissue regeneration. In the kidney, primary cultures of human tubular progenitors became available only recently and allow dissection of the functional properties of tubular progenitors vs. differentiated tubular epithelia. Toll-like receptor-mediated activation now appears as a previously unknown mechanism of progenitor-mediated tubular regeneration, implying that proinflammatory factors activate regenerative processes in the kidney.

Human renal stem/progenitor cells repair tubular epithelial cell injury through TLR2-driven inhibin-A and microvesicle-shuttled decorin.

Acute kidney injury (AKI) is emerging as a worldwide public health problem. Recent studies have focused on the possibility of using human adult renal stem/progenitor cells (ARPCs) to improve the repair of AKI. Here we studied the influence of ARPCs on the healing of cisplatin-injured renal proximal tubular epithelial cells. Tubular, but not glomerular, ARPCs provided a protective effect promoting proliferation of surviving tubular cells and inhibiting cisplatin-induced apoptosis. The recovery effect was specific to tubular ARPCs, occurred only after damage sensing, and was completely cancelled by TLR2 blockade on tubular ARPCs. Moreover, tubular, but not glomerular, ARPCs were resistant to the apoptotic effect of cisplatin. Tubular ARPCs operate mainly through the engagement of TLR2, the secretion of inhibin-A protein, and microvesicle-shuttled decorin, inhibin-A, and cyclin D1 mRNAs. These factors worked synergistically and were essential to the repair process. The involvement of tubular ARPC-secreted inhibin-A and decorin mRNA in the pathophysiology of AKI was also confirmed in transplant patients affected by delayed graft function. Hence, identification of this TLR2-driven recovery mechanism may shed light on new therapeutic strategies to promote the recovery capacity of the kidney in acute tubular damage. Use of these components, derived from ARPCs, avoids injecting stem cells.

Insertion of Inhbb into the Inhba locus rescues the Inhba-null phenotype and reveals new activin functions.

The activins (dimers of betaA or betaB subunits, encoded by the genes Inhba and Inhbb, respectively) are TGF-beta superfamily members that have roles in reproduction and development. Whereas mice homozygous for the Inhba-null allele demonstrate disruption of whisker, palate and tooth development, leading to neonatal lethality, homozygous Inhbb-null mice are viable, fertile and have eye defects. To determine if these phenotypes were due to spatiotemporal expression differences of the ligands or disruption of specific ligand-receptor interactions, we replaced the region of Inhba encoding the mature protein with Inhbb, creating the allele Inhbatm2Zuk (hereafter designated InhbaBK). Although the craniofacial phenotypes of the Inhba-null mutation were rescued by the InhbaBK allele, somatic, testicular, genital and hair growth were grossly affected and influenced by the dosage and bioactivity of the allele. Thus, functional compensation within the TGF-beta superfamily can occur if the replacement gene is expressed appropriately. The novel phenotypes in these mice further illustrate the usefulness of insertion strategies for defining protein function.

Effect of xylazine sedation on testicular blood flow, testicular echotexture, and circulating hormones in Shiba goats.

Under field conditions, sedation may be required for a full assessment of the reproductive potential of farm animals. The present study aimed to investigate the effect of xylazine sedation on testicular hemodynamics (TBF), echotexture, testicular volume (TV), and circulating hormones in goats. Sixteen male Shiba goats were sedated using the recommended dose of xylazine (0.05 mg/Kg BW). Testicular hemodynamics were evaluated using color-pulsed Doppler ultrasonography before and after sedation. Echotexture of the testicular parenchyma and TV were assessed using computerized image analysis. Concentrations of testosterone, estradiol (E2), inhibin, cortisol, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were measured using radioimmunoassay. There were no effects of xylazine sedation in TBF, TV, testicular parenchyma parameters, and concentrations of testosterone, inhibin, FSH, and LH (P ˃ 0.05). However, after sedation, there was significantly (P ˂ 0.05) lower cortisol and E2 concentration (42.88 ± 6.79 ng/ml and 2.47 ± 0.58 pg/ml, respectively) than before sedation (94.89 ± 13.74 ng/ml and 8.65 ± 1.79 pg/ml, respectively). The required time to perform the full scanning of the testis was significantly lower (8.50 ± 0.38 min) after xylazine sedation compared to the non-sedated goats (25.75 ± 1.14 min). In conclusion, xylazine sedation may be practically recommended for the evaluation of TBF in goats because it did not significantly alter velocities parameters and Doppler indices of blood flow within the testicular arteries. Most plasma hormones did not significantly change; however, E2 and cortisol were significantly reduced after xylazine administration.

The FSH-inhibin axis in prader-willi syndrome: heterogeneity of gonadal dysfunction.

BACKGROUND: We characterized the spectrum and etiology of hypogonadism in a cohort of Prader-Willi syndrome (PWS) adolescents and adults. METHODS: Reproductive hormonal profiles and physical examination were performed on 19 males and 16 females ages 16-34 years with PWS. Gonadotropins, sex-steroids, inhibin B (INB) and anti-Mullerian hormone (AMH) were measured. We defined 4 groups according to the relative contribution of central and gonadal dysfunction based on FSH and INB levels: Group A: primary hypogonadism (FSH >15 IU/l and undetectable INB (<10 pg/ml); Group B: central hypogonadism (FSH <0.5 IU/l, INB <10 pg/ml); Group C: partial gonadal & central dysfunction (FSH 1.5-15 IU/l, INB >20 pg/ml); Group D: mild central and severe gonadal dysfunction (FSH 1.5-15 IU/l, INB < 10 pg/ml. RESULTS: There were 10, 8, 9 and 8 individuals in Groups A-D respectively; significantly more males in group A (9, 4, 4 and 2; P = 0.04). Significant differences between the groups were found in mean testosterone (P = 0.04), AMH (P = 0.003) and pubic hair (P = 0.04) in males and mean LH (P = 0.003) and breast development (P = 0.04) in females. Mean age, height, weight, BMI and the distribution of genetic subtypes were similar within the groups. CONCLUSIONS: Analysis of FSH and inhibin B revealed four distinct phenotypes ranging from primary gonadal to central hypogonadism. Primary gonadal dysfunction was common, while severe gonadotropin deficiency was rare. Longitudinal studies are needed to verify whether the individual phenotypes are consistent.

A phase plane graph based model of the ovulatory cycle lacking the "positive feedback" phenomenon.

When hormones during the ovulatory cycle are shown in phase plane graphs, reported FSH and estrogen values form a specific pattern that resembles the leaning "&" symbol, while LH and progesterone (Pg) values form a "boomerang" shape. Graphs in this paper were made using data reported by Stricker et al. [Clin Chem Lab Med 2006;44:883-887]. These patterns were used to construct a simplistic model of the ovulatory cycle without the conventional "positive feedback" phenomenon. The model is based on few well-established relations:hypothalamic GnRH secretion is increased under estrogen exposure during two weeks that start before the ovulatory surge and lasts till lutheolysis.the pituitary GnRH receptors are so prone to downregulation through ligand binding that this must be important for their function.in several estrogen target tissue progesterone receptor (PgR) expression depends on previous estrogen binding to functional estrogen receptors (ER), while Pg binding to the expressed PgRs reduces both ER and PgR expression.Some key features of the presented model are here listed:High GnRH secretion induced by the recovered estrogen exposure starts in the late follicular phase and lasts till lutheolysis. The LH and FSH surges start due to combination of accumulated pituitary GnRH receptors and increased GnRH secretion. The surges quickly end due to partial downregulation of the pituitary GnRH receptors (64% reduction of the follicular phase pituitary GnRH receptors is needed to explain the reported LH drop after the surge). A strong increase in the lutheal Pg blood level, despite modest decline in LH levels, is explained as delayed expression of pituitary PgRs. Postponed pituitary PgRs expression enforces a negative feedback loop between Pg levels and LH secretions not before the mid lutheal phase.Lutheolysis is explained as a consequence of Pg binding to hypothalamic and pituitary PgRs that reduces local ER expression. When hypothalamic sensitivity to estrogen is diminished due to lack of local ERs, hypothalamus switches back to the low GnRH secretion rate, leading to low secretion of gonadotropins and to lutheolysis. During low GnRH secretion rates, previously downregulated pituitary GnRH receptors recover to normal levels and thus allow the next cycle.Possible implications of the presented model on several topics related to reproductive physiology are shortly discussed with some evolutionary aspects including the emergence of menopause.

New targets for old hormones: inhibins clinical role revisited.

Inhibins are gonadal peptide hormones belonging to the transforming growth factor-ß (TGF-ß) superfamily that regulate the pituitary follicle stimulating hormone (FSH) secretion by negative feedback mechanisms. It is evident that the understanding of inhibins function in the hypothalamic-pituitary-gonadal axis will provide insights into physiology and pathology of the gonadal function. In recent years, a great deal of attention has been focussed on clinical relevance of measuring circulating inhibins in normal and disease state. The past few years also have witnessed the emergence and discovery of extra pituitary action of inhibins that might provide further insights into the underlying diseases like cancer especially in the reproductive axis and various other new endocrine target organs. In this review after systematic analysis of literature, we discuss briefly the known and recent advances in function of these hormones highlighting also its structure, production and mechanisms of signal transduction. Also this review discusses about the physiological relevance of inhibin association in the normal function to the development of reproductive cancers. Finally, we describe evidence from various emerging studies that inhibins make an important contribution to other physiological functions apart from reproduction which reveals new endocrine target organs of inhibins. The emerging view is inhibin participates in multiple ways to regulate the function in different cell types and still complete repertoire of its actions is under investigation.

Loss of inhibin alpha uncouples oocyte-granulosa cell dynamics and disrupts postnatal folliculogenesis.

Targeted disruption of the inhibin alpha gene (Inha(-)(/)(-)) in mice results in an ovarian phenotype of granulosa cell tumors that renders the animals infertile. Little is known about the reproductive defects prior to tumor development. Here, we report novel data on early follicle dynamics in Inha(-)(/)(-) mice, which demonstrate that inhibin alpha has important consequences upon follicle development. Morphological changes in both germ and somatic cells were evident in postnatal day 12 ovaries, with Inha(-/-) mice exhibiting numerous multilayered follicles that were far more advanced than those observed in age-matched controls. These changes were accompanied by alterations in follicle dynamics such that Inha(-/-) ovaries had fewer follicles in the resting pool and more committed in the growth phase. Absence of inhibin alpha resulted in advanced follicular maturation as marked by premature loss of anti-Müllerian hormone (AMH) in secondary follicles. Additionally, gene expression analysis revealed changes in factors known to be vital for oocyte and follicle development. Together, these data provide key evidence to suggest that regulation of the inhibin/activin system is essential for early folliculogenesis in the prepubertal mouse ovary.

Changes in expression of bone morphogenetic proteins (BMPs), their receptors and inhibin co-receptor betaglycan during bovine antral follicle development: inhibin can antagonize the suppressive effect of BMPs on thecal androgen production.

We reported previously that bone morphogenetic proteins (BMPs) potently suppress CYP17 expression and androgen production by bovine theca interna cells (TC) in vitro. In this study, real-time PCR was used to analyse gene expression in TC and granulosa cell (GC) layers from developing bovine antral follicles (1-18 mm). Abundance of mRNA transcripts for four BMPs (BMP2, BMP4, BMP6, and BMP7) and associated type I (BMPR1A, BMPR1B, ACVR1 and ACVR1B) and type II (BMPR2, ACVR2A and ACVR2B) receptors showed relatively modest, though significant, changes during follicle development. BMP2 was selectively expressed in GC, while BMP6, BMP7 and betaglycan (TGFBR3) were more abundant in TC. Abundance of betaglycan mRNA (inhibin co-receptor) in TC increased progressively (fivefold; P<0.001) as follicles grew from 1-2 to 9-10 mm. This suggests a shift in thecal responsiveness to GC-derived inhibin, produced in increasing amounts as follicles achieve dominance. This prompted us to investigate whether inhibin can function as a physiological antagonist of BMP action on bovine TC in vitro, in a manner comparable to that for activin signalling. BMP4, BMP6 and BMP7 abolished LH-induced androstenedione secretion and suppressed CYP17 mRNA >200-fold (P<0.001), while co-treatment with inhibin-A reversed the suppressive action of BMP in each case (P<0.001). Results support a physiological role for granulosa-derived inhibin as an antagonist of BMP action on thecal androgen synthesis. A shift in intrafollicular balance between thecal BMP signalling (inhibitory for androgen synthesis) and betaglycan-dependent inhibin signalling (stimulatory for androgen synthesis) accords with the physiological requirement to deliver an adequate supply of aromatase substrate to GC of developing follicles.

Anti-Müllerian hormone (Amh/amh) plays dual roles in maintaining gonadal homeostasis and gametogenesis in zebrafish.

Anti-Müllerian hormone (AMH/Amh) plays a role in gonadal differentiation and function across vertebrates. In zebrafish we demonstrated that Amh deficiency caused severe gonadal dysgenesis and dysfunction. The mutant gonads showed extreme hypertrophy with accumulation of early germ cells in both sexes, namely spermatogonia in the testis and primary growth oocytes in the ovary. In amh mutant females, the folliculogenesis was normal in young fish but receded progressively in adults, which was accompanied by progressive decrease in follicle-stimulating hormone (fshb) expression. Interestingly the expression of fshb increased in the pituitary of juvenile amh mutant males but decreased in adults. The upregulation of fshb in mutant male juveniles was likely one of the mechanisms for triggering gonadal hypergrowth, whereas the downregulation of fshb in adults might involve a negative feedback by gonadal inhibin. Further analysis using mutants of fshb and growth differentiation factor 9 (gdf9) provided evidence for a role of FSH in triggering ovarian hypertrophy in young female amh mutant as well. In summary, the present study provided comprehensive genetic evidence for dual roles of Amh in controlling zebrafish gonadal homeostasis and gametogenesis in both sexes. Amh suppresses proliferation or accumulation of early germ cells (spermatogonia in testis and primary growth oocytes in ovary) while promoting their exit to advanced stages, and its action may involve both endocrine and paracrine pathways.

Activins and inhibins: novel regulators of thymocyte development.

Activins and inhibins are members of the transforming growth factor-beta superfamily that act on different cell types and regulate a broad range of cellular processes including proliferation, differentiation, and apoptosis. Here, we provide the first evidence that activins and inhibins regulate specific checkpoints during thymocyte development. We demonstrate that both activin A and inhibin A promote the DN3-DN4 transition in vitro, although they differentially control the transition to the DP stage. Whereas activin A induces the accumulation of a CD8+CD24(hi)TCRbeta(lo) intermediate subpopulation, inhibin A promotes the differentiation of DN4 to DP. In addition, both activin A and inhibin A appear to promote CD8+SP differentiation. Moreover, inhibin alpha null mice have delayed in vitro T cell development, showing both a decrease in the DN-DP transition and reduced thymocyte numbers, further supporting a role for inhibins in the control of developmental signals taking place during T cell differentiation in vivo.

Characterization of inhibin alpha subunit (inha) in the zebrafish: evidence for a potential feedback loop between the pituitary and ovary.

Inhibin and activin are closely related disulphide-linked dimers that belong to the transforming growth factor beta superfamily. Although inhibin has been extensively studied in mammals, the information about its existence and function in lower vertebrates is very scarce. Using zebrafish as a model, the present study demonstrated that the inhibin-specific alpha subunit (inha) was predominantly expressed in the gonads and no transcript could be detected in other tissues including the pituitary and brain. In the ovary, the expression of inha was restricted to the somatic follicle cells surrounding the oocyte, together with the beta subunits (inhbaa and inhbb). This was further supported by the absence of its expression in the ovulated unfertilized eggs. During folliculogenesis, inha expression in the follicles slightly but steadily increased from primary growth to the mid-vitellogenic stage; however, its expression surged dramatically at the full-grown stage. Interestingly, the expression level of inha decreased significantly in the follicles whose oocytes were undergoing spontaneous maturation or germinal vesicle breakdown. When tested on cultured ovarian fragments, both goldfish pituitary extract and forskolin significantly stimulated inha expression. Further experiments showed that recombinant zebrafish FSH but not LH significantly increased inha expression in the same assay system. When tested in vitro, human inhibin A exhibited a slight but significant inhibitory effect on 17alpha, 20beta-dihydroxyprogesterone-induced oocyte maturation after 4 h incubation. The stimulation of inha expression by FSH and the potential inhibition of FSH by inhibin suggest a possible existence of a negative feedback loop between the pituitary and ovary in the zebrafish.

Xenopus embryonic cell adhesion to fibronectin: position-specific activation of RGD/synergy site-dependent migratory behavior at gastrulation.

During Xenopus laevis gastrulation, the basic body plan of the embryo is generated by movement of the marginal zone cells of the blastula into the blastocoel cavity. This morphogenetic process involves cell adhesion to the extracellular matrix protein fibronectin (FN). Regions of FN required for the attachment and migration of involuting marginal zone (IMZ) cells were analyzed in vitro using FN fusion protein substrates. IMZ cell attachment to FN is mediated by the Arg-Gly-Asp (RGD) sequence located in the type III-10 repeat and by the Pro-Pro-Arg-Arg-Ala-Arg (PPRRAR) sequence in the type III-13 repeat of the Hep II domain. IMZ cells spread and migrate persistently on fusion proteins containing both the RGD and synergy site sequence Pro-Pro-Ser-Arg-Asn (PPSRN) located in the type III-9 repeat. Cell recognition of the synergy site is positionally regulated in the early embryo. During gastrulation, IMZ cells will spread and migrate on FN whereas presumptive pre-involuting mesoderm, vegetal pole endoderm, and animal cap ectoderm will not. However, animal cap ectoderm cells acquire the ability to spread and migrate on the RGD/synergy region when treated with the mesoderm inducing factor activin-A. These data suggest that mesoderm induction activates the position-specific recognition of the synergy site of FN in vivo. Moreover, we demonstrate the functional importance of this site using a monoclonal antibody that blocks synergy region-dependent cell spreading and migration on FN. Normal IMZ movement is perturbed when this antibody is injected into the blastocoel cavity indicating that IMZ cell interaction with the synergy region is required for normal gastrulation.

Cloning of a second type of activin receptor and functional characterization in Xenopus embryos.

A complementary DNA coding for a second type of activin receptor (ActRIIB) has been cloned from Xenopus laevis that fulfills the structural criteria of a transmembrane protein serine kinase. Ectodermal explants from embryos injected with activin receptor RNA show increased sensitivity to activin, as measured by the induction of muscle actin RNA. In addition, injected embryos display developmental defects characterized by inappropriate formation of dorsal mesodermal tissue. These results demonstrate that this receptor is involved in signal transduction and are consistent with the proposed role of activin in the induction and patterning of mesoderm in Xenopus embryos.

Pattern formation during animal development.

At the beginning of this century, embryologists defined the central problems of developmental biology that remain today. These questions include how differentiated cells arise and form tissues and organs and how pattern is generated. In short, how does an egg give rise to an adult? In recent years, the application of molecular biology to embryological problems has led to significant advances and recast old problems in molecular and cellular terms. Although not necessarily comprehensive, this idiosyncratic review is intended to highlight selected findings and indicate where there are important gaps in our knowledge for those less than familiar with developmental biology.

Secretion of activin by interstitial cells in the testis.

Activin, a dimer formed by the beta subunits of inhibin, has an effect that is opposite to that of inhibin in a number of biological systems. Which cell types secrete activin in vivo is not known. TM3 cells, a Leydig-derived cell line, contained messenger RNAs that hybridized with human beta A and beta B complementary DNA probes and were similar in size to the porcine messenger RNA for the beta subunits of inhibin. No hybridization to the inhibin alpha subunit was detectable in the TM3 cells. Conditioned medium from TM3 cells and from primary cultures of rat and porcine interstitial cells stimulated the release of follicle-stimulating hormone in a pituitary cell culture assay. It is likely that, in the testis, the Leydig cells secrete activin and the Sertoli cells produce inhibin, or a combination of both.

N-Cadherin, a cell adhesion molecule involved in establishment of embryonic left-right asymmetry.

Within the bilaterally symmetric vertebrate body plan, many organs develop asymmetrically. Here, it is demonstrated that a cell adhesion molecule, N-cadherin, is one of the earliest proteins to be asymmetrically expressed in the chicken embryo and that its activity is required during gastrulation for proper establishment of the left-right axis. Blocking N-cadherin function randomizes heart looping and alters the expression of Snail and Pitx2, later components of the molecular cascade that regulate left-right asymmetry. However, the expression of other components of this cascade (Nodal and Lefty) was unchanged after blocking N-cadherin function, suggesting the existence of parallel pathways in the establishment of left-right morphogenesis. Here, the results suggest that N-cadherin-mediated cell adhesion events are required for establishment of left-right asymmetry.

Localization, secretion, and action of inhibin in human placenta.

Inhibin is a gonadal glycoprotein hormone that regulates the production of follicle-stimulating hormone (FSH) by the anterior pituitary gland and exhibits intragonadal actions as well. The present study shows that inhibin-like immunoreactivity (inhibin-LI) is present in cells of the cytotrophoblast layer of human placenta at term and in primary cultures of human trophoblasts. Human chorionic gonadotropin (hCG) stimulated secretion of inhibin-LI from these cultured placental cells. This effect was mimicked by 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP), forskolin, and cholera toxin, suggesting that the mechanism of hCG induction of placental inhibin-LI secretion is cAMP-dependent. Incubation with an antiserum that binds the alpha-subunit of human inhibin increased the secretion of hCG and gonadotropin-releasing hormone-like immunoreactivity (GnRH-LI) from trophoblast cells in culture, suggesting a local tonic inhibitory action of endogenous inhibin on hCG and GnRH-LI release. The action of inhibin on hCG secretion may partially require the presence of placental GnRH, as suggested by evidence that a synthetic GnRH antagonist partially reverses the hCG increase induced by inhibin immunoneutralization. Results suggest paracrine roles for both inhibin and GnRH in the regulation of placental hCG production.