Miriam Verhoef-Post

Leydig cell hypoplasia: Cases with new mutations, new polymorphisms and cases without mutations in the luteinizing hormone receptor gene

background Defective male sex differentiation in patients with hypoplasia of Leydig cells (LCH) is caused by deficient LH receptor signal transduction. To further investigate the variety of LH receptor gene mutations present in LCH patients and their influence on the phenotype, we examined 10 nonrelated patients with the clinical presentation of LCH.

EFFECT OF PRENATAL EXPOSURE TO DIETHYLSTILBESTROL ON MULLERIAN DUCT DEVELOPMENT IN FETAL MALE MICE

The clinical use of diethylstilbestrol (DES) by pregnant women has resulted in an increased incidence of genital carcinoma in the daugh- ters born from these pregnancies. Also, in the so-called DES-sons abnormalities were found, mainly, the presence of Mullerian duct remnants, which indicates that fetal exposure to DES may have an effect on male sex differentiation. Fetal regression of the Mullerian ducts is under testicular control through anti-Mullerian hormone (AMH). In male mice, treated in utero with DES, the Mullerian ducts do not regress completely, although DES-exposed testes do produce AMH. We hypothesized that incomplete regression in DES-exposed males is caused by a diminished sensitivity of the Mullerian ducts to AMH. Therefore, the effect of DES on temporal aspects of Mullerian duct regression and AMH type II receptor (AMHRII) messenger RNA (mRNA) expression in male mouse fetuses was studied. It was observed that Mullerian duct regression was incomplete at E19 (19 days post coitum), upon DES administration during preg- nancy from E9 through E16. Furthermore, analysis of earlier time points of fetal development revealed that the DES treatment had clearly delayed the onset of Mullerian duct formation by approxi- mately 2 days; in untreated fetuses, Mullerian duct formation was complete by E13, whereas fully formed Mullerian ducts were not observed in DES-treated male fetuses until E15. Using in situ hybridization, no change in the localization of AMH and AMHRII mRNA expression was observed in DES-exposed male fetuses. The mRNA expression was quantified using ribonuclease protection assay, showing an increased expression level of AMH and AMHRII mRNAs at E13 in DES-exposed male fetuses. Furthermore, the mRNA expression levels of Hoxa 11 and steroidogenic factor-1 (SF-1) were determined as a marker for fetal development. Prenatal DES exposure had no effect on Hoxa 11 mRNA expression, indicating that DES did not exert an overall effect on the rate of fetal develop- ment. In DES-exposed male fetuses, SF-1 showed a similar increase in mRNA expression as AMH, in agreement with the observations that the AMH gene promoter requires an intact SF-1 DNA binding site for time- and cell-specific expression, although an effect of DES on SF-1 expression in other tissues, such as the adrenal and pituitary gland, cannot be excluded. However, the increased expression levels of AMH and AMHRII mRNAs do not directly explain the decreased sensitivity of the Mullerian ducts to AMH. Therefore, it is concluded that prenatal DES exposure of male mice delays the onset of Mulle- rian duct development, which may result in an asynchrony in the timing of Mullerian duct formation, with respect to the critical period of Mullerian duct regression, leading to persistence of Mullerian duct remnants in male mice. (Endocrinology 139: 4244 - 4251, 1998)

Asp330 and Tyr331 in the C-terminal cysteine-rich region of the luteinizing hormone receptor are key residues in hormone-induced receptor activation

The luteinizing hormone (LH) receptor plays an essential role in male and female gonadal function. Together with the follicle-stimulating hormone (FSH) and thyroid stimulating hormone (TSH) receptors, the LH receptor forms the family of glycoprotein hormone receptors. All glycoprotein hormone receptors share a common modular topography, with an N-terminal extracellular ligand binding domain and a C-terminal seven-transmembrane transduction domain. The ligand binding domain consists of 9 leucine-rich repeats, flanked by N- and C-terminal cysteine-rich regions. Recently, crystal structures have been published of the extracellular domains of the FSH and TSH receptors. However, the C-terminal cysteine-rich region (CCR), also referred to as the “hinge region,” was not included in these structures. Both structure and function of the CCR therefore remain unknown. In this study we set out to characterize important domains within the CCR of the LH receptor. First, we mutated all cysteines and combinations of cysteines in the CCR to identify the most probable disulfide bridges. Second, we exchanged large parts of the LH receptor CCR by its FSH receptor counterparts, and characterized the mutant receptors in transiently transfected HEK 293 cells. We zoomed in on important regions by focused exchange and deletion mutagenesis followed by alanine scanning. Mutations in the CCR specifically decreased the potencies of LH and hCG, because the potency of the low molecular weight agonist Org 41841 was unaffected. Using this unbiased approach, we identified Asp330 and Tyr331 as key amino acids in LH/hCG mediated signaling.

A new LH receptor splice mutation responsible for male hypogonadism with subnormal sperm production in the propositus, and infertility with regular cycles in an affected sister

BACKGROUND Inactivating LH receptor (LHR) mutations have been described so far in men as well as in women. Phenotypes in men have been variable with in nearly all cases impairment of sex differentiation or azoospermia. We report a milder reproductive phenotype both in a male patient and his sister. METHODS AND RESULTS We describe a family that carries a homozygous mutation G-->A at position -1 at the intron 10-exon 11 boundary of the LHR gene. The male patient presented with delayed puberty, micropenis and oligospermia. Two of his sisters were homozygous for the same mutation and were infertile. Surprisingly, one of them was found to have had regular ovarian cycles for years and showed normal LH values (6.5 and 10.6 mIU/ml for LH and FSH, respectively). In vitro analysis showed that this altered splicing resulted in an LHR from which eight amino acids are deleted from the extracellular domain (Delta Tyr(317)-Ser(324)). In vitro expression has shown that the receptor was expressed and capable of LH-induced signaling, albeit with reduced potency (P < 0.001). CONCLUSIONS LHR mutations may represent an underestimated cause of infertility in women, in addition to being responsible for male hypogonadism with reduced spermatogenesis.

Polymorphic variations in exon 10 of the luteinizing hormone receptor: functional consequences and associations with breast cancer.

Polymorphic variation of the LHR gene may affect receptor function and accordingly may influence ovarian steroid hormone action, including steroid hormone-dependent clinical outcome. The functional effects of two single nucleotide polymorphisms (SNPs), i.e. LHR 291Asn/Ser (rs12470652) and 312Ser/Asn (rs2293275) in the biologically interesting exon 10 of the LHR gene are described. Furthermore, ethnic diversity in allele frequencies and genotype distributions of both SNPs was determined. In addition associations with breast cancer were studied in 751 breast cancer patients. In vitro transfection studies revealed altered glycosylation status and increased receptor sensitivity for the 291Ser LHR variant. No functional consequences were observed for the 312SerAsn LHR SNP. The LHR 312Asn allele was slightly more often present in two independent breast cancer patient cohorts as compared to controls (OR=1.15; p=0.03 and 1.26; p=0.001, respectively). In conclusion, although functional changes of the LHR 291Ser candidate allele were observed, no associations with breast cancer were found, while the LHR 312Asn allele can be regarded as a weak breast cancer risk allele.

Mutation analysis of the LH receptor gene in Leydig cell adenoma and hyperplasia and functional and biochemical studies of activating mutations of the LH receptor gene

CONTEXT Germline and somatic activating mutations in the LH receptor (LHR) gene have been reported. OBJECTIVE Our objective was to perform mutation analysis of the LHR gene of patients with Leydig cell adenoma or hyperplasia. Functional studies were conducted to compare the D578H-LHR mutant with the wild-type (WT)-LHR and the D578G-LHR mutant, a classic cause of testotoxicosis. The three main signal transduction pathways in which LHR is involved were studied. PATIENTS We describe eight male patients with gonadotropin-independent precocious puberty due to Leydig cell adenoma or hyperplasia. RESULTS The D578H-LHR mutation was found in the adenoma or nodule with hyperplasia in all but two patients. D578H-LHR displayed a constitutively increased but noninducible production of cAMP, led to a very high production of inositol phosphates, and induced a slight phosphorylation of p44/42 MAPK in the absence of human chorionic gonadotropin. The D578G-LHR showed a response intermediate between WT-LHR and the D578H-LHR. Subcellular localization studies showed that the WT-LHR was almost exclusively located at the cell membrane, whereas the D578H-LHR showed signs of internalization. D578H-LHR was the only receptor to colocalize with early endosomes in the absence of human chorionic gonadotropin. CONCLUSIONS Although several LHR mutations have been reported in testotoxicosis, the D578H-LHR mutation, which has been found only as a somatic mutation, appears up until now to be specifically responsible for Leydig cell adenomas. This is reflected by the different activation of the signal transduction pathways, when compared with the WT-LHR or D578G-LHR, which may explain the tumorigenesis in the D578H mutant.

LH receptor gene expression is essentially absent in breast tumor tissue: implications for treatment

Worldwide, breast cancer is the most frequently occurring malignancy in women. Early age at full term pregnancy has a protective effect against breast cancer. Evidence coming from a rat breast cancer model suggests a possible role for the pregnancy hormone hCG, a ligand of the LH receptor, as a mediator for this effect. In a previous study, we found that a common polymorphism in the LH receptor associates with tumor progression in premenopausal breast cancer patients, as carriers of the variant receptor showed a shorter disease free survival compared to non-carriers. How hCG and its receptor exert their effects on breast cancer, however, is unclear. One possibility is that these effects take place through LH receptors present in the ovaries, thereby influencing steroid hormone production. Another possibility is that the effects take place through LH receptors present in breast tumor cells themselves, as some studies have detected the receptor in both normal and neoplastic breast tissues and in breast cancer cell lines. To investigate whether a direct effect of LH signaling in breast cancer is likely, we measured LH receptor mRNA expression levels in 1551 breast tumors and 42 different human breast cancer cell lines using a qRT-PCR with a wide dynamic range. In addition, associations between LH receptor expression and clinico-pathologic factors were investigated. Assay validation showed that as little as ?10 copies per reaction volume of LH receptor cDNA could still be detected by our assay. We show that LH receptors are undetectable in 62% of breast tumor samples and 41 of 42 breast cancer cell lines. For the remaining samples we found expression levels to be very low. Although low, expression of the LH receptor appears to be associated with normal breast cells, favorable tumor characteristics and low tumor percentage. Since expression of the LH receptor in breast cancer cells is very low, it almost excludes the possibility of direct signaling effects. We therefore conclude that signaling effects of the LH receptor on breast cancer most likely take place by an indirect pathway through the ovaries.

Unsaturated fatty acids prevent desensitization of the human growth hormone secretagogue receptor by blocking its internalization

The composition of the plasma membrane affects the responsiveness of cells to metabolically important hormones such as insulin and vasoactive intestinal peptide. Ghrelin is a metabolically regulated hormone that activates the G protein-coupled receptor GH secretagogue receptor type 1a (GHSR) not only in the pituitary gland but also in peripheral tissues such as the pancreas, stomach, and T cells in the circulation. We have investigated the effects of lipids and altered plasma membrane composition on GHSR activation. Oligounsaturated fatty acids (OFAs) disrupt the structure of membranes and make them more fluid. Prolonged (96 h), but not acute, treatment of the GHSR cells with the 18C OFAs oleic and linoleic acid caused a significant increase in sensitivity of the receptor to ghrelin (EC(50) reduced by a factor of 2.4 and 2.9 at 60 and 120 microM OFAs, respectively). OFAs were found to block the inhibitory effects of ghrelin pretreatment on subsequent ghrelin responsiveness, suggesting that OFAs suppress desensitization of GHSR. Radioligand displacement studies did not show a significant shift in receptor binding after incubation with OFAs. However, it was found that OFA treatment suppressed GHSR internalization, likely explaining OFA-induced refractoriness to ligand-induced desensitization. The involvement of lipid rafts in this process was indicated by the altered responsiveness of GHSR under conditions that alter membrane cholesterol. In conclusion, our findings demonstrate the importance of membrane composition for GHSR activation and desensitization and indicate at least part of the mechanism through which OFAs and cholesterol could affect ghrelins activity in vivo.