Biomedical subjects

Manuela Simoni

Publications and source records attributed to Manuela Simoni.

At least 19 recordsLinked to original sources

PubMed2006

Elevated follicle-stimulating hormone levels and the chances for azoospermic men to become fathers after retrieval of elongated spermatids from cryopreserved testicular tissue.

OBJECTIVE: To assess individual chances for a live-born child in azoospermic men by performance of testicular sperm extraction (TESE) followed by intracytoplasmatic sperm injection (ICSI). DESIGN: A retrospective cohort study. SETTING: An academic fertility care center and research unit. PATIENT(S): Two hundred three couples who wished to have a child; all men had azoospermia. INTERVENTION(S): All men were operated for TESE; 112 men were found to have elongated spermatids (ES), and 209 ICSI cycles were performed in these men using cryopreserved tissue. MAIN OUTCOME MEASURE(S): Predictors for the chances to obtain live sperm and for probabilities of fertilization, clinical pregnancies, and live births. RESULT(S): Testicular volume, FSH, and inhibin B levels were predictors for the presence of ES. Intracytoplasmic sperm injection resulted in 23 pregnancies, leading to 20 live births. Despite the presence of ES and performance of ICSI in cases of FSH levels >or=20 IU/L, no pregnancy resulted in these men (n = 21). Receiver operating characteristics revealed FSH levels of >or=20 IU/L as cutoff for treatment success. The number of testicular tubuli containing ES served as a predictor for clinical pregnancy as well as for live birth. Cigarette smoking by the male partner exerted a significant negative influence on treatment success. CONCLUSION(S): The degree of completely maintained spermatogenesis within the biopsy appears to reflect intrinsic abilities of spermatozoa to induce normal embryo development. Charts based on regression models are presented for counseling patients before TESE; these explain chances of finding ES and probability of successful ICSI. Obtaining offspring is unlikely in cases of azoospermia and of FSH levels of >or=20 IU/L.

Adult↗

PubMed2006

Changes in endocrine profile and reproductive organs during puberty in the male marmoset monkey (Callithrix jacchus).

Data on pubertal maturation in male marmoset, a model for human reproduction, are scant and conflicting. We collected data on novel parameters to characterize puberty. Twenty-five marmoset monkeys were assigned to five age groups by weeks (wk): 21 (pre-pubertal), 43 (onset of puberty), 52 (fully pubertal), 70 (mature), and 116 (fully adult). Serum and intratesticular testosterone and pituitary bioactive chorionic gonadotropin (bioCG) were measured. Testicular development was assessed by ultrasonography, histology, and flow cytometry. Three consecutive blood samples revealed extreme fluctuations in testosterone concentrations, suggesting an erratic secretion. Age-related changes in serum testosterone and pituitary bioCG concentrations were observed. Intratesticular androgens (ITAs) showed high fluctuations within groups at all ages and were high in some animals by 21 wk. Unexpectedly, no correlation between pituitary bioCG and serum testosterone or ITAs was found, but these parameters significantly correlated with testicular weight and volume. These observations were consistent a dependence on the testis growth on bioCG. Unfortunately, the low serum levels of bioCG were not measurable in this study. At 43 wk, the animals reached puberty. At 52 wk of age, animals attained maximum body and epididymal weights and qualitatively normal spermatogenesis, but testes continued growing, reaching a maximum of all parameters at 70 wk of age, without further major changes at the age of 116 wk. It is concluded that (1) gonadal activation is evident at wk 21, (2) the male marmoset reaches the pubertal threshold around 43 wk of age, attains qualitative parameters at 52 wk, matures further to sexual maturity at 70 wk, and (3) serum testosterone and ITAs are highly variable without any identifiable correlation with pituitary bioCG.

Animals↗

PubMed2006

Tissue expression of the nuclear progesterone receptor in male non-human primates and men.

In females, progesterone is associated with reproductive functions. In males, its role and the expression of its genomic receptor are not very well understood. In attempts to achieve a hormonal male contraceptive method, gestagens are used to downregulate gonadotropin and sperm production. It is therefore essential to understand the mechanism of action of progesterone at the molecular level in males, especially in primates. This investigation was undertaken: (a) to determine whether the genomic progesterone receptor is expressed in males; and (b) to locate it in various organs that are potential targets of gestagens. Human tissues were obtained at surgery for benign prostatic hyperplasia or prostate cancer and at autopsy. Non-human primate tissues were obtained at autopsy. This study was performed by analyzing the genomic progesterone receptor by immunohistochemistry, Western blot and RT-PCR. The nuclear progesterone receptor was expressed in pituitary and hypothalamus of both monkeys and men. In the testis progesterone receptor expression was found in a few peritubular and interstitial cells, but not in germ cells. In addition, expression was detected in the epididymis, prostate and male mammary gland. Reverse transcriptase (RT)-PCR experiments indicated that progesterone receptor A and B are expressed in all tissues analyzed. These data exclude direct genomic effects of gestagens at the spermatogenic level but indicate that a male contraceptive based on gestagens might have some effects on other tissues, such as the epididymis, prostate and mammary gland.

Animals↗

PubMed2005

Meiosis in autologous ectopic transplants of immature testicular tissue grafted to Callithrix jacchus.

Grafting of immature testicular tissue provides a tool to examine testicular development and may offer a perspective for preservation of fertility in prepubertal patients. Successful xenografting in mice, resulting in mature spermatids, has been performed in several species but has failed with testicular tissues from the common marmoset, Callithrix jacchus. Previous data indicate that the hormonal milieu provided by the mouse host might cause this failure. We conducted autologous ectopic transplantation of testicular fragments under the back skin in newborn marmoset monkeys. Seventeen months after transplantation, we found viable transplants in 2 out of the 4 grafted animals. In the transplants, tubules developed up to a state intermediate between the pregraft situation and adult controls. Dividing spermatogonia and primary spermatocytes were present. Boule-like positivity and CDC25A negativity indicated that spermatogenesis was arrested at early meiosis. Immunohistochemistry revealed normal maturation of Sertoli cells, Leydig cells, and peritubular cells. Serum testosterone values were not restored to the normal range and bioactive chorionic gonadotropin levels increased to castrate levels. Meiotic arrest could have occurred in the grafts because of lack of sufficient testosterone or because of hyperthermia caused by the ectopic position of the grafts. We conclude that autologous transplants of immature testicular tissues in the marmoset can mature up to meiosis but that normal serum testosterone levels are not restored. Further studies have to be performed to overcome the meiotic arrest to explore the model further and to develop therapeutic options.

Animals↗

PubMed2005

Hypothalamic and gonadal components of hypogonadism in boys with Prader-Labhart- Willi syndrome.

CONTEXT: The specific form of hypogonadism in Prader-Labhart-Willi syndrome (PWS), central or peripheral, remains unexplained. OBJECTIVES: The objectives of this study were to investigate the cause of hypogonadism in PWS and determine whether human chorionic gonadotropin (hCG) treatment can restore pubertal development. DESIGN: This was a clinical follow-up study, divided into two samples, over a duration of 1.5 and 4.5 yr. PATIENTS: Eight male infants and six peripubertal boys (age at start of observation, 0.06-0.93 and 8.1-10.8 yr, respectively) with genetically confirmed PWS were studied. INTERVENTION: hCG (500-1500 U twice weekly) was given from age 13.5 yr to the present. MAIN OUTCOME MEASURES: Serum FSH, LH, inhibin B, and testosterone levels and pubertal development were the main outcome measures. RESULTS: Infants with PWS presented normal LH (2.3 +/- 0.7 U/liter) and testosterone (2.5 +/- 0.9 nmol/liter) levels (mean +/- sem at 5 months) compared with the reference range. However, two thirds of the boys displayed cryptorchidism. Inhibin B levels were at the lowest level of the normal range and decreased significantly between infancy and puberty (at 13 yr, 72 +/- 17 pg/ml), whereas FSH secretion increased (9.9 +/- 2.6 U/liter). Pubertal maturation stopped at an average bone age of 13.9 yr. hCG therapy increased testosterone (11 +/- 2 nmol/liter) and reduced FSH (at 16 yr, 1.1 +/- 0.9 U/liter) levels. Testicular volume (5.6 +/- 1 ml) and inhibin B (26.5 +/- 11.9 pg/ml) remained low. CONCLUSION: Children with PWS display a specific form of combined hypothalamic (low LH) and peripheral (low inhibin B and high FSH) hypogonadism, suggesting a primary defect in Sertoli and/or germ cell maturation or an early germ cell loss. hCG therapy stimulates testosterone production and virilization.

Child↗

PubMed2005

Endogenous progesterone and the exogenous progestin norethisterone enanthate are associated with a proinflammatory profile in healthy men.

CONTEXT: Inflammatory processes are related to atherosclerosis. Identification of inflammation triggers may furnish new therapeutic pathways. In women, progestins can have a marked inflammatory capacity. OBJECTIVE AND DESIGN: We investigated the effects of progesterone in men within the setting of two independent trials. First, the relation of endogenous progesterone levels to inflammation markers was assessed in 67 healthy nonsmoking Caucasian men (age, 20-50 yr) on a cross-sectional basis. Second, in a longitudinal controlled trial (52 wk) involving 28 healthy men receiving i.m. medication, we determined the effects of an exogenous progestin (norethisterone enanthate 200 mg) in combination with a long-acting testosterone preparation (testosterone undecanoate 1000 mg) administered to avoid androgen deficiency caused by pituitary-hypothalamic feedback. Controls received testosterone plus placebo. RESULTS: In the cross-sectional study, progesterone levels were positively related to concentrations of IL-6 (r = 0.41; P < 0.001), C-reactive protein (r = 0.37; P = 0.007), soluble vascular cell adhesion molecule 1 (r = 0.28; P = 0.02), E-selectin (r = 0.45; P < 0.001), leptin (r = 0.42; P < 0.001), neutrophils (r = 0.62; P < 0.001), and serum protein fractions alpha-1 (r = 0.44; P < 0.001) and alpha-2 (r = 0.36; P = 0.002). During the pharmacological trial, the testosterone/progestin group exhibited a marked increase of IL-6 concentrations (P < 0.001), whereas these decreased in the testosterone/placebo group (P = 0.03). Antiinflammatory IL-10 levels decreased in the group receiving testosterone/progestin (P = 0.01) but did not change in the testosterone/placebo group. CONCLUSION: Progesterone concentrations correspond to an inflammatory profile in healthy men, and external progestins elicit a similar effect. Men receiving regimens for hormonal male contraception involving progestins should be monitored for inflammatory effects. Speculatively, testosterone treatment decreasing endogenous progesterone production may facilitate beneficial effects on inflammation profiles even in eugonadal men.

Adult↗

PubMed2005

Single-nucleotide polymorphisms in the promoter region influence the expression of the human follicle-stimulating hormone receptor.

OBJECTIVE: To characterize novel single-nucleotide polymorphisms (SNPs) in the human FSH receptor (FSHR) promoter region. DESIGN: Retrospective and basic research study. SETTING: University hospital. PATIENTS: Women (202 from Germany and 55 from Indonesia) with male or tubal factor infertility undergoing controlled ovarian stimulation for IVF treatment. INTERVENTIONS: None. MAIN OUTCOME MEASURE(S): Frequency, distribution, and correlation with clinical data of the SNPs. Dual luciferase assays and electrophoretic mobility shift assays (EMSA). RESULT(S): We identified two SNPs and three mutations in the promoter region of the human FSHR which could be allocated to positions -29, -37, -114, -123, and -138 upstream of the translational initiation codon. One SNP showed a high incidence (-29: 44%, n = 202), but no correlation with basal FSH serum levels or ovarian response with the SNP at position -29 was found. Luciferase reporter assays, using pGL3 vector constructs, showed that mutations at positions -37 and -138 lead to significantly higher promoter activity. EMSA indicate that putative binding sites for transcription factors are affected by the SNPs. CONCLUSIONS: The newly identified SNPs do not seem to influence clinical parameters substantially, but modulate expression of the FSHR via changes in transcription factor binding sites.

Analysis of Variance↗

PubMed2005

Genetic complexity of FSH receptor function.

The interaction between follicle-stimulating hormone (FSH) and the FSH receptor (FSHR) is essential for normal oogenesis and spermatogenesis. Recently, single-nucleotide polymorphisms (SNPs) have been assigned to the FSHR gene. These give rise to different FSHR haplotypes that modify the action of FSH. In women, FSH sensitivities during the menstrual cycle and different cycle lengths are observed, depending on the FSHR haplotype. Thus, SNPs of the FSHR determine the ovarian response and should, therefore, be considered in controlled ovarian hyperstimulation during assisted-reproduction techniques in women with normal ovarian function. In men, the impact of the FSHR SNPs is unclear. The genetic complexity of FSHR should be considered when studying FSH action. These SNPs are one of the first examples in which genetic changes contribute to fine-tuning the endocrine regulation of reproduction. A rational pharmacogenetic approach that combines FSH dose according to the FSHR haplotype is envisaged.

Animals↗

PubMed2005

Pharmacogenetics in ovarian stimulation - current concepts and future options.

Tailoring ovarian stimulation to the individual patient can be challenging because the ovarian response varies substantially between patients. Pharmacogenetics has emerged as a new area of research to improve the balance between desired and undesired actions of drugs, based upon the genetic predisposition of the individual patient. Clinical studies have demonstrated that the p.N680S polymorphism of the FSH-receptor gene determines the ovarian response to FSH stimulation in patients undergoing IVF. In homozygous Ser(680)/Ser(680) type women, the FSH receptor appears to be more resistant to FSH action even in normal menstrual cycles. Therefore, genotyping of patients scheduled for ovarian stimulation could be an attractive tool to individualize FSH dosing according to genetic differences in ovarian sensitivity. More clinical studies are warranted to investigate the usefulness of genotyping for the p.N680S polymorphism as a routine diagnostic test before ovarian stimulation.

Dose-Response Relationship, Drug↗

PubMed2005

Significance of a common single nucleotide polymorphism in exon 10 of the follicle-stimulating hormone (FSH) receptor gene for the ovarian response to FSH: a pharmacogenetic approach to controlled ovarian hyperstimulation.

The p.N680S sequence variation of the follicle-stimulating hormone (FSH) receptor gene was previously shown to influence the ovarian response to FSH in normo-ovulatory women undergoing controlled ovarian hyperstimulation. In this prospective, randomized, controlled study, we tested whether the same daily dose of FSH results in lower levels of oestradiol in women homozygous for the p.N680S sequence variation, and whether the difference can be overcome by higher FSH doses. Women undergoing controlled ovarian hyperstimulation for in vitro fertilization or intracytoplasmic sperm injection and homozygous for the wild-type or for the p.N680S FSH receptor were randomly assigned to group I (Ser/Ser, n=24), receiving an FSH dose of 150 U/day, or group II (Ser/Ser, n=25), receiving an FSH dose of 225 U/day. In group III (Asn/Asn, n=44), the FSH dose was 150 U/day. Age and basal FSH levels were not different between groups. At ovulation induction, total FSH doses were comparable in group I (1631+/-96 U) and group III (1640+/-57 U) but significantly higher in group II (2421+/-112 U) (P<0.001). Peak oestradiol levels on the day of human chorionic gonadotrophin (hCG) administration were significantly lower in group I (5680+/-675 pmol/l) compared to group III (8679+/-804 pmol/l) (P=0.028). Increasing the FSH dose from 150 to 225 U/day overcame the lower oestradiol response in women with Ser/Ser (group II, 7804+/-983 pmol/l). In women undergoing controlled ovarian hyperstimulation, the p.N680S sequence variation results in lower oestradiol levels following FSH stimulation. This lower FSH receptor sensitivity can be overcome by higher FSH doses.

Adolescent↗

PubMed2005

Pharmacokinetics and pharmacodynamics of injectable testosterone undecanoate in castrated cynomolgus monkeys (Macaca fascicularis) are independent of different oil vehicles.

Testosterone undecanoate (TU) dissolved in soybean oil was developed in China to improve the pharmacokinetics of this testosterone ester in comparison with TU in castor or tea seed oil. As a pre-clinical primate model, three groups of five castrated cynomolgus macaques received either a single intramuscular injection of 10 mg/kg body weight TU in soybean oil, in tea seed oil, or in castor oil (equals 6.3 mg pure T/kg body weight for all preparations). Testosterone, estradiol, luteinizing hormone, and follicle-stimulating hormone as well as prostate volume, body weight and ejaculate weight were evaluated. After injection supraphysiological testosterone levels were induced. There were no significant differences in the pharmacokinetics of the three TU preparations for testosterone and estradiol. The gonadotropin levels showed a high individual variation. Prostate volumes increased equally in all groups after administration and declined to castrate level afterwards. The results suggest that TU in soybean oil produces similar effects as TU in the other vehicles. This study in non-human primates provides no objection to testing of this new preparation in humans.

Animals↗

PubMed2005

Norethisterone enanthate has neither a direct effect on the testis nor on the epididymis: a study in adult male cynomolgus monkeys (Macaca fascicularis).

OBJECTIVE: Norethisterone enanthate (NETE) is evaluated in trials of hormonal male contraception. It has been speculated that progestins may exert their contraceptive effects not only by suppressing gonadotropins but also by direct effects on male organs. NETE was given to monkeys in which endogenous gonadotropin secretion was suppressed by a gonadotropin releasing hormone (GnRH) antagonist, and replaced by human follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). If NETE has a direct effect on spermatogenesis and/or epididymal function, some changes in testicular histology, sperm motility and/or morphology should occur soon after exposure to NETE. METHODS: Fifteen adult intact male monkeys were grouped and treated for a 38-day period. Group I received GnRH antagonist, FSH, hCG and NETE while group II received a regime identical to group I without NETE and group III received only NETE and vehicle. Ejaculates, body weight, testicular biopsies and volume, and hormones were evaluated. RESULTS: There was a similar pattern of serum FSH and testosterone in groups I and II. Testicular volume and the proportion of tubuli exhibiting spermatids was significantly decreased in group III. There were no significant differences between group I and group II in any parameters measured. The forward progression of sperm was not affected by NETE treatment. The consistently low percentages of grade c sperm indicated no sign of hyperactivation. No changes in the gross morphology of the acrosome were detected. CONCLUSIONS: Short-term NETE treatment has neither a direct effect on the testis nor on the epididymis in this nonhuman primate model and its contraceptive effects appear to be exerted exclusively through gonadotropin suppression.

Animals↗

PubMed2005

Role of sequence variations of the GnRH receptor and G protein-coupled receptor 54 gene in male idiopathic hypogonadotropic hypogonadism.

OBJECTIVE: To determine the frequency of mutations of the gonadotropin-releasing hormone receptor (GnRHR) and of the G protein-coupled receptor 54 (GPR54) genes in normosmic idiopathic hypogonadotropic hypogonadism (IHH). METHODS: In a retrospective study we analyzed the GnRHR and the GPR54 genes of 45 IHH patients and 50 controls. Genomic DNA was amplified by PCR to obtain partially overlapping amplicons encompassing the exon-intron boundaries of the GnRHR and GPR54 genes and analyzed by single-stranded conformation polymorphism gel electrophoresis and/or DNA sequencing. RESULTS: One heterozygous R262Q mutation of the GnRHR gene was identified in one patient with familial IHH. The silent single-nucleotide polymorphism (SNP) 453C > T occurred at the same frequency in patients and controls. One patient with sporadic IHH and consanguineous parents showed a novel homozygous sequence variation of the GPR54 gene (1001_1002insC) resulting in an open reading frame shift and elongation of 43 amino acids with an increased number of proline residues in the intracellular receptor domain. This patient had delayed puberty, low testosterone (3.4 nmol/l), and low-normal LH and FSH levels responsive to GnRH. Pulsatile GnRH administration normalized testosterone levels and induced spermatogenesis sufficiently to induce a pregnancy with assisted reproduction. Two common SNPs in exon 1 and exon 5 of the GPR54 gene showed similar frequency distribution and hormonal profiles in IHH and controls. CONCLUSIONS: Mutations of the GnRHR and of the GPR54 gene are rare in IHH and should be investigated especially in cases with autosomal recessive transmission. Common SNPs of the GnRHR and GPR54 genes do not play any role in IHH.

Adolescent↗

PubMed2004

Serum adiponectin levels in hypogonadal males: influence of testosterone replacement therapy.

OBJECTIVE: Adiponectin is an adipocyte-specific secretory protein which exhibits antiatherogenic, anti-inflammatory and antidiabetic properties. We hypothesized that testosterone plays an important role in the regulation of its secretion in humans, as adiponectin concentrations are higher in women than in men and as testosterone administration is accompanied by a reduction in serum adiponectin in animals and by reduced protein secretion in cultured adipocytes. This study aimed to evaluate adiponectin levels in hypogonadal men prior to and during testosterone replacement therapy. SUBJECTS AND METHODS: In a retrospective study, adiponectin, total and free testosterone, oestradiol, SHBG, total cholesterol and triglyceride levels were evaluated in 31 hypogonadal men [HM; age, mean +/- SEM: 36.5 +/- 2.4 years; body mass index (BMI) 24.6 +/- 0.8 kg/m2] and 29 weight-matched eugonadal men (EM; age 30.8 +/- 1.5 years; BMI 23.4 +/- 0.6 kg/m2). In 13 HM (age 33.9 +/- 3.2 years; BMI 24.2 +/- 0.9 kg/m2) the same parameters were also evaluated after 6 months of testosterone replacement therapy. Correlation analysis between adiponectin and hormonal, biochemical and anthropometric parameters was performed in all subjects. RESULTS: Testosterone, free testosterone and oestradiol concentrations were significantly lower in HM than in EM (4.4 +/- 0.4 nmol/l, 78.4 +/- 10.9 pmol/l and 36.1 +/- 3.0 pmol/l, respectively, in HM vs. 21.9 +/- 0.7 nmol/l, 507.9 +/- 13.8 pmol/l and 65.2 +/- 1.8 pmol/l, respectively, in EM, P < 0.0001), while SHBG levels in HM were higher than in EM (54.4 +/- 7.5 vs. 30.9 +/- 2.2 nmol/l, P < 0.005). Serum adiponectin levels in HM were significantly higher than in EM (9.53 +/- 0.73 vs. 6.80 +/- 0.55 microg/ml, P < 0.01). Calculation of the Pearson coefficient showed that adiponectin levels in HM were not correlated with any of the anthropometric and hormonal parameters examined, but showed a significant negative correlation with serum triglycerides (r = -0.38, P < 0.05). Serum adiponectin levels were negatively correlated with body weight (r = -0.41, P < 0.05) in EM but not with other anthropometric, hormonal or biochemical parameters. Six months after initiation of testosterone replacement therapy, which increased testosterone and free testosterone levels to the normal range, adiponectin levels were significantly reduced in HM (6.37 +/- 0.93 vs. 9.26 +/- 1.01 microg/ml, P < 0.01) and similar to those recorded in EM. CONCLUSIONS: Compared to eugonadal subjects, hypogonadal men show higher adiponectin levels which are reduced by testosterone replacement therapy. This study indicates that testosterone exerts a regulatory role on adiponectin secretion in humans.

Adiponectin↗

PubMed2004

Epidermal growth factor receptor pathway substrate 8 (Eps8) expression in maturing testis.

AIM: Although epidermal growth factor receptors are expressed in the testes, whether they signal through epidermal growth factor receptor pathway substrate 8 (Eps8) is unknown. Here we evaluated the expression pattern of Eps8 in the maturing testis. METHODS: The expression of Eps8 was analysed by Northern blotting, immunocytochemistry and Western blotting in primary Sertoli cell cultures and in testicular tissue of rodents. RESULTS: Eps8 is specifically expressed in gonocytes, Leydig and Sertoli cells of the neonatal rats and in Leydig and Sertoli cells of the adult rats and mice. Although gonocytes express Eps8, no signal was found in prepubertal or mature spermatogonia and the expression level of Eps8 in Sertoli cells increases with age. No regulation of Eps8 expression in primary immature rat Sertoli cells by Follicle stimulating hormone (FSH) was detected by Western blotting. CONCLUSION: Eps8 seems to be involved in the growth factor-controlled regulation of cell proliferation and differentiation in the seminiferous epithelium. Eps8 is a possible marker for gonocytes and in Sertoli cells it could be involved in crosstalk with other growth factor pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Gene expression profiling of mouse Sertoli cell lines.

The proliferation and differentiation of Sertoli cells is regulated by follicle-stimulating hormone (FSH). The molecular events following FSH stimulation are only partially known. To investigate FSH action in Sertoli cells, we established two novel FSH-responsive mouse Sertoli-cell-derived lines expressing human wild-type (WT) FSH receptor (FSHR) or overexpressing mutated (Asp567Gly) constitutively active FSHR (MUT). Gene expression profiling with commercially available cDNA arrays, including 588 mouse genes, revealed 146 genes expressed in both cell lines. Compared with the expression pattern of WT cells, 20 genes were identified as being either up- or down-regulated (>two-fold) in the MUT cells. We observed a strong differential expression of factors involved in cellular proliferation, e.g. cyclin D2 (repressed to nearly undetectable levels), proliferating cell nuclear antigen (2.5-fold repression) and Eps-8 (six-fold repression), and in genes involved in cellular differentiation, e.g. cytokeratin-18 (13-fold induction). The cDNA array results for six representative genes were confirmed by Northern blotting, which also included the parental SK-11 cell line devoid of FSHR expression. We found no further acute FSH- or forskolin-induced change in expression levels after 3-h stimulations, suggesting that the observed differences between the two cell lines is a consequence of mild, chronically increased, cAMP production in MUT cells. These results provide a platform for the further investigation of selected candidate genes in primary cultures and/or in vivo.

Animals↗

PubMed2003

A new subclass of the luteinizing hormone/chorionic gonadotropin receptor lacking exon 10 messenger RNA in the New World monkey (Platyrrhini) lineage.

The luteinizing hormone receptor (LHR) plays an essential role as a mediator of LH and CG action during embryonic sexual differentiation and in gametogenesis. In a hypogonadal male patient, we recently demonstrated that a genomic deletion of exon 10, located in the hinge region of the extracellular domain, results in discrimination of LH and hCG action. In the common marmoset (Calltithrix jacchus), exon 10 of the LHR is naturally missing at the mRNA level. In order to investigate whether this is an isolated species-specific phenomenon, we performed a phylogenetic screening, searching for the presence of LHR exon 10 mRNA in a number of primate species representative for the major lineages of primate evolution. The expressed LHR region encompassing exon 10 was amplified from testicular tissue by RT-PCR, cloned, and sequenced. In addition, we performed Southern blot analysis of the LHR of selected New World and Old World primates. The results revealed that exon 10 mRNA is lacking in the complete New World monkey (Platyrrhini) lineage but is present in both more primitive and more advanced primates. However, exon 10 seems to be present at the genomic level, arguing for a splicing failure possibly due to a genomic mutation or the lack of appropriate splicing factors. Considering that, in the human, LH is far less active than hCG on the LHR lacking exon 10, we addressed the question whether the existence of such a receptor has any consequences on the dual hormone LH/CG system present in Platyrrhini. Using primers specific for the known marmoset CG beta cDNA, we amplified the CG beta subunit cDNA from male common marmoset pituitaries by RT-PCR, while LH beta could not be amplified, suggesting a possible physiological role of pituitary CG in this species. In conclusion, we demonstrated for the first time that the LH mRNA without exon10 is the natural wild-type LHR in the Platyrrhini lineage. We propose that this LHR represents a new subclass of receptors that should be named LHR type II. In addition, the high expression of CG beta in the marmoset pituitary suggests a physiological role of CG in the reproductive function of these primates beyond pregnancy.

Amino Acid Sequence↗

PubMed2003

Follicle-stimulating hormone receptor polymorphisms in women with normogonadotropic anovulatory infertility.

OBJECTIVE: To assess the incidence of different FSH receptor genotypes in normogonadotropic anovulatory infertile women (World Health Organization class II) and normo-ovulatory controls and to correlate these genotypes with baseline characteristics and ovarian responsiveness during ovulation induction. DESIGN: Cross-sectional study. SETTING: University hospital. PATIENT(S): Thirty normo-ovulatory controls and 148 normogonadotropic anovulatory infertile women. INTERVENTION(S): All participants underwent a standardized evaluation that included cycle history, body mass index measurement, and transvaginal ultrasonography of ovaries. Fasting blood samples were obtained for endocrine evaluation. Ovarian responsiveness to FSH in normogonadotropic anovulatory infertile women was assessed during ovulation induction, and DNA was analyzed to determine the FSH receptor genotype. MAIN OUTCOME MEASURE(S): Prevalence of FSH receptor polymorphisms, baseline serum FSH levels, amount of FSH administered, duration of stimulation, and ovarian response dose. RESULT(S): The Thr/Thr 307 genotype was significantly less prevalent (52% vs. 23%) and the Ser/Ser 680 polymorphism was significantly more prevalent (40% vs. 16%) in patients compared with controls. Normogonadotropic anovulatory infertile women with the Ser/Ser 680 polymorphism presented with higher median FSH serum levels (5.2 IU/L [range, 2.4-9.7 IU/L]) than did those with the Asn/Asn 680 (4.6 IU/L [range, 1.4-5.8 IU/L) and Asn/Ser 680 (4.5 IU/L [range, 1.8-9.7 IU/L) variants. However, ovarian responsiveness to FSH was similar among anovulatory women with the various polymorphisms. CONCLUSION(S): Normogonadotropic anovulatory infertile patients have a different FSH receptor genotype than do normo-ovulatory controls. Although this characteristic is associated with increased baseline FSH serum levels, altered ovarian sensitivity to exogenous FSH during ovulation induction could not be established.

Anovulation↗