Derek K. Lobb

Membrane receptors: Structure and function of the relaxin family peptide receptors

The receptors for members of the relaxin peptide family have only recently been discovered and are G-protein-coupled receptors (GPCRs). Relaxin and insulin-like peptide 3 (INSL3) interact with the leucine-rich-repeat-containing GPCRs (LGRs) LGR7 and LGR8, respectively. These receptors show closest similarity to the glycoprotein hormone receptors and contain large ectodomains with 10 leucine-rich repeats (LRRs) but are unique members of the LGR family (class C) as they have an LDL class A (LDLa) module at their N-terminus. In contrast, relaxin-3 and INSL5 interact with another class of type I GPCRs which lack a large ectodomain, the peptide receptors GPCR135 and GPCR142, respectively. These receptors are now classified as relaxin family peptide (RXFP) receptors, RXFP1 (LGR7), RXFP2 (LGR8), RXFP3 (GPCR135) and RXFP4 (GPCR142). This review outlines the identification of the peptides and receptors, their expression profiles and physiological roles and the functional interactions of the peptides with their unique receptors.

Melatonin receptor mRNA expression in human granulosa cells.

We have shown that the melatonin receptor agonist, 2-[125I] iodomelatonin, binds to high-affinity guanine nucleotide-sensitive sites on human granulosa (HG) cell membranes. In order to confirm the presence of melatonin receptors in HG cells, we have now used a reverse transcriptase-polymerase chain reaction (RT-PCR) procedure to examine receptor subtype expression. RT-PCR studies revealed the presence of the mt1 (Mel1alpha) melatonin receptor subtype in ten single or pooled HG cell samples which were obtained from 14 patients. In contrast, expression of MT2 ( Mel1b) mRNA was observed in only two of these HG samples. DNA sequencing of the mt1 PCR product confirmed its identity with the reported human mt1 melatonin receptor. The expression of mt1 and MT2 receptor mRNA in HG cells and the reported presence of melatonin in human follicular fluid indicate a potentially important role for this hormone in regulating human ovarian and reproductive function.

Intraovarian regulation of follicular development

Abstract Pituitary gonadotrophins are required for normal follicular growth and development; the proliferation of granulosa cells, however, cannot be attributed to the direct actions of these hormones. Since polypeptide growth factors are known to have mitogenic actions on granulosa cells, the possibility that the surrounding thecal cells may be a source of growth factors that are physiologically important in regulating granulosa cell proliferation was examined in the bovine ovary. In support of this hypothesis bovine thecal cells in culture were shown secrete products that stimulated [ 3 H]thymidine incorporation into bovine granulosa cell DNA, anwiled to an increase in cell number. To characterize the growth-promoting factor thecal cell conditioned medium was concentrated and the peptides fractionated on a Bio-Gel P-60 column in 1.0 M atic acid. Fractions were tested for their ability to stimulate [ 3 H]thymidine incorporation into bovine granulosa cell DNA. Growth-promoting activity was located in the 6–9 K and the 16 K molecular weight fractions. The peak fractions inhibited FSH-induced aromatase activity in rat granulosa cells, indicating the presence of EGF-like or TGF-α-like peptides. A specific radioimmunoassay for TGF-α indicated that the active fractions that promoted both growth and aromatase activity contained TGF-α. An immunohistochemical approach as also used to localize TGF-α in the bovine ovary throughout the various stages of folliular, development. Immunoperoxidase staining was most intense in the theca of follicles at the discrete physiological stages known to show rapid granulosa cell growth. Staining intensity for TGF-α declined in large pre-ovulatory follicles, coincident with the known decline in granulosa cell mitosis. Northern analysis of bovine tneca mRNA showed the presence of the TGF-α mRNA transcript. A growth inhibitory activity was observed in the fractions with a molecular weight of approx. 25 K. The inhibitory activity had the molecular weight of TGF-β, and TGF-β was found to inhibit [ 3 H]thymidine incorporation into bovine granulosa cell DNA. The presence of TGF-β-like activity was also induced by the stimulation of FSH-induced aromatase activity in rat granulosa cells. In suppossedly, bovine thecal cells secrete both TGF-α and TGF-β in vitro. As shown in the bioassay, the TGF-α-like and the TGF-β-like factors have pronounced effects on the proliferation of bovine granulosa cells; TGF-α promotes cell growth whereas TGF-β inhibits cell growth. The relative concentrations of TGF-α and TGF-β may determine the rates of proliferation of granulosa cells during follicular development.

Dichlorodiphenylchloroethylene Elevates Cytosolic Calcium Concentrations and Oscillations in Primary Cultures of Human Granulosa-Lutein Cells

Abstract 1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), a metabolite of DDT (1,1-dichlorodiphenyltrichloroethane), is a persistent hormonally active environmental toxicant that has been found in human serum and follicular fluid. The objective of this study was to determine whether DDE can alter free calcium ion concentrations in the cytosol ([Ca2+]cyt) of human granulosa cells. Changes in [Ca2+]cyt in single cells loaded with Fura-2 were studied using a dynamic digital Ca2+ imaging system. At a concentration of 100 ng/ml, DDE stimulated small elevations of [Ca2+]cyt accompanied by Ca2+ oscillations. At 1 μg DDE/ml, there was a biphasic Ca2+ response with marked elevations of [Ca2+]cyt over time. In Ca2+-free medium, cells showed an initial small elevation of [Ca2+]cyt, which was magnified after addition of Ca2+ to the medium. Washing the cells after DDE treatment failed to remove the elevated [Ca2+]cyt and oscillations, both of which were eliminated by addition of EGTA. ATP also induced [Ca2+]cyt elevations and oscillations, and these effects were potentiated when DDE was added. FSH induced transient [Ca2+]cyt elevations, whereas hCG caused a prolonged elevation and marked oscillations in [Ca2+]cyt. These results suggest that DDE at concentrations normally found in human tissues induces elevations in [Ca2+]cyt in granulosa-lutein cells. Our data therefore highlight a novel mechanism through which DDE can alter endocrine homeostasis and possibly act as an endocrine toxicant.

Identification of a transforming growth factor alpha-like molecule in human seminal plasma

OBJECTIVE To characterize the putative seminal growth promoting factor serendipitously observed when human seminal plasma was analyzed for bioactive FSH. DESIGN A pool of human seminal plasma was subjected to sequential Sephadex G-75 (superfine) chromatography and high-performance size exclusion liquid chromatography. The fractions were tested for mitogenic activity using a rat granulosa cell assay and normal rat kidney (NRK) cells. Properties of the factor were established and characterization by gel electrophoresis and neutralization with antibody were accomplished. SETTING Reproductive Biology Research Laboratory at McMaster University Medical Centre. MAIN OUTCOME MEASURES Ability of purified fractions of human seminal plasma to augment the uptake of tritiated thymidine into cell DNA. RESULTS Mitogenic activity of human seminal plasma was augmented in the presence of FSH but not hCG, PRL, E2, T, P, or dihydrotestosterone. The putative growth factor synergized with insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), and transforming growth factor beta (TGF-beta) but not with TGF-alpha. Mitogenic activity was neutralized by a specific TGF-alpha antibody in a dose-dependent manner. The molecular weight of the factor as assessed by gel electrophoresis is 6 kd. CONCLUSIONS Human seminal plasma contains a mitogen that is similar to TGF-alpha.

Resistin expression in human granulosa cells

Resistin, also known as found in inflammatory zone 3 (FIZZ 3), is a cysteine-rich low molecular weight member of the FIZZ family of proteins. Resistin levels are elevated in obese mice, and those treated with the recombinant protein exhibit increased insulin resistance, whereas treatment with an antibody against resistin attenuated this condition [1]. Moreover, resistin expression is inhibited by thiazolidinediones which exert insulin-sensitizing effects via the peroxisome proliferator-activated receptor c (PPARc). On the basis of these findings, it was suggested that resistin may be a link between obesity and type-2 diabetes [1]. However, subsequent studies aimed at establishing a similar link between obesity, resistin levels, and type-2 diabetes in humans have been inconclusive [2]. Recent studies show that resistin is produced in the human placenta and its expression in the term placenta is significantly higher than that observed in the first trimester [3]. We have examined for the first time, the mRNA and protein expression of resistin in human granulosa cells obtained from patients undergoing fertility treatment. The mRNA expression of PPARc, which is involved in the regulation of resistin gene transcription [4], was also examined. Materials and methods

ONTARIO, CANADA: A simplified method for preparing IVF granulosa cells for culture

Purpose: To validate an expedited method for the removal of erythrocytes when preparing IVF granulosa-luteal cells for culture. Methods: Granulosa cells were enriched for culturing from follicular aspirates by density gradient centrifugation and by hypo-osmotic lysis treatments. Results: Cells prepared by either method showed the same cell viability and produced progesterone in similar quantities. Conclusions: Using hypo-osmotic lysis to remove erythrocytes does not impair granulosa cell viability or steroidogenesis. It avoids multiple density gradient centrifugations and washings, and yields IVF granulosa cells ready for culture efficiently.

Binding of progesterone to cell surfaces of human granulosa-lutein cells

Progesterone is produced by granulosa cells under the influence of luteinizing hormone. Nuclear progesterone receptors have been found in rat granulosa cells. Human granulosa-lutein cells rapidly respond to progesterone with an increase in intracellular calcium suggesting the existence of a nongenomic mechanism. This study was conducted to determine whether binding of progesterone to granulosa cells could occur at the membrane. Granulosa cells were obtained from an in vitro fertilization program and examined immunohistochemically with an antiserum to membrane progesterone receptors. Approximately 14-70% of freshly harvested or cultured granulosa cells of six patients showed a positive reaction to the antiserum, limited to the cell membrane. Western blot analysis of homogenates of granulosa cells and a granulosa cell tumour confirmed the presence of progesterone receptors A, B and C and low amounts of a putative membrane receptor. These results demonstrate that the plasma membranes of human granulosa cells possess binding components for progesterone which may be involved in its nongenomic mechanism of action.

Expression and actions of transforming growth factors during human follicular development

OBJECTIVE To correlate the temporal expression of transforming growth factor-alpha (TGF-alpha) and TGF-beta genes in granulosa cells (GC) and thecal cells from human follicles at various developmental stages and to determine their trophic effects on GCs. DESIGN Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of extracted RNA from follicular GCs and theca cells. SETTING Academic endocrinology laboratory. PATIENT(S) Premenopausal women undergoing total abdominal hysterectomy and bilateral salpingo-oophorectomy for nonovarian reasons. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Confirmation by the RT-PCR product for TGF-alpha and TGF-beta gene expression in GC and theca cells from human follicles at various developmental stages and (3)H-thymidine uptake in vitro to assess growth effects on GCs. RESULT(S) The RT-PCR product indicating the presence of TGF-alpha messenger RNA (mRNA) was found consistently in theca from healthy antral follicles. In theca from large follicles (>1.0 cm) the TGF-alpha PCR product was of reduced intensity. The TGF-alpha was absent or undetectable in granulosa cells from all follicle sizes. The PCR product for TGF-beta was generated by all GC and thecal cell RNAs from all follicle sizes examined. The TGF-alpha promoted and TGF-beta inhibited human GC growth. CONCLUSION(S) The presence of TGF-alpha gene expression in thecal cells coincides with periods of follicular growth. The expression of TGF-beta occurs in both cell types throughout antral follicle development. The TGF-alpha and TGF-beta have opposing trophic effects on GCs.

Student attrition in the Ontario midwifery education programme

OBJECTIVE to identify the factors associated with student withdrawal during their university training. DESIGN an Internet-based survey questionnaire was designed and administered. SETTING two universities in Ontario, Canada. PARTICIPANTS senior level students in years three and four, graduates of the programme and those students that withdrew prior to graduation. FINDINGS students who withdrew from the programme were more likely to report not feeling academically supported and not being prepared for the time commitments required. Students with the greatest risk for leaving the programme were those that took a leave of absence, over half of which were maternity leaves. CONCLUSION having identified those significant factors associated with student attrition, we can now begin to develop specific interventions to improve retention rates.