Clinical Applications of Gonadotropins in the Male.

The pituitary gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) play a pivotal role in reproduction. The synthesis and secretion of gonadotropins are regulated by complex interactions among several endocrine, paracrine, and autocrine factors of diverse chemical structure. In men, LH regulates the synthesis of androgens by the Leydig cells, whereas FSH promotes Sertoli cell function and thereby influences spermatogenesis. Gonadotropins are complex molecules composed of two subunits, the α- and ß-subunit, that are noncovalently associated. Gonadotropins are decorated with glycans that regulate several functions of the protein including folding, heterodimerization, stability, transport, conformational maturation, efficiency of heterodimer secretion, metabolic fate, interaction with their cognate receptor, and selective activation of signaling pathways. A number of congenital and acquired abnormalities lead to gonadotropin deficiency and hypogonadotropic hypogonadism, a condition amenable to treatment with exogenous gonadotropins. Several natural and recombinant preparations of gonadotropins are currently available for therapeutic purposes. The difference between natural and the currently available recombinant preparations (which are massively produced in Chinese hamster ovary cells for commercial purposes) mainly lies in the abundance of some of the carbohydrates that conform the complex glycans attached to the protein core. Whereas administration of exogenous gonadotropins in patients with isolated congenital hypogonadotropic hypogonadism is a well recognized therapeutic approach, their role in treating men with normogonadotropic idiopathic infertility is still controversial. This chapter concentrates on the main structural and functional features of the gonadotropin hormones and how basic concepts have been translated into the clinical arena to guide therapy for gonadotropin deficit in males.

Expression of progranulin (Acrogranin/PCDGF/Granulin-Epithelin Precursor) in benign and malignant ovarian tumors and activation of MAPK signaling in ovarian cancer cell line.

It has been recently demonstrated that progranulin is overexpressed in ovarian cancer and that this protein is involved in the stimulation of cell proliferation, malignancy, and chemoresistance in ovarian cancer. The goal of the present study was to establish the differences in progranulin expression among normal, benign, and malignant ovarian tissues and to identify the signal transduction pathways activated by progranulin in an ovarian cancer cell line. Compared with benign tumors and normal ovarian tissue, progranulin mRNA and protein were overexpressed in malignant tumors. Survival analysis by the Kaplan-Meier method showed a correlation between high mRNA expression levels with poor survival outcome. Progranulin activated the MAPK-signaling pathway in NIH-OVCAR-3 cells. Progranulin expression may be potentially involved in the pathogenesis and malignant progression of ovarian cancer, and thus may represent a therapeutic target for this particular malignancy.

Hormonal regulation of pituitary FSH sialylation in male rats.

Sialic acid content in FSH is modulated by GnRH and sexual steroids. Galbeta1,3GlcNAcalpha2,3-sialyltransferase (ST3Gal III) and Galbeta1,4GlcNAcalpha2,6-sialyltransferase (ST6Gal I) incorporate sialic acid residues into FSH oligosaccharides. The aim of the present study was to assess pituitary FSH molecular microheterogeneity and ST3Gal III/ST6Gal I expression during sexual development and after castration in male rats. Preparative isoelectric focusing and lectin chromatography were used to isolate FSH glycosylation variants according to charge and complexity of their oligosaccharides; RT-PCR and immunohistochemistry were employed to analyse sialyltransferase expression. Sexual development was associated with a progressive shift towards more acidic/sialylated FSH glycoforms concomitantly with an increment in ST6Gal I gene and protein expression. After castration, a transient decrease followed by a marked increase in ST6Gal I expression were observed. Less acidic/sialylated FSH glycoforms bearing incomplete oligosaccharides increased after castration, despite high ST6Gal I expression. ST3Gal III expression remained unchanged in all the experimental conditions examined. These results show that the synthesis of FSH isoforms possessing alpha2,6-linked sialic acid is hormonally regulated in male rats.

Anti-prolactin autoantibodies in pregnant women with systemic lupus erythematosus: maternal and fetal outcome.

The aim of this study was to determine in pregnant women with systemic lupus erythematosus (SLE) the frequency of anti-prolactin autoantibodies and to compare the outcome of pregnancy in SLE women with and without anti-prolactin autoantibodies. Ninety-nine consecutive SLE pregnant women and 151 healthy pregnant women were studied prospectively. Patients with or without anti-prolactin autoantibodies were identified by gel filtration chromatography and affinity chromatography for IgG. Serum total and free prolactin (PRL) levels and molecular heterogeneity of PRL at each trimester of pregnancy were determined. The frequency of anti-PRL autoantibodies in SLE pregnant women was 13.1%. Serum total PRL levels were significantly higher in women with anti-PRL autoantibodies compared with SLE women without anti-PRL autoantibodies and in healthy pregnant women; and serum free PRL levels were lower in the third trimester in women with anti-PRL autoantibodies than in healthy pregnant women. In contrast, serum total and free PRL levels were significantly lower in the second and third trimester in SLE pregnant women without anti-PRL autoantibodies compared with healthy pregnant women. All adverse outcomes of pregnancy studied were more frequent in SLE women without anti-PRL autoantibodies than anti-PRL autoantibody-positive SLE women. Moreover, both maternal and fetal main complications were significantly higher in SLE women without anti-PRL autoantibodies than anti-PRL autoantibody-positive SLE women (P

Effects of human pituitary FSH isoforms on mouse follicles in vitro.

The effects of human FSH glycoforms on mouse follicle development and function in vitro were analysed, and an attempt was made to relate markers of follicular maturation to the expression of immunolocalized connexin (Cx) 43 and Cx26-based gap junctions. Three FSH fractions comprising discrete pI ranges [7.10-5.99 (pool I), pI 5.62-4.95 (pool II) and <3.75 (pool III)] were studied. Pool I produced the strongest effect on preantral granulosa cell proliferation and oestradiol production, and was highly effective for stimulating antral formation; this isoform also evoked a peripheral distribution of Cx43-containing gap junctions. Pool II was effective in promoting preantral granulosa cell proliferation but required higher FSH doses. This particular isoform provoked a more central distribution of Cx43-containing gap junctions, which was associated with a lower oestradiol production and less effective antral formation. Pool III was the least active for all markers of follicle development, and this was associated with minimal induction of Cx43-based gap junctions. The effects of the three FSH isoform pools on Cx26 expression were similar. The pattern of differences strongly suggests that FSH isoforms have complementary and specific actions on developing follicles, and that a shifting stage specific balance of isoforms is required for optimal follicle development.

Basal and gonadotropin-releasing hormone-releasable serum follicle-stimulating hormone charge isoform distribution and in vitro biological-to-immunological ratio in male puberty.

Follicle-stimulating hormone is synthesized and secreted as a mixture of heterogeneous isoforms that differ from each other in carbohydrate structure, biological potency, and plasma half-life. The relative abundance of the FSH isoforms will depend on the endocrine status of the donor at the time of sample collection. In the present study, we attempted to define the impact of the changing endocrine milieu characteristic of male puberty on the charge heterogeneity and plasma half-life of the serum FSH isoforms released under endogenous and exogenous GnRH drives, and examined whether such a varying hormone milieu modifies the capability of the circulating hormone to trigger intracellular signal transduction at the human FSH receptor level. Forty healthy male subjects at Tanner stages (Ts) 1 to 5 were sampled at 10 min intervals for 10 h. Serum from successive samples collected across 2-4 h intervals containing FSH released under basal, low-dose (10 microg), and high-dose (90 microg) exogenous GnRH-stimulated conditions was subjected to preparative chromatofocusing and tested for bioactivity employing a homologous cell in vitro bioassay system. Deconvolution analysis was applied to estimate the apparent endogenous FSH plasma half-life in samples obtained after administration of low-dose exogenous GnRH. Under all conditions studied, serum FSH charge isoforms were distributed along a pH range of 7.0 to less than 3.0. Comparisons across the different Tanner stages revealed a significant and selective increase in the ratio of FSH isoforms with elution pH values <4.50 relative to those with values >/=4.50 at Ts-2. At Ts-3, this ratio returned to that present at Ts-1, to decline thereafter during the ensuing pubertal stages. Serum bioactive FSH concentrations progressively increased (from 3.72 +/- 1.3 to 16.2 +/- 5.3 IU/L) throughout puberty, and in all conditions bioactive FSH concentrations exceeded those detected by a specific radioimmunoassay. The biological to immunological (B:I) FSH ratio at baseline was significantly (p < 0.05) lower at Ts-1 and Ts-2 (1.33 +/- 0.30 and 1.62 +/- 0.34, respectively) than at more advanced stages of pubertal development (2.28 +/- 0.20, 2.96 +/- 0.38, and 2.77 +/- 0.63 at Ts-3-, 4-, and -5, respectively) Similar differences were detected in samples containing FSH molecules released after low- and high-dose GnRH administration. The apparent endogenous FSH half-life of the deconvolved GnRH-induced FSH pulses was similar in the five study groups. These results demonstrate that the transition from infancy to sexual maturity in men is accompanied by qualitative changes in the circulating FSH isoform mixture. Although the changes in FSH glycosylation occurring throughout puberty are not of sufficient magnitude to alter the survival of the gonadotropin in circulation, they allow preferential secretion of bioactive FSH. The enrichment of the circulating mix of FSH isoforms with highly bioactive variants throughout spontaneous puberty may potentially favor the development of spermatogenesis and acquisition of reproductive competence.

The phosphatidylinositol 3-kinase inhibitor LY294002 binds the estrogen receptor and inhibits 17beta-estradiol-induced transcriptional activity of an estrogen sensitive reporter gene.

Estrogen receptors (ERs) are members of the superfamily of ligand-activated transcription factors. In addition to the classical, hormone-mediated activation, ERs may alternatively be activated in a ligand-independent manner by a variety of agents including growth factors, neurotransmitters and cAMP. It has been demonstrated that the phosphatidylinositol 3 (PI3)-dependent kinase/Akt pathway may activate the ER alpha by increasing the activity of both estrogen independent activation function-1 and estrogen-dependent activation function-2 domains. The Akt phosphorylation site in the ER is Ser167. Phosphorylation of this residue is inhibited by LY294002, which blocks the PI3-kinase/Akt pathway. In the course of studies examining the effects of LY294002 on ligand-independent activation of ERs in L cells, we found that LY294002 exhibits antiestrogenic effects in a dose-dependent manner. By competition binding assays, we found that LY294002 specifically displaced radiolabelled estradiol from ERs with an IC(50) of 11+/-0.06 nM, being an estradiol competitor as effective as the antiestrogens ICI182,780 (IC(50), 21+/-0.13) and 4-OH-tamoxifen (IC(50), 15+/-0.09). Further, LY294002 irreversibly blocked estrogen-induced transactivation of an estradiol-sensitive reporter gene. These findings are of particular importance in the interpretation of studies demonstrating ERs inactivation by the PI3-kinase inhibitor. Our studies show that an apparent block of ER activation cannot be dissociated from inhibition of ligand-mediated events. Thus, this effect can be the result of the ability of LY294002 to bind the ERs and inhibit transactivation of estrogen-regulated genes.

Assessment of the in vitro and in vivo biological activities of the human follicle-stimulating isohormones.

Gonadotropins are synthesized and released in different molecular forms. In this article, we present evidence that the glycosylation variants of human pituitary FSH exhibit differential and divergent effects at the target cell level and that less sialylated, short-lived variants may exert significant effects in in vivo conditions. Less acidic/sialylated glycoforms (elution pH value 6.60-4.60 as disclosed by high resolution chromatofocusing of anterior glycoprotein extracts), induced higher cAMP release, estrogen production and tissue-type plasminogen activator (tPA) enzyme activity as well as cytochrome P450 aromatase and tPA mRNA expression in cultured rat granulosa cells than the more acidic analogs (pH<4.76). By contrast, the more acidic/sialylated glycoforms induced higher alpha-inhibin subunit mRNA expression than their less acidic counterparts. In cumulus enclosed oocytes isolated from mice ovaries, addition of less acidic isoforms induced resumption of meiosis more efficiently than the more acidic analogs. Interestingly, the least acidic isoform (pH>7.10) behave as a strong antagonist of several FSH-mediated effects. Assessment of the in vivo effects of the isoforms on granulosa cell proliferation in follicles from immature rats, revealed that short-lived isoforms were equally or even more efficient than their more acidic counterparts in maintaining granulosa cell proliferation when administered immediately after hypophysectomy. These results show that the naturally occurring human FSH isoforms may exhibit differential or even unique effects at the target cell level and that factors other than the metabolic clearance rate of the molecule (including receptor-binding affinity and capability of the ligand to activate its receptor and trigger intracellular signaling) also play an important role in determining the net in vivo effects of a particular FSH variant.

Endocrine regulation of gonadotropin glycosylation.

The pituitary gonadotropins--luteinizing hormone and follicle-stimulating hormone--as well as the placental choriogonadotropin belong to the family of glycoprotein hormones. These structurally related hormones, which regulate several major reproductive functions of the body, are heterodimers consisting of a common alpha-subunit noncovalently bound to a beta-subunit. The N- and O-linked oligosaccharide chains of these gonadotropins play an important role in intracellular folding, assembly, secretion, metabolic clearance, and biological activity of the hormone. Gonadotropin glycosylation is a highly complex process; within the gonadotropes it is modulated by a variety of extrapituitary factors of hypothalamic and gonadal origin. In particular, estrogens and androgens appear to regulate terminal sialylation and/or sulfation of the oligosaccharide attachments and hence some functional properties of the gonadotropin molecule determined by these residues, i.e., metabolic clearance and in vivo biopotency. Through these extrapituitary inputs, the anterior pituitary may not only regulate the quantity but also the quality of the gonadotropin signal delivered to the gonads in a given physiologic or pathologic condition.

Eight novel mutations of the androgen receptor gene in patients with androgen insensitivity syndrome.

Androgen insensitivity syndrome (AIS) is an X-linked genetic disorder of male sexual differentiation caused by mutations in the androgen receptor (AR) gene. A reliable genotype-phenotype correlation in these patients does not exist as yet. Here we report the molecular studies performed on eight individuals with AIS. Exon-specific polymerase chain reaction (PCR), single-strand conformation polymorphism, and sequencing analyses, were performed in exons 2 to 8 of the AR gene. In one case, total cellular RNA was extracted from genital skin fibroblasts and reverse transcriptase-PCR was performed. Six different point mutations leading to amino acid substitutions (P682T, Q711E, G743E, F827V, H874R, D879Y), one splice-junction mutation (g-->c at +5, exon 6/intron 6), and a missense mutation without amino acid substitution (S888S) were identified. All mutations, including a de novo mutation, were previously undescribed on the steroid binding domain. Of the eight mutations identified, four led to a complete female phenotype (codons 743, 827, 874 and the donor splice site +5), two were detected in phenotypic females with partial virilization (codons 682 and 711), and two were present in phenotypic male subjects with undervirilized external genitalia, thus indicating that all of these sites determine AR functional activity.

Changes in the biological:immunological ratio of basal and GnRH-releasable FSH during the follicular, pre-ovulatory and luteal phases of the human menstrual cycle.

BACKGROUND: Significant changes in charge isoform distribution of serum FSH occur throughout the human menstrual cycle. In the present study, we analysed the impact of the changing endocrine milieu characteristic of the menstrual cycle on the capability of basal and gonadotrophin-releasing hormone (GnRH)-releasable FSH to trigger intracellular signal transduction via the human FSH receptor. METHODS: Seven normal women underwent blood sampling every 10 min for 10 h during the early follicular phase (FP), pre-ovulatory phase (PO) and mid- to late luteal phase (LP) of the menstrual cycle. Serum from successive samples collected across 2 h intervals containing FSH released under baseline and exogenous GnRH-stimulated conditions was tested for bioactivity employing a homologous in-vitro assay. RESULTS: The biological to immunological (B:I) ratio of basal and GnRH-releasable FSH was significantly (P < 0.05 ) higher at LP (range, 0.83 +/- 0.07 to 1.35 +/- 0.30) than during the FP (0.43 +/- 0.02 to 0.65 +/- 0.04) and PO (0.49 +/- 0.05 to 0.62 +/- 0.06). In all phases, the B:I FSH ratio in baseline samples was similar to those exhibited by samples collected after 10 and 90 microg GnRH administration. CONCLUSIONS: The selective increase in the capability of the admixture of FSH isoforms circulating during the LP to activate the FSH receptor, apparently represents an additional mechanism through which the anterior pituitary may regulate the maturation of those follicles destined to ovulate during the coming cycle.

A novel homozygous mutation in the second transmembrane domain of the gonadotrophin releasing hormone receptor gene.

BACKGROUND AND OBJECTIVE: Mutations in the GnRH receptor (GnRH-R) gene cause hypogonadotrophic hypogonadism. Here, we present the molecular studies of the GnRH-R gene in three families with isolated hypogonadotrophic hypogonadism. PATIENTS: Three unrelated families, with at least two members diagnosed with isolated hypogonadotrophic hypogonadism were included. MEASUREMENTS: DNA sequencing was performed after polymerase chain reaction amplification of each of the three exons of the gene. RESULTS: A novel homozygous missense mutation, at nucleotide 268, turning glutamic acid into lysine, located at the second transmembrane domain of the GnRH-R gene was found in two patients pertaining to one of the families studied. Both parents and an unaffected brother were heterozygous carriers of one mutant allele, an unaffected sister was homozygote wild type. In the other two affected families no mutations were found in the GnRH-R gene. CONCLUSIONS: This constitutes the first description of an spontaneous mutation located at the second transmembrane domain (Glu90Lys) of the GnRH-R, indicating that the integrity of glutamic acid at this position is crucial for receptor function. Also this report, complementing others, demonstrates that mutations are distributed throughout the GnRH-R gene and that as in the only other homozygous mutation previously described, affected patients present a complete form of hypogonadotrophic hypogonadism. Due to the fact that apparently consanguinity was present in our affected family, we presume that the mutation derived from a common ancestor, by a founder gene effect.

Human FSH isoforms: carbohydrate complexity as determinant of in-vitro bioactivity.

Differences in sialic acid content of the hormone have been considered the main determinant of FSH polymorphism. The aim of the present study was to investigate the effect of variations in the oligosaccharide structure of the intrapituitary human FSH (hFSH) glycosylation variants on their intrinsic biological activity. FSH charge isoforms obtained after chromatofocusing were further separated by lectin affinity chromatography [Concanavalin A (ConA), Wheat germ agglutinin (WGA), Lentil lectin (LcH)]. Isolated isoforms were separately tested for in-vitro bioactivity in a rat Sertoli cell aromatization bioassay. Our results show that: (1) FSH microheterogeneity is due not only to variations in the sialic acid content of the hormone but also to differences in the internal structure of the carbohydrate chains, and (2) variations in the sialic acid content as well as differences in the complexity of the glycans determine the full biological expression of FSH glycosylation variants.

Is there any physiological role for gonadotrophin oligosaccharide heterogeneity in humans? I. Gondatrophins are synthesized and released in multiple molecular forms. A matter of fact.

Carbohydrates attached to the protein core of all glycoprotein hormones play an essential role in the function of the molecule, influencing a number of intracellular and extracellular processes. As with other members of the glycoprotein hormone family, pituitary gonadotrophins are not produced as single or unique molecules but rather as arrays of isoforms that differ from each other mainly in the structure of their oligosaccharide attachments. In both experimental animals and in humans, the abundance of the different isoforms varies depending on the endocrine status of the donor present at the time of collection of the tissue or sample. Conditions characterized by an oestrogen-enriched hormonal milieu (eg. the preovulatory phase of the menstrual cycle) promote the formation and secretion of variants with relatively low sialic acid and/or sulphate content, whereas physiological deficiency of this sex steroid (as in the postmenopause) favours the production of highly sialylated, long-lived gonadotrophin variants. When tested individually, less sialylated isoforms exhibit higher receptor-binding and in-vitro biological activity but shorter plasma half-life than their more sialylated counterparts. Both the hormonal regulation and the functional differences among the naturally occurring isoforms strongly suggest that gonadotrophin heterogeneity represents a distinctly different mechanism through which the pituitary gland may regulate the intensity and duration of the gonadotrophic stimulus. Nevertheless, whereas the existence of the alternatively glycosylated variants of gonadotrophins in both the pituitary and in serum is currently without doubt, the physiological role of this phenomenon is still a controversial issue and a matter of debate.

Effect of different FSH isoforms on cyclic-AMP production by mouse cumulus-oocyte-complexes: a time course study.

The ability of different isoforms of follicle stimulating hormone (FSH) to induce accumulation of cAMP in cultured mouse cumulus-oocyte-complexes (COC) was evaluated in a time course study. Using isoform fractions representing less acidic (pI 6.43-5.69), mid-acidic (pI 5.62-4.96) and acidic (pI 4.69-3.75) isoforms, the accumulation of cAMP was monitored after an exposure time of 0, 5, 10, 15, 30, 60, 120 and 180 min. In addition, cAMP production was monitored for 0, 5, 10, 15 and 30 min following a 5 min exposure to FSH isoform fractions. Based on FSH measurements using radioimmunoassays, the less and mid-acidic isoforms caused almost twice as much cAMP to be accumulated than the acidic isoform fraction, thereby confirming an enhanced biological activity of FSH isoforms with a isoelectric point (pI) of >5.0. For all isoform fractions, maximal accumulation of cAMP was achieved after 30 min of exposure, after which the production declined to background levels. After a 5 min exposure to isoform fractions, levels of cAMP were significantly higher in the less acidic isoform fractions, but after isoform removal, the decline in cAMP production to background levels followed a similar time course. The results demonstrate that FSH isoforms with a pI of >5.0 induced significant biological responses within a period of 30 min and that prolonged exposure caused attenuated signal transduction. The present results, set in the context of the pulsatile characteristics of FSH release from the pituitary and the reported half-life of less acidic isoforms of approximately 35 min, make it conceivable that isoforms with a pI >5.0 actually possess important physiological functions during the periovulatory period.

No evidence of mutations in the follicle-stimulating hormone receptor gene in Mexican women with 46,XX pure gonadal dysgenesis.

In the ovary FSH is necessary for normal follicular development, binding to its receptor (FSHR) that pertains to the superfamily of G-protein coupled receptors. In the FSHR gene, which consists of 10 exons, an homozygous mutation was reported in six Finnish families with gonadal dysgenesis; whereas two isolated French patients exhibited compound heterozygous mutations. Several groups, however, have searched for FSHR mutations, although in most cases the gene has been studied partially, not finding any genetic abnormalities in German, English, North American or Brazilian women. We performed direct sequencing of all 10 exons of the FSHR gene in seven sporadic patients and two sisters with 46,XX pure gonadal dysgenesis, to investigate the cause of their disorder. No heterozygous or homozygous mutant alleles were present in any of the patients. Although the number of patients evaluated was small, considering all the other previous reports, it seems that except in the Finnish population, the proportion of women with mutations in the encoding region of this gene is very low. Other possibilities for the presence of 46,XX gonadal dysgenesis, such as defects in the regulatory regions of the FSHR gene promoter, in the untranslated regions of exons 1 and 10, and within introns, or the existence of other genes likely to be important for normal ovarian function on the X chromosome or on autosomes, should be considered. In contrast with other studies, we did not find polymorphisms of the FSHR gene, indicating that apparently in Mexicans this gene is not highly polymorphic.

Transactivation of progestin- and estrogen-responsive promoters by 19-nor progestins in African Green Monkey Kidney CV1 cells.

New and more potent progestins and antiprogestins suitable for reproductive therapy and contraception are currently the target of intensive research. The design of such drugs has been hampered by the complex technology required for screening these compounds at the molecular level. To solve this problem, we developed an in vitro cell system that allows detection of the progestagenic effects of a given compound using a PRE2-TATA-CAT reporter vector transiently introduced in a cell line stably transfected with the rabbit progesterone receptor (PR). The African Green Monkey Kidney CV1 (AGMK-CV1) cell line was chosen because these cells do not express endogenous steroid receptors; the selected clone stably expressing the rabbit PR has been maintained in our laboratory for more than 2 yr without detectable losses in PR content and progestagenic response. The presence and function of the PR were assessed by immunohistochemical and saturation analyses as well as by monitoring transactivation of the PRE2-TATA-CAT reporter gene. In this cell line, the PR is expressed at a concentration of 0.170 fmol/mg of protein, and the receptor is localized within the cell nucleus in either the presence or absence of the potent synthetic progestin R5020. This PR-expressing cell system allowed study of the in vitro progestational activity of several 19-nor progestins. The antiprogestin RU486 inhibited CAT activity induced by R5020; norethisterone (NET), levonorgestrel (LNG), and gestodene (GSD) induced PRE2-TATA-CAT activity at concentrations similar to those of R5020, whereas NET A-ring-reduced metabolites induced CAT activity at an extent lower than (5alpha-NET) or similar (3beta,5alpha-NET) to that of the precursor compound. The PRE2-TATA-CAT induction by 17beta-estradiol was also analyzed and no crossreactivity was detected. However, when the ERE-VitA2-TK-CAT (estrogen-responsive element-vitellogenin A2-thymidine kinase promoter-CAT) reporter vector and the estradiol receptor alpha or beta were cotransfected, CAT activity was induced in the presence of 17beta-estradiol, and NET tetrahydro-reduced derivatives. The results indicate that this AGMK-CV1-PR cell assay system appears to be suitable for measuring the effects of different synthetic progestins at the transcriptional level. In this assay system, NET, LNG, and GSD exhibit potent progestational effects at the transcriptional level. In the particular case of NET, the assay system allowed us to determine that the single or multiple hormonal transcriptional effects of this compound are partially mediated by its A-ring-reduced derivatives.

Reactivity of different LH and FSH standards and preparations in the world health organization matched reagents for enzyme-linked immunoassays of gonadotrophins.

The immunoreactivity of various LH and FSH calibration standards and recombinant preparations in the enzyme-linked immunoassay (EIA) systems for gonadotrophins developed for the Special Programme of Research in Human Reproduction of the World Health Organization (WHO) were compared. The preparations tested included three LH and two FSH pituitary standards (calibrated against LH 80/552 and 68/40 and FSH 78/549 respectively) provided with the EIA or radioimmunoassay WHO matched reagent kits, the pituitary preparation LER-907, and recombinant human LH (rhLH) and FSH (rhFSH). Simultaneous curve fitting of the EIA dose-response curves revealed no significant differences among the slopes generated by the WHO LH standards and LER-907; in contrast, no parallelism was found between the curves of rhLH and the pituitary-derived LH standards. No significant differences were found among the slopes of the curves elicited by the pituitary and recombinant FSH preparations. Each LH preparation exhibited a high degree of charge heterogeneity. Considerable variations in charge isoform distribution among the WHO LH standards, rhLH and LER-907 were also evident. In contrast, the FSH preparations were less heterogeneous and exhibited minor differences in charge distribution. Despite the existing differences in charge isoform distribution, all the pituitary-derived preparations as well as rhFSH seem appropriate for using as calibration standards in this particular EIA system.

A mutation in the 5' non-high mobility group box region of the SRY gene in patients with Turner syndrome and Y mosaicism.

In Ullrich-Turner syndrome (UTS) patients, the presence of a Y-chromosome or Y-derived material has been documented in frequencies ranging from 4-61%. Mutations of SRY (testis-determining gene) constitute the cause of XY sex reversal in approximately 10-15% of females with pure gonadal dysgenesis. Most of these mutations have been described in the HMG (high mobility group) box of the gene, which is the region responsible for DNA binding and bending; however, various mutations outside the HMG box have been reported. We carried out molecular studies of the SRY gene in three patients with a UTS phenotype and bilateral streaks; two presented a 45,X/46,XY mosaic, and the third a Y marker chromosome. In two patients a missense mutation, S18N, was identified in the 5' non-HMG box region in DNA from blood and both streaks; this mutation was not identified in 75 normal males. Sequencing of the DNA region of interest was normal in the father and older brother of patient 1, demonstrating that in this patient the mutation was de novo. A previous report of a 46,XY patient with partial gonadal dysgenesis who presented the same mutation as our patients indicates the probable existence of a hot spot in this region of the SRY gene and strengthens the possibility that all gonadal dysgeneses constitute part of a spectrum of the same disorder. It also demonstrates that a single genetic abnormality can result in a wide range of phenotypic expression.

Differential effects of the charge variants of human follicle-stimulating hormone.

FSH is synthesized and secreted by the anterior pituitary gland in multiple molecular forms; the release of these isoforms depends on the endocrine status of the donor at the time of sample collection. In the present study, we analysed the possibility that the FSH charge isoforms may exert differential effects at the target cell. Seven FSH isoform mixes were isolated from pooled anterior pituitary glycoprotein extracts by high resolution chromatofocusing, followed by affinity chromatography, which removed nearly 90% of the LH that co-eluted with the FSH isoforms during chromatofocusing. The isoforms (isoform I, pH >7.10; II, pH range 6.60-6.20; III, pH 5. 47-5.10; IV, pH 5.03-4.60; V, pH 4.76-4.12; VI, pH 4.05-3.82 and VII, pH <3.80) were then tested for their capacity to stimulate cAMP release, androgen aromatization and tissue-type plasminogen activator (tPA) enzyme activity and cytochrome P450 aromatase, tPA and inhibin alpha-subunit mRNA production by rat granulosa cells in culture. cAMP and oestradiol production were determined by RIA, tPA enzyme activity by SDS-PAGE and zymography and all mRNAs by northern blot hybridization analysis and semiquantitative RT-PCR. All isoforms, with the exception of isoform I, stimulated synthesis and release of cAMP, oestrogen and tPA enzyme activity in a dose-dependent manner; the potency of the less acidic isoforms (pH 6. 60-4.60) was greater than that exhibited by the more acidic/sialylated analogs (pH 4.76 to <3.80; potencies II>III>IV>V>VII>VI). A similar trend was observed in terms of cytochrome P450 aromatase and tPA mRNA production. In contrast, when FSH-stimulated production of alpha-inhibin mRNA was analysed, isoforms V-VII were significantly more potent (two- to threefold) than the less acidic/sialylated counterparts (II-IV). In contrast to isoforms II-VII (which behaved as FSH agonists), isoform I (elution pH >7.10) completely blocked P450 aromatase and tPA mRNA expression, without altering that of a constitutively expressed gene (glyceraldehyde-3-phosphate dehydrogenase). These results show for the first time that the naturally occurring human FSH isoforms may exhibit differential or even unique effects at the target cell level.