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.

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.

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.

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.

Receptor binding activity and in vitro biological activity of the human FSH charge isoforms as disclosed by heterologous and homologous assay systems: implications for the structure-function relationship of the FSH variants.

Follicle-stimulating hormone (FSH) is produced and secreted in multiple molecular forms. These isoforms differ in their oligosaccharide structures, which determine the particular behavior of a given variant in in vitro and in vivo systems. Employing heterologous cell assay systems, this and other laboratories have shown that highly sialylated human FSH variants exhibit lower receptor binding/immunoactivity as well as in vitro bioactivity/immunoactivity relationships than their less sialylated counterparts. It is not known, however, whether this characteristic behavior of the FSH isoforms is reproduced by homologous assay systems, in which unique variants of the receptor are presumptively expressed. To gain further insights into the structure-activity relationship of the various FSH isoforms, we analyzed the capacity of nine charge isoforms obtained after high-resolution chromatofocusing (pH window, 7.10 to <3.80) of anterior pituitary glycoprotein extracts to bind and activate their cognate receptor expressed by naturally occurring heterologous cell systems (rat granulosa cells and seminiferous tubule homogenates) as well as by human embryonic kidney-derived 293 (HEK-293) cells transfected with the human FSH (FSH-R) receptor cDNA. In both (heterologous and homologous) receptor assay systems, the isoforms displaced 125I-labeled FSH from the receptor in a dose-response manner; however, whereas in the heterologous systems, the receptor binding activity varied according to the elution pH value/sialic content of the isoforms, with the less acidic variants exhibiting higher receptor binding activity (r = 0.851 and 0.495 [p < 0.01 and p < 0.05] for the granulosa cell and testicular homogenate receptor assay systems, respectively) than the more acidic/sialylated analogs, in the homologous assay, this relationship was practically absent (r = 0.372, p N.S.). The capacity of the isoforms to induce androgen aromatization by rat granulosa cells followed the same trend shown by its corresponding receptor assay system (r = 0.864, p < 0.01). Interestingly and in contrast to the results observed in the homologous receptor binding assay, the ability of the isoforms to induce cAMP production by HEK-293 cells varied according to their elution pH value, with the more sialylated isoforms exhibiting lower potency than their less acidic counterparts (r = 0.852, p < 0.01). The results yielded by the heterologous assays suggest that the different potency of the isoforms to elicit a biological effect in a naturally occurring receptor system depends primarily on the particular affinity of the receptor molecule for each isoform. The existence of a clear dissociation between receptor binding and signal transduction in the homologous system indicate that this later function is rather related to the different ability of the FSH glycosylation variants to induce and/or stabilize distinct receptor conformations that may permit preferential or different degrees of activation/inhibition of a given signal transduction pathway. Thus, the human FSH receptor-transducer system apparently possesses sufficient versatility to respond in a different manner to glycosylation-dependent diverse FSH signals.

FSH-induced resumption of meiosis in mouse oocytes: effect of different isoforms.

The ability of different isoforms of follicle stimulating hormone (FSH) to induce the resumption of meiosis in cultured mouse oocytes was evaluated. Oocytes were cultured in the presence of hypoxanthine to prevent spontaneous resumption of meiosis. Using serial dilutions of the isoform fractions representing less acidic isoforms (pI 6. 43-5.69), mid-acidic (pI 5.62-4.96) and acidic (pI 4.69-3.75), the concentration which caused 50% of the oocytes to resume meiosis and undergo germinal vesicle breakdown (GVBD) after a culture period of 24 h (i.e. ED(50%) GVBD) was determined. The FSH concentration of the isoform fractions was determined by radioimmunoassay, radio-receptor assay or through cAMP release in a Chinese hamster ovary-cell line expressing the human FSH-receptor. Determined by radioimmunoassay, the (ED(50%) GVBD) values were: less acidic 6.4 +/- 0.3 IU/l (mean +/- SD), mid-acidic 6.1 +/- 0.7 IU/l and acidic 12.2 +/- 0.7 IU/l. The less and mid-acidic isoforms were significant lower than the acidic (P < 0.0005). Similar relationships between the isoform fractions were obtained by the two other FSH assays. The results demonstrate that FSH isoforms with a pI of >5.0 induced resumption of meiosis significantly more efficiently than acidic isoforms. Less and mid-acidic isoforms may exert an important physiological function by inducing the resumption of meiosis in oocytes from pre-ovulatory follicles during the mid-cycle gonadotrophin surge.

A naturally occurring basically charged human follicle-stimulating hormone (FSH) variant inhibits FSH-induced androgen aromatization and tissue-type plasminogen activator enzyme activity in vitro.

It is well known that deglycosylation of gonadotropins by enzymatic or chemical procedures or by deletion of sites for N-linked glycosylation produces antagonistic analogs which are able to interact strongly with the receptor and to inhibit binding of the wild-type hormone. In the present study, we analyzed the antagonistic properties of a naturally occurring basic follicle-stimulating hormone (FSH) charge isoform obtained after high-resolution chromatofocusing of human anterior pituitary glycoprotein extracts. Coincubation of increasing amounts of this isoform with a highly purified human pituitary FSH preparation or with recombinant human FSH at doses equivalent to their corresponding ED50 for estradiol and tissue-type plasminogen activator (tPA) production, inhibited FSH-induced estrogen production and tPA enzyme activity by cultured rat granulosa cells in a dose-dependent manner. These inhibitory effects were apparently exerted at steps following 3',5'-cyclic adenosine monophosphate (cAMP) formation and did not involve activation of the protein kinase C pathway since: (a) at low doses, this basic FSH isoform moderately increased FSH-induced cAMP production by cultured rat granulosa cells; (b) coincubation of the antagonist isoform with dibutyryl cAMP completely inhibited the effects of this cAMP analog on estrogen and tPA production; (c) the isoform was able to stimulate production of cAMP in a human fetal cell line expressing the recombinant human FSH receptor, and (d) the inhibitory effects of the isoform were not affected by staurosporine, a protein kinase C inhibitor. The effects of this isoform upon dibutyryl cAMP-induced estrogen and tPA production were blocked by the addition of a highly specific antibody directed against human FSH, further demonstrating that the antagonistic effects observed were due to FSH-like molecules. In contrast to the inhibitory effects exhibited by this basic FSH isoform, a more acidic FSH charge variant consistently acted as an agonist of pituitary and recombinant FSH on both estrogen production and induction of tPA enzyme activity. These results indicate that the anterior pituitary gland normally produces FSH isoforms which act as either agonists or antagonists of FSH at the target cell level.

More in-vitro bioactive, shorter-lived human chorionic gonadotrophin charge isoforms increase at the end of the first and during the third trimesters of gestation.

In the present study we analysed the dynamics of serum human chorionic gonadotrophin (HCG) charge isoform distribution throughout normal gestation and characterized some of the biological features of the several HCG glycoforms present in the circulation of pregnant women. Blood samples were obtained from normal pregnant women at 10-11, 12-15, 23-26 and 35-38 weeks of gestation. The sera were fractionated by preparative chromatofocusing and the separated HCG isoforms were identified and quantified by radioimmunoassay. The in-vitro biological activity and the plasma half-life of the several circulating HCG isoforms were determined by conventional methods. HCG isoforms became less acidic as pregnancy advanced. In samples taken at 10-11 weeks of gestation, the most acidic HCG molecules (pH < 3.7) comprised > 80% of total HCG recovered after chromatofocusing; this proportion decreased to 58, 60 and 47% in samples taken from weeks 12.1 to 38.4 of gestation. Meanwhile, the relative proportion of less acidic isoforms recovered within pH values 6.49-4.50 increased at the end of the first trimester (12-15 weeks), remained constant until weeks 23-26 and then increased further by the end of the third trimester. Less acidic isoforms had higher in-vitro biological potency per immunological unit than the more acidic analogues. Regardless of the trimester of pregnancy, the plasma half-life of the highly acidic (elution pH < 3.7) isoforms varied from 84.4 to 150 min (116.3 +/- 23.0; mean +/- SD), whereas the corresponding half-life of mid-acidic (pH 4.25-5.31) and low-acidic (pH 5.74-6.50) HCG isoforms ranged from 31.0 to 115.3 (75.5 +/- 20.6) and 15.3 to 58.3 (41.2 +/- 14.3) min respectively (P < 0.01, highly acidic versus mid- and low-acidic analogues and mid-acidic versus least acidic isoforms). The overall data indicate that the human trophoblast is able to regulate the exact intensity, biochemical composition and duration of the gonadotrophic stimulus secreted during the course of normal gestation. They also suggest that the decrease and maintenance of low serum HCG concentrations during the second and third trimesters of gestation may be partially caused by changes in the carbohydrate structure of the HCG molecule.

Studies on the relative in-vitro biological potency of the naturally-occurring isoforms of intrapituitary follicle stimulating hormone.

In the present study, we analysed and compared the relative in-vitro biological activity of the various intrapituitary human follicle stimulating hormone (FSH) isoforms employing two different bioassay systems. FSH was fractionated by chromatofocusing (pH range 7.10 to < 3.80) and the several isoforms isolated were quantified at multiple dose levels by three highly specific immunoassay systems: radioimmunoassay (RIA), enzyme-immunoassay (EIA) and immunoradiometric assay (IRMA), as well as by two in-vitro bioassays, one that measures the amount of oestrogen produced by rat granulosa cells in culture and the other that determines the amount of cAMP produced by a human fetal cell line (293) expressing the recombinant human FSH receptor. The relative in-vitro biological activity of each FSH isoform, expressed as the bioassay/ immunoassay (B/I) activity ratio (B/RIA, B/EIA and B/IRMA ratios) varied with its elution pH value. Regardless of the immunoassay or bioassay method employed, less acidic FSH isoforms exhibited higher B/I ratios than their more acidic counterparts [B/RIA, B/EIA and B/IRMA ratios for isoforms with elution pH values > 4.5 = 1.05 +/- 0.13, 0.99 +/- 0.10 and 1.15 +/- 0.08 (rat oestrogen bioassay), and 2.75 +/- 0.34, 2.20 +/- 0.25 and 2.96 +/- 0.35 (human cAMP production bioassay) respectively. Ratios for isoforms with pH values < 4.5 = 0.71 +/- 0.06, 0.47 +/- 0.05 and 0.63 +/- 0.06 (rat oestrogen assay), and 1.80 +/- 0.26, 1.10 +/- 0.09 and 1.44 +/- 0.13 (cAMP assay) respectively (P < 0.05 for isoforms with pH < 4.5 compared with those isoforms with pH > 4.5)]. Furthermore, statistically significant direct relationships between the B/RIA, B/EIA and B/IRMA ratios and elution pH value of each isoform was identified by regression analysis [rat assay: r = 0.844, 0.800 and 0.780 (P < 0.01); human assay: r = 0.730, 0.845 and 0.821 (P < 0.01), for their corresponding B/RIA, B/EIA and B/IRMA ratios respectively]. The finding of significant differences in relative in-vitro biological potency among the various intrapituitary FSH isoforms strongly suggests that the shifts towards the production and secretion of more basic or acidic FSH molecules occurring in certain specific physiological conditions (e.g. puberty and menstrual cycle), may represent an important mechanism through which the anterior pituitary regulates gonadal function.

Amplitude regulation of episodic release, in vitro biological to immunological ratio, and median charge of human chorionic gonadotropin in pregnancy.

In the present study, we examined the regulation of 24-h serum immunoreactive levels, in vitro biological to immunological (B/I) ratio, and median charge of circulating CG at the end of the first, second, and third trimesters of human gestation. Seven pregnant women were prospectively studied at 12-15, 23-26, and 35-38 weeks of gestation. Blood was sampled every 20 min over a 24-h period, and serum CG concentrations were determined by RIA. Pulse detection and analysis of the 24-h rhythm of serum immunoreactive CG concentrations were carried out by the program Cluster and cosine curve fitting, respectively. The in vitro biological activity of circulating CG was determined by the mouse Leydig cell-testosterone production bioassay, and the median charge of its isoforms was determined by zone electrophoresis in agarose suspension. The immunoreactive levels of CG present at the end of each trimester of gestation fluctuated over a 24-h period; such variability exceeded that of the within-assay coefficient of variation of the CG RIA and could be resolved into a series of CG peaks and valleys. Although no trend in the number of peaks or valleys was systematically found in relation to gestational age, comparisons between the amplitude and area of the CG peaks revealed that these pulse parameters were significantly higher at 12-15 weeks than at 23-26 and 35-38 weeks of gestation. Cosine fits for 24-h rhythms revealed the existence of significant nyctohemeral profiles of serum CG levels in all women studied at 12-15 weeks, in four subjects at 23-26 weeks, and in six women at 35-38 weeks gestation. The time of acrophase was highly homogeneous only between 12-15 weeks of gestation, occurring between 1057-1452 h in six of the women. The in vitro B/I ratio of CG contained in serum pools from 12-15 weeks was significantly (P < 0.05) higher than that exhibited by CG during later gestational periods (B/I ratio at the end of first trimester, 1.14 +/- 0.14; second trimester, 0.87 +/- 0.22; third trimester, 0.79 +/- 0.12). hCG isoforms at 12-15 weeks were more negatively charged than those circulating at 23-26 and 35-38 weeks of gestation. There were no significant differences between the B/I ratio and the median charge of CG molecules from the second and third trimesters. We conclude that serial serum concentrations of CG throughout pregnancy show significant amplitude-modulated pulsatile release and nyctohemeral variations.(ABSTRACT TRUNCATED AT 400 WORDS)