The influence of sex steroid hormones on ferrochelatase gene expression in Harderian gland of hamster (Mesocricetus auratus).

Ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the insertion of ferrous iron into protoporphyrin IX to form protohaem. The Syrian hamster Harderian gland (HG) is known for its ability to produce and accumulate large amounts of protoporphyrins. In this species, the female gland contains up to 120 times more porphyrin than the male gland. Data from biochemical studies suggest that this gland possesses the enzymatic complex for haem biosynthesis but lacks ferrochelatase activity. The abundance of intraglandular haem proteins does not support this idea. To gain more insight into this process, we isolated cDNA for ferrochelatase from hamster liver, using the 5'- and 3'- rapid amplification of complementary DNA ends (RACE), and investigated its expression in HG from males and females. The full-length cDNA comprises an open reading frame of 1269 bp encoding a polypeptide of 422 amino-acid residues. Hamster DNA sequence exhibits 92% identity to mouse and 87% identity to human sequences. The predicted hamster enzyme was shown to have structural features of mammalian ferrochelatase, including a putative NH2- terminal presequence, a central core of about 330 amino-acid residues and an extra 30-50-amino-acid stretch at the carboxyl-terminus. RNA blotting experiments indicated that this cDNA hybridized to a liver mRNA of about 2.1 kb, while a weak hybridization signal was observed with mRNA from HG preparations. RT-PCR assays confirmed the expression of specific transcripts in both tissues. Male glands contained approximately twofold more enzyme mRNA than female glands. Likewise, the intraglandular content of mRNA varied during the oestrous cycle, with the highest levels found in the oestrous phase. These cyclic variations were less evident in liver. Ovariectomy plus treatment with progesterone or 17beta-oestradiol plus progesterone increased ferrochelatase mRNA of the gland. In HG of short- or long-term castrated males, the administration of testosterone did not affect the ferrochelatase mRNA concentration. Based on mRNA expression levels, we conclude that Harderian ferrochelatase may play an active role in maintaining the physiological pool of haem required for processing cytochromes and other glandular haem proteins. Likewise, the sex-steroid hormones appear to have only a modest influence upon Harderian ferrochelatase.

The cDNA cloning and tissue expression of the cytochrome P450scc from Syrian hamster (Mesocricetus auratus).

In this study we report the cloning and sequencing of a cDNA for cholesterol side chain cleavage cytochrome P450scc from Syrian hamster adrenal glands. Isolation of P450scc mRNA was carried out with degenerate primer PCR together with 5' and 3' RACE protocol. The full-length cDNA comprises an open reading frame of 1563 bp encoding a polypeptide of 520 amino acid residues. The predicted protein sequence exhibits well-preserved heme- and steroid-binding domains and shares 89% amino acid sequence identity with rat and mouse enzymes. Transient transfection of HEK-293 cells with the cloned cDNA leads to the formation of pregnenolone from 25-hydroxycholesterol. Northern blot analysis showed expression of mRNAs for P450scc in the major steroidogenic tissues, namely, the adrenal cortex, testis, and ovary. In addition, tissue distribution analysis using the coupled reaction of RT-PCR and Southern blotting revealed that the mRNA of the enzyme is also expressed in various nonendocrine tissues, including the epididymis, Harderian gland, and lungs. The relative abundance of specific transcripts at these novel sites suggests that P450scc could potentially play an important role in regulating local steroid hormone synthesis.

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.

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.

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.

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.

Biochemical and functional aspects of gonadotrophin-releasing hormone and gonadotrophins.

Reproductive function in mammals is governed by the hypothalamic-pituitary-gonadal axis, which conforms a functional unit. Sexual maturation and the subsequent development of reproductive competence depend on the precise and coordinated function of this axis. The components of the reproductive axis communicate each other through endocrine signals. The hypothalamus synthesizes gonadotrophin-releasing hormone or GnRH, which in turn stimulates synthesis and secretion of the pituitary gonadotrophins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The ovarian follicles and the interstitial and Sertoli cells of the testis are the targets for these pituitary signals. Under gonadotrophic stimulation, the gonads produce and secrete several steroid and non-steroid (polypeptide) factors, which in turn regulate in different ways the function of the hypothalamic-pituitary axis. An episodic and pulsatile mode of secretion of hormonal signals characterize (as in other endocrine systems) the function of the reproductive axis, particularly that of the hypothalamic-pituitary unit. The target cell response, and consequently the harmonic function of the corresponding gland, will depend on the adequate dynamics of this pulsatile secretion. The function of each component of the reproductive axis is strongly influenced by locally-produced signals acting either in a paracrine or autocrine manner; these particular signals represent fine-tuning regulation systems that eventually amplify or restrain the magnitude of response to a particular endocrine signal, providing additional mechanisms for tissue homeostasis and a better functional plasticity of the target gland. The design and rational use of novel therapeutic strategies for an optimal exogenously-controlled reproductive function largely depend on the detailed knowledge of the hypothalamic-pituitary-gonadal axis function and the structure and mechanism of action of those factors and signals involved in its regulation.

Oestrogens regulate pituitary alpha2,3-sialyltransferase messenger ribonucleic acid levels in the female rat.

Follicle-stimulating hormone (FSH) is synthesized by the anterior pituitary gland in multiple molecular forms. Increased acidic/sialylated FSH charge isoforms are associated with conditions characterized by a low oestrogen output. In the present study, we analysed the dynamics of the changes in mRNA levels of the enzyme Galbeta1,3[4]GlcNAc alpha2,3-sialyltransferase (2,3-STase) (one of the enzymes that incorporate sialic acid residues into the FSH molecule) in intact and ovariectomized rats. The anterior pituitaries of 4-day regularly cyclic adult female Wistar rats were obtained at 1000 h on the days of pro-oestrus (P), oestrus (O), dioestrus 1 (D1) and dioestrus 2 (D2), at 0200 h, 1400 h, 1800 h and 2200 h on D1, at 1800 h on day of O and at 1000 h after 7, 14, 21, 28 and 45 days of oophorectomy performed on the morning of P. Total RNA was isolated from each gland and the 2,3-STase levels were measured by Northern blot hybridization analysis employing a 346-base pair cDNA probe encoding for a non-conserved amino acid sequence of the catalytic domain of the enzyme. Maximal levels of the enzyme mRNA were detected at 1000 h on D1; thereafter, they progressively decreased by 60% during the ensuing 24 h, reaching the lowest concentration values (26% of the maximally observed level on D1) at 1000 h on day of P and remaining unchanged during the morning of O. Administration of the potent oestradiol receptor antagonist ICI 182,780 at 1000 h on D1 completely reverted the time-dependent decrease in 2,3-STase mRNA levels observed during the afternoon of D1, whereas oestradiol benzoate administered at 1000 h on day of O significantly reduced the enzyme mRNA levels (to 21% of the levels detected in vehicle-treated controls). In ovariectomized rats, the alpha2,3-STase mRNA progressively increased from day 21 to day 45 post castration. Administration of oestradiol benzoate on day 28 after oophorectomy significantly reduced the 2,3-STase mRNA levels (to 36% of the levels detected in vehicle-injected controls); ICI 182,780 partially counteracted this oestradiol-mediated effect. The dynamics of these changes in 2,3-STase mRNA levels partially correlated with changes in the relative abundance of the FSH charge isoforms separated by preparative chromatofocusing of anterior pituitary extracts, particularly in glands obtained during the morning of P and O. These data demonstrate for the first time that pituitary 2,3-STase is a hormonally-regulated enzyme and that the changes in transcription and/or stability of its mRNA may be involved, in part, in the post-translational processing of the FSH molecule during certain physiological conditions.

Role of glycosylation in function of follicle-stimulating hormone.

The oligosaccharide structures of heterodimeric glycoprotein hormones, such as follicle-stimulating hormone (FSH), have been shown to play an important role in the biosynthesis, secretion, metabolic fate, and regulation of potency of the hormone. The oligosaccharide structures attached to each subunit of the protein seem to exhibit distinct roles in some of these functions. Glycans attached to the alpha-subunit are critical for dimer assembly, integrity, and secretion, as well as for signal transduction; although beta-subunit glycans are also important for dimer assembly and secretion, they play a crucial role in clearance of the dimer from the circulation. Alternative glycosylation on FSH and other glycoprotein hormones not only may affect the metabolic clearance and net in vivo biopotency of the hormone, but also offers the interesting possibility that some glycosylation variants of the hormone may provoke differential or even unique effects at the target cell level. Glycosylation of FSH is regulated by hypothalamic and/or end products from the glands under the control of this hormone. In particular, estrogens regulate terminal sialylation and thus some functional properties of the gonadotropin influenced by sialic acid. Through these extrapituitary inputs, the gonadotroph may regulate not only the amount but also the intensity of the gonadotropin signal to be secreted by the pituitary in a given physiological condition.

A less acidic human follicle-stimulating hormone preparation induces tissue-type plasminogen activator enzyme activity earlier than a predominantly acidic analogue in phenobarbital-blocked pro-oestrous rats.

Follicle-stimulating hormone (FSH) exists in multiple molecular forms. In both experimental animals and in humans the production and secretion of less acidic, short-lived FSH glycoforms significantly increase during the peri-ovulatory period. To gain further insights on the physiological role of these FSH variants, we analysed the ability of two FSH compounds, recombinant FSH (rFSH) and purified FSH from urinary origin (uFSH), (less acidic and acidic pattern of FSH charge isoform distributors respectively) to induce ovarian tissue-type plasminogen activator (tPA) enzyme activity in vivo. FSH produced by Chinese hamster ovary cells and highly purified uFSH were injected at 15:00 h on the pro-oestrous day into phenobarbital-blocked rats and the ovaries were analysed for tPA enzyme activity and tPA mRNA concentrations at different times after FSH injection. Induction of tPA enzyme activity by uFSH and rFSH showed distinct dynamics depending on the particular preparation administered. In animals treated with uFSH, maximum tPA enzyme activity was detected at 20:00 h, and maximum tPA mRNA concentrations were detected at 17:00 h. tPA enzyme activity induction by rFSH was at the maximum at 17:00 h, and maximum tPA mRNA concentration was at 16:00 h (P< 0.05 for uFSH versus rFSH). All animals in the uFSH- and rFSH-treated groups and none in phenobarbital-blocked, saline-treated controls ovulated. No significant differences were present in the number of ova shed by rats treated with uFSH or rFSH and spontaneously ovulating rats (10.7+/-1.7, 10.0+/-2.6 and 11.3+/-1.6 respectively). These data indicate that the increased biological activity exhibited by less acidic FSH glycovariants at the target cell level may compensate for the drawback imposed by their relatively short plasma half-life.

Altered luteinizing hormone pulsatility in infertile patients with idiopathic oligoasthenozoospermia.

In a previous study, we demonstrated that oligoasthenozoospermic (OAZ) patients had two types of testosterone response to human chorionic gonadotrophin (HCG) administration: group 1 (OAZ-1) had an altered, monophasic (no first peak) response, and group 2 (OAZ-2) had a normal biphasic response. The objective of the present work was to study the luteinizing hormone (LH) pulsatility in OAZ-1 compared with both OAZ-2 and men of proven fertility (PF), in order partly to determine the possible aetiology of the blunted acute testosterone response to HCG in these patients. LH pulsatility was measured in 10 PF, 10 OAZ-1 and 10 OAZ-2 patients, in blood samples taken every 5 min for 6 h in PF, and for 4 h in OAZ patients. LH values were determined by a time-resolved immunofluorometric assay. Frequency and amplitude of the LH pulses were determined by a computer program. LH pulse frequency, expressed as pulses/4 h, was significantly lower in OAZ-1 (1.5+/-0.97) than in PF (2.4+/-0.63) and OAZ-2 (2.4+/-0.84) patients. In six OAZ-1 and two OAZ-2 patients, LH pulsatility was diminished, as they showed less than two pulses/4 h. No statistically significant differences in LH pulse amplitude were found. These results, together with a higher number of OAZ-1 cases found with decreased LH pulsatility, suggest that, at least in a subset of these men, quantitative and/or qualitative alterations of LH secretion might have occurred.

Structure-function relationship of follicle-stimulating hormone and its receptor.

Follicle stimulating hormone (FSH) is one of the two pituitary gonadotrophins involved in the regulation of gonadal function. Structurally, this gonadotrophin is a heterodimer composed of two non-covalently associated subunits containing several heterogenous oligosaccharide residues which play an important role in both the in-vivo and in-vitro bioactivity of the hormone. Its cognate receptor, which belongs to the superfamily of the G protein-linked cell surface receptors, also displays a high degree of functional and molecular complexity. Studies employing monoclonal antibodies, synthetic peptides and/or site directed mutagenesis, have unveiled some of the multiple structural determinants involved in FSH and FSH receptor function and interaction. Despite their structural complexity, both molecules exhibit a high degree of plasticity and diversity that allows formation of distinct ligand-receptor complexes capable of selectively activating or deactivating a variety of signalling pathways. Knowledge and mapping of the structural determinants and functional epitopes for intra- and extracellular hormone action are of paramount importance not only for a better and more detailed understanding of the molecular basis of FSH action and FSH receptor function but also for the rational design of analogues with predicted properties and effects.

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.

Estrogenic effects of p-hydroxybenzoic acid in CD1 mice.

Xenobiotic estrogens in the environment or diet have received much attention as a possible source of certain hormonal disease states in human and wildlife. Therefore, the detection of estrogenic activity of any substance, especially those related to the food industry, is important. The estrogenic activity of p-hydroxybenzoic acid (PHBA), a compound related to a commonly used group of preservatives in food, cosmetic, and pharmaceutical preparations, was evaluated with immature and adult ovariectomized female mice (CD1) using two well-known bioassays. Subcutaneous administrations (s.c.) of different doses of PHBA were compared with estradiol (E2), and their effects on vaginal cornification and uterotrophic activities were evaluated. Different groups of animals were treated s.c. daily for 3 days with vehicle (corn oil, 0.3 ml/100 g), E2 (1 microgram/100 g), and PHBA (0.5, 5, 50, and 500 micrograms/100 g). Four days after treatment, PHBA produced a dose-dependent response on vaginal cornification and uterotrophic activity in both immature and adult ovariectomized mice. The relative uterotrophic potency of PHBA (500 micrograms/100 g) to E2 (1 microgram/100 g) was 0.0011 in immature mice and 0.0018 in ovariectomized animals.

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.

On the nature of the follicle-stimulating signal delivered to the ovary during exogenously controlled follicular maturation. A search into the immunological and biological attributes and the molecular composition of two preparations of urofollitropin.

In the present study, we analyzed the immunological and biological potencies as well as the molecular composition of urinary follicle-stimulating hormone (FSH) present in determined lots of regular and highly purified (HP) commercial preparations of urofollitropin in order to obtain additional insights on the particular type of gonadotropin signal received by the ovary during exogenously regulated ovarian stimulation. In both preparations, a high degree of FSH charge heterogeneity was detected as disclosed by chromatofocusing analysis (pH range 7.5 to < 4.0). Urinary FSH present in the HP compound was consistently more acidic and exhibited a longer survival in rat circulation than the regular formulation. Inter-batch variability for FSH heterogeneity and in vitro bioactivity was higher in the partially purified preparation than in the HP analog. In the regular preparation, the amount of immunoreactive and bioactive FSH per ampule was two times higher than that present in the HP preparation; the resultant in vitro B/I ratios were similar. Although both urinary FSH preparations showed detectable amounts of immunoreactive and bioactive luteinizing hormone and choriogonadotropin hormone material, the degree of activity present in the less purified formulation was considerably higher than that shown by the HP analog. When the capability of each urinary FSH preparation to induce ovarian tissue-type plasminogen activator enzyme activity in hypophysectomized rats was determined, both formulations exhibited similar potencies despite the existing differences in plasma clearance rate and charge distribution profile. The present study indicates that the isoform composition of urinary FSH in the two commercial preparations analyzed differs according to the degree of purity of the formulation. More FSH material is needed in the partially purified FSH preparation to induce biological effects similar in magnitude to those exhibited by the highly purified analog. The possible impact of these variations in the molecular composition of the FSH signal on other biological functions of the ovary during the course of exogenously controlled follicular growth and maturation still remains to be ascertained.