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1.
Mol Cell Endocrinol ; 405: 42-51, 2015 Apr 15.
Article in English | MEDLINE | ID: mdl-25661536

ABSTRACT

Previously, our laboratory demonstrated the existence of a ß-subunit glycosylation-deficient human FSH glycoform, hFSH(21). A third variant, hFSH(18), has recently been detected in FSH glycoforms isolated from purified pituitary hLH preparations. Human FSH(21) abundance in individual female pituitaries progressively decreased with increasing age. Hypo-glycosylated glycoform preparations are significantly more active than fully-glycosylated hFSH preparations. The purpose of this study was to produce, purify and chemically characterize both glycoform variants expressed by a mammalian cell line. Recombinant hFSH was expressed in a stable GH3 cell line and isolated from serum-free cell culture medium by sequential, hydrophobic and immunoaffinity chromatography. FSH glycoform fractions were separated by Superdex 75 gel-filtration. Western blot analysis revealed the presence of both hFSH(18) and hFSH(21) glycoforms in the low molecular weight fraction, however, their electrophoretic mobilities differed from those associated with the corresponding pituitary hFSH variants. Edman degradation of FSH(21/18)-derived ß-subunit before and after peptide-N-glycanase F digestion confirmed that it possessed a mixture of both mono-glycosylated FSHß subunits, as both Asn(7) and Asn(24) were partially glycosylated. FSH receptor-binding assays confirmed our previous observations that hFSH(21/18) exhibits greater receptor-binding affinity and occupies more FSH binding sites when compared to fully-glycosylated hFSH(24). Thus, the age-related reduction in hypo-glycosylated hFSH significantly reduces circulating levels of FSH biological activity that may further compromise reproductive function. Taken together, the ability to express and isolate recombinant hFSH glycoforms opens the way to study functional differences between them both in vivo and in vitro.


Subject(s)
Follicle Stimulating Hormone/physiology , Animals , CHO Cells , Carbohydrate Conformation , Carbohydrate Sequence , Cattle , Cell Line, Tumor , Cricetinae , Cricetulus , Follicle Stimulating Hormone/chemistry , Follicle Stimulating Hormone/isolation & purification , Glycosylation , Humans , Molecular Sequence Data , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/isolation & purification , Protein Isoforms/physiology , Protein Processing, Post-Translational , Rats , Receptors, FSH/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sequence Analysis, Protein
2.
Biosens Bioelectron ; 42: 592-7, 2013 Apr 15.
Article in English | MEDLINE | ID: mdl-23261694

ABSTRACT

A new sensitive gold-linked electrochemical immunoassay (GLEIA) for the detection of the pregnancy marker human chorionic gonadotropin (hCG) has been developed using the direct electrochemical detection of Au nanoparticles. We utilized single-walled carbon nanotube (SWCNT) microelectrodes; 24 SWCNT microelectrodes were arrayed on a single Si substrate 25×30 mm² in size, for the development of a new GLEIA (SWCNT-GLEIA). This SWCNT-GLEIA provided convenient and cost-effective tests with the required antibody and antigen sample volumes as small as 2.0 µL for a group of 4 SWCNT microelectrodes. In addition, this assay also exhibited properties of high sensitivity and selectivity benefitting from the intrinsic extraordinary features of SWCNTs. Using scanning electron microscopy, we also observed Au nanoparticle-labeled antigen-antibody complexes immobilized on the surface of the SWCNT microelectrodes. The concentration of the pregnancy marker (hCG) showed a linear relationship with the current intensity obtained from differential pulse voltammetry measurements with a limit of detection (LOD) of 2.4 pg/mL (0.024 mIU/mL) hCG. This LOD is 15 times more sensitive than a previous GLEIA, which used screen-printed carbon electrodes.


Subject(s)
Antibodies, Monoclonal/immunology , Antigen-Antibody Complex/isolation & purification , Biosensing Techniques/methods , Chorionic Gonadotropin/isolation & purification , Nanotubes, Carbon/chemistry , Chorionic Gonadotropin/immunology , Female , Follicle Stimulating Hormone/immunology , Follicle Stimulating Hormone/isolation & purification , Gold/chemistry , Humans , Immunoassay , Metal Nanoparticles , Microscopy, Electron, Scanning , Pregnancy
3.
Rev. lab. clín ; 5(4): 151-154, oct.-dic. 2012.
Article in Spanish | IBECS | ID: ibc-107848

ABSTRACT

Mujer que presentó un importante incremento de la hormona estimulante del tiroides (TSH) (62,2 mU/L) con hormonas tiroideas dentro de los intervalos de referencia. La paciente se encontraba eutiroidea y no presentaba bocio. Se realizó un estudio inicial para determinar la posible causa del incremento en la concentración de TSH. La recuperación de TSH tras precipitación con polietilenglicol fue del 1%, sugiriendo la presencia de alguna molécula de elevado peso molecular que podría interferir en la determinación. Mediante cromatografía de exclusión, se confirmó la presencia de macro-TSH, un complejo autoinmune formado por TSH unido a una Inmunoglobulina G que es inmunorreactivo pero biológicamente inactivo, por lo que, si no se detecta, induce a una interpretación errónea de la concentración de TSH (AU)


Woman showing an important increase of serum TSH (62.2 mU/L) with thyroid hormones within the reference interval. The patient was clinically euthyroid and without goitre. Investigations were carried out to determine the origin of the unexpected high TSH. Polyethylene glycol precipitation test showed low TSH recovery (1%), indicating the presence of large molecules that could interfere with the measurement. The serum sample was fractionated by gel filtration chromatography and the presence of a macro-TSH form was confirmed, an immunoreactive but biologically inactive TSH-Immunoglobulin G autoantibody complex. Its detection is important to avoid a misleading interpretation of the TSH concentration (AU)


Subject(s)
Humans , Female , Adult , Follicle Stimulating Hormone/analysis , Follicle Stimulating Hormone/isolation & purification , Thyrotropin/analysis , Chromatography/methods , Chromatography, Gel/instrumentation , Chromatography, Gel/methods , Immunoglobulin G/analysis , Immunoglobulin G , Hyperpituitarism/diagnosis , Thyroid Diseases/diagnosis , Primary Health Care/methods , Primary Health Care/trends , Thyroid Hormones/analysis , Chromatography, Gel , Thyroid Hormones , Receptors, Thyrotropin/biosynthesis
4.
Reprod Biol Endocrinol ; 10: 71, 2012 Sep 05.
Article in English | MEDLINE | ID: mdl-22950645

ABSTRACT

BACKGROUND: The gonadotropins (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are produced in the pituitary gland and regulates gametogenesis through production of gonadal steroids. However, respective roles of two GtHs in the teleosts are still incompletely characterized due to technical difficulties in the purification of native GtHs. METHODS: Native FSH and LH were purified from the pituitaries of adult chub mackerel, Scomber japonicus by anion-exchange chromatography and immunoblotting using specific antisera. The steroidogenic potency of the intact chub mackerel FSH (cmFSH) and LH (cmLH) were evaluated in mid- and late-vitellogenic stage follicles by measuring the level of gonadal steroids, estradiol-17beta (Ε2) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P). In addition, we evaluated the maturation-inducing potency of the GtHs on same stage follicles. RESULTS: Both cmFSH and cmLH significantly stimulated E2 production in mid-vitellogenic stage follicles. In contrast, only LH significantly stimulated the production of 17,20beta-P in late-vitellogenic stage follicles. Similarly, cmLH induced final oocyte maturation (FOM) in late-vitellogenic stage follicles. CONCLUSIONS: Present results indicate that both FSH and LH may regulate vitellogenic processes, whereas only LH initiates FOM in chub mackerel.


Subject(s)
Follicle Stimulating Hormone/pharmacology , Luteinizing Hormone/pharmacology , Perciformes/metabolism , Pituitary Gland/chemistry , Animals , Estradiol/analysis , Estradiol/biosynthesis , Female , Follicle Stimulating Hormone/isolation & purification , Hydroxyprogesterones/analysis , Hydroxyprogesterones/metabolism , Luteinizing Hormone/isolation & purification , Ovarian Follicle/chemistry , Ovarian Follicle/drug effects , Ovarian Follicle/metabolism , Vitellogenesis/drug effects
5.
Fertil Steril ; 95(6): 1937-42, 1942.e1-3, 2011 May.
Article in English | MEDLINE | ID: mdl-21429486

ABSTRACT

OBJECTIVE: To compare the pregnancy rates (PRs) in intrauterine insemination (IUI) using recombinant FSH (rec-FSH) or highly purified urinary FSH (HP-FSH). DESIGN: Systematic review and metaanalysis. SETTING: University hospital. PATIENT(S): None. INTERVENTION(S): Electronic and manual searches. MAIN OUTCOME MEASURE(S): PR, per first cycle PR and per woman PR. RESULT(S): Six randomized trials (713 women, 1,581 cycles) were identified. In three the same doses of rec-FSH and HP-FSH were used ("equal dose" group), whereas in the other three the ratio HP-FSH:rec-FSH dose was 1.5. The global metaanalysis showed no differences in PRs. The PR per cycle was similar across the 1.5 ratio group (14.51% vs. 14.93%; relative risk [RR], 0.970; 95% confidence interval [CI], 0.687-1.369). However, the metaanalysis of the equal dose group, showed differences in the PR in favor of rec-FSH (16.36% vs. 12.31%; RR, 1.394; 95% CI, 1.004-1.936). Per woman PR analysis showed similar results (41.44% vs. 31.55%; RR, 1.273; 95% CI, 0.987-1.643). Per first cycle PR analysis showed a similar trend, although the difference did not reach significance (RR, 1.434; 95% CI, 0.934-2.203). CONCLUSION(S): Rec-FSH was associated with higher per cycle PR than HP-FSH, when used at the same dose, whereas the PR were similar when the dose of rec-FSH was 50% lower.


Subject(s)
Follicle Stimulating Hormone/therapeutic use , Insemination, Artificial/methods , Female , Fertility Agents, Female/administration & dosage , Fertility Agents, Female/adverse effects , Follicle Stimulating Hormone/administration & dosage , Follicle Stimulating Hormone/adverse effects , Follicle Stimulating Hormone/isolation & purification , Humans , Male , Ovulation Induction/methods , Pregnancy , Randomized Controlled Trials as Topic/statistics & numerical data , Recombinant Proteins/administration & dosage , Recombinant Proteins/adverse effects , Recombinant Proteins/therapeutic use , Uterus
6.
J Pharm Biomed Anal ; 54(1): 27-36, 2011 Jan 05.
Article in English | MEDLINE | ID: mdl-20800406

ABSTRACT

Biological drugs represent an important and rapidly growing class of therapeutics useful in the treatment of a variety of disorders ranging from cancer to inflammation to infectious diseases. Unlike single chemical entities, the recombinant production of these drugs in living cells confers considerable structural and chemical heterogeneity to the biologically derived protein product that constitutes the active pharmaceutical ingredient (API). In mammalian based expression systems, much of this diversity is conferred through heterogeneous protein glycosylation. These post-translational modifications can have significant effects on the structure, biological function, and pharmacological properties of the API. In addition, the bulk proteins that comprise the API are further formulated through the use of multiple excipients designed to ensure product stability, solubility, and lot-to-lot consistency. Unfortunately, these matrices can interfere with commonly available analytical methods used in the thorough chemical characterization of the biological drug product. At the same time, a demonstration of the suitable extraction of the bulk drug substance in a manner and form that does not destabilize the active ingredient or introduce any structural bias with direct reference to the original drug product is both critical and necessary. Here, we use recombinant human follicle stimulating hormone (follitropin alpha for injection) from a pharmaceutical source as an example to illustrate a suitable purification strategy to effectively extract the bulk drug substance from the formulated drug product with high purity and yield. We assess the suitability of this extraction method in preserving the structural integrity and overall quality of the drug substance relative to the formulated drug product, placing a particular emphasis on glycosylation as a key product attribute. In so doing, we demonstrate that it is possible to effectively extract the active pharmaceutical ingredient from a formulated biological drug product in a manner that is consequently sufficient for its use in comparability studies.


Subject(s)
Biological Products/analysis , Glycoproteins/chemistry , Pharmaceutical Preparations/analysis , Biological Products/chemistry , Chemistry, Pharmaceutical/methods , Chromatography, High Pressure Liquid/methods , Chromatography, Ion Exchange/methods , Follicle Stimulating Hormone/analysis , Follicle Stimulating Hormone/isolation & purification , Follicle Stimulating Hormone, Human/analysis , Follicle Stimulating Hormone, Human/chemistry , Glycosylation , Gonadotropins/chemistry , Humans , Isoelectric Focusing/methods , Pharmaceutical Preparations/chemistry , Polysaccharides/chemistry , Protein Isoforms , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
7.
J Reprod Immunol ; 85(2): 172-9, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20452035

ABSTRACT

This study investigated the in vitro immune-modulating activities of recombinant versus highly purified urinary follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) at the cellular level. CD4(+) T cells were isolated from peripheral blood mononuclear cells obtained from ten healthy women (aged 19-30 years) with regular menstrual cycles during the follicular phase of their cycle. CD4(+) T cells were stimulated with anti-CD3/CD28 monoclonal antibodies as a T cell-specific mitogen. Proliferative and cytokine responses were analyzed at standard time points (72h). Recombinant FSH (r-FSH) and LH (r-LH) alone showed a modest capacity to influence proliferation and cytokine release by CD4(+) T cells. Conversely, their addition to T cells in combination with recombinant hCG (r-hCG) induced a powerful down-modulation of T cell proliferation, decreased interferon-gamma (IFN-gamma) secretion and increased interleukin-10 (IL-10) production. These immune-modulating activities were not present when CD4(+) T cells were stimulated either in the presence of urinary-purified FSH (u-FSH) or human menopausal gonadotropin (HMG), alone or in combination with recombinant hCG. We are the first to suggest that urinary-purified gonadotropins do not display profound immune-modulating activities as compared with the recombinant preparations, despite their endocrine effects. Therefore, the use of the recombinant preparations in assisted reproductive techniques might be relevant not only for their well-documented endocrine actions but also for their impact on the transient immune tolerance known to favour embryo implantation and progression of pregnancy.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Chorionic Gonadotropin/metabolism , Follicle Stimulating Hormone/metabolism , Luteinizing Hormone/metabolism , Recombinant Proteins/metabolism , Adult , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , Cell Proliferation , Cells, Cultured , Chorionic Gonadotropin/genetics , Chorionic Gonadotropin/immunology , Chorionic Gonadotropin/isolation & purification , Female , Follicle Stimulating Hormone/genetics , Follicle Stimulating Hormone/immunology , Follicle Stimulating Hormone/isolation & purification , Humans , Immune Tolerance , Immunomodulation , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Interleukin-10/biosynthesis , Interleukin-10/genetics , Luteinizing Hormone/genetics , Luteinizing Hormone/immunology , Luteinizing Hormone/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Urine/chemistry , Urine/physiology
8.
Biocell ; 33(2): 91-7, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19886036

ABSTRACT

Porcine pituitary follicle stimulating hormone (pFSH) is known to regulate the production of growth factors that have an essential role in early foliculogenesis. However, the effects of different preparations of pFSH on the survival and development of caprine follicles are not yet known. The aim of this study was to evaluate the effects of different pFSH (Stimufol and Folltropin) on the in vitro survival and growth of caprine preantral follicles. Pieces of caprine ovarian tissues were cultured for either one or seven days in a supplemented Minimum Essential Medium, alone or containing either Stimufol (50 ng/mL) or Folltropin (10, 50, 100 and 1000 ng/mL). Fresh control ovarian tissues as well as cultured tissued were processed for histological and ultrastructural studies. The results showed that after seven days, only Stimufol maintained follicular morphology similar to control. Moreover, follicular degeneration was higher in medium alone or with Folltropin at 50, 100 and 1000 ng/mL. However, at day seven, the percentage of growing follicles was higher in 100 ng/mL of Folltropin than Stimufol. In conclusion, FSH preparations affect differently the performance of in vitro culture of caprine preantral follicles. Stimufol was better to preserve follicular morphology while Folltropin was more efficient to promote follicular growth.


Subject(s)
Follicle Stimulating Hormone/isolation & purification , Follicle Stimulating Hormone/pharmacology , Morphogenesis/drug effects , Ovarian Follicle/drug effects , Ovarian Follicle/growth & development , Pituitary Gland/metabolism , Animals , Cell Survival/drug effects , Culture Media , Female , Goats , Oocytes/cytology , Oocytes/drug effects , Ovarian Follicle/cytology , Ovarian Follicle/ultrastructure , Swine , Tissue Culture Techniques
9.
Biocell ; 33(2): 91-97, Aug. 2009. ilus, tab, graf
Article in English | BINACIS | ID: bin-127210

ABSTRACT

Porcine pituitary follicle stimulating hormone (pFSH) is known to regulate the production of growth factors that have an essential role in early foliculogenesis. However, the effects of different preparations of pFSH on the survival and development of caprine follicles are not yet known. The aim of this study was to evaluate the effects of different pFSH (Stimufol and Folltropin) on the in vitro survival and growth of caprine preantral follicles. Pieces of caprine ovarian tissues were cultured for either one or seven days in a supplemented Minimum Essential Medium, alone or containing either Stimufol (50 ng/mL) or Folltropin (10, 50, 100 and 1000 ng/mL). Fresh control ovarian tissues as well as cultured tissued were processed for histological and ultrastructural studies. The results showed that after seven days, o nly Stimufol maintained follicular morphology similar to control. Moreover, follicular degeneration was higher in medium alone or with Folltropin at 50, 100 and 1000 ng/mL. However, at day seven, the percentage of growing follicles was higher in 100 ng/mL of Folltropin than Stimufol. In conclusion, FSH preparations affect differently the performance of in vitro culture of caprine preantral follicles. Stimufol was better to preserve follicular morphology while Folltropin was more efficient to promote follicular growth.(AU)


Subject(s)
Animals , Female , Cell Survival , Follicle Stimulating Hormone/isolation & purification , Follicle Stimulating Hormone/pharmacology , Morphogenesis , Pituitary Gland/metabolism , Oocytes/cytology , Oocytes , Culture Media , Goats , Swine , Ovarian Follicle/cytology , Ovarian Follicle , Ovarian Follicle/growth & development , Ovarian Follicle/ultrastructure
10.
Biocell ; 33(2): 91-97, Aug. 2009. ilus, tab, graf
Article in English | LILACS | ID: lil-595033

ABSTRACT

Porcine pituitary follicle stimulating hormone (pFSH) is known to regulate the production of growth factors that have an essential role in early foliculogenesis. However, the effects of different preparations of pFSH on the survival and development of caprine follicles are not yet known. The aim of this study was to evaluate the effects of different pFSH (Stimufol and Folltropin) on the in vitro survival and growth of caprine preantral follicles. Pieces of caprine ovarian tissues were cultured for either one or seven days in a supplemented Minimum Essential Medium, alone or containing either Stimufol (50 ng/mL) or Folltropin (10, 50, 100 and 1000 ng/mL). Fresh control ovarian tissues as well as cultured tissued were processed for histological and ultrastructural studies. The results showed that after seven days, o nly Stimufol maintained follicular morphology similar to control. Moreover, follicular degeneration was higher in medium alone or with Folltropin at 50, 100 and 1000 ng/mL. However, at day seven, the percentage of growing follicles was higher in 100 ng/mL of Folltropin than Stimufol. In conclusion, FSH preparations affect differently the performance of in vitro culture of caprine preantral follicles. Stimufol was better to preserve follicular morphology while Folltropin was more efficient to promote follicular growth.


Subject(s)
Animals , Female , Pituitary Gland/metabolism , Follicle Stimulating Hormone/pharmacology , Follicle Stimulating Hormone/isolation & purification , Morphogenesis , Oocytes/cytology , Oocytes , Cell Survival , Culture Media , Ovarian Follicle/cytology , Ovarian Follicle/growth & development , Ovarian Follicle , Ovarian Follicle/ultrastructure , Goats , Swine
11.
Gen Comp Endocrinol ; 158(1): 68-76, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18558403

ABSTRACT

Follicle-stimulating hormone (FSH) was purified from pituitaries of sea bass (Dicentrarchus labrax), and its biochemical and biological properties were studied. Sea bass FSH (sbsFSH) was purified by ethanol extraction-precipitation (40-85%), followed by anion-exchange chromatography on a LKB Ultropac TSK-DEAE column using a linear gradient of ammonium bicarbonate (50-1000 mM) and reverse phase chromatography on a RESOURCE 15RPC column with a linear gradient of acetonitrile (0-50%), using a FPLC system. The molecular mass of the purified sbsFSH, estimated by mass spectrometry, was of 28.5 kDa for the dimer, 12.6 kDa for the glycoprotein alpha (GPalpha) and 13.6 kDa for FSHbeta subunits. After separation by SDS-PAGE under reducing condition, the intact sbsFSH was dissociated in the respective subunits (GPalpha and FSHbeta). Subunit identity was confirmed by immunological detection and N-terminal amino acid sequencing. Deglycosylation treatment with N-glycosidase F, decreased the molecular mass of both subunits. Intact sbsFSH activated the sea bass FSH receptor stably expressed in the cell line HEK 293, in a dose dependent manner. Purified sbsFSH showed gonadotropic activity, by stimulating the release of estradiol-17beta (E2) from sea bass ovary and testosterone (T) and 11-ketotestosterone (11KT) from testicular tissue cultured in vitro, in a dose and time dependent manner. These results showed that the purified sbsFSH is a heterodimeric hormone, composed of two distinct glycoprotein subunits (GPalpha and FSHbeta), and has biological activity judged by its ability to stimulate its receptor in a specific manner and to promote steroid release from gonadal tissue fragments.


Subject(s)
Bass , Follicle Stimulating Hormone/isolation & purification , Pituitary Gland/chemistry , Amino Acid Sequence , Animals , Bass/metabolism , Female , Follicle Stimulating Hormone/chemistry , Follicle Stimulating Hormone/metabolism , Follicle Stimulating Hormone/physiology , Male , Molecular Sequence Data , Pituitary Gland/metabolism , Protein Binding , Protein Subunits/chemistry , Protein Subunits/isolation & purification , Protein Subunits/metabolism , Receptors, FSH/analysis , Receptors, FSH/metabolism , Receptors, LH/analysis , Receptors, LH/metabolism , Sequence Homology, Amino Acid
12.
Biochemistry ; 47(6): 1708-20, 2008 Feb 12.
Article in English | MEDLINE | ID: mdl-18197704

ABSTRACT

Follicle-stimulating hormone (FSH) glycosylation is regulated by feedback from the gonads, resulting in an array of glycans associated with FSH preparations derived from pools of pituitary or urine extracts. FSH glycosylation varies due to inhibition of FSHbeta N-glycosylation, elaboration of 1-4 branches possessed by mature N-glycans, and the number and linkage of terminal sialic acid residues. To characterize FSH glycosylation, FSH isoforms in pituitary gland extracts and a variety of physiological fluids are commonly separated by chromatofocusing. Variations in the ratios of immunological and biological activities in the resulting FSH isoform preparations are generally attributed to changes in glycosylation, which are most often defined in terms of sialic acid content. Using Western blotting to assess human FSHbeta glycosylation inhibition revealed 30-47% nonglycosylated hFSHbeta associated with four of six hFSH isoform preparations derived by chromatofocusing. Glycopeptide mass spectrometry assessment of glycan branching in these isoforms extensively characterized two N-glycosylation sites, one at alphaAsn52, the critical glycan for FSH function, and the other at betaAsn24. With two to four N-glycans per FSH molecule, many combinations of charges distributed over these sites can provide the same isoelectric point. Indeed, several glycans were common to all isoform fractions that were analyzed. There was no trend showing predominantly monoantennary glycans associated with the high-pI fractions, nor were predominantly tri- and tetra-antennary glycans associated with low-pI fractions. Thus, differences in receptor binding activity could not be associated with any specific glycan type or location in the hormone. FSH aggregation was associated with reduced receptor binding activity but did not affect immunological activity. However, as gel filtration indicated sufficient heterodimer was present in each isoform preparation to generate complete inhibition curves, the near total loss of receptor binding activity in several preparations could not be explained by aggregation alone, and the mechanism remains unknown.


Subject(s)
Follicle Stimulating Hormone/isolation & purification , Polysaccharides/chemistry , Protein Isoforms/isolation & purification , Animals , Blotting, Western , CHO Cells , Carbohydrate Conformation , Cricetinae , Cricetulus , Follicle Stimulating Hormone/chemistry , Humans , Mass Spectrometry , Protein Isoforms/chemistry , Radioimmunoassay , Rats
13.
Mol Biotechnol ; 34(1): 37-44, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16943569

ABSTRACT

In this article, we describe the use of a caprine beta-lactoglobulin (betaLG) expression cassette previously obtained by our group to target the expression of the human follicle-stimulating hormone (hFSH) to the mammary gland of transgenic mice. The hFSH is a pituitary glycoprotein composed of two subunits (alpha and beta). At present, this protein is obtained from mammalian cellular fermentors, and it is extensively used in the treatment of human infertility. Four lines of double (hFSHalpha/beta) transgenic mice that stably transmitted the transgenes were obtained, and hFSHalpha and hFSHbeta mRNA was detected by reverse transcriptase-polymerase chain reaction in the mammary gland of lactating females from all four transgenic lines. The hFSH protein was present in the mammary gland of the lactating females, but could not be detected in the milk by Western blot, probably as a result of low levels of transgene expression.


Subject(s)
Follicle Stimulating Hormone/biosynthesis , Follicle Stimulating Hormone/isolation & purification , Mammary Glands, Human/metabolism , Mice, Transgenic/metabolism , Milk/chemistry , Protein Engineering/methods , Animals , Follicle Stimulating Hormone/genetics , Gene Expression/physiology , Humans , Mice , Recombinant Proteins/biosynthesis
14.
J Biotechnol ; 122(1): 73-85, 2006 Mar 09.
Article in English | MEDLINE | ID: mdl-16198015

ABSTRACT

Complex glycoprotein biopharmaceuticals, such as follicle stimulating hormone (FSH), erythropoietin and tissue plasminogen activator consist of a range of charge isoforms due to the extent of sialic acid capping of the glycoprotein glycans. Sialic acid occupies the terminal position on the oligosaccharide chain, masking the penultimate sugar residue, galactose from recognition and uptake by the hepatocyte asialoglycoprotein receptor. It is therefore well established that the more acidic charge isoforms of glycoprotein biopharmaceuticals have higher in vivo potencies than those of less acidic isoforms due to their longer serum half-life. Current strategies for manipulating glycoprotein charge isoform profile involve cell engineering or altering bioprocesss parameters to optimise expression of more acidic or basic isoforms, rather than downstream separation of isoforms. A method for the purification of a discrete range of bioactive recombinant human FSH (rhFSH) charge isoforms based on Gradiflowtrade mark preparative electrophoresis technology is described. Gradiflowtrade mark electrophoresis is scaleable, and incorporation into glycoprotein biopharmaceutical production bioprocesses as a potential final step facilitates the production of biopharmaceutical preparations of improved in vivo potency.


Subject(s)
Biotechnology/methods , Chemical Fractionation/methods , Electrophoresis/methods , Follicle Stimulating Hormone/isolation & purification , Protein Engineering/methods , Animals , CHO Cells , Cricetinae , Cricetulus , Follicle Stimulating Hormone/genetics , Humans , Protein Isoforms/genetics , Protein Isoforms/isolation & purification
15.
Prep Biochem Biotechnol ; 35(4): 331-45, 2005.
Article in English | MEDLINE | ID: mdl-16239197

ABSTRACT

An improved and cost effective method to isolate FSH from buffalo pituitary glands is described here. The buFSH activity was monitored throughout by a highly sensitive heterologous radioimmunoassay (sensitivity 0.2 ng oFSH/mL) and the in vivo biological activity of the final preparation was also established. A biologically active buFSH-enriched preparation with a moderate recovery (42%) was obtained. The yield of the final buFSH-enriched preparation was 26.5 mg/kg of buffalo pituitary gland. In SDS-PAGE, the purified buFSH resolved as a heterodimer of 30 kDa molecular size, with a 21 kDa presumptive alpha-subunit. This preparation was also characterized in terms of biological and some of its physicochemical properties. A high-titer antiserum to buFSH was also raised in rabbit using this preparation. The reagents generated, buFSH and buFSH-specific polyclonal antisera, have possible diagnostic and therapeutic usage for improvement of reproductive health of water buffaloes.


Subject(s)
Buffaloes , Follicle Stimulating Hormone/isolation & purification , Pituitary Gland/chemistry , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Blotting, Western , Chromatography, Affinity/methods , Chromatography, Gel , Female , Follicle Stimulating Hormone/immunology , Follicle Stimulating Hormone/pharmacology , Immune Sera/immunology , Immune Sera/isolation & purification , Molecular Weight , Organ Size/drug effects , Ovary/drug effects , Ovary/growth & development , Rabbits , Radioimmunoassay , Rats , Sepharose/analogs & derivatives , Vaccination
16.
Curr Med Res Opin ; 21(6): 899-905, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15969890

ABSTRACT

OBJECTIVE: Pharmaceutical preparations of human menopausal gonadotrophin (hMG), urine-derived follicle-stimulating hormone (u-FSH) and highly purified u-FSH (u-FSH-HP) have been available since the early 1960s and the mid 1980s and 1990s, respectively. Another commercial preparation of u-FSH-HP, Folyrmon P, was launched in Japan in 1999. The aim of this study is to assess the purity of Folyrmon P and to compare it with Fertinorm-P, another commercial preparation of u-FSH-HP that has been available since 1993. METHODS: Folyrmon P and Fertinorm-P were assessed for total protein content, biological activity, immunological activity, specific activity, purity and levels of protein contamination. RESULTS: Folyrmon P, which is extracted from the urine of post-menopausal women, has a specific activity of between 4000 and 5000 IU/mg, while Fertinorm-P, which is also manufactured from the urine of post-menopausal women, has a specific activity of at least 10,000 IU/mg. It has been well documented that commercially available hMG and u-FSH preparations can contain a number of urine-derived protein contaminants. This also proves to be the case for Folyrmon P, in which contaminant proteins other than FSH were shown to be present. It was also demonstrated that both preparations, Folyrmon P and Fertinorm-P, contained high levels of oxidized FSH. CONCLUSIONS: The low specific activity and high level of contaminants in Folyrmon P indicate that this u-FSH is not highly purified. Overall, Fertinorm-P, with higher specific activity and lower levels of contaminant proteins, appears to be of higher quality compared with Folyrmon P.


Subject(s)
Follicle Stimulating Hormone/analysis , Pharmaceutical Preparations/analysis , Female , Follicle Stimulating Hormone/isolation & purification , Follicle Stimulating Hormone/urine , Humans , Postmenopause
17.
Gen Comp Endocrinol ; 143(3): 257-66, 2005 Sep 15.
Article in English | MEDLINE | ID: mdl-15894317

ABSTRACT

Follicle-stimulating hormone (FSH) was purified, for the first time, from immature Japanese eel, Anguilla japonica, and its biochemical properties were investigated. FSH was extracted from immature eel pituitaries and purified by gel-filtration on Sephadex G-100, and two step anion-exchange chromatography: stepwise elution on DE-52, followed by gradient elution on TSK-gel Super-Q using HPLC. Purification was performed using its molecular mass and the positive reaction with anti-Japanese eel (je) FSHbeta antiserum. Purified eel FSH was detected as a single band after separation by SDS-PAGE under a non-reducing condition, showing positive reaction with both anti-je glycoprotein (GP) alpha and anti-jeFSHbeta antisera. The molecular mass of purified eel FSH was estimated to be approximately 33 kDa. After separation by SDS-PAGE under reducing condition, the intact molecule was detected as distinct proteins, whose N-terminus amino acid sequences coincided with those predicted from cDNA sequences for jeGPalpha and jeFSHbeta mature peptides. Deglycosylation of these subunits led to a decrease in their molecular mass. These results suggest that eel FSH is a heterodimeric molecule which consists of distinct glycoprotein subunits, GPalpha and FSHbeta. Cells reacting with anti-jeFSHbeta antiserum were observed in the proximal pars distalis of an immature eel pituitary, while jeLHbeta-immunoreactive cells were not detected. Gonadotropic activities of eel FSH were demonstrated in vitro by stimulating testosterone and 11-ketotestosterone secretions in immature eel testes. Purified eel FSH stimulated the secretion of both androgens from the immature eel testis in a dose-dependent manner, similar to immature eel pituitary homogenate and recombinant eel FSH produced by yeast. These results show that endogenous and recombinant FSH in this species possess similar activities, presumably stimulating the gametogenesis through the sex steroid secretion during the early stages of gonadal development.


Subject(s)
Anguilla/physiology , Follicle Stimulating Hormone/chemistry , Follicle Stimulating Hormone/isolation & purification , Gametogenesis/physiology , Animals , Electrophoresis, Polyacrylamide Gel , Female , Follicle Stimulating Hormone/physiology , Glycoproteins/chemistry , Male , Pituitary Gland/physiology , Sexual Maturation , Testis/physiology , Testosterone/analogs & derivatives , Testosterone/metabolism
18.
Hum Reprod Update ; 10(6): 453-67, 2004.
Article in English | MEDLINE | ID: mdl-15388674

ABSTRACT

The 20th century witnessed the steady development of knowledge about the reproductive process in animals and humans. These advances led to the identification of higher centres governing the dynamics of ovarian function and to the discovery of gonadotrophic hormones. As the mechanisms of action of these hormones became increasingly understood, they began to be used in the management of infertility during the early 1930s. Hormone extracts were originally prepared from animal pituitaries and pregnant mare serum, as well as from human pituitaries, placenta and urine, with pregnancies reported following their use in the late 1930s. This review traces the constant quest to reduce risks and improve safety and efficacy of hormone preparations for patients. It describes the complex path and perils leading to the pure hormone preparations that are available today, concluding with an optimistic glimpse towards the future. Small molecules that are orally active and specific are currently being investigated, some with the capacity to bypass many parts of the receptor conformation. Here lies the immediate future of this field, utilizing low-cost, small, defined molecules to stimulate follicle growth, ovulation and corpus luteum formation. Perhaps one day the classical gonadotrophins will no longer be required in clinical treatment.


Subject(s)
Gonadotropins/therapeutic use , Animals , Chorionic Gonadotropin/history , Dogs , Drug Industry/methods , Drug Industry/trends , Female , Follicle Stimulating Hormone/isolation & purification , Follicle Stimulating Hormone/urine , Gonadotropins/history , Gonadotropins/physiology , Gonadotropins, Equine/pharmacology , Gonadotropins, Equine/standards , History, 20th Century , Humans , Hypothalamo-Hypophyseal System/physiology , Menopause , Ovary/physiology , Pregnancy , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Sheep
19.
Exp Anim ; 53(4): 395-7, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15297716

ABSTRACT

We investigated whether refined follicle stimulating hormone (FSH) with only a little contaminating LH can promote the responsiveness of rabbits to multiple-ovulation treatment. One group of female rabbits was stimulated with refined porcine FSH (pFSH), an FSH source with low LH activity, and another group was treated with pFSH. The mean number of eggs recovered from donors stimulated with refined pFSH (27 +/- 3) was significantly greater (P<0.05) than that with pFSH (20 +/- 2). Furthermore, the mean number of remaining follicles of donors stimulated with refined pFSH (19 +/- 4) was significantly greater (P<0.05) than that with pFSH (12 +/- 1). To decrease the number of remaining follicles in donors treated with refined pFSH, the dose of human chorionic gonadotropin (hCG) was increased from 75 to 150. However, there were no differences in the numbers of eggs and remaining follicles. The results of the present study suggest that refined pFSH with little contaminating LH promotes the responsiveness of rabbits to multiple-ovulation treatment compared with pFSH.


Subject(s)
Follicle Stimulating Hormone/pharmacology , Ovulation/drug effects , Superovulation/physiology , Animals , Female , Follicle Stimulating Hormone/isolation & purification , Pregnancy , Rabbits
20.
Int J Androl ; 26(4): 215-25, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12846797

ABSTRACT

Sertoli cell functional reserve was assessed in normozoospermic men and oligozoospermic patients and its prognostic potential was evaluated for patient selection and treatment. For the first objective, three groups of normo-follicle-stimulating hormone (FSH)/normozoospermic fertile men (n:12), normo-FSH/oligozoospermic (n:21) and hyper-FSH/oligozoospermic subfertile men participated in the study whereas for the second objective 24 normo-FSH oligozoospermic patients volunteered for a pilot therapeutic trial. For the first part, high purity (hp) FSH (225 i.u., i.m.), human chorionic gonadotropin (hCG) (1500 i.u., i.m.) or their combination was given separately at weekly intervals, with samplings at 0, 3, 24 and 48 h. For the pilot trial, rec-FSH (150 i.u./48 h, i.m.) or placebo were prescribed for 6 months. The main outcome measures for the study were inhibin-B (inh-B), insulin-like growth factor (IGF)-I, testosterone and oestradiol concentrations and the main sperm parameters. Bolus administration of hp-FSH or hp-FSH/hCG combination in normozoospermic men resulted in a significant rise of inh-B in normozoospermic men (mean +/- SD, basal: 183.8+/-24.2 pg/mL in hp-FSH and 175.2+/-23.5 in hp-FSH/hCG treatment; 48 h: 256.1+/-34.2 and 246.3+/-19.0, respectively, p<0.001 for both). In oligozoospermic groups basal inh-B concentration was lower than in normozoospermic men (normo-FSH: 117.4+/-16.5, hyper-FSH: 81.2+/-19.8, p<0.001 for both) with a post-stimulation increase noted only in normo-FSH patients (hp-FSH 24-h: 132.8+/-19.7, p<0.01; hp-FSH/hCG 0 min: 105.7+/-20.1, 24-h: 119.5+/-20.6, p<0.05). Total sperm number and progressive motility showed significant improvements (p<0.05 for both) after 6 months of rec-FSH treatment in the group of patients with a satisfactory response to hp-FSH stimulation. In conclusion, the basal and reserve activity of Sertoli cells, as judged by inh-B secretion, was higher in normozoospermic than in dyspermic men, with a better therapeutic outcome noted in those patients with an adequate response to hp-FSH stimulation.


Subject(s)
Oligospermia/physiopathology , Sertoli Cells , Spermatozoa , Adult , Case-Control Studies , Chorionic Gonadotropin/pharmacology , Drug Combinations , Estradiol/blood , Follicle Stimulating Hormone/blood , Follicle Stimulating Hormone/isolation & purification , Follicle Stimulating Hormone/pharmacology , Hormones/pharmacology , Humans , Inhibins/blood , Insulin-Like Growth Factor I/metabolism , Male , Middle Aged , Oligospermia/blood , Oligospermia/therapy , Pilot Projects , Prognosis , Prospective Studies , Sperm Count , Sperm Motility , Spermatozoa/drug effects , Spermatozoa/physiology , Testosterone/blood
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