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ABSTRACT: Recent studies have found anti-Müllerian hormone (AMH) to be a potentially important marker for the assessment of ovarian reserve and prediction of the success of in vitro fertilization (IVF) treatment. The objectives of this study were to develop a sensitive and specific assay for AMH and to evaluate the potential application of the assay. This assay will be then available to our collaborators in the UK and overseas.
Samples obtained as part of another prospective cross-sectional study from infertility patients and another prospective longitudinal study from pregnant women were used in this study to measure AMH using a new double-antibody enzyme-linked immunosorbent assay (ELISA).
AMH levels were evaluated in (i) serum and seminal fluid from males (normal and male factor infertility males), (ii) serum and follicular fluid from females (normal and female with unexplained infertility) and (iii) serum, amniotic fluid (AF) and coelomic fluid (CF) from pregnant women. AMH levels in the samples were measured by a newly developed ELISA.
The assay had a detection limit of<0.078 ng/ml. High recoveries of spiked recombinant protein were observed from male and female sera and also from follicular, seminal, coelomic and amniotic fluids. The intra- and interassay coefficients of variation (CVs) were 3.6% and 4.0%, respectively. Serially diluted human samples gave dose-response curves parallel to the standard curve. Immunoreactivity was stable to sample storage at room temperature for several days and to multiple cycles of freezing and thawing. In seminal fluid, the AMH concentrations in a group of men with male factor infertility were insignificantly different from those in fertile men. By contrast, serum AMH concentrations were lower in the male factor infertility group than the normal group of patients. Women with unexplained infertility had similar concentrations of AMH in serum and follicular fluid compared to controls. Pregnant women had higher concentrations of AMH in the circulation in early pregnancy compared with nonpregnant women, suggesting a foeto-placental contribution and a possible biological role for this molecule in early pregnancy.
We have developed a sensitive and specific assay for AMH. Serum AMH in men with male factor infertility is lower than in normal men. Levels of AMH in pregnancy are higher than normal menstrual cycle levels suggesting a foeto-placental contribution.
Clinical Endocrinology 10/2005; 63(3):267-73. · 3.17 Impact Factor
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ABSTRACT: The mRNA expression of two activin growth factor subunits (betaA- and betaC-activin), activin receptor subunits (ActRIIA, ActRIIB) and the activin-binding protein follistatin, and peptide expression of betaA-activin and betaC-activin subunits, were examined in regenerating rat liver after partial hepatectomy (PHx). Liver samples were collected from adult, male Sprague-Dawley rats, 12-240 h (n=3-5 rats per time point) after PHx or from sham-operated controls at the same time points. Hepatocyte mitosis and apoptosis were assessed histologically and by in situ cell death detection. RT and PCR were used to assess relative gene expression. betaA- and betaC-activin peptide immunoreactivity was assessed in liver and serum samples by western blotting, whereas cellular expression was investigated by immunohistochemistry, using specific monoclonal antibodies. betaA- and betaC-activin mRNA dropped to < 50% of sham control values 12 h after PHx and remained at this level until 168 h post-PHx, when betaA-activin expression increased to three times sham control values and betaC-activin mRNA returned to pre-PHx levels. A peak in follistatin expression was observed 24-48 h post-PHx, coincident with an increase in hepatocyte mitosis. No changes were observed in ActRIIA mRNA, whereas ActRIIB expression paralleled that of betaA-activin mRNA. betaC-activin immunoreactive homo- and heterodimers were observed in regenerating liver and serum. Mitotic hepatocytes frequently contained betaC-activin immunoreactivity, whereas apoptotic hepatocytes were often immunoreactive for betaA-activin. We conclude that betaA- and betaC-activin subunit proteins are autocrine growth regulators in regenerating liver and when expressed independently lead to hepatocyte apoptosis or mitosis in a subset of hepatocytes.
Journal of Molecular Endocrinology 04/2005; 34(2):505-15. · 3.48 Impact Factor
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R B Gilchrist,
L J Ritter, M Cranfield,
L A Jeffery,
F Amato,
S J Scott,
S Myllymaa,
N Kaivo-Oja,
H Lankinen,
D G Mottershead,
N P Groome,
O Ritvos
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ABSTRACT: Paracrine factors secreted by oocytes play a pivotal role in promoting early ovarian follicle growth and in defining a morphogenic gradient in antral follicles, yet the exact identities of these oocyte factors remain unknown. This study was conducted to determine the extent to which the mitogenic activity of mouse oocytes can be attributed to growth differentiation factor 9 (GDF9). To do this, specific anti-human GDF9 monoclonal antibodies were generated. Based on epitope mapping and bioassays, a GDF9 neutralizing antibody, mAb-GDF9-53, was characterized with very low cross-reactivity with related transforming growth factor (TGF)beta superfamily members, including BMP15 (also called GDF9B). Pep-SPOT epitope mapping showed that mAb-GDF9-53 recognizes a short 4-aa sequence, and three-dimensional peptide modeling suggested that this binding motif lies at the C-terminal fingertip of mGDF9. As predicted by sequence alignments and modeling, the antibody detected recombinant GDF9, but not BMP15 in a Western blot and GDF9 protein in oocyte extract and oocyte-conditioned medium. In a mouse mural granulosa cell (MGC) bioassay, mAb-GDF9-53 completely abolished the mitogenic effects of GDF9, but had no effect on TGFbeta1 or activin A-stimulated MGC proliferation. An unrelated IgG at the same dose had no effect on GDF9 activity. This GDF9 neutralizing antibody was then tested in an established oocyte-secreted mitogen bioassay, where denuded oocytes cocultured with granulosa cells promote cell proliferation in a dose-dependent manner. The mAb-GDF9-53 dose dependently (0-160 microg/ml) decreased the mitogenic activity of oocytes but only by approximately 45% at the maximum dose of mAb. Just 5 microg/ml of mAb-GDF9-53 neutralized 90% of recombinant mGDF9 mitogenic activity, but only 15% of oocyte activity. Unlike mAb-GDF9-53, a TGFbeta pan-specific neutralizing antibody did not affect the mitogenic capacity of the oocyte, but completely neutralized TGF beta 1-induced DNA synthesis. This study has characterized a specific GDF9 neutralizing antibody. Our data provide the first direct evidence that the endogenous GDF9 protein is an important oocyte-secreted mitogen, but also show that GDF9 accounts for only part of total oocyte bioactivity.
Biology of Reproduction 10/2004; 71(3):732-9. · 4.01 Impact Factor
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K P McNatty,
J L Juengel,
T Wilson,
S M Galloway,
G H Davis,
N L Hudson,
C L Moeller, M Cranfield,
K L Reader,
M P E Laitinen,
N P Groome,
H R Sawyer,
O Ritvos
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ABSTRACT: The physiological mechanisms controlling ovulation rate in mammals involve a complex exchange of endocrine signals between the pituitary gland and the ovary, and a localized exchange of intraovarian hormones between the oocyte and its adjacent somatic cells. The discoveries in sheep of mutations in bone morphogenetic protein 15 (BMP15) and bone morphogenetic protein receptor type IB (BMPR-IB) together with recent findings on the physiological effects of growth differentiation factor 9 (GDF9) and BMP15 on follicular development and ovulation rate highlight some important differences in the way in which the oocyte may function in mammals with different ovulation rate phenotypes. In sheep, BMP15 and GDF9 have each been shown to be essential for the early and later stages of follicular development. In addition, ovulation rate is sensitive to changes in the dose of either of these two oocyte-derived growth factors. These findings are in contrast to those reported for mice in which GDF9, but not BMP15, is essential for follicular development. The evidence to date is consistent with the hypothesis that the oocyte plays a central role in regulating key events in the process of follicular development and hence, is important in determining ovulation rate. Moreover, it appears that the mechanisms that the oocyte uses to control these processes differ between species with low and high ovulation rate phenotypes.
Reproduction (Cambridge, England) Supplement 02/2003; 61:339-51.
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ABSTRACT: Previous studies have shown that changes in the plasma concentrations of immunoreactive inhibin measured by radioimmunoassay occur in parallel with growth and regression of the testes during a reproductive cycle in adult Soay rams induced by exposure to an artificial lighting regimen of alternating 16 week periods of long days and short days. With the development of new two-site ELISAs for sheep inhibin A and inhibin B, we have re-examined the relationship between FSH and dimeric, biologically active inhibin in the reproductive cycle in adult Soay rams. No signal was generated by sheep testicular extract, ram or ewe plasma, or sheep ovarian follicular fluid in the inhibin B ELISA. In contrast, ram plasma contained significant activity in the inhibin A ELISA, which diluted in parallel to the inhibin A standard, and was abolished by preincubation of ram plasma with monoclonal antibodies specific for the betaA, but not the betaB, subunit. These results indicate that the ram is the first adult male mammalian species identified to date in which the testes produce and secrete dimeric inhibin A and not inhibin B. Northern blot analysis and immunocytochemistry confirmed the presence of alpha, betaA and betaB inhibin/activin subunit mRNA and protein in the testes of adult rams. Changes in plasma inhibin A concentrations occurred in parallel with the growth and regression of the testes during the long day: short day: long day lighting regimen in adult Soay rams, confirming our previous observations with immunoreactive inhibin. During the growth phase of the testes in the first 8 weeks of exposure to short days there was a positive correlation between plasma FSH and inhibin A concentrations, indicating that during this phase the secretion of inhibin A is stimulated by FSH and that inhibin A did not act as a negative feedback hormone on FSH secretion. From week 8.5 to week 16.0 of exposure to short days, there was a negative correlation between FSH and testosterone concentrations, but not inhibin, indicating that when inhibin concentrations are high, testosterone acts as the negative regulator of FSH secretion. Thus, in intact adult rams, when the testes are fully active it appears that inhibin A may sensitize the pituitary to the negative feedback effects of testosterone, at which time they act synergistically to maintain plasma concentrations of FSH.
Reproduction (Cambridge, England) 07/2002; 123(6):827-35. · 3.09 Impact Factor
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ABSTRACT: Activins are cytokines of the transforming growth factor beta family, which plays a central role in the determination of cell fate and the regulation of tissue balance. Family members are composed of two subunits and this dimerization is critical for liganding their cognate receptors and execution of proper functions. In the current study we focused on the localization of activin betaA, betaB, betaC and betaE subunits in the adult rat and analyzed the composition of putative activin beta dimers. By dissecting tissue distribution of various activins, we found that the liver, in particular the hepatocytes, is the major source for activin betaC and betaE transcripts, since other tissues almost failed to express these isoforms. In sharp contrast, the emergence of activin betaA and betaB appeared ubiquitous. Using a highly selective proteome approach, we were able to identify homo- as well as heterodimers of individual activin subunits, indicating a high redundancy of ligand composition. Certainly, this broad potential to homo- and heterodimerize has to be considered in future studies on activin function.
Journal of Molecular Endocrinology 05/2002; 28(2):137-48. · 3.48 Impact Factor
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ABSTRACT: The biosynthesis of oestrogens from androgens is catalysed by the aromatase complex, an essential component of which is the aromatase cytochrome P450 (P450 arom) protein. Expression of a functional P450 arom is essential for normal fertility in males and females and the sequence of the protein is highly conserved. We have raised a new monoclonal antibody against a conserved peptide and validated it on fixed tissue sections of the rat, common marmoset (Callthrix jacchus) and human. The monoclonal antibody was used successfully for Western analysis and specifically reacted with a 55 kDa protein in microsomal extracts. On sections of ovaries in all three species, expression in follicles was specific to the mural granulosa cells of antral follicles and was present in corpora lutea. In the human and marmoset, staining of luteal cells was markedly heterogeneous and did not appear to vary consistently with the stage of the cycle. The intensity of immunostaining was elevated in corpora lutea from pregnant rats and following human chorionic gonadotropin rescue in the human. In the testis, the highest levels of expression were observed in the Leydig cells within the interstitium. In adult rat and marmoset, and possibly also in the human, some P450 arom was associated with the cytoplasm surrounding elongate spermatids but other germ cells were immunonegative. In conclusion, a new monoclonal antibody specific for P450 arom recognises the protein in rodent, primate and human. Its ability to work on fixed tissue sections will facilitate identification of individual cells expressing P450 arom within complex tissues.
Journal of Endocrinology 02/2002; 172(1):21-30. · 3.55 Impact Factor
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ABSTRACT: In this short review, the authors summarise the inhibin, activin and follistatin assays developed by the Oxford group and collaborators, and some of the main purposes for which they have been applied. Over 500 research publications have used these assays. We also discuss new assays recently developed at the request of our collaborators for particular applications, and comment on outstanding assay problems.
Molecular and Cellular Endocrinology 07/2001; 180(1-2):73-7. · 4.19 Impact Factor
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ABSTRACT: Activin ligands are formed by dimerization of activin ss(A)- and/or ss(B)-subunits to produce activins A, AB, or B. These ligands are members of the transforming growth factor-ss superfamily and act as growth and differentiation factors in many cells and tissues. New additions to this family include activin ss(C)-, ss(D)-, and ss(E)-subunits. The aim of this investigation was to examine the localization of and dimerization among activin subunits; the results demonstrate that activin ss(C) can form dimers with activin ss(A) and ss(B) in vitro, but not with the inhibin alpha-subunit. Using a specific antibody, activin ss(C) protein was localized to human liver and prostate and colocalized with ss(A)- and ss(B)-subunits to specific cell types in benign and malignant prostate tissues. Activin C did not alter DNA synthesis of the prostate tumor cell line, LNCaP, or the liver tumor cell line, HepG2, in vitro when added alone or with activin A. Therefore, the capacity to form novel activin heterodimers (but not inhibin C) resides in the human liver and prostate. Activin A, AB, and B have diverse actions in many tissues, including liver and prostate, but there is no known biological activity for activin C. Thus, the evidence of formation of activin AC or BC heterodimers may have significant implications in the regulation of levels and/or biological activity of other activins in these tissues.
Journal of Clinical Endocrinology & Metabolism 01/2001; 85(12):4851-8. · 6.50 Impact Factor
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ABSTRACT: Within 2 days of birth, the mouse ovary is mainly composed of oocytes surrounded by a few pregranulosa cells forming primordial follicles that remain quiescent until they are recruited by intraovarian or other unknown factors to initiate growth of the oocyte and proliferation of the attendant granulosa cells. However, the role of the oocyte in this early development and organization of the follicle is poorly understood. The Dazl knockout (-/-) mouse in which there is total ablation of oocytes in fetal life has allowed us to address this issue. Ovaries from -/- females lack any follicular structure and have no cells positive for either Mullerian inhibiting factor or sulfated glycoprotein-1, indicating a lack of small follicles or corpora lutea. However, by immunocytochemistry, there are cells positive for 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase, and aromatase, indicating the presence of steroidogenically active cells capable of producing estrogen. This was confirmed by the presence of hypertrophied uterine endometrium expressing both estrogen receptor alpha (ER alpha) and ER beta together with normal levels of plasma estradiol. In addition, these steroidogenically active cells contain ER beta, inhibin alpha, and betaB-subunits, and -/- mice have low measurable plasma inhibin A and B levels. The ovarian steroids and inhibins had no significant effect on either plasma or pituitary gonadotropin levels, with significantly (P < 0.01) lower LH and FSH in intact +/+ and +/- females. However, significantly (P < 0.05) increased plasma inhibin B together with significantly (P < 0.05) lower FSH were observed in the +/- females. In conclusion, our data showed that despite oocyte loss in fetal life, the adult ovaries contained steroidogenically active cells capable of producing estradiol and inhibin. Furthermore, in the +/- mice, the enhanced plasma inhibin B implies a role for Dazl protein within the oocyte either from more small follicles or increased inhibin B production from each follicle.
Endocrinology 11/2000; 141(11):4284-94. · 4.46 Impact Factor
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ABSTRACT: In this short review, the authors summarise the inhibin, activin and follistatin assays developed by the Oxford group and collaborators, and some of the main purposes for which they have been applied. Over 500 research publications have used these assays. We also discuss new assays recently developed at the request of our collaborators for particular applications, and comment on outstanding assay problems.
Molecular and Cellular Endocrinology.
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ABSTRACT: The mRNA expression of two activin growth factor subunits (ßA- and ßC-activin), activin receptor subunits (ActRIIA, ActRIIB) and the activin-binding protein follistatin, and peptide expression of ßA-activin and ßC-activin subunits, were examined in regenerating rat liver after partial hepatectomy (PHx). Liver samples were collected from adult, male Sprague Dawley rats, 12 240 h (n=3 5 rats per time point) after PHx or from sham-operated controls at the same time points. Hepatocyte mitosis and apoptosis were assessed histologically and by in situ cell death detection. RT and PCR were used to assess relative gene expression. ßA- and ßC-activin peptide immunoreactivity was assessed in liver and serum samples by western blotting, whereas cellular expression was investigated by immunohistochemistry, using specific monoclonal antibodies. ßA- and ßC-activin mRNA dropped to < 50% of sham control values 12 h after PHx and remained at this level until 168 h post-PHx, when ßA-activin expression increased to three times sham control values and ßC-activin mRNA returned to pre-PHx levels. A peak in follistatin expression was observed 24 48 h post-PHx, coincident with an increase in hepatocyte mitosis. No changes were observed in ActRIIA mRNA, whereas ActRIIB expression paralleled that of ßA-activin mRNA. ßC-activin immunoreactive homo- and heterodimers were observed in regenerating liver and serum. Mitotic hepatocytes frequently contained ßC-activin immunoreactivity, whereas apoptotic hepatocytes were often immunoreactive for ßA-activin. We conclude that ßA- and ßC-activin subunit proteins are autocrine growth regulators in regenerating liver and when expressed independently lead to hepatocyte apoptosis or mitosis in a subset of hepatocytes Yes Yes
