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P G Knight

Publications and source records attributed to P G Knight.

At least 19 recordsLinked to original sources
PubMed1999

Demonstration of a suppressive effect of inhibin alpha-subunit on the developmental competence of in vitro matured bovine oocytes.

Changes in intrafollicular concentrations of different forms of inhibin (free alpha-subunits and alpha beta dimers) occur during follicle development and may influence the oocyte maturation process. The aim of this study was to investigate the effects of inhibin A and free alpha-subunit (pro-alpha C) isolated from bovine follicular fluid on maturation of bovine cumulus-oocyte complexes, as reflected by their competence for embryo development after in vitro fertilization. Bovine cumulus-oocyte complexes were isolated from ovaries obtained from an abattoir and were cultured for 22-24 h at 38.5 degrees C in TCM-199 medium supplemented with 10% oestrous cow serum, pregnant mares' serum gonadotrophin (2.5 iu ml-1) and either inhibin A (0, 0.2 and 1.0 microgram ml-1) or pro-alpha C (0, 2 and 10 micrograms ml-1). Neither inhibin A nor free alpha-subunit affected the cleavage rate of cumulus-oocyte complexes after fertilization (approximately 60%). Inhibin A reduced the proportion of cleaved oocytes reaching the eight-cell stage by 19% (P < 0.05), but did not affect the yield of blastocysts. However, pro-alpha C decreased the proportion of cleaved oocytes that reached the eight-cell (25%; P < 0.05) and blastocyst (28%; P < 0.05) stages. In addition, a negative correlation (r = -0.55, P < 0.001) was found between concentrations of total immunoreactive (ir) alpha-inhibin (measured by radioimmunoassay) produced by untreated control cumulus-oocyte complexes and their post-cleavage development to the blastocyst stage. In a second experiment, mouse monoclonal antibodies (20 micrograms ml-1) against two different regions of the inhibin alpha-subunit precursor (pro-region and alpha C fragment) were tested for their ability to neutralize endogenous inhibin alpha-subunit-related molecules produced by cumulus cells; control cumulus-oocyte complexes were treated with normal mouse IgG (20 micrograms ml-1). Although the cleavage rate was not affected, the yield of blastocysts was significantly higher in the presence of mouse monoclonal antibodies to both pro-alpha (77% increase; P < 0.05) and alpha C (48% increase; P < 0.05). None of the treatments tested affected endogenous production of activin-A or follistatin by cumulus-oocyte complexes. Overall, these results indicate that the inhibin alpha-subunit (pro-alpha C) has an inhibitory role in oocyte maturation which is independent of the modulatory effects of activin and follistatin.

Analysis of Variance↗
PubMed1998

A longitudinal study of maternal serum inhibin-A, inhibin-B, activin-A, activin-AB, pro-alphaC and follistatin during pregnancy.

Maternal serum concentrations of inhibin-A, inhibin-B, activin-A, activin-AB, pro-alphaC-related inhibin forms, total follistatin, steroids and gonadotrophins were measured longitudinally in six normal singleton pregnancies. Maternal venous blood was collected randomly during a spontaneous follicular phase prior to donor insemination, at 5, 7, 9, 11, 16, 20, 24, 28, 32 and 36 weeks after the first missed menses and in the early puerperium. Steroid and gonadotrophin profiles conformed to previous reports. While at week 5 of gestation inhibin-A, activin-A and follistatin concentrations were similar to those at the follicular phase, all three increased progressively (P < 0.001) to maximal concentrations in week 36: approximately 48-fold (3740 +/- 1349 ng inhibin-A/ml), approximately 22-fold (6109 +/- 1443 ng activin-A/ml) and approximately 10-fold (3563 +/- 418 ng follistatin/ml) higher. Pro-alphaC concentrations reached a maximum in weeks 5 (approximately 5-fold, P < 0.001) and 36 (1027 +/- 174 pg/ml, P < 0.01). Inhibin-B (71 +/- 23 pg/ml prior to pregnancy) was undetectable (<12 pg/ml) between week 5-16 of gestation but increased slightly in the third trimester (26 +/- 7 pg/ml in week 36). Activin-AB was undetectable throughout pregnancy. Post-partum concentrations of inhibin-A (41 +/- 12 ng/ml), inhibin-B (<12 pg/ml), activin-A (950 +/- 149 pg/ml), pro-alphaC (128 +/- 22 pg/ml) and follistatin (990 +/- 79 ng/ml) were substantially lower than at week 36 of gestation. The activin-A:follistatin ratio increased from 0.5 in week 5 to 1.8 in week 36, suggesting that more free activin-A is available in the maternal circulation during late pregnancy.

Activins↗
PubMed1998

Modulatory actions of activin-A and follistatin on the developmental competence of in vitro-matured bovine oocytes.

The presence of activin receptors on oocytes and granulosa cells suggests that activin and its binding protein, follistatin, may regulate oocyte maturation. The aim of the present study was to investigate whether activin-A and follistatin can influence the in vitro maturation of bovine oocytes as assessed by their competence to form blastocysts after in vitro fertilization. Bovine cumulus oocyte complexes (COCs) were cultured for 22-24 h at 38.5 degrees C in tissue culture medium-199 supplemented with 10% estrous cow serum, eCG (2.5 IU/ml), and either no treatment (control), activin-A (0.1 or 0.5 microg/ml), follistatin (0.1, 1, or 10 microg/ml), or activin-A (0.5 microg/ml) in combination with follistatin (0.5 or 5 microg/ml). In separate experiments, the same treatments were also tested on cumulus-free oocytes, which had a much reduced developmental capacity when compared to COCs. Neither activin-A nor follistatin affected the postfertilization cleavage rate of either COCs (approximately 60%) or cumulus-free oocytes (approximately 40%). Activin increased (p < 0.05) the proportion of cleaved oocytes that reached the blastocyst stage when added to both COCs (38% increase) and cumulus-free oocytes (160% increase), with the magnitude of response much greater with the latter. Follistatin had a dose-dependent inhibitory effect on blastocyst yield from COCs (67% reduction, p < 0.05) and opposed the stimulatory effect of activin (p < 0.05). With cumulus-free oocytes, however, follistatin did not further decrease the low developmental potential of oocytes. A positive correlation (r = 0.57, p < 0.001) was found between endogenous levels of activin-A produced by COCs and their postcleavage development to the blastocyst stage. No such correlation was found between endogenous follistatin level and postcleavage development (r = 0.10, p = 0.46). Endogenous levels of activin-A and follistatin secreted by cumulus-free oocytes were undetectable. These in vitro observations support the hypothesis that activin-A and follistatin, both secretory products of cumulus cells, contribute to the regulation of oocyte maturation in vivo.

Activins↗
PubMed1998

Differential changes in inhibin A, activin A, and total alpha-subunit levels in granulosa and thecal layers of developing preovulatory follicles in the chicken.

Accumulating evidence implicates inhibins and activins as endocrine and local regulators of follicular development in mammals, and it was recently confirmed that inhibin/activin alpha and betaA genes are also expressed in the avian ovary. To investigate the potential involvement of these proteins in the chicken ovary, thecal and granulosa layers of the four largest follicles (F1-F4) and the most recent postovulatory follicle were collected from hens (10/group) killed 4, 12, and 20 h before the expected time of F1 ovulation. Inhibin A and activin A concentrations of tissue extracts (expressed per mg DNA) were measured using validated two-site enzyme-linked immunosorbent assays; total immunoreactive inhibin alpha-subunit (ir-alpha) was also measured by heterologous RIA (Monash assay). Inhibin A and ir-alpha were largely confined to the granulosa layer, whereas activin A was much more abundant in the thecal layer. Granulosa inhibin A contents were similar in F4 and F3, but increased approximately 40-fold from F3-F1 (P < 0.0001). As such, the F1 granulosa layer was by far the richest source of inhibin A in the chicken ovary, but contained very little activin A. Total ir-alpha in granulosa was much more abundant than inhibin A and increased only 3-fold from F4-F1 (P < 0.001). Activin A in both granulosa and theca showed little variation between F1 and F4 follicles (by ANOVA, P > 0.05). The inhibin A content of F1 granulosa was maximal 12 h before ovulation and had fallen approximately 6-fold (P < 0.0001) within 8 h, suggesting an inhibitory effect of the preovulatory LH surge on the F1 capacity to synthesize inhibin A. Inhibin A, activin A, and ir-alpha were all less in the postovulatory follicle compared with F1 before ovulation (P < 0.0001). In conclusion, application of the present two-site enzyme-linked immunosorbent assays to the chicken ovary revealed 1) divergent tissue distribution of inhibin A and activin A within preovulatory follicles, and 2) differential regulation of granulosa cell production of inhibin A and activin A dimers during preovulatory follicular development. These findings of dynamic changes in inhibin A, activin A, and total ir-alpha support the hypothesis that these proteins subserve regulatory roles during preovulatory follicular development in the hen.

Activins↗
PubMed1998

Measurement of inhibin-A (alpha beta A dimer) during the oestrous cycle, after manipulation of ovarian activity and during pregnancy in ewes.

A new two-site ELISA was validated for ovine plasma and used to measure circulating inhibin-A concentrations during a synchronized oestrous cycle in four ewes and throughout pregnancy in six ewes. Inhibin A concentrations were also determined in four ewes during chronic treatment with a GnRH agonist and after subsequent exposure to pregnant mares' serum gonadotrophin (PMSG) to stimulate ovarian follicular development. Concentrations of FSH, LH, oestradiol and progesterone were determined by radioimmunoassay. The detection limit of the inhibin-A ELISA was approximately 50 pg ml-1 and no significant crossreaction was observed with a range of related molecules including activin-A, inhibin-B, activin-B, follistatin and alpha 2-macroglobulin. Inhibin-A concentrations were below the detection limit in plasma from hypophysectomized and ovariectomized ewes. During the oestrous cycle, plasma inhibin-A concentrations (approximately 0.3-0.4 ng ml-1) did not vary during the follicular phase whereas plasma oestradiol increased approximately tenfold. After the preovulatory LH/FSH surge, inhibin-A fell to a nadir (approximately 0.15 ng ml-1) coincident with the peak of the postovulatory FSH rise. During the next 2 days, FSH concentrations fell to basal values as inhibin-A concentrations increased (P < 0.05) to a peak (approximately 0.5 ng ml-1) 3 days after the preovulatory LH/FSH surge. Over the following 3 days, FSH values increased again (P < 0.05) as inhibin-A concentrations fell to approximately 0.25 ng ml-1 (P < 0.05). Chronic GnRH agonist treatment suppressed FSH concentrations by about 50%, while inhibin-A and oestradiol concentrations fell below detection limits. Within 2 days after the PMSG injection, concentrations of inhibin-A (approximately 4.5 ng ml-1) and oestradiol (approximately 20 pg ml-1) had increased to very high values, while FSH concentrations had been reduced by a further 50%. Plasma concentrations of inhibin-A and FSH were similar to those in nonpregnant ewes during the first 60 days of gestation, but inhibin-A values fell markedly (sevenfold; P < 0.01) between days 60 and 90, coincident with a twofold decrease in FSH (P < 0.05). Inhibin A and FSH concentrations remained low for the remainder of gestation and were positively correlated throughout pregnancy (r = 0.48; P < 0.005). These observations support an endocrine feedback role for ovarian inhibin-A and oestradiol in controlling the secondary (postovulatory) FSH surge in ewes, but indicate that an increase in oestradiol is responsible for the characteristic reduction in FSH during the early to mid-follicular phase. The reduced secretion of FSH from mid- to late pregnancy cannot be attributed to increased inhibin-A secretion by the feto-placental unit, but most likely reflects increased steroid secretion from this source.

Analysis of Variance↗
PubMed1998

Effects of active immunization of sheep against an amino terminal peptide of the inhibin alpha C subunit on intrafollicular levels of activin A, inhibin A and follistatin.

Active immunization of ewes against inhibin (IMM) consistently increases ovulation rate but this response is not always accompanied by the expected rise in plasma FSH. Inhibin-related molecules also have local auto/paracrine effects within the ovary and the ovulatory response to IMM could be due to neutralization of one of these effects, independent of changing FSH levels. To investigate this, ovaries were collected from long-term IMM (n = 6) and control (CON; n = 8) ewes killed 48 h after progestagen withdrawal (late follicular phase) and all follicles > or = 3 mm were recovered to determine intrafollicular levels of inhibin A, activin A and follistatin by specific two-site immunoassay and oestradiol and testosterone by radioimmunoassay. Blood samples were collected to assess plasma FSH, oestradiol and inhibin antibody titres. Although plasma FSH levels were similar in IMM and CON ewes, IMM ewes had approximately 3-fold more follicles > or = 3 mm (P < 0.0001) and approximately 3-fold more oestrogenic follicle (P < 0.001) than CON ewes. Compared with CON ewes, follicles from IMM ewes had much higher concentrations of activin A (approximately 6-fold; P < 0.001) and inhibin A (approximately 3-fold; P < 0.001) but only slightly more follistatin (approximately 1.4-fold; not significant). The activin A:follistatin ratio in follicles from IMM ewes (approximately 1:1) was significantly higher (P < 0.001) than in follicles from CON ewes (approximately 0.3:1). Levels of inhibin antibody measured in follicular fluid (FF) from IMM ewes were similar to plasma levels. Given that activin A has been shown previously to up-regulate FSH receptors and aromatase activity in rat granulosa cells, the increase in intrafollicular activin A, unaccompanied by a rise in the concentration of its binding protein (follistatin), could explain how long-term IMM enhances follicle development and ovulation rate without necessarily promoting a sustained increase in FSH secretion.

Activins↗
PubMed1997

Activin A and inhibin A as possible endocrine markers for pre-eclampsia.

BACKGROUND: Inhibin A and activin A are produced by the placenta during human pregnancy. This study aimed to measure circulating concentrations of inhibin A, pro alpha C-containing inhibins, and activin A in the serum of women with pre-eclampsia and of healthy matched control pregnant women, and to establish the molecular-weight forms of circulating inhibin A and activin A in pre-eclampsia. METHODS: In a retrospective cross-sectional study, blood samples were taken from 20 women in hospital with established pre-eclampsia, and from 20 control pregnant women attending antenatal clinics, who were matched for duration of gestation (pre-eclampsia mean 29.15 [SD 3.75] weeks; controls 29.30 [3.93] weeks), parity, and maternal age. Serum samples were analysed for inhibin A, inhibin B, pro alpha C, and activin A. Pooled samples of control (n = 3) and pre-eclampsia serum (n = 3) subsequently underwent fast protein liquid chromatographic analysis to assess the molecular-weight forms of inhibin A and activin A. Results are expressed as mean and SD for all variables measured. FINDINGS: Serum concentrations of inhibin A, activin A, and pro alpha C were significantly higher in pre-eclampsia than in control normal pregnancy (inhibin A 3.05 [1.8] vs 0.36 [0.14] ng/mL, p < 0.001; activin A 38.08 [25.88] vs 3.95 [2.32] ng/mL, p < 0.001; pro alpha C-containing inhibins 2.2 [0.81] vs 0.71 [0.33] ng/mL, p < 0.001). Inhibin B concentrations in maternal serum were not increased. Molecular-weight forms of inhibin A (32 kDa) and activin A (> 100 kDa) were similar in pre-eclampsia and normal pregnancy. The mean concentrations of hCG were 59.05 [43.98] and 16.3 [8.72] ng/mL, respectively. INTERPRETATION: Higher maternal serum concentrations of inhibin A, pro alpha C, and total activin A in pre-eclampsia than in control pregnancies could be helpful in the diagnosis of pre-eclampsia. These changes are interpreted as further evidence for trophoblast dysfunction in pre-eclampsia.

Activins↗
PubMed1997

Decline in serum follicle-stimulating hormone concentrations alters key intrafollicular growth factors involved in selection of the dominant follicle in heifers.

Declining FSH after a transient rise coincides with selection of a dominant follicle (DF) and atresia of the remaining cohort follicles (subordinates) in cattle. The objectives of this study were to determine 1) whether intrafollicular amounts of inhibins, activin-A, insulin-like growth factor I (IGF-I), and IGF-I-binding proteins (IGFBP) are altered during selection of the first-wave dominant follicle (DF1) and 2) whether these biochemical markers are FSH dependent. Beef heifers received six or eight 6-h injections of saline (controls) or eight 6-h injections of recombinant bovine FSH (1 mg/injection) at 38 to 42 h after estrus (Day 0). Daily ultrasound scanning was used to define selection of DF1. Controls (n = 6 per group) were ovariectomized 1) on Day 3 of the estrous cycle before DF1 selection (preselection follicles) and 2) after DF1 selection on Day 4.8 +/- 0.5. In controls, FSH declined between Days 2 and 3 and selection of DF1 occurred between Days 3 and 5. During this interval, intrafollicular estradiol concentrations increased > 5-fold in DF1, yet declined 4-fold in subordinates (p < 0.05). In DF1, total IGF-I increased 1.3-fold (p < 0.05), whereas the amounts of the 40- to 47-kDa and the 35-kDa IGFBP (ligand hybridization) decreased 2.4- and 2.5-fold, respectively (p < 0.05), compared to values in preselection follicles on Day 3; total dimeric inhibin-A decreased 1.8-fold (p < 0.05). In contrast, amounts of the 30- to 32-kDa IGFBP increased 12.4-fold (p < 0.05) in subordinates on Day 4.8 compared with preselection follicles on Day 3, while the amount of inhibins > 34 kDa decreased 4- to 9-fold (p < 0.05). In FSH-treated heifers, both selection of DF1 and atresia of subordinates were delayed by 2.2 days. Preselection follicles recovered on Day 4.9 +/- 0.1 from FSH-treated heifers were similar (p > 0.05) in almost all biochemical parameters to preselection follicles from control heifers; however, they differed markedly from both DF1 and subordinate follicles recovered from control heifers on Day 4.8 +/- 0.5. In conclusion, the decline in FSH beginning after Day 2 of the heifer estrous cycle causes differential alterations in FSH-dependent growth factors and hormones within the cohort of preselection follicles, simultaneously inducing growth and enhanced estradiol-producing capacity of the DF and atresia of subordinate follicles.

Activins↗
PubMed1997

Effects of supplementary treatment with bovine growth hormone on hormonal and ovulatory responses to inhibin immunization in ewes.

The aim of this study was to determine whether supplementary treatment with recombinant bovine growth hormone(rbGH) can enhance the ovulatory response of ewes to inhibin immunization. Crossbred ewes (n = 20) were actively immunized against bovine inhibin a1-29 peptide conjugate while 20 ewes served as controls. Oestrus was synchronized using progestagen sponges and ewes were allocated to four groups: control ewes (n = 10); control ewes given rbGH (n = 10); inhibin-immunized ewes (n = 10) and inhibin-immunized ewes given rbGH (n = 10). A single s.c. dose of rbGH (50 mg) was given 7 days before sponge removal. Blood was collected for measurement of inhibin antibody titre, and concentrations of insulin-like growth factor I (IGF-I), FSH, oestradiol and progesterone. Ovulation, pregnancy and lambing rates were also recorded. All inhibin-immunized ewes produced antibodies that bound 125I-labelled (32 kDa) inhibin. The concentration of FSH in the plasma of the ewes after the second booster inhibin immunization was higher than that in control ewes (P < 0.005). Treatment with rbGH promoted a 2-3-fold increase in plasma concentration of IGF-I (P < 0.001); the response was less (P < 0.01) in immunized compared with control ewes. Treatment with rbGH alone had no significant effect on the concentration of FSH or oestradiol or on ovulation rate or litter size. Overall, inhibin-immunized ewes had higher mean FSH concentrations (P < 0.002), higher preovulatory oestradiol surges (P < 0.05) and higher progesterone concentrations in the luteal phase (P < 0.0001). Treatment with rbGH reduced the effects of immunization on FSH (P < 0.01) and progesterone (P < 0.02) concentrations. Immunized ewes showed a threefold increase in ovulation rate (P < 0.001) and a 1.8-fold increase in litter size (P < 0.05) compared with control ewes. In immunized ewes given rbGH, ovulation rate was increased by a factor of 2.2 and litter size by a factor of 1.8. In conclusion, these data do not support the hypothesis that supplementary treatment of ewes with rbGH to raise plasma IGF-I concentrations (and presumably intraovarian IGF-I) can enhance the ovulatory response to inhibin immunization.

Animals↗
PubMed1997

Development, validation and application of a two-site enzyme-linked immunosorbent assay for activin-AB.

Monoclonal antibodies, specific for the beta A and beta B subunits of activin, were used to develop a new two-site ELISA for activin-AB. The assay had a detection limit of 0.19 ng/ml. High concentrations of activin-AB were found in bovine, ovine and porcine follicular fluids (FF), with less in human FF (1310, 1730, 688 and 7 ng/ml respectively). Recovery of spiked activin-AB standard from human, bovine and ovine FFs and from homogenized human placental extracts averaged 91%, 115%, 115% and 94% respectively. Within-plate coefficients of variation for different concentration of activin-AB were between 1.3% and 2.67%. The between-plate coefficient of variation was 5.5%. Cross-reactivity experiments showed the high specificity of the assay for activin-AB, with inhibin-A, inhibin-B, follistatin, activin-A and activin-B all cross-reacting < 0.2%. Incubation with high concentrations of follistatin (500 ng/ml) prior to assay did not affect the recovery of activin-AB. Samples of bovine, porcine, ovine and human FF gave dose responses parallel to that of the standard, as did bovine granulosa cell-conditioned media. In human and porcine FF, levels of activin-A and activin-AB were similar whereas, in bovine and ovine FF, activin-A levels were approximately threefold higher than activin-A, nearly all of the endogenous activin-AB in bovine FF was detected in the eluate from gel permeation chromatography with an M(r) of > 700000 indicating its association with higher molecular weight binding protein(s). By contrast, after denaturation, immunoreactive activin-AB was detected with an M(r) of approximately 25000 consistent with the complete dissociation from binding proteins. Activin-A was detected in relatively high concentrations in human FF (approximately 5 ng/ml), homogenized placental extracts (4.35-95.5 ng/g), sera from pregnant women (> 4 ng/ml) and amniotic fluid (3-13 ng/ml), and in much lower concentrations in postmenopausal serum (500 pg/ ml), normal cycle serum (100-200 pg/ml), serum from gonadotrophin-treated women (200 pg/ml), and normal adult male serum (225 pg/ml). Activin-A was also found in the culture media from explants of human amnion, chorion, maternal decidua and placenta. In marked contrast, activin-AB was undetectable (< 0.19 ng/ml) in all of these samples with the exception of human FF (approximately 7 ng/ml). In conclusion, we have developed a sensitive and specific ELISA to measure total (bound+free) activin-AB. Preliminary results show a more restricted distribution of this isoform compared with activin-A. The presence of high levels of both activin-A and activin-AB in FF suggests a function for both isoforms in the developing ovarian follicle.

Activins↗
PubMed1996

Prenatal screening for Down's syndrome using inhibin-A as a serum marker.

The value of measuring inhibin-A (a beta A dimer) with human chorionic gonadotrophin (total or the sub-units free a-hCG and free beta-hCG separately), alpha-fetoprotein (AFP), and unconjugated oestriol (uE3) was examined to determine the effect on the performance of serum screening for Down's syndrome between 15 and 22 weeks of pregnancy. The study was based on stored serum samples from 77 Down's syndrome singleton pregnancies and 385 unaffected singleton pregnancies, matched for maternal age, gestational age, and duration of storage of the sample, supplemented by data from 970 white women with unaffected pregnancies. Inhibin-A was elevated in the serum of women with Down's syndrome pregnancies with a median of 1.79 multiples of the median (MOM). Using the four serum markers AFP, uE3, total hCG, and inhibin-A, in addition to maternal age, 70 per cent of Down's syndrome pregnancies were detected for a 5 per cent false-positive rate compared with 59 per cent with the conventional triple test (AFP, uE3, and total hCG with maternal age). If the estimate of gestational age were based on an ultrasound scan examination, the detection rate would be 77 per cent [95 per cent confidence interval (CI) 69-85 per cent] using the four serum markers including inhibin-A, compared with 67 per cent with the triple test or 79 per cent (95 per cent CI 71-87 per cent) if marker values were adjusted for maternal weight. If the detection rate were kept at 70 per cent and the gestational age were estimated by an ultrasound scan examination, the four-marker test would reduce the false-positive rate from 6-1 per cent using the triple test to 2-9 per cent. The results were virtually the same if free beta-hCG was used instead of total hCG. The inhibin-A-based four-marker test is the most effective method of prenatal screening for Down's syndrome suitable for routine use. If the extra cost required to carry out the inhibin-A test were less than about [symbol: see text]3 per woman screened, the four-marker test including inhibin-A would be financially cost-effective.

Biomarkers↗
PubMed1996

Roles of inhibins, activins, and follistatin in the female reproductive system.

Some 10 years have elapsed since inhibins were first isolated from ovarian follicular fluid and characterized as disulphide-linked dimeric glycoproteins capable of selectively suppressing the synthesis and secretion of follicle-stimulating hormone (FSH) by pituitary gonadotropes. There have been numerous surprises subsequent to this break-through, including the discovery and molecular characterization of activins and follistatins, proteins which share with inhibin an ability to modulate pituitary FSH secretion. It has also emerged that (i) inhibins and activins are members of the diverse but structurally related transforming growth factor beta (TGF beta) peptide family; (ii) follistatin, although structurally unrelated to the TGF beta family, modulates activin bioactivity by acting as a specific high-affinity binding protein; (iii) inhibins, activins, and follistatin function as intragonadal autocrine/paracrine regulators of follicle cell differentiation and steroidogenesis; (iv) inhibins, activins, and follistatin are expressed and subserve local regulatory roles in numerous extragonadal tissues, including brain, adrenal, bone marrow, and placenta but perhaps most notably in anterior pituitary--the classical target tissue for inhibin; (v) the activin-follistatin system may play a key role in early embryogenesis. This article focuses on these developments of the past decade with particular reference to the distribution and functional roles of inhibins, activin, and follistatin in tissues related to the female reproductive system, including hypothalamus, anterior pituitary, ovary, and placenta.

Activins↗
PubMed1996

Circulating inhibins and activin A during GnRH-analogue down-regulation and ovarian hyperstimulation with recombinant FSH for in-vitro fertilization-embryo transfer.

OBJECTIVE: We have investigated serial changes in plasma concentrations of inhibin A, inhibin B, pro alpha C and activin A in women undergoing stimulation with recombinant FSH in 'long-protocol' down-regulated cycles of IVF treatment. DESIGN: Blood samples were collected during the entire IVF treatment cycle at points coinciding with the early follicular phase of the cycle preceding treatment, pituitary down-regulation, stimulation with recombinant FSH, ovulatory triggering, and the luteal phase of the cycle. In patients who achieved conception, blood samples were also taken during the first 2 weeks of pregnancy. All samples were analysed for inhibin A, inhibin B, pro alpha C, activin A and oestradiol. PATIENTS: Fifteen women with normal ovarian function undergoing IVF treatment with tubal factor, mild endometriosis or idiopathic infertility. RESULTS: During pituitary desensitization, both inhibin A and inhibin B were significantly (P < 0.001, P = 0.002, respectively) reduced whereas levels of pro alpha C and activin A were largely unaltered. Levels of both inhibins rose markedly (P < 0.01) during FSH stimulation and a further rise in inhibin A was detected on the day after ovulatory trigger. Levels of both inhibin A and inhibin B then fell during and after oocyte pickup and continued to fall during the luteal phase. Activin A levels rose less markedly during gonadotrophin stimulation. Statistical analysis showed a high degree of correlation between the number of follicles (> 10 mm) and serum inhibin A (r = 0.65, P < 0.01) and pro alpha C (r = 0.65, P < 0.01) concentrations during the late follicular phase. CONCLUSIONS: These results indicate that ovarian production of dimeric inhibin A and B are gonadotrophin dependent, whereas activin A may have a significant gonadotrophin independent or extra-gonadal source. Inhibin A and pro alpha C may be useful markers for monitoring the effects of gonadotrophin stimulation.

Activins↗
PubMed1996

Effect of mifepristone (RU486) on the pituitary response to gonadotrophin releasing hormone in women.

Mifepristone interrupts folliculogenesis in women but the mechanism is not clear. Previous studies have investigated the effect of this compound on gonadotrophin secretion and have provided conflicting results. To study further the effect of mifepristone on basal and gonadotrophin-releasing hormone (GnRH)-induced gonadotrophin secretion, 12 normally ovulating women were investigated during two consecutive menstrual cycles, comprising an untreated cycle (control) and a cycle treated with mifepristone. All women were treated with mifepristone on days 2-8 at the dose of 100 mg (group 1, eight women) or 10 mg per day (group 2, six women). Two women were treated with both regimens in two different cycles. On day 8 of both cycles, the women received two GnRH pulses of 10 micrograms each 2 h apart. Blood samples in relation to the first GnRH pulse were taken at-15, 0, 30, 60, 120, 150, 180 and 240 min. In group 1, the increase in luteinizing hormone (delta LH) in response to GnRH was significantly attenuated from 30 to 180 min, while the increase in follicle stimulating hormone (delta FSH) was attenuated only in response to the second GnRH pulse. No significant decrease in delta LH and delta FSH response to GnRH was seen during treatment with the 10 mg dose (group 2). In group 1, serum oestradiol and inhibin-A concentrations after day 8 were lower than in the control cycles and the LH peak was postponed by 7 days on average. Basal LH values increased significantly on day 8 in both groups, while FSH values did not change significantly compared with the control cycles. A significant increase in serum progesterone and cortisol values occurred during the treatment only in group 1. Mid-luteal values of inhibin-A were significantly lower in cycles treated with 100 mg mifepristone than in the control cycles. We conclude that the disruption of folliculogenesis by mifepristone cannot be explained by a decrease in basal FSH concentrations during the critical period of follicle recruitment and selection. It is possible that mifepristone exerts its effect at the level of the ovary. It is also suggested that progesterone during the follicular phase of the cycle may participate in the control of the self-priming action of GnRH on the pituitary.

Estradiol↗
PubMed1996

Alterations in intrafollicular levels of different molecular mass forms of inhibin during development of follicular- and luteal-phase dominant follicles in heifers.

The experiment reported here characterized changes in serum concentrations of FSH, follicular fluid (FF) concentrations of immunoreactive alpha inhibins and dimeric inhibins, and different molecular forms of inhibin during development of dominant follicles in both luteal and follicular phases of the estrous cycle in heifers. Follicular status was determined daily by ultrasound examination of ovaries, and blood samples were taken at 4-6-h intervals. Heifers (5-6 per treatment) were ovariectomized on Days 0 (follicular phase, pre-LH surge), 1-Ov (post-LH surge, preovulation), 1 + Ov (postovulation), 3 (selection phase), 6 (dominance phase), and 12 (atretic phase) of the estrous cycle. Follicles were classified on the basis of FF estradiol (E) to progesterone (P) ratio and by ultrasound morphology. Concentrations of E, P, immunoreactive alpha inhibin, LH, and FSH were determined by RIA, and concentrations of dimeric inhibins were determined by a two-site dimeric-specific immunoradiometric assay (IRMA); the different molecular mass forms of inhibin were determined by immunoblot analysis. Serum FSH was transiently elevated during Days 0.5-1.5 and 8-10.5, in association with emergence of each new wave of follicular growth. The follicular-phase dominant follicle of heifers before the preovulatory LH/FSH surge had less immunoreactive alpha inhibin and 29-kDa inhibin but more 110-kDa inhibin compared with concentrations after the surge (p < 0.05). During growth of the dominant follicle between Days 3 and 6, there were no changes in either proportions or amounts of any inhibin forms (p > 0.05). However, while concentrations of dimeric inhibins decreased between Days 1 and 6, the concentration of immunoreactive alpha inhibin increased (p < 0.05). Atresia of the luteal-phase dominant follicle during Days 6-12 was associated with a decrease in amounts and proportions of the 110- and > 160-kDa forms, an increase in amounts and proportions of the 34-kDa form, and an increase in dimeric inhibin concentration (p < 0.05). Alterations in FF E concentrations were positively correlated with changes in amounts of the 77- and 110-kDa precursor forms of inhibin, but negatively correlated with 34-kDa inhibin. The results of this experiment demonstrate that 1) seven different molecular mass forms of inhibin were present in bovine FF during all stages of development of follicular- and luteal-phase dominant follicles examined, 2) amounts and proportions of most inhibin forms are altered differently in follicular- and luteal-phase dominant follicles, 3) FF inhibins and E are regulated by different factors, and 4) RIA, IRMA, and immunoblot measurements of inhibin in bovine FF produced divergent results.

Animals↗
PubMed1996

Changes in peripheral serum levels of total activin A during the human menstrual cycle and pregnancy.

The main objective of this study was to determine whether activin A concentrations in peripheral blood fluctuate during the normal human menstrual cycle and pregnancy. Blood samples were collected longitudinally from five regularly cycling volunteers (22-30 yr) throughout a spontaneous menstrual cycle and cross-sectionally from normal pregnant women attending the antenatal clinic (8-38 weeks gestation: 3-20 subjects/time point). Total (i.e. bound plus free) activin A concentrations were measured using a recently developed two-site enzyme immunoassay that employs an analyte denaturation/oxidation step to eliminate interference due to endogenous activin-binding proteins. During the menstrual cycle, mean serum activin A levels varied in a biphasic manner (by ANOVA, P = 0.02), with highest levels around midcycle (approximately 220 pg/mL) and the late luteal/early follicular phase (approximately 310 pg/mL) and nadirs in both midfollicular (approximately 125 pg/mL) and midluteal (approximately 120 pg/mL) phases. Between the mid- to late luteal phase, the activin A level increased progressively (approximately 2.5-fold; P < 0.05), whereas inhibin A, estradiol, and progesterone all decreased progressively (approximately 10-fold; P < 0.001). During pregnancy, serum activin A levels were much higher than those in nonpregnant subjects, with a value of 2.12 +/- 0.31 ng/mL recorded in week 8. Levels remained at approximately 2 ng/mL between weeks 8-24, but increased thereafter to reach 25.5 +/- 6 ng/mL by week 38, a value approximately 100 times greater than that during the normal menstrual cycle. Serum activin A levels during pregnancy were significantly correlated with inhibin A (r = 0.69; P < 0.001), estradiol (r = 0.55; P < 0.001), and progesterone (r = 0.74; P < 0.001) values. Gel permeation chromatography indicated that all of the detectable activin A in human follicular fluid, pregnancy serum, and term placental extract eluted with an apparent molecular mass between 70-200 kDa, indicating that little, if any, free activin (molecular mass, 25 kDa) is present in these samples. Although these results support a possible endocrine role for circulating activin A during the human menstrual cycle and pregnancy, the observation that all detectable activin A is associated with binding protein(s) raises questions about its relative bioavailability for action on peripheral target cells.

Activins↗
PubMed1996

Development and application of a two-site enzyme immunoassay for the determination of 'total' activin-A concentrations in serum and follicular fluid.

The performance of existing immunoassays and bioassays for activins is compromised by the presence of activin-binding proteins such as follistatin and alpha 2 macroglobulin (alpha 2M) in biological fluids. To overcome this problem we have developed a novel two-site enzyme immunoassay procedure for activin-A which incorporates an analyte denaturation and oxidation step. The optimized assay is sensitive (detection limit approximately 10 pg/well), precise (mean within- and between-plate coefficients of variation 4.9 and 9.1% respectively) and accurate (activin-A recovery values of 102 +/- 3 and 96 +/- 5% for bovine follicular fluid (FF) and human serum respectively). In specificity tests, high concentrations of follistatin (500 ng/ml) and alpha 2M (100 microgram/ml) did not interfere with the response signal to activin-A. In addition, no significant cross-reactivity was observed with a range of related molecules including inhibin-A, inhibin-B, activin-B (all < 0.5%), bovine pro-alpha C and follistatin (both < 0.1%). Response curves parallel to the activin-A standard curve were obtained for a variety of test samples including bovine, human, ovine and porcine FF, human sera and conditioned medium from cultured bovine and human granulosa cells. Fractionation of bovine FF by SDS-PAGE confirmed assay specificity since only one peak of activin-A immunoreactivity was detected (M(r) approximately 25 k) in eluted gel slices. However, gel-permeation chromatography showed that under physiological conditions all of the detectable activin-A in bovine FF eluted with apparent M(r) values of > 700 and 60-200 k reflecting its association with binding protein(s). Analysis of bovine FF samples (n = 76) from morphologically dominant follicles during the luteal phase showed that activin-A levels were positively correlated with inhibin-A (r = +0.54; P < 0.001) and total beta subunit immunoreactivity (r = +0.32; P < 0.005) but not with total alpha subunit immunoreactivity (r = -0.09). Classification of these follicles according to oestrogenic status showed that activin-A, inhibin-A and total beta subunit levels were highest in oestrogen-inactive follicles (P < 0.01) whereas total alpha subunit levels were lowest in these follicles (P < 0.001). Activin-A levels were measurable in all human serum samples analysed, ranging from 128 pg/ml during the normal menstrual cycle, 210 pg/ml in women undergoing ovarian hyperstimulation and approximately 500 pg/ml in postmenopausal women to over 4000 pg/ml during pregnancy. In conclusion, the present assay provides a reliable method for quantitating total (i.e. bound+free) activin-A concentrations in a variety of biological samples and should prove useful for further in vivo and in vitro studies in a range of species including man.

Activins↗