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Biomedical subjects

P G Knight

Publications and source records attributed to P G Knight.

At least 73 records · Page 4Linked to original sources
PubMed1994

Active immunization of heifers against inhibin: effects on plasma concentrations of gonadotrophins, steroids and ovarian follicular dynamics during prostaglandin-synchronized cycles.

We reported previously that active immunization of heifers using a synthetic peptide-based inhibin vaccine (bI alpha(1-29)Tyr30) can enhance ovarian follicular development and ovulation rate during spontaneous oestrous cycles. To extend this study, we investigated the effect of inhibin immunization more closely by monitoring plasma hormone profiles and ovarian activity in bI alpha(1-29)Tyr30-immunized and control (ovalbumin-immunized) heifers (n = 6 per group) over three consecutive oestrous cycles, which were synchronized and shortened by administering a PGF2 alpha analogue at intervals of 14 days. Blood samples were collected at 2-8 h intervals for 40 days and the ovaries were examined daily using ultrasonography. Repeated-measures ANOVA showed that inhibin immunization significantly increased plasma FSH concentration (by 52% overall; P < 0.01) and ovulation rate (by 58%; P < 0.01). Both immunized and control heifers showed the same cyclic pattern of plasma FSH (treatment x time interaction; not significant), indicating that the increase in plasma FSH was sustained throughout the cycle. Immunization did not affect the concentration or pattern of secretion of LH, oestradiol or progesterone and had no influence on the timing of the LH surge or ovulation after PG injection. While inhibin immunization increased the number of 'large' (i.e. growing to > or = 10 mm diameter) follicles that developed during both the preovulatory (by 90%, P < 0.02) and postovulatory (by 190%, P < 0.01) period, there was no difference between groups in the temporal pattern of growth or regression of large follicles or of corpora lutea.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Effect of chronic exposure to a gonadotrophin-releasing hormone agonist on in vitro responsiveness of ovine pituitary cells to inhibin, activin and oestradiol.

To investigate the extent to which the direct actions of inhibin, activin and oestradiol on pituitary output of FSH and LH are dependent on the presence of functional gonadotrophin-releasing hormone (GnRH) receptors, we have compared the effects of these agents on cultured ovine pituitary cells derived from control and GnRH agonist-suppressed ewes. Chronic treatment with GnRH agonist reduced plasma LH and FSH levels (P < 0.01) and abolished GnRH-induced release of LH and FSH both in vivo and in vitro. As expected, basal LH release and LH cell content in vitro were drastically reduced in GnRH agonist-suppressed cells (P < 0.001). However, basal FSH release and FSH cell content were approximately twofold higher than in control cells (P < 0.001). Irrespective of whether the cells had been desensitized to GnRH, inhibin and oestradiol were both found to suppress basal FSH release and FSH cell content in a dose-dependent fashion (P < 0.001). Although inhibin had no effect on basal release of LH from control cells, it markedly enhanced GnRH-induced release (P < 0.001). In contrast, inhibin increased (P < 0.001) basal LH release from GnRH agonist-suppressed cells (which were unresponsive to the GnRH challenge). Inhibin had no overall effect on total LH content/well for either control or GnRH agonist-suppressed cells. Treatment with oestradiol, on the other hand, reduced total LH content/well, an effect which was more pronounced with GnRH agonist-suppressed cells (-44%; P < 0.001) than with control cells (-14%, P < 0.01). Whereas in control cells activin had no significant effect on any aspect of FSH production examined, in GnRH agonist-treated cells activin enhanced basal FSH release, residual cell content and total FSH content/well (P < 0.001). Altering GnRH receptor status also modified the LH response to activin. With control cells activin increased basal release (P < 0.001), decreased GnRH-induced release (P < 0.001) and increased total LH content/well (P < 0.001). With GnRH agonist-treated cells, however, activin had a uniform inhibitory effect on each aspect of LH production examined (P < 0.001 in each case). It was concluded that desensitization of ovine gonadotrophs to GnRH by chronic agonist treatment results in a paradoxical enhancement of FSH output in vitro but has little effect on the responsiveness of the cells (in terms of gonadotrophin release and content) to either inhibin or oestradiol. In contrast, GnRH agonist treatment leads to qualitative changes in cellular responsiveness to activin.

Activins↗
PubMed1994

Measurement of dimeric inhibin using a modified two-site immunoradiometric assay specific for oxidized (Met O) inhibin.

Several years ago we developed a novel two-site immunoradiometric assay (IRMA) for dimeric inhibin. However, relative to the purified 32 kDa bovine inhibin standard used at that time, the immunopotencies of crude inhibin-containing samples were much less than their biopotencies estimated by pituitary cell bioassay. In attempting to improve assay performance and resolve this discrepancy we recently discovered that introduction of a preassay oxidation step to the IRMA results in a dramatic increase in the immunopotencies of inhibin-containing test samples (e.g.: bovine, human, porcine follicular fluid (FF)) and of a new (purified in 1993) 32 kDa bovine inhibin standard. However, the oxidation step did not affect the immunopotency of our original standard (purified in 1987), indicating that this material had undergone spontaneous oxidation during long-term storage, thus accounting for its higher immunopotency in our original IRMA and providing an explanation for the discrepancy between immunoactivity and bioactivity referred to above. These findings, together with other observations on the behaviour of oxidized and non-oxidized samples of inhibin, related peptide fragments and inhibin-containing samples in the IRMA and alpha subunit radioimmunoassay (RIA), indicate that the anti-beta A82-114 monoclonal antibody (E4) used as tracer in the IRMA binds selectively to the oxidized (Met O89,91,108) form of the peptide. This property of the antibody can be exploited to advantage by incorporating simple modifications to existing inhibin/activin immunoassays to ensure that all samples and standards are fully oxidized before antibody addition.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Higher gonadotrophin surge-attenuating factor bioactivity is found in small follicles from superovulated women.

Ovine and rat pituitary bioassays for gonadotrophin surge-attenuating factor (GnSAF) were utilized to determine whether the level of GnSAF bioactivity in pooled human follicular fluid (hFF) from superovulated women varied according to follicle diameter (< or = 11 mm, 12-15 mm and 16-21 mm follicles examined using the ovine bioassay, or < or = 10 mm, 11-13 mm, 14-17 mm, 18-20 mm, 21-24 mm and > or = 25 mm follicles examined using the rat bioassay). When tested using dispersed ovine pituitary cells, GnSAF bioactivity, expressed in terms of the reduction in gonadotrophin-releasing hormone (GnRH)-induced LH secretion, was inversely related to follicle diameter (P < 0.01). In response to 5 microliters hFF/well from follicles of < or = 11, 12-15 and 16-21 mm diameter, GnRH-induced LH secretion was reduced to 40.5 +/- 6.9%, 65.2 +/- 6.6% and 83.7 +/- 7.9% of control respectively. A similar inverse relationship was observed when a second batch of hFF samples from different sized follicles was tested using rat pituitary cell monolayers. Expressing GnSAF bioactivity in terms of the dose required to suppress GnRH-induced LH secretion by rat pituitary cells to 50% of the maximal suppression observed (ED50), the three smallest follicle size pools contained the most GnSAF (ED50 values of 0.13, 2.79 and 5.36 microliters hFF/well from follicles of < or = 10, 11-13 and 14-17 mm respectively). The ED50 values for follicles of 18-20, 21-24 and > or = 25 mm were 8.81, 27.1 and 60.0 microliters hFF/well respectively. Thus hFF from follicles < or = 11 mm was over 450 times more potent than hFF from follicles > or = 25 mm in suppressing GnRH-induced LH release. The ED50 values for inhibin bioactivity (measured as the suppression of basal FSH secretion from rat pituitary monolayers) were much less variable than those for GnSAF bioactivity (between 0.85 and 0.13 microliters hFF/well). Inhibin immunoreactivity, measured by a two-site immunoradiometric assay, followed the same pattern as inhibin bioactivity with lowest concentrations in the smallest follicles (41.96 ng/ml) and highest concentrations in the three largest follicle size groups (56.48-64.48 ng/ml). The specific effects of inhibin on GnRH-induced LH and basal FSH release in these pituitary bioassays were determined by incubating culture dishes with pure recombinant human inhibin at doses of 0.025-25 ng/well. In both the sheep and rat pituitary monolayers, basal FSH was suppressed (ED50 = 0.02 and 0.16 ng/well respectively).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Production of immunoactive inhibin by bovine granulosa cells in serum-free culture: effects of exogenous steroids and FSH.

Granulosa cells from pooled bovine follicles were cultured under chemically-defined (serum-free) conditions to study the effects of exogenous steroids and FSH on production of immunoactive (ia) inhibin, oestradiol and progesterone. Levels of ia-inhibin in media samples and cell lysates were measured by radioimmunoassay (RIA) using an antiserum raised against a synthetic fragment of human inhibin alpha-subunit [hI alpha (1-32)]. Cells secreted measurable amounts of ia-inhibin, oestradiol and progesterone for at least 7 d of culture, although intracellular levels of inhibin were very low, indicating that newly-synthesized ia-inhibin is rapidly released from the cells. Treatment with androstenedione (0.2 mumol/l) or testosterone (0.2 mumol/l) increased ia-inhibin secretion markedly; levels on Day 5 of culture were approximately 6-fold (P < 0.005) higher than control values. In contrast, treatment with the non-aromatizable androgen dihydrotestosterone (DHT; 0.2 mumol/l) resulted in only a one- to two-fold increase (P < 0.05) over control values (Day 5). Addition of exogenous oestradiol (8 nmol/l) markedly increased ia-inhibin secretion (8-9 fold on Day 5; P < 0.05) compared with basal levels, whereas progesterone had no effect. Secretion of oestradiol, undetectable in the absence of exogenous androgens, rose daily in the presence of either androstenedione or testosterone, levels rising approximately 6-fold and 9-fold respectively over a 4-d treatment period. Progesterone secretion increased approximately 2-fold over the culture period and was unaffected by any steroid treatment. Treatment with ovine FSH (10ng/ml) alone stimulated secretion of progesterone over basal levels (3-fold higher on Day 6; P < 0.005), but did not affect output of either ia-inhibin or oestradiol. However, exposure to FSH in the presence of androstenedione not only promoted a further 4-fold increase in progesterone output but also led to a dose-dependent suppression of both ia-inhibin (approximately 90% lower on Day 6; P < 0.001) and oestradiol (approximately 80% lower on Day 6; P < 0.001) secretion compared to cells treated with androstenedione alone. These observations indicate that the secretion of ia-inhibin by bovine granulosa cells in culture is positively regulated by oestradiol, implying an autocrine/paracrine role for this hormone in control of ovarian inhibin production. The ability of aromatizable androgens to stimulate secretion of inhibin, coupled with the inability of the non-aromatizable androgen DHT to elicit such an effect, suggests that inhibin output is largely unaffected by androgens prior to their conversion to oestradiol.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of Variance↗
PubMed1993

Relationships between the level of atresia and inhibin contents (alpha subunit and alpha-beta dimer) in morphologically dominant follicles during their growing and regressing phases of development in cattle.

The objectives were to determine the relationships between changes in the levels of histological and biochemical (estradiol [E2]:androstenedione [A], E2:A ratio) atresia and changes in inhibin contents of morphologically dominant follicles collected during the growing or the regressing phase of the first wave of follicular development in cycling crossbred beef heifers. Heifers were slaughtered either when the dominant follicle (> or = 9 mm; diameter of the antral cavity as assessed by ultrasonography) of the first wave was still growing (n = 7) or when the first dominant follicle (> or = 9 mm; n = 7) was regressing or was at the end of the plateau phase. Following ovary collection, the dominant follicle was dissected and level of histological atresia was determined on sections of follicular walls. Aliquots of follicular fluid from each of the dominant follicles were collected to measure concentrations of E2, A, and inhibin alpha subunit by RIA and to measure concentrations of dimeric (alpha-beta dimer) inhibin by a two-site immunoradiometric assay. Heifers were slaughtered on Days 5.4 +/- 0.5 (growing phase) and 11.8 +/- 0.5 (regressing phase) of the estrous cycle, and mean diameter of the dominant follicles was similar in both phases (9.9 +/- 0.4 vs. 10.8 +/- 0.4 mm; p > 0.09). All morphologically dominant follicles collected during the growing phase (7/7) were histologically healthy and estrogen-active (E2:A ratio > 1), while those collected during the regressing phase (7/7) were histologically atretic and estrogen-inactive (E2:A ratio < 1; chi 2 = 14.0, p < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1993

The ability of steroid-free bovine follicular fluid to suppress FSH secretion and delay ovulation persists in heifers actively immunized against inhibin.

Previous reports indicate that administration of steroid-free bovine follicular fluid (bFF) to intact heifers suppresses plasma FSH levels and delays the timing of ovulation. In addition, cessation of bFF treatment is associated with a rebound hypersecretion of FSH. To test the hypothesis that these effects of bFF are mediated by inhibin, we have compared the responses to bFF treatment in heifers actively immunized against the N-terminal sequence of inhibin alpha subunit (bI alpha(1-29)Tyr30-ovalbumin) with those in ovalbumin-immunized controls. Oestrous cycles were synchronized, and inhibin-immune (n = 10) and control (n = 10) heifers were subdivided into two groups which received either 5 ml bFF (n = 5) or 5 ml bovine serum (n = 5) every 4 h for a 60 h period starting 8 h before prostaglandin (PG)-induced luteolysis. Blood was withdrawn every 4 h for 10 days starting 30 h before luteolysis and ovaries were examined daily by ultrasonography. Overall, mean ovulation rate in bI alpha(1-29)Tyr30-immunized heifers was 44% higher (P < 0.02) than in controls. Inhibin antibody titres tested in bI alpha(1-29)Tyr30-immunized heifers before (19 +/- 3%), during (19 +/- 3%) and after (20 +/- 3%) bFF treatment did not differ. In the pretreatment period (i.e. mid-luteal phase), plasma FSH levels were 32% (P < 0.05) higher in inhibin-immunized than in control heifers. During administration of bFF to control heifers, plasma FSH was suppressed to a level 40% lower than in serum-treated heifers (P < 0.02). Unexpectedly, bFF suppressed FSH to a similar extent in inhibin-immunized heifers (37% lower than in the serum-treated group; P < 0.025). Similarly, a post-bFF rebound hypersecretion of FSH was observed in both control (P < 0.05) and inhibin-immunized (P < 0.05) heifers, although the FSH rebound lasted about 24 h longer in the latter group (P < 0.001). The timing of the preovulatory LH surge in control (86 +/- 7 h post-PG) and immunized (81 +/- 6 h post-PG) groups treated with serum was similar as was the timing of the preovulatory rise in plasma oestradiol and the subsequent rise in plasma progesterone. However, bFF treatment delayed (P < 0.001) the preovulatory surges of LH and oestradiol and the subsequent rise in plasma progesterone to a similar extent (> 4 days) in both control and inhibin-immunized groups.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Evidence that most of the radioimmunoassayable inhibin secreted by the corpus luteum of the common marmoset monkey is of a non-dimeric form.

Although recent data for several species of primate, including human and marmoset, indicate that the corpus luteum secretes high levels of radioimmunoassayable inhibin, the nature of the immunoreactive (ir) inhibin detected has not been established. In this study, plasma ir-inhibin levels during the ovarian cycle of the marmoset (n = 12 animals) were measured by alpha-subunit-directed inhibin RIA, and values were compared with those estimated by a recently developed two-site immunoradiometric assay (IRMA) specific for inhibin alpha-beta dimer. Consistent with earlier data, plasma levels of ir-inhibin measured by RIA (overall mean value 133 +/- 7 ng/ml; n = 171) reached values 4-fold higher (p less than 0.001) during the luteal phase (222 +/- 20 ng/ml) than during the follicular phase (58 +/- 8 ng/ml), being directly correlated with plasma progesterone levels (r = 0.65; p less than 0.001). In contrast, plasma ir-inhibin levels estimated by IRMA were substantially lower than those measured by RIA (overall mean value 9.62 +/- 1.08 ng/ml; n = 171) and did not vary significantly during the cycle. Administration of a luteolytic dose of cloprostenol during the late luteal phase/early pregnancy led to an abrupt fall in plasma concentrations of progesterone (95%) and alpha-inhibin measured by RIA (82%), whereas dimeric inhibin levels remained unchanged. Analysis of marmoset luteal extracts (n = 5) by RIA, IRMA, and inhibin bioassay yielded inhibin estimates of 102.6 +/- 21.0, 0.632 +/- 0.103, and less than 2.0 ng/mg, respectively, thus confirming that only a very small proportion of the inhibin produced was dimeric (i.e., bioactive) in nature.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Effects of active immunization against a synthetic peptide sequence of the inhibin alpha-subunit on plasma gonadotrophin concentrations, ovulation rate and lambing rate in ewes.

Thirty adult Mule (Blue-faced Leicester x Swaledale) ewes were actively immunized against a synthetically produced peptide corresponding to the N-terminus of the alpha-subunit of bovine inhibin conjugated to tuberculin purified protein derivative (PPD). Primary immunization in the late anoestrous period was followed by two booster injections at 5 week intervals. Control groups were either not immunized (n = 15) or received PPD only (n = 15). Ten days after the second booster, oestrus was synchronized using progestagen sponges and ovulation rate was assessed by laparoscopy on days 9-10 of the cycle. Blood samples were taken at the time of each immunization and immediately before laparoscopy. Ewes were mated with fertile rams in mid-November and the resulting conception, pregnancy and lambing rates monitored. All inhibin-immunized ewes generated antibodies that bound 125I-labelled native bovine inhibin (M(r) 32,000), and their plasma follicle-stimulating hormone (FSH) concentrations after the second booster were significantly higher than the preimmunization values (30%; P less than 0.001) and the corresponding value in the controls (25%; P less than 0.025). Inhibin immunization was associated with a 90% increase in ovulation rate (P less than 0.005) and had no adverse effect on conception rate (100%), pregnancy rate (100%) or length of gestation (146 days). However, only a 37% increase (P less than 0.05) in lambing rate was recorded for inhibin-immunized ewes, indicating a higher incidence of wastage of ova, or embryos, or both, in these ewes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Stimulation of immunoreactive inhibin production by preimplantation embryos during early pregnancy in the marmoset monkey (Callithrix jacchus).

The role of the embryo in promoting increased plasma concentrations of immunoreactive inhibin after conception in the marmoset monkey was determined by flushing embryos from the uterus between days 5 and 9 after ovulation (implantation commences on days 11-12). Blood samples were taken from each animal (three times a week) after ovulation until the end of the luteal phase. Plasma inhibin concentrations were measured using a radioimmunoassay based on antisera against a synthetic fragment of the alpha-subunit of human inhibin. When embryos were flushed on days 5 and 6 (n = 6) after ovulation inhibin concentrations did not exceed 250 ng ml-1 for the duration of the luteal phase. In contrast when embryos were flushed on days 7 (n = 4), 8 (n = 4) and 9 (n = 3) maximum concentrations of inhibin always exceeded 250 ng ml-1, reaching > 400 ng ml-1 when embryos were flushed on days 8 and 9. Inhibin concentrations remained high for the duration of the luteal phase, which varied in length between 20 and 32 days. Significantly (P < 0.01) higher mean plasma concentrations of immunoreactive inhibin were first recorded on days 7-8 after ovulation in animals that had embryos flushed on days 7, 8 and 9 compared with concentrations in animals that had embryos flushed on days 5 and 6. Inhibin could not be detected in the medium of embryos cultured for up to 2 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Effect of active immunization of heifers against inhibin on plasma FSH concentrations, ovarian follicular development and ovulation rate.

To study the effects of immunoneutralization of endogenous inhibin on gonadotrophin secretion and ovarian function, prepubertal heifers (n = 6) were actively immunized against a synthetic peptide replica of the N-terminal sequence of bovine inhibin alpha subunit (bI alpha(1-29)Tyr30) coupled to ovalbumin. In contrast to ovalbumin-immunized controls (n = 6), bI alpha(1-29)Tyr30-immunized heifers had detectable inhibin antibody titres (% binding to 125I-labelled bovine inhibin at 1:2000 dilution of plasma) of 17 +/- 3% (S.E.M.) at puberty, rising to 31 +/- 5% by the end of the study period 7 months later. Neither age (immunized: 295 +/- 8 days; controls: 300 +/- 5 days) nor body weight (immunized: 254 +/- 13 kg; controls 251 +/- 9 kg) at onset of puberty differed between groups. Although the difference did not reach statistical significance, mean plasma FSH concentrations recorded in inhibin-immunized heifers remained 35-40% higher than in controls throughout the 12-week period leading up to puberty (P = 0.14) and during nine successive oestrous cycles studied after puberty (P = 0.10). Plasma LH concentrations did not differ between groups at any time during the study. Inhibin immunization had no effect on oestrous cycle length (immunized: 19.8 +/- 0.5 days; controls: 19.9 +/- 0.5 days).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Expression of bovine inhibin beta subunit in Escherichia coli.

1. A DNA fragment encoding the beta subunit of bovine inhibin was amplified using the polymerase chain reaction and was cloned in plasmids pUC8 and pUR291. 2. Cultures of Escherichia coli TG2 harbouring pKDK37, a pUR291-derived recombinant plasmid, produced a novel protein with a molecular weight of 130,000 corresponding to a beta-galactosidase-inhibin beta fusion protein. 3. The fusion protein was purified from inclusion bodies by solubilization in 8 M urea followed by an ion-exchange and gel permeation chromatography. 4. Analysis by immunoblotting and competitive radioimmuno assay revealed that the fusion protein was recognized by a monoclonal antibody raised against a chemically synthesized peptide for amino acid residues from +82 to +114 of the beta subunit of the bovine inhibin thereby confirming its identity.

Activins↗
PubMed1991

Inverse effects of activin and inhibin on the synthesis and secretion of FSH and LH by ovine pituitary cells in vitro.

Little information is available on the effects of activin and inhibin on the synthesis and secretion of pituitary gonadotrophins in species other than the rat. In this in-vitro study, ovine pituitary cell cultures derived from immature sheep were used to investigate the effects of recombinant human activin-A and native Mr 32,000 bovine inhibin on basal and gonadotrophin-releasing hormone (GnRH)-induced release of FSH and LH. Residual cellular contents of FSH and LH were also determined, allowing total content/well to be calculated. Activin-A promoted a dose-dependent increase in basal (+72%; P less than 0.001) and GnRH-induced (+25%; P less than 0.001) release of FSH as well as in the residual cell content (+114%; P less than 0.001) and total FSH content/well (+67%; P less than 0.001). Conversely, inhibin significantly (P less than 0.001) suppressed each aspect of FSH production examined, confirming that in sheep, as in rats, activin and inhibin exert opposing effects on pituitary FSH production. In contrast to the rat, however, in which activin is reported to have no effect on LH secretion, exposure of sheep pituitary cells to activin-A promoted a dose-dependent suppression (-42%; P less than 0.001) of GnRH-induced LH release. This was associated with a corresponding increase (P less than 0.001) in residual cellular content of LH. Consistent with a previous report from this laboratory, inhibin had the opposite effect and significantly enhanced (+47%; P less than 0.001) GnRH-induced LH release. This was associated with a corresponding fall (P less than 0.01) in residual cellular content of LH.(ABSTRACT TRUNCATED AT 250 WORDS)

Activins↗
PubMed1991

Effects of bovine follicular fluid on the secretion of LH and FSH in inhibin-immunized seasonally anoestrous ewes.

It has been shown previously that treatment of seasonally anoestrous ewes with steroid-free bovine follicular fluid (FF), a crude inhibin-containing preparation, leads to a decrease in plasma FSH level which is accompanied by a marked increase in pulsatile LH secretion. Since FF contains several factors (e.g. activin, follistatin, unidentified components) other than inhibin, which might act to modify gonadotrophin secretion, it was of interest to establish whether these concurrent effects of FF on FSH and LH secretion persisted in ewes which had been actively immunized against a synthetic peptide replica of the alpha subunit of bovine inhibin. In June 1989 (anoestrous period) groups of inhibin-immune and control ewes (n = 5 per group) received 6-hourly s.c. injections of either bovine serum (2 ml) or one of two doses of FF (0.5 ml or 2 ml) for 3 days. Blood was withdrawn at 6-h intervals for 6 days beginning 24 h before the first injection. On the final day of treatment, additional blood samples were withdrawn at 15-min intervals for 8 h to monitor pulsatile LH secretion. Ewes were then challenged with exogenous gonadotrophin-releasing hormone (GnRH; 2 micrograms i.v. bolus) to assess pituitary responsiveness. In control ewes, FF promoted a dose-dependent suppression of basal (maximum suppression 65%; P less than 0.01) and post-GnRH (maximum suppression 72%; P less than 0.01) levels of FSH in plasma. This was accompanied by an increase (P less than 0.01) in LH pulse frequency from 1.40 +/- 0.24 (S.E.M.) to 3.20 +/- 0.37 pulses/8 h. In contrast, FF did not affect secretion of either FSH or LH in inhibin-immunized ewes.(ABSTRACT TRUNCATED AT 250 WORDS)

Anestrus↗
PubMed1991

Increased concentrations of immunoreactive inhibin during conception cycles in the marmoset monkey: suppression with an LHRH antagonist and cloprostenol.

Peripheral concentrations of immunoreactive (ir) inhibin have been measured during the ovarian cycle and early pregnancy in the marmoset monkey. Blood samples were taken (three per week) during conception (n = 6) and non-conception (n = 5) cycles. Ir-inhibin was measured by radioimmunoassay using an antiserum raised against a synthetic peptide fragment of the alpha subunit of human inhibin. Monomeric bovine alpha subunit and 32 kDa bovine inhibin were used as tracer and standard respectively. In all animals low concentrations of ir-inhibin were recorded during the follicular phase (40-60 micrograms/l) of the cycle. After ovulation, ir-inhibin concentrations increased but the peak concentrations attained differed between conception and non-conception cycles. In non-pregnant animals ir-inhibin concentrations reached a maximum of 242 +/- 16 micrograms/l on days 12/13 after ovulation. In pregnant animals ir-inhibin concentrations were significantly (P less than 0.05) higher (1.8-fold) than in non-pregnant animals on days 8/9 after ovulation, and reached a maximum value of 636 +/- 141 micrograms/l on days 20/21 after ovulation. Administration of an LHRH antagonist during the luteal phase on days 6-8 after ovulation resulted in a significant (P less than 0.05) decrease in progesterone and ir-inhibin concentrations within 4 and 8 h respectively. This was prevented by co-administration with human chorionic gonadotrophin. Administration of cloprostenol to pregnant animals between days 17 and 20 after ovulation halved the initial concentrations of both inhibin and progesterone within 1.5 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Development of a two-site immunoradiometric assay for dimeric inhibin using antibodies against chemically synthesized fragments of the alpha and beta subunit.

A two-site (liquid-phase) immunoradiometric assay (IRMA) for dimeric inhibin has been developed using antibodies raised against synthetic peptide sequences corresponding to the N-terminus (1-32) of the alpha subunit and the C-terminal region (82-114) of the beta A subunit of Mr approximately 30,000 human inhibin. Highly-purified Mr 32,000 bovine inhibin (standard) gave a dilution curve parallel to those for bovine follicular fluid (bFF), human (h)FF and rat ovary extract. Whilst the assay detected both Mr 56,000 and 32,000 inhibin forms in bFF, little reaction with higher Mr forms was evident. Cross-reaction of 'free' inhibin subunit (Mr 25,000 form) and recombinant human activin A in the IRMA were minimal (less than 0.1 and less than 2% respectively). Although the detection limit of the IRMA (approximately 50 pg/tube) was similar to that of several reported radioimmunoassays (RIA), the IRMA was unable to detect dimeric inhibin in jugular or utero-ovarian vein plasma of heifers. Similarly, when assayed by IRMA, bFF, hFF and rat ovary extract contained 8-58 times less inhibin than was indicated by RIA. These observations are consistent with earlier evidence that the ovary secretes a substantial excess of 'free' inhibin alpha subunit and that this material reaches the peripheral circulation. Surprisingly, however, the inhibin contents of bFF, hFF and rat ovary extract determined by in vitro bioassay were 8-23 times greater than the corresponding IRMA values, being similar to those derived by RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Attenuation by an opioid agonist of the oestradiol-induced LH surge in anoestrous ewes and its reversal by naloxone.

The effects of a potent opioid peptide agonist [D-ala2-Phe4, Met(0)ol5-enkephalin (FK 33-824) on the magnitude of the oestradiol-induced LH surge and on basal plasma LH concentrations were examined in intact and chronically-ovariectomized ewes during the late-anoestrous period. In intact ewes, treatment with FK 33-824 (0.5 mg i.v. every 3 hr) for a 24 hr period commencing at the time of oestradiol-17 beta administration (25 micrograms i.m. bolus) was associated with non-significant 65% reduction in the peak plasma LH level observed and a significant (P less than 0.05) 58% reduction in the total amount of LH released during the surge (calculated from the area under the curve). Concurrent treatment with the opioid antagonist naloxone (10 mg i.v. every 3 hr) partially reversed this suppressive effect on the magnitude of the LH-surge. In ovariectomized ewes no significant effects on the oestradiol-induced LH surge of either FK 38-824 alone or FK 33-824 in combination with naloxone were observed. Administration of FK 33-824 at a 6-fold higher dose rate (0.5 mg every 30 min) failed to modify basal plasma LH concentration in intact ewes. In ovariectomized ewes, however, a significant (P less than 0.05) 25% fall in basal plasma LH was observed, an effect which was completely reversed by combined treatment with naloxone (10 mg every 30 min). These results support the conclusion that endogenous opioid peptides may contribute to the neuroendocrine mechanism through which oestradiol promotes a preovulatory-like surge in the anoestrous ewe.

Anestrus↗