Biomedical subjects

A S McNeilly

Publications and source records attributed to A S McNeilly.

At least 19 recordsLinked to original sources

PubMed1992

Purification, partial characterization, and heterologous radioimmunoassay of growth hormone (cGH) in red deer.

Red deer growth hormone (cGH; 3.3 mg) was purified from an aqueous extract of seven pituitary glands (4.01 g wet weight) by preparative gel filtration on Sephadex G-100, gel filtration on Sephadex G-100 SF, and anion exchange chromatography on DEAE-Sepharose CL-6B. Purified cGH gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight under reducing conditions of 20,000 Da and gave a single peak on reverse-phase high-performance liquid chromatography. N-Terminal amino acid determination of 42 residues gave a sequence identical with those published for bovine and ovine GH. In a radioreceptor assay based on binding of iodinated recombinant bovine GH (rbGH) to liver microsomes prepared from a pregnant ewe, cGH was equipotent with an ovine GH (oGH) standard. In an oGH radioimmunoassay, cGH diluted in parallel with oGH and rbGH. Using this assay plasma GH concentrations were determined in adult nonpregnant red deer hinds over a 12-month period. There was a significant seasonality in plasma GH concentrations with concentrations consistently low between mid-May and mid-September. This is the period when voluntary food intake and liveweight gain are greatest. It is suggested that in the presence of low plasma GH concentrations nutrients may be diverted toward lipogenesis and hence promote fat deposition.

Amino Acid Sequence↗

PubMed1992

Purification, partial characterization, and radioimmunoassay of prolactin and growth hormone from the Bennett's wallaby.

Bennett's wallaby prolactin (wPRL) and growth hormone (wGH) were purified from an aqueous extract of pituitary glands. The extract from 202 glands (6.5 g wet wt) was processed by gel filtration on Sephadex G-100, gel filtration on Sephadex G-100 SF, and then anion-exchange chromatography on DEAE-Sepharose CL-6B. The yields of wPRL and wGH were 5.2 and 15.7 mg, respectively. Since recovery of wPRL from the anion exchange column was 10%, anion exchange was performed in the presence of 20% acetonitrile in a subsequent purification. Recovery from this column was markedly increased to 42%. The purified hormones each gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight under reducing conditions of 21,000 and 23,000 for GH and PRL, respectively. Each hormone was positively identified by its N-terminal amino acid sequence, which showed high sequence identity with the equivalent eutherian hormone. Semianalytical gel filtration of purified hormone was used to demonstrate that each hormone remained as a monomer in aqueous solution. Each purified hormone was tested in the heterologous PRL radioimmunoassay (RIA) which has been used in many earlier studies to measure marsupial PRL. Highly purified wPRL was less potent than ovine prolactin (5.3 compared with 1.5 ng/ml at 50% displacement) and the cross-reaction of wGH was < 0.01%. Antibodies were raised against wPRL and wGH and a homologous RIA was developed for each hormone. The sensitivity of the wPRL assay was 0.8 ng/ml which is similar to that of the heterologous PRL assay. Cross-reaction with a number of eutherian pituitary hormones or wGH was < 0.07%. The wGH assay detected 0.8 ng/ml which is similar to that of the heterologous PRL assay. Cross-reaction with a number of eutherian pituitary hormones or wGH was < 0.07%. The wGH assay detected 0.8 ng/ml, cross-reacted with GH from several eutherian species, and showed low cross-reaction with wPRL (< 0.5). In both the wPRL and wGH assays, pituitary homogenates from several species of marsupial diluted in parallel with the wallaby standard, suggesting that these assays will be of use in studies of a number of marsupial species.

Amino Acid Sequence↗

PubMed1992

The 24 h pattern of pulsatile luteinizing hormone, follicle stimulating hormone and prolactin release during the first 8 weeks of lactational amenorrhoea in breastfeeding women.

In women, breastfeeding results in a variable period of ovarian inactivity which is apparently related to suppression of the normal pulsatile release of luteinizing hormone (LH). However, pulse profiles had only been studied during the daytime. Since resumption of pulsatile LH secretion during puberty is initiated at night, the present study determined the pattern of pulsatile LH secretion in relation to that of follicle stimulating hormone (FSH) and prolactin, and suckling and ovarian activity at 4 and 8 weeks postpartum in 20 fully breastfeeding women with lactational amenorrhoea. Blood samples were withdrawn at 10 min intervals for 24 h from 0900 h to 0900 h at either 4 weeks (n = 9) or 8 weeks (n = 11) postpartum, while the mothers and babies continued their normal pattern of suckling activity. At 4 weeks postpartum, no LH pulses occurred over 24 h in six of the nine women while one (n = 1) or two (n = 2) LH pulses occurred in three of the nine women. In contrast, LH pulses were present in nine of the 11 women at 8 weeks postpartum. The pulse frequency varied considerably from two to eight pulses over the 24 h and there was no influence of the time of day or sleep on the time of the pulse release. Lactational amenorrhoea was maintained for at least 10 weeks afterwards and there was no relationship between the time of resumption of ovarian activity and the presence or absence of pulsatile LH secretion at 4 or 8 weeks postpartum.(ABSTRACT TRUNCATED AT 250 WORDS)

Amenorrhea↗

PubMed1992

Inhibitory effects of a luteinizing-hormone-releasing hormone agonist implant on ovine fetal gonadotrophin secretion and pituitary sensitivity to luteinizing-hormone-releasing hormone.

Sheep fetuses at day 70 of gestation (term = 145 days) were implanted subcutaneously with a biodegradable implant containing a luteinizing-hormone-releasing hormone (LHRH) agonist (buserelin) to investigate whether treatment with LHRH agonist would induce a state of desensitization of the fetal gonadotrophs and thus influence fetal gonadal development. Treatment with the LHRH agonist for 35-40 days caused a significant reduction in mean fetal plasma concentrations of LH and follicle-stimulating hormone (FSH) compared with control fetuses. LH pulses were evident in control fetuses but were completely abolished by buserelin treatment. Furthermore, the pituitary content of LH and FSH was significantly depleted in fetuses implanted with LHRH agonist. A bolus intravenous injection of 500 ng LHRH given to control fetuses caused a rapid and significant increase in plasma LH and FSH concentrations which was sustained for at least 60 min after injection. Pretreatment with buserelin completely abolished the LH and FSH responses to a bolus injection of LHRH. There were no differences between the sexes in fetal gonadotrophin concentrations or pituitary sensitivity to LHRH in control or agonist-treated fetuses. Furthermore, buserelin treatment for 35-40 days had no effect on the morphological appearance of the fetal gonads when compared with control fetuses, at least to day 110 of pregnancy. These results provide evidence for the induction of a state of desensitization of the LHRH receptors of the fetal pituitary gonadotrophs following long-term treatment with an LHRH agonist, but provide no evidence for a role for gonadotrophin secretion in gonadal development at this stage in fetal life.

Analysis of Variance↗

PubMed1992

Suppression of pulsatile luteinizing hormone secretion by gonadotrophin-releasing hormone antagonist does not affect episodic progesterone secretion or corpus luteum function in ewes.

Progesterone secretion has been observed to be episodic in the late luteal phase of the oestrous cycle of ewes and is apparently independent of luteinizing hormone (LH). This study investigated the effects of suppressing the pulsatile release of LH in the early or late luteal phase on the episodic secretion of progesterone. Six Scottish Blackface ewes were treated i.m. with 1 mg kg-1 body weight of a potent gonadotrophin-releasing hormone (GnRH) antagonist on either day 4 or day 11 of the luteal phase. Six ewes received saline at each time and acted as controls. Serial blood samples were collected at 10 or 15 min intervals between 0 and 8 h, 24 and 32 h, and 48 and 56 h after GnRH antagonist treatment and daily from oestrus (day 0) of the treatment cycle for 22 days. Oestrous behaviour was determined using a vasectomized ram present throughout the experiment. Progesterone secretion was episodic in both the early and late luteal phase with a frequency of between 1.6 and 3.2 pulses in 8 h. The GnRH antagonist abolished the pulsatile secretion and suppressed the basal concentrations of LH for at least 3 days after treatment. This suppression of LH, in either the early or late luteal phase, did not affect the episodic release of progesterone. Daily concentrations of progesterone in plasma showed a minimal reduction on days 11 to 14 after GnRH antagonist treatment on day 4, although this was significant (P < 0.05) only on days 11 and 13. There was no effect of treatment on day 11 on daily progesterone concentration, and the timing of luteolysis and the duration of corpus luteum function was unaffected by GnRH antagonist treatment on either day 4 or day 11. These results indicate that the episodic secretion of progesterone during the luteal phase of the oestrous cycle in ewes is independent of LH pulses and normal progesterone secretion by the corpus luteum can be maintained with minimal basal concentrations of LH.

Analysis of Variance↗

PubMed1992

Relationship between gonadotrophin subunit gene expression, gonadotrophin-releasing hormone receptor content and pituitary and plasma gonadotrophin concentrations during the rebound release of FSH after treatment of ewes with bovine follicular fluid during the luteal phase of the cycle.

The modulation of FSH secretion at the beginning and middle of the follicular phase of the cycle represents the key event in the growth and selection of the preovulatory follicle. However, the mechanisms that operate within the pituitary gland to control the increased release of FSH and its subsequent inhibition in vivo remain unclear. Treatment of ewes with bovine follicular fluid (bFF) during the luteal phase has been previously shown to suppress the plasma concentrations of FSH and, following cessation of treatment on day 11, a rebound release of FSH occurs on days 12 and 13. When luteal regression is induced on day 12, this hypersecretion of FSH results in an increase in follicle growth and ovulation rate. To investigate the mechanisms involved in the control of FSH secretion, ewes were treated with twice daily s.c. injections of 5 ml bFF on days 3-11 of the oestrous cycle and luteal regression was induced on day 12 with prostaglandin (PG). The treated ewes and their controls were then killed on day 11 (luteal), or 16 or 32 h after PG and their pituitaries removed and halved. One half was analysed for gonadotrophin and gonadotrophin-releasing hormone (GnRH) receptor content. Total pituitary RNA was extracted from the other half and subjected to Northern analysis using probes for FSH-beta, LH-beta and common alpha subunit. Frequent blood samples were taken and assayed for gonadotrophins. FSH secretion was significantly (P less than 0.01) reduced during bFF treatment throughout the luteal phase and then significantly (P less than 0.01) increased after cessation of treatment, with maximum secretion being reached 18-22h after PG, and then declining towards control values by 32h after PG. A similar pattern of LH secretion was seen after bFF treatment. Pituitary FSH content was significantly (P less than 0.05) reduced by bFF treatment at all stages of the cycle. No difference in the pituitary LH content was seen. The increase in GnRH receptor content after PG in the controls was delayed in the treated animals. Analysis of pituitary mRNA levels revealed that bFF treatment significantly (P less than 0.01) reduced FSH-beta mRNA levels in the luteal phase. Increased levels of FSH-beta, LH-beta and alpha subunit mRNA were seen 16h after PG in the bFF-treated animals, at the time when FSH and LH secretion from the pituitary was near maximum.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Hormone production in vivo and in vitro from follicles at different stages of the oestrous cycle in the sheep.

This experiment was undertaken in order to investigate the production of inhibin, oestradiol and androstenedione by ovarian follicles at different stages of the oestrous cycle in sheep. Twenty-four Scottish Blackface ewes were allocated to four groups of six ewes, i.e. those operated on during the luteal phase (day 10), and those operated on during the follicular phase 24-30, 36 and 60 h after the induction of luteal regression by an injection of 125 micrograms cloprostenol on day 10 of the luteal phase. Samples of jugular and ovarian venous blood were collected under anaesthesia and ovaries were then removed and all follicles larger than 3 mm diameter dissected out and incubated in medium for 2 h. After injection of cloprostenol, luteal regression occurred as indicated by a fall in the secretion rate of progesterone. The ovarian secretion rate of inhibin was similar at all stages of the follicular phase and during the luteal phase while, in contrast, the secretion rate of oestradiol was significantly (P less than 0.05) elevated in the group 24 h after injection of cloprostenol. There was good correlation between the in-vivo ovarian secretion rate and production rate during incubation in vitro for both inhibin (r = 0.57) and oestradiol (r = 0.60). When follicle diameter was compared with in-vitro hormone production there was good correlation for inhibin (r = 0.72) with larger follicles producing more inhibin, while the value for oestradiol was somewhat lower (r = 0.57) owing to the presence of large atretic follicles with low oestradiol production. Androstenedione production showed a lower correlation with follicle diameter (r = 0.39). When the four time periods were compared separately, there were significantly (P less than 0.05) more follicles with high in-vitro oestradiol production (greater than 90 fmol/min) in the group at 36 h than in the other three groups, while inhibin release in relation to follicle size was similar in the four groups. Large oestrogenic follicles were responsible for 90% of the total oestradiol production during culture while only providing 55% of the total inhibin production, with large non-oestrogenic and small follicles contributing 33% and 12% of inhibin production respectively. From the results of this study we conclude that while oestradiol is mainly produced by the large oestrogenic follicles, a considerable amount of inhibin is also produced by large non-oestrogenic and small follicles.(ABSTRACT TRUNCATED AT 400 WORDS)

Androstenedione↗

PubMed1992

Immunoneutralization and immunocytochemical localization of inhibin alpha subunit during the mid-luteal phase in the stump-tailed macaque.

The putative endocrine role of inhibin in the control of FSH secretion during the luteal phase in the primate was investigated by immunoneutralization. Antisera against the 1-23 amino acid sequence of the N-terminus of the human inhibin alpha subunit were raised in a ewe and three macaques. Antisera (10-20 ml) were administered to macaques on day 8/9 of the luteal phase and serum samples collected during the treatment cycle and post-treatment cycle for determination of FSH, oestradiol and progesterone. In addition, localization of inhibin within the macaque ovary at this stage of the luteal phase was investigated using the ovine antiserum. Intense immunostaining was localized within the granulosa-lutein cells of the corpus luteum with absence of staining in the theca-lutein cells or other ovarian compartments. Administration of antisera was without significant effect on serum concentrations of FSH when compared with control animals, either during the first 24 h of detailed observation or for the following 10-day period of the late luteal phase and subsequent early follicular phase. These results provide further evidence that the corpus luteum is the major source of inhibin immunoreactivity during the primate menstrual cycle, but fail to support an endocrine role for inhibin in the suppression of FSH secretion.

Animals↗

PubMed1992

The role of inhibin and oestradiol in the control of FSH secretion in the sheep.

The relative importance of inhibin and oestradiol in the control of FSH and LH secretion in the ewe was investigated by passive immunization in intact animals and by hormone replacement therapy following acute ovariectomy, in the same experiment. Mature Scottish Blackface ewes on day 10 of the luteal phase were allocated to nine groups of four to five animals. Four groups were ovariectomized and immediately treated with either progesterone alone or in combination with steroid-stripped ovine follicular fluid ('inhibin') and/or oestradiol. Three further groups of ewes were left intact and injected with antibodies to the 1-26 alpha peptide fragment of porcine inhibin and/or oestradiol-17 beta. Two groups of animals were either ovariectomized alone with no further treatment, or were left intact and treated with normal sheep plasma to act as controls. Blood samples were collected at 2 h intervals from 12 h before until 48 h after ovariectomy/immunization, and from 12 to 24 h after treatment, blood samples were collected at 10-min intervals. After ovariectomy there was a large rise in the peripheral concentration of LH (P less than 0.001) which was not affected by treatment with progesterone alone but was completely prevented by treatment with progesterone and oestradiol. Treatment with inhibin had no effect on this post-castrational rise in LH. In intact ewes, immunization against oestradiol, alone or in combination with inhibin, resulted in a rise in the concentration of LH, while immunization against inhibin had no effect on LH concentration. The peripheral concentration of FSH showed a significant (P less than 0.001) increase after ovariectomy which was not affected by treatment with progesterone alone. Treatment with inhibin or oestradiol alone caused a significant (P less than 0.01) reduction in this rise, while treatment with inhibin and oestradiol together completely prevented this post-castrational rise in FSH concentration. Passive immunization against inhibin or oestradiol alone resulted in a transitory (P less than 0.01) rise in the peripheral concentration of FSH, while immunization against the two hormones in combination resulted in a significantly (P less than 0.01) larger rise. During the 14-h period after treatment, the rise in the concentration of FSH in this combined immunization group was not significantly different from that seen in the control ovariectomized group. These results provide evidence that FSH secretion is under the control of both oestradiol and inhibin, while reinforcing the hypothesis that inhibin is not involved in the regulation of LH production, which is under the dual control of oestradiol and progesterone.

Animals↗

PubMed1992

3 beta-hydroxysteroid dehydrogenase inhibitor reduces ovarian steroid production but increases ovulation rate in the ewe: interactions with gonadotrophins and inhibin.

Two experiments were carried out during the breeding season in ewes, first to investigate the effects of oral administration of a 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) inhibitor (epostane) on the number of corpora lutea, and secondly to investigate the mechanism through which epostane acts. In the first experiment Dorset Horn ewes were treated orally with 25, 50, 100 or 200 mg epostane twice daily between days 10 and 15 of the oestrous cycle. All doses of epostane resulted in an increase in the number of corpora lutea per ewe, although the response was curvilinear, with the 25 mg dose showing the largest response and the 200 mg group the smallest response. Although there was no difference between groups in the number of ewes showing oestrus, the higher doses of epostane had a detrimental effect on fertility. In the second experiment Welsh Mountain ewes were treated twice daily with 25 mg epostane from day 10 of the oestrous cycle and the ovaries were removed for analysis during either the luteal or the follicular phases. Treatment significantly increased the number of follicles greater than 6 mm in diameter, but significantly reduced in-vitro follicular oestradiol and testosterone production. Despite a marked increase in peripheral inhibin concentrations there was no effect on in-vitro inhibin production. Epostane treatment also caused a significant reduction in peripheral FSH concentrations and an increase in mean LH concentration. The latter was due to an increase in LH pulse frequency during the luteal phase and LH pulse amplitude during the follicular phase. These results confirm that treatment of ewes with epostane orally has a significant effect on follicular steroidogenesis and causes a significant increase in the number of corpora lutea per ewe. This effect on ovulation rate is not via an increase in peripheral FSH concentration, but may be caused by a reduction in follicular steroid activity either directly on the ovary or via an alteration in the pattern of LH secretion.

3-Hydroxysteroid Dehydrogenases↗

PubMed1992

Apparent alpha-inhibin subunit immunoactivity in porcine and ovine luteal extracts is due to interference by cytosolic proteases in the assay.

Immunoreactive alpha-inhibin (ir-inhibin) was measured in luteal homogenates and subcellular fractions of ovine and porcine corpora lutea (CL) and in pig granulosa cells (GCs), using a sensitive radioimmunoassay specific for the 1-26 amino acid sequence of the N-terminus of the alpha chain of porcine inhibin (p1-26 alpha-inhibin). Inclusion of N-ethylmaleimide (N-EM) and/or EDTA in the immunoassay had no effect on the measurement of p1-26 alpha-inhibin peptide standards, on ir-inhibin levels in ovine follicular fluid and serum, or on ir-inhibin in subcellular fractions of pig GC. Fractionation of porcine GC homogenates on sucrose gradients demonstrated a major particular peak of ir-inhibin (buoyant density, 1.15-1.21 g/cm3) with variable activity in the cytosol. The particulate ir-inhibin peak was released into the cytosol by pretreatment of GC homogenates with the saponin, digitonin, prior to fractionation. Porcine GC extracts contained a protein (M(r) 45,000) which immunoblotted against p1-26 alpha-inhibin antibody. In the absence of inhibitors of proteolysis, apparent ir-inhibin activity was very high in extracts of sheep and pig CL. However, inclusion of N-EM or EDTA in the radioimmunoassay significantly reduced ir-inhibin levels in porcine and ovine CL extracts in a dose-dependent manner. Measurements of peptide tracer integrity indicated that porcine luteal cytosol degraded 125I-labelled p1-26 alpha-inhibin peptide. Subcellular fractionation studies demonstrated high levels of apparent ir-inhibin in luteal cytosol fractions, with only minor activity peaks associated with particulate fractions; however, this material was not releasable by digitonin. Immunoblotting of detergent extracts of porcine luteal particulate fractions failed to demonstrate alpha-inhibin material, and immunocytochemical localization studies of alpha-inhibin in porcine and ovine luteal sections were negative. Our results are consistent with the intracellular packaging/storage of a form of alpha-inhibin (M(r) similar to that of alpha-inhibin subunit precursor) in the porcine granulosa cell. However, luteinization of the porcine follicle was associated with a dramatic fall in ir-inhibin content, and the loss of immunostaining for alpha-inhibin peptides. We conclude that porcine and ovine CL contain little, if any, authentic inhibin. These studies emphasize the importance of excluding proteolytic artefacts when measuring biological peptides in luteal tissue extracts by radioimmunoassay.

Animals↗

PubMed1992

Luteinizing hormone pulses, follicle-stimulating hormone and control of follicle selection in sheep.

The growth of large oestrogenic follicles that have the potential to ovulate if given an appropriate luteinizing hormone (LH) signal is dictated by the plasma concentration of follicle-stimulating hormone (FSH). Basal amounts of LH are essential for this FSH-induced follicle growth, but pulses of LH do not appear to be essential. The fall in FSH concentration during the follicular phase of the oestrous cycle in sheep, and the subsequent withdrawal of FSH from other developing follicles may not be sufficient to explain follicle selection. There is little evidence to support an active suppression of the growth of other follicles by a factor(s) produced by the dominant, or selected follicle. It is possible that LH pulses cause active atresia of non-selected follicles. The selected follicle is the one that can survive the fall in plasma FSH and the large increase in LH pulses during the follicular phase and selection may simply be a case of being the follicle(s) that is present at the correct time to receive adequate FSH and develop sufficiently to survive the inhibitory effects of LH pulses. Several mechanisms whereby LH pulses may inhibit follicle growth are suggested, and avenues for future research, particularly related to the expression of the LH receptor on granulosa cells of the developing follicle, are outlined.

Animals↗

PubMed1991

Effects of mifepristone in vivo on decidual prostaglandin synthesis and metabolism.

Mifepristone is an effective abortifacient in combination with an exogenous prostaglandin but its mechanism of action is unknown. Mifepristone stimulates prostaglandin production from decidua in tissue culture. To determine whether this effect also operates in vivo, we treated women with mifepristone 24, 36 and 48 hours prior to surgical termination. Decidua was removed at operation and the ability of the tissue to generate prostaglandin in culture subsequently assessed. Pretreatment with mifepristone 36 hours prior to termination of pregnancy resulted in an increased production of PGF2 alpha in tissue culture (p less than 0.01). A significant decrease in PGFM production was seen 24 hours after pretreatment with mifepristone in vivo (p less than 0.01). These results suggest that the increased uterine activity observed after administration of mifepristone may be due to stimulation of endogenous prostaglandin production and inhibition of prostaglandin metabolism.

Culture Techniques↗

PubMed1991

The ovarian follicle and fertility.

The precise roles of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in the control of preovulatory follicle growth has been re-examined. Suppression of both pulsatile LH secretion and FSH or specific suppression of FSH results in an inhibition of preovulatory follicle growth beyond 2.5 mm dia. Infusion of sheep FSH alone in physiological amounts in the presence of basal, non-pulsatile LH results in the growth of preovulatory follicles. Co-infusion of large amplitude pulses of LH reduced or abolished this effect of FSH. It is suggested that: (1) FSH controls the number of follicles which develop; (2) selection of the large follicle destined to ovulate is directly related to the decline in the plasma concentration of FSH occurring during the period of follicle selection--thus, only the follicle(s) which can withstand this withdrawal of FSH will continue to develop; and (3) pulses of LH may directly affect the action of FSH on the follicle and play an important, hitherto unrecognized role in the selection of the ovulatory follicle by actively inducing atresia.

Animals↗

PubMed1991

Chronic treatment with long-acting bromocriptine does not affect duration of the breeding season, voluntary food intake, body weight, or wool growth in the Scottish blackface ewe.

The aim of this study was to determine whether suppression of the seasonal increase in prolactin concentrations by chronic treatment with the dopamine agonist bromocriptine would affect onset of anoestrus, voluntary food intake, body weight, and wool growth in a seasonal breed of sheep. Groups of eight Scottish Blackface ewes were injected i.m. each week with either the vehicle (Group A) or 2.0 mg (Group B), 6.0 mg (Group C), or 18.0 mg (Group D) of bromocriptine in a long-acting formulation, commencing on 18 January and terminating on 25 July (midwinter to midsummer in the northern hemisphere). Immediately before the bromocriptine injection, blood samples were taken for progesterone and prolactin determination. Voluntary food intakes were measured daily, and body weights were recorded every fortnight. Estimates of wool growth were made by weighing wool clipped from a measured area of skin once a month. Treatment had no effect on onset of anoestrus, voluntary food intake, body weight, or wool growth. Plasma prolactin concentrations increased significantly in all groups during the treatment period. From January to April, all doses of bromocriptine significantly reduced prolactin concentrations but later in the study (May and June) prolactin was significantly suppressed in Group D only, although even in this group prolactin concentrations increased between March and June. Pituitary prolactin content, measured at the end of the study in July, was also suppressed by bromocriptine. The gradual increase in prolactin concentrations in ewes receiving chronic bromocriptine was further investigated by treating a fifth group of ewes (Group E) with 18.0 mg of long-acting bromocriptine each week, commencing on 20 June.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

The effect of stage of estrous cycle and follicular maturation on ovarian inhibin production in sheep.

Twenty-four Scottish Blackface ewes (mean weight 50.0 +/- 0.1 kg with ovulation rate 1.3 +/- 0.1) were randomly divided into 4 groups of 6 animals. Under general anesthesia, following the collection of a timed sample of ovarian venous blood, the ovaries of these animals were collected either on Day 10 of the luteal phase or 12, 24, and 48 h after a luteolytic dose of a prostaglandin (PG) F2 alpha analogue (cloprostenol 100 micrograms i.m.) administered on Day 10. All follicles greater than 3 mm were dissected from the ovaries and incubated in Medium 199 (M199) at 37 degrees C for 2 h, following which the granulosa cells were harvested and incubated in triplicate for 24 h in M199 with or without ovine FSH or ovine LH. Plasma and culture media samples were assayed for inhibin, estradiol (E2), androstenedione (A4), and testosterone (T) by specific RIA. After correcting for hematocrit, ovarian secretion rates were calculated from the product of the plasma concentration and flow rate. The rate of ovarian inhibin secretion during the luteal phase was similar from ovaries categorized on the basis of presence of luteal tissue (1.0 +/- 0.3 and 0.9 +/- 0.5 ng/min for CL present and absent, respectively), confirming that the ovine CL does not secrete appreciable amounts of inhibin. Inhibin secretion was higher (p less than 0.05) at 12 h after PG-induced luteolysis but not at 24 or 48 h compared to values for luteal phase control ewes. Although ovaries containing large estrogenic follicles (greater than or equal to 4 mm in diameter and classified as estrogenic from in vitro criteria) secreted the most inhibin (55%; p less than 0.05), both ovaries containing large nonestrogenic follicles (33%) and small (11%; less than 4 mm in diameter) follicles secreted appreciable amounts of inhibin. This contrasted strongly with E2 where greater than 80% of the steroid was secreted by large estrogenic follicles. The rate of ovarian inhibin secretion was positively correlated (p less than 0.05) with the rate of E2, A4, and T secretion. Overall, there was no significant effect of stage of cycle on follicular inhibin content after 2 h incubation in vitro, release of inhibin by follicles incubated in vitro, or synthesis of inhibin by granulosa cells cultured in vitro. FSH and LH had no effect on the production of either inhibin or estradiol by cultured granulosa cells. Follicular diameter was positively correlated (p less than 0.001) with follicular inhibin and steroid release. Follicular inhibin content after 2 h incubation in vitro was more highly correlated with inhibin release by incubated follicles (r = 0.7; p less than 0.001) than with inhibin synthesis by granulosa cells in vitro (0.4; p less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Control of immunoactive inhibin production by human granulosa cells.

OBJECTIVE: The aim was to determine the relation between stage of antral follicular development and granulosa cell production of immunoactive inhibin. DESIGN: Primary granulosa cell cultures in serum-free Medium 199 were incubated at 37 degrees C for 96 hours with a change of medium at 48 hours. Inhibin and steroid levels in culture medium were determined by radioimmunoassay. The inhibin assay was based on the N-terminal 1-26 amino acid sequence of the alpha-chain of porcine 32 kDa inhibin using pl alpha 1-26-GLY27-TYR28 as the immunogen, tracer and standard. PATIENTS: Granulosa cells were obtained from the ovaries of women with regular menstrual cycles undergoing hysterectomy with unilateral or bilateral oophorectomy to treat non-malignant gynaecological disease. RESULTS: Basal production of immunoactive inhibin by granulosa cells from presumptive preovulatory follicles (greater than 15 mm diameter) was 5-13 times higher than that by granulosa cells from immature (less than 10 mm diameter) or intermediately mature (10-15 mm diameter) follicles. Basal production of progesterone and oestradiol followed a qualitatively similar pattern, establishing a positive relation between functional granulosa cell maturity and inhibin production. Treatment of granulosa cell cultures from immature follicles with follicle-stimulating hormone (FSH), but not luteinizing hormone (LH), increased inhibin production, time and dose dependently. FSH, but not LH, also brought about similar increases in steroid hormone synthesis by granulosa cells from immature follicles. The stimulatory effect of FSH on granulosa cell inhibin production was augmented at least twofold by the presence of testosterone or 5 alpha-dihydrotestosterone (1.0 mumol/l) but was unaffected by oestradiol. Granulosa cells from intermediately mature follicles undertook variable degrees of both FSH and LH-responsive inhibin production which generally corresponded with gonadotrophin-responsive steroid production. Granulosa cells from presumptive preovulatory follicles showed inconsistent inhibin responses to FSH. However, LH caused marked (at least twofold) increases in inhibin production, paralleling LH-responsive steroid production. CONCLUSION: These results show that for human beings, granulosa cell capacity to produce immunoactive inhibin in vitro increases with follicular maturity. FSH, but not LH, stimulates inhibin production by immature granulosa cells and this response to FSH is subject to modulation by androgen. During preovulatory follicular development, production of inhibin, like steroids, becomes increasingly responsive to LH. Such a development-related pattern of granulosa cell inhibin production helps explain how, post-ovulation, the corpus luteum is able to secrete inhibin as well as steroids. It is also compatible with the concept that locally produced inhibin could participate in the paracrine control of follicular development during the human menstrual cycle.

Adult↗

PubMed1991

Prolactin short-loop feedback and prolactin inhibition of luteinizing hormone secretion during the breeding season and seasonal anoestrus in the ewe.

In seasonally breeding mammals, plasma prolactin (PRL) concentrations vary on an annual basis with levels high in summer and low in winter. In this study of the ewe, we determined, first, whether PRL secretion is regulated by short-loop feedback and, second, whether the high summer levels of PRL are due to a change in sensitivity or loss of this feedback loop. Because the high summer levels of PRL coincide with the period of seasonal anoestrus in the ewe and could therefore be involved in the seasonal suppression of gonadotrophins, the effects of intracerebroventricular PRL on pulsatile LH secretion were also determined. Ovary intact ewes received intracerebroventricular injections of ovine PRL (oPRL; 50 micrograms) or anti-PRL serum. From 3 to 13 h after central administration of oPRL, plasma PRL concentrations were significantly reduced compared with the vehicle-treated controls. In contrast, following injection of anti-PRL serum, plasma PRL levels increased significantly. To determine whether there was a seasonal change in sensitivity to PRL feedback, a series of experiments were conducted in July and November when PRL concentrations are high and low, respectively. At each time of year, ovariectomized oestradiol-implanted ewes were injected intracerebroventricularly with 10 and 50 micrograms oPRL with control animals receiving the vehicle. At both times of year there was clear evidence of PRL short-loop feedback with no indication that sensitivity was reduced in the July trial. Luteinizing hormone (LH) pulse frequency, pulse amplitude and mean LH were not affected by intracerebroventricular oPRL at either time of year.(ABSTRACT TRUNCATED AT 250 WORDS)

Anestrus↗