Biomedical subjects

J K Findlay

Publications and source records attributed to J K Findlay.

At least 145 records · Page 8Linked to original sources

PubMed1986

Cloning and sequence analysis of cDNA species coding for the two subunits of inhibin from bovine follicular fluid.

The primary amino acid structures of the 43-kDa (A) and 15-kDa (B) subunits of the 58-kDa form of the hormone inhibin have been elucidated by cloning and analysis of cDNA species derived from bovine granulosa cell mRNA. The A subunit (Mr = 32,298) is a protein of 300 amino acids with two potential N-glycosylation sites and two potential proteolytic processing sites and has a pre-pro region of 60 amino acids. The mature B subunit (Mr = 12,977) is a protein of 116 amino acids synthesized from a separate mRNA. These data establish that a 31-kDa form of inhibin also isolated from bovine follicular fluid, with subunits of 20 kDa (Ac) and 15 kDa (B), is derived from the 58-kDa form by proteolytic processing of the A subunit.

Amino Acid Sequence↗

PubMed1986

The effects of the preimplantation blastocyst in vivo and in vitro on protein synthesis and secretion by cultured epithelial cells from sheep endometrium.

Epithelial endometrial cells were isolated on day 13 from nonpregnant (NPr) and pregnant (Pr) ewes and cultured in the presence or absence of conditioned media from 15-day blastocysts (BM) under optimized conditions in the presence of [35S]methionine (S-met). The incorporation of S-met into secreted protein was analyzed, and individual proteins were identified by autoradiography of two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Incorporation of S-met into secreted protein was higher (P less than 0.05) for cells from Pr than NPr animals. Secretion by cells from NPr ewes was increased in three of four cases by addition of BM to the culture medium. Autoradiography of two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis showed five secreted proteins [mol wt range 74,000-120,000; isoelectric point (pI) less than 6.5] which were either absent from or present in only small amounts in secretions from cells from NPr animals. The proportion of these proteins was greatly increased in secretions from cells from Pr animals. The presence of BM in cultures from NPr ewes enhanced the secretion of these same proteins, this effect being maintained even after heat treatment of the BM. One of these proteins had previously been shown to be maximally induced by the presence of estrogen and progesterone. Three other similarly controlled proteins were also enhanced, though to a lesser extent, by the presence of a blastocyst in vivo but were not stimulated by BM in vitro. It is concluded that endometrial epithelial cells from Pr ewes are metabolically more active than those from ewes on day 13, and the blastocyst and its secretions induce the secretion of several specific proteins by epithelial cells, some of these proteins being the same as those controlled by the combination of estrogen and progesterone.

Animals↗

PubMed1986

Effect on plasma gonadotropins of cyclic steroid replacement in women with premature ovarian failure.

A sequential regimen of steroid replacement of oral estradiol valerate and progesterone (P) by intravaginal suppository was developed for women with premature ovarian failure or ovarian agenesis. The regimen, based on a 28-day cycle, resulted in peripheral plasma concentrations of estradiol and P within the normal range of the menstrual cycle and endometrial differentiation consistent with the normal secretory phase. Pregnancy has now been successfully established in four patients following this regimen of steroid treatment and transfer of donated embryos. Plasma concentrations of LH were within the normal range by the end of the first cycle of treatment with exogenous steroids. However, plasma FSH remained above the normal range, even during the third treatment cycle, consistent with the necessity of a gonadal feedback factor (inhibin?) other than estradiol and P for maintaining FSH in the normal range. Although 7/8 patients had a surge of LH at midcycle, only 3/8 patients had concomitant FSH surges, supporting a role for progesterone in facilitating the midcycle FSH surge.

Administration, Oral↗

PubMed1986

Permeability of rat ovarian follicles to LH during development and luteinization.

A 'double isotope' technique has been used to describe the temporal relationship between plasma and follicular concentrations of LH after injection of 51Cr and 125I-rat LH into immature rats. Radiolabelled LH was detectable in all follicles 1 min after injection. Concentrations in small antral and large preovulatory follicles were not significantly different at any time and reached a maximum of 34.2 +/- 3.0% of plasma concentrations at 40 min. Concentrations of LH in preovulatory follicles exposed to an ovulatory dose of hCG 4 h previously were significantly greater (P less than 0.05) than those in small antral and preovulatory follicles at all times, and reached a maximum of 46.2 +/- 1.7% of plasma concentrations after 1 h. Polyacrylamide gel electrophoresis and immunoprecipitation with an antibody specific for rat LH indicated that radioactivity in plasma and follicular fluid represented radio-iodinated LH. Steroidogenic activities, light microscopy and measurements of follicular volume of each class of follicle confirmed that small antral, preovulatory follicles and preovulatory follicles exposed to an ovulatory dose of hCG in vivo could be isolated specifically. Based on these findings it is possible to calculate that, during an endogenous pulse of LH secretion, follicular concentrations of LH never exceed 20% of peak plasma concentrations. Pronounced increases in functional activities during antral growth were not correlated with increased follicular permeability. Only after acute exposure to an ovulatory dose of hCG in vivo was permeability significantly increased. We conclude that entry of LH into antral follicles is restricted and that exposure to an ovulatory dose of hCG results in greater amounts of LH entering preovulatory follicles.

Animals↗

PubMed1986

Inhibin secretion by the sheep ovary.

An in-vitro bioassay for inhibin based on FSH content or release by rat pituitary cells was validated for measuring inhibin activity in ovine plasma and lymph. Dose-dependent increases in inhibin activity were detected in peripheral plasma of 4 ovariectomized ewes 1 min after i.v. injections of ovine follicular fluid, and the half-life of inhibin in plasma for 2 ewes was 45 and 50 min, respectively. Inhibin was detected in ovarian lymph but not in ovarian or jugular venous plasma, even after treatment of ewes with PMSG to induce folliculogenesis. Destruction of visible follicles (greater than 0.5 mm diameter) on the ovaries of 4 PMSG-treated ewes by electrocautery was followed by a rapid and sustained decline in secretion of inhibin in ovarian lymph for up to 4 h. Ovarian lymph flow rates were either unchanged or slightly increased after cautery. Oestrogen concentrations in peripheral venous plasma declined within 15-30 min of cautery, but concentrations remained well above baseline. There was a significant decrease in peripheral progesterone concentrations in these same samples, but not until 2-3 h after cautery. FSH in peripheral plasma was depressed or non-detectable in PMSG-treated ewes and neither FSH nor LH concentrations in peripheral plasma were significantly altered up to 4 h after cautery of ovarian follicles. It is concluded that (a) antral follicles (greater than 0.5 mm) are the source of inhibin present in ovarian lymph, and (b) the ovarian lymphatic system is a route by which inhibin could reach the peripheral circulation, particularly in the luteal phase when ovarian lymph flow rates are high.

Animals↗

PubMed1986

Increase in ovulation rate after immunization of Merino ewes with a fraction of bovine follicular fluid containing inhibin activity.

Affinity chromatography of bovine follicular fluid using Matrix gel Red A resulted in a 20-fold increase in inhibin-like specific activity assessed in a mouse ovulation inhibition test system. When this material was emulsified with Freund's adjuvant and injected into adult Merino ewes their mean ovulation rate was increased from 1.2 to 2.3 (P less than 0.01). Follicles of diameter greater than or equal to 3.5 mm and 2-3.4 mm were also increased (4.33 vs 2.25 and 5.39 vs 2.44 per ewe respectively). The ovulation rate response was variable and transient. Length of oestrous cycles, number of granulosa cells per follicle and seasonal oestrous patterns were not affected. Plasma from the immunized ewes contained antibodies to the immunogen and reversed the ovulation-inhibiting effects of bovine follicular fluid in mice.

Animals↗

PubMed1986

Effects of ovine follicular fluid on plasma LH and FSH secretion in ovariectomized ewes to indicate the site of action of inhibin.

Ovariectomized ewes were given 2 ml s.c. injections of ovine follicular fluid (oFF) (N = 3) or serum (N = 3) and blood samples were collected each day for 3 days. Follicular fluid caused a significant (P less than 0.005) reduction in FSH within 1 day, but did not affect mean LH values. Two groups of 3 ewes were treated as above but sampled intensively (each 10 min for 6 h) on Days 1 (before treatment) and 4; mean plasma FSH concentration and plasma LH pulse frequency and amplitude were ascertained. Significant (P less than 0.005) reduction of FSH concentration was seen in the oFF-treated ewes. A non-specific reduction in LH pulse amplitude, but not pulse frequency, was noted in the control ewes. This experiment was repeated with 2 groups of 4 ewes that were conditioned to the experimental environment and effects on LH secretion were not observed in the controls given serum. Treatment with oFF caused a 70% reduction (P less than 0.005) in plasma FSH and a small (30%) but significant (P less than 0.005) reduction in mean LH concentrations. The latter was probably associated with a reduction in LH pulse amplitude in 3/4 animals (N.S.) with no change in LH pulse frequency. Treatment with oFF, as in Exp. 1, caused a 95% reduction in FSH values and significant (P less than 0.01) reduction (32%) of LH pulse amplitude in ovariectomized ewes that had been subjected to hypothalamo-pituitary disconnection and in which gonadotrophin secretion was reinstated with pulses of 250 ng GnRH every 2 h. These results suggest that proteins from the sheep follicular fluid, including inhibin, act at the pituitary level to inhibit FSH secretion and may have some effects on LH pulse amplitude.

Animals↗

PubMed1986

Angiogenesis in reproductive tissues.

Angiogenesis is the process of generating new capillaries and leads, therefore, to vascularization of tissues. This process occurs during embryological development and during pathological and physiological conditions in adult life, including those involving the reproductive organs. Recent studies, in the field of tumour biology in particular, have led to the identification of several factors responsible for inducing angiogenesis and the elucidation of ways of modulating their activity. This review summarizes our knowledge of angiogenesis in the ovary, testis, endometrium and placenta, and suggests ways in which further research might contribute to a better understanding of the processes controlling reproduction and identify new approaches to the regulation of fertility.

Endometrium↗

PubMed1985

Distribution of specific androgen binding sites within the ovine ovarian follicle.

Two specific androgen binding sites were characterized in the ovine follicle with [3H]DHT, [3H]T and [3H]R-1881 as ligands, different incubation times and a charcoal separation step: the first, with characteristics very similar to testicular ABP in terms of its capacity, affinity, association and dissociation rates and specificity for natural and synthetic androgens, was found in serum, follicular fluid and the 27000 X g particulate and cytosol fractions of granulosa cells; the second, classic androgen receptors, were found in the cytosol with high affinity and low capacity for the synthetic androgen R-1881 and a very slow steroid-protein rate of dissociation. Saturation analysis on purified nuclei showed only the presence of the androgen receptor binding R-1881 with capacity similar to cytosol receptor. Isolated follicles showed a direct correlation between the total concentrations of androgen ([3H]-[3H]R-1881) binding sites and the follicular diameter. The complex actions which androgens exert on granulosa cell function may be mediated by interactions in vivo between these extra- and intracellular specific androgen binding proteins.

Animals↗

PubMed1985

The effects of estrogen and progesterone in vivo on protein synthesis and secretion by cultured epithelial cells from sheep endometrium.

Ovariectomized ewes were treated with either nothing or implants of estrogen (E), progesterone (P), or E + P. Epithelial and stromal cells from caruncular and intercaruncular regions of sheep endometrium were dispersed by collagenase digestion and enriched by Ficoll gradient separation. Verification of cell types was by electron microscopy, keratin staining (epithelial cells), cell size, and appearance in culture. Epithelial cells were cultured under optimized conditions with [35S]methionine (S-met) and uptake of label by cells and its incorporation into cellular and secreted protein determined. Protein in the medium and lysed cells was analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cells from E-treated animals had higher S-met uptake and incorporation into proteins (cellular and secreted) than cells from ewes treated with nothing and P-treated animals. E effects were not significantly reduced in the presence of P. When secreted protein was expressed as a percent of total incorporated S-met, P treatment either alone or with E increased the proportion of labeled protein secreted by cells. There were no significant differences between caruncular and intercaruncular. Two-dimensional polyacrylamide-gel electrophoresis of secreted proteins showed one major glycoprotein (mol wt, 46,000, isoelectric point, 5.8-6.5) and four minor proteins induced by E + P greater than E, and five minor proteins inhibited by the steroids. Both induction and inhibition of cellular proteins were also apparent, though of lesser magnitude. Overall, whereas E treatment in vivo influenced the rate of incorporation of S-met into proteins by epithelial cells in vitro, P treatment increased the proportion of newly synthesized protein which was secreted. Steroids caused significant alterations in the individual proteins secreted by ovine endometrium.

Animals↗

PubMed1985

Influence of season and sex on the inhibitory effect of ovine follicular fluid on plasma gonadotrophins in gonadectomized sheep.

The inhibitory effects of follicular fluid on FSH secretion were similar in gonadectomized male and female sheep, and in the anoestrous and breeding seasons. Significant suppression of LH was variable and was observed only at the highest dose of follicular fluid when suppression rarely exceeded 50% of pretreatment values. Basal plasma FSH and LH concentrations were higher in castrated males than in ovariectomized females in both seasons. Plasma FSH concentrations in gonadectomized males and females and LH concentrations in the males were lower in the anoestrous than the breeding season. Therefore, in the absence of the gonads, sex and photoperiod can influence hypothalamic control of basal pituitary gonadotrophin secretion in males and females, whereas the feedback effect of non-steroidal factors in follicular fluid (inhibin) on FSH secretion is not influenced by photoperiod or sex.

Anestrus↗

PubMed1985

Do small and large luteal cells of the sheep interact in the production of progesterone?

Corpora lutea from cyclic ewes were dissociated by collagenase and trypsin/EGTA treatments, and enriched fractions of small and large luteal cells were prepared on gradients of Ficoll. These fractions were incubated separately or remixed before incubation. Colchicine, cytochalasin B and the calcium channel-blocker verapamil significantly reduced progesterone production by both small and large luteal cell fractions, while isoprenaline stimulated an increase in progesterone production by large luteal cell fractions only. When fractions of small and large luteal cells were remixed, no more and no less progesterone was produced than would have been predicted from equivalent fractions incubated separately. There was therefore no evidence of synergism between small and large luteal cells in the production of progesterone. Prostaglandin F-2 alpha, which can inhibit LH-stimulated progesterone production by ovine luteal tissue in vitro, had no effect on LH-stimulated progesterone production by small luteal cell fractions, but significantly inhibited that by enriched fractions of large luteal cells. Since large luteal cell fractions were contaminated with small luteal cells, which are probably responsible for the progesterone-secretory response of these fractions to LH, it was concluded that the inhibition of LH-stimulated progesterone production by small luteal cells is dependent on the presence of large luteal cells. Oxytocin added to large and small luteal cell fractions did not affect progesterone production by either fraction. It was therefore concluded that the inhibitory action of PGF-2 alpha on LH-stimulated progesterone production may require the interaction of large and small luteal cells, but that oxytocin is not likely to be an intermediary in this interaction.

Animals↗

PubMed1985

Role of intrafollicular regulators and FSH in growth and development of large antral follicles in sheep.

Manipulation of circulating concentrations of hormones and ovarian follicle status was carried out on Day 11-12 of the oestrous cycle in sheep. All follicles visible on the ovary were ablated by cautery and ewes were treated with oestradiol or ovine follicular fluid (oFF) to suppress FSH or with PMSG to increase circulating gonadotrophic activity. One group underwent unilateral ovariectomy which greatly increased endogenous FSH and was the only treatment which significantly affected LH pulse frequency. The size distribution of antral follicles, the extent of atresia and the mitotic index of granulosa cells of follicles on Day 15 showed that (a) treatment with oFF inhibited the growth of follicles beyond 2 mm diameter by suppressing the mitotic index of the granulosa cells and (b) the concentration of FSH in peripheral plasma was related to the ability of small antral follicles to grow during the late luteal-early follicular phase of the cycle. Subsequently, it was demonstrated that oFF inhibits, in a dose-dependent manner, folliculogenesis sustained by PMSG in ewes on Days 12-15. Inhibition of folliculogenesis was represented by a decrease in those follicles greater than 4 mm, an increase in the relative proportion of follicles less than 2 mm, and minimal change in the average number of follicles visible on the ovarian surface, and a decrease in the mitotic index of granulosa cells of follicles less than 2 mm. There was no change in the extent of atresia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Plasma FSH and LH in prepubertal Booroola ewe lambs.

Basal plasma concentrations (four 30-min samples) and GnRH-induced release of gonadotrophins were measured every 15 days between 30 and 90 days and at 110 days of age in Merino ewe lambs from the prolific Booroola ('B') flock (n = 18-23), the medium prolificacy ('T') flock (n = 14-20), and the 'O' flock (n = 4-8) of low prolificacy. At ages of 30 and 45 days B ewe lambs had mean basal plasma FSH concentrations of 145 and 122 ng/ml which were significantly higher (P less than 0.01) than those seen in T (45 and 53 ng/ml), and O (39 and 38 ng/ml) flock ewes. Between 60 and 110 days of age there were no significant differences between genotypes. The increment in FSH concentrations above basal levels induced by the subcutaneous injection of 100 micrograms synthetic GnRH was only significantly (P less than 0.05) greater in B than T and O genotype ewe lambs at 110 days of age but not at other ages. The basal plasma FSH differences between the B, T and O genotypes at 30 and 45 days of age were not consistently related to the size of litter in which lambs were born. At 30 days of age the mean plasma LH concentration of B, T, and O flock lambs were 2.6 +/- 0.5, 1.2 +/- 0.6 and 0.7 +/- 0.8 ng/ml respectively. These differences were not significant. At later ages there were also no significant differences between the genotypes with respect to basal LH, and the increase in LH induced by exogenous GnRH was always similar for the three genotypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1985

Oocyte and embryo donation in IVF programmes.

We have described a cyclic steroid replacement regimen of oestradiol and progesterone which is able to produce physiological concentrations of these steroids in plasma indicative of a normal menstrual cycle. The stimulation of a secretory endometrium at the appropriate time in this artificial menstrual cycle bears testament to the suitability of the treatment and has led to the establishment of pregnancy in three women with complete ovarian failure or ovarian agenesis when used in combination with the established techniques of in vitro fertilization and embryo transfer. The steroid replacement regimen can easily be adjusted to maintain pregnancy until the time of luteoplacental shift, based on the close monitoring of the plasma steroid and hCG levels in the pregnant individual. The implications of this form of treatment for other disease states and for our understanding of the mechanisms involved in ovarian hormone action, pregnancy maintenance and parturition are immense.

Chorionic Gonadotropin↗

PubMed1984

Effects on plasma luteinizing hormone and follicle-stimulating hormone of varying the frequency and amplitude of gonadotropin-releasing hormone pulses in ovariectomized ewes with hypothalamo-pituitary disconnection.

The effects on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion of various regimens of pulsatile gonadotropin-releasing hormone (GnRH) replacement were examined in ovariectomized (OVX) ewes after hypothalamo-pituitary disconnection (HPD). Hourly pulses of 500 ng GnRH restored gonadotropin secretion in OVX-HPD sheep. Replacement beginning 2 days after HPD gave consistent responses of LH and FSH within a week. Replacement beginning 61-96 days after HPD caused more gradual re-establishment of LH and FSH secretion with LH responses appearing immediately and FSH responses appearing 2 weeks later. When hourly GnRH pulses were increased in amplitudes from 250 to 500 ng the plasma LH baseline, peak values and pulse amplitudes were increased. There was no significant change in plasma FSH levels over 10 pulses at the higher dose. Decreases in GnRH pulse frequency led to increases in LH pulse amplitude and decreases in plasma LH baseline. In contrast, immediately after a change from a 2-hourly to an hourly mode, an increase in LH baseline occurred without an immediate reduction in LH pulse amplitude. Mean plasma FSH concentrations increased when the frequency was reduced from hourly to 2-hourly or 4-hourly. However, a change from 4-hourly to hourly pulses did not reduce FSH values within 7 days. It is concluded that changes in the pattern of LH secretion observed during the ovine estrous cycle could be accounted for, in part, by changes in GnRH pulse frequency.

Animals↗

PubMed1984

Identification at the onset of luteolysis of follicles capable of ovulation in the ewe.

Follicles of various sizes at the surface of the ovary were ablated by electrocautery at the time of cloprostenol-induced luteolysis in ewes and the interval from cloprostenol treatment to the onset of the LH surge determined as an index of the time from luteolysis to ovulation. When follicles 2-4 mm or greater than 4 mm diameter remained in the ovaries, the interval from cloprostenol treatment to the onset of the LH surge was similar to that in sham-operated (control) ewes (55-60 h), whereas when the only follicles remaining were less than 2 mm, the interval was extended by 24 h (P less than 0.05). This study demonstrates that follicles capable of ovulating can be selected from those greater than or equal to 2 mm diameter at luteolysis, emphasizing the flexibility of the sheep ovary in its final selection of the ovulatory follicle.

Animals↗

PubMed1984

Induction of plasma LH surges and normal luteal function in acyclic post-partum ewes by the pulsatile administration of LH-RH.

Plasma LH surges, similar to preovulatory surges in cyclic ewes (N = 9), were induced in 11/15 acyclic ewes, 19-26 days post partum, treated with LH-RH (100 ng i.v. every 3 h for 24 h, every 2 h for 24 h, then every 1 h for 24 h) (Group PPrh ), and in 11/15 LH-RH-treated acyclic ewes pretreated with progesterone (s.c. implant for 11 days) (Group PPprh ). Plasma progesterone, indicating subsequent luteal function, was detected in 10/11 Group PPrh ewes and in 9/11 Group PPprh ewes. The mean and peak plasma progesterone concentrations in these ewes were similar to those in cyclic ewes (2.0 +/- 0.2 and 3.1 +/- 0.4 ng/ml). The periods over which plasma progesterone was detected were similar in cyclic (11.7 +/- 0.5 days) and in Group PPprh ewes (12.2 +/- 0.8 days), but less in Group PPrh ewes (9.6 +/- 0.4 days). After regression of the induced corpus luteum more Group PPprh ewes (6/9) than Group PPrh ewes (2/10) showed oestrus and continued cycling. These results show that an appropriate pattern of pulsatile LH-RH treatment of post-partum acyclic ewes, pretreated with progesterone, can lead to plasma LH surges, luteal function, and subsequent ovarian cyclicity similar to that found in cyclic ewes. This suggests that ovarian acyclicity in post-partum ewes is a reflection of an inadequate pattern of pulsatile release of LH-RH from the hypothalamus.

Animals↗