Corpora lutea induced by gonadotrophin-releasing hormone treatment of anoestrous Welsh Mountain ewes: reduced sensitivity to luteinizing hormone in vivo and to chorionic gonadotrophin in vitro.

Seasonally anoestrous Welsh Mountain ewes received 250 ng gonadotrophin-releasing hormone (GnRH) every 2 h, with (Group 1; n=13) or without (Group 2; n=14) progesterone priming for 48 h. Fourteen control ewes (Group 3) were studied during the luteal phase in the breeding season. Animals in Group 4 (n=12) received progesterone priming followed by 250 ng GnRH at increasing frequency for 72 h, while ewes in Group 5 (n=13) were given three bolus injections of 30 microg GnRH at 90-min intervals. All treatment regimens induced ovulation. However, only corpora lutea (CL) from ewes in Group 3 (breeding season) or Group 4 exhibited normal luteal function. Luteal luteinizing hormone (LH) receptor levels were significantly higher on day 12 than day 4, and CL from groups with adequate CL (3 and 4) had significantly higher (125)I-human chorionic gonadotrophin (hCG)-binding levels than the three groups with inadequate CL on day 12. LH-binding affinity was unchanged. Exogenous ovine LH (10 microg) in vivo on days 3 or 11 after ovulation induced a pulse of progesterone in ewes with adequate CL: however, ewes in Groups 1, 2 and 5 showed no significant response. Basal progesterone secretion in vitro was significantly greater on day 4 than on day 12. Maximal steroidogenic responses of adequate and inadequate CL to hCG and to dibutyryl cyclic-3',5'-AMP were similar at both stages of the luteal phase. However, the EC50 for hCG on days 4 and 12 was 10-fold lower for groups with an adequate CL (0.1 IU hCG/ml) than for inadequate-CL groups (1 IU hCG/ml; P <0.05). Thus, in addition to the well-characterized premature sensitivity of GnRH-induced inadequate CL to endometrial luteolysin, we have shown (1) a marked decrease in total number of cells in the CL, a profound reduction in vascular surface area, and a decrease in mean large luteal cell volume (with no change in large luteal cell numbers), (2) decreased luteal LH receptor and progesterone content compared with adequate CL and (3) that CL that were becoming, or were destined to become, inadequate failed to respond to ovine LH in vivo and were 10-fold less sensitive to hCG in terms of luteal progesterone secretion in vitro.

Reduced LH sensitivity in vivo and in vitro of corpora lutea induced during anoestrus by GnRH, and during the late breeding season, in Scottish Blackface ewes.

Scottish Blackface ewes were synchronised in mid-breeding (November; group 1; n=12 ewes) or late-breeding season (March; group 2; n=16). Anoestrous ewes (May) were treated with progestagen sponges for 7 days and then given 250 ng GnRH 3-hourly for 24 h, 2-hourly for 24 h and hourly for a further 24 h (group 3; n=12). A second group of anoestrous ewes (group 4, n=19) received three bolus injections (30 microg) of GnRH at 90-min intervals without progestagen pretreatment. After ovulation, ewes were bled twice daily until slaughter (day 4 or day 12: oestrus=day 0). Mid-breeding season (group 1) and anoestrous ewes in group 3 formed 'adequate' corpora lutea (CL) with high plasma progesterone levels (3-4 ng/ml) maintained for at least 12 days, and responded in vivo to ovine LH (oLH) (10 microg) with a rise in plasma progesterone on day 11 (group 3, but not group 1, ewes also responded on day 3). CL minces from these ewes responded to human chorionic gonadotrophin (hCG) in vitro with a dose-dependent increase in progesterone secretion. Ewes in group 4 had a foreshortened luteal phase (8-10 days) and low plasma progesterone levels (approximately 1 ng/ml), consistent with formation of inadequate CL. LH injection failed to induce a significant plasma progesterone increase. Furthermore, although progesterone secretion in vitro in response to maximally stimulating doses of hCG or dibutyryl cAMP (dbcAMP) was similar to that in adequate CL, the sensitivity of these CL to hCG (EC (effective concentration)50, 1 IU hCG/ml) was reduced 10-fold compared with adequate CL (EC50, 0.1 IU hCG/ml; P<0.01). Ewes that ovulated in the late breeding season (group 2) had high plasma progesterone, although levels began to decrease after day 10. Injection of oLH in vivo increased plasma progesterone. However, sensitivity to hCG in vitro (EC50, 0.5 IU hCG/ml) was intermediate between that of adequate luteal tissue (groups 1 and 3; EC50, 0.1 IU/ml) and that of group 4 ewes (EC50, 1 IU hCG/ml). Our data demonstrate a markedly reduced luteal sensitivity to LH in vivo and hCG in vitro in Scottish Blackface ewes with inadequate CL, and suggest that a similar loss of sensitivity to LH may occur in the late breeding season.

Human placental GnRH-like factors: II. Inhibition of enzymatic degradation of GnRH-II and [D-Trp6]GnRH ethylamide tracers by human term placental cytosol fractions reveals the presence of GnRH-binding protein(s).

We describe the preliminary characterization of GnRH-binding protein(s) in human placental cytosol. Samples were analysed by chromatography on Sephadex G25. Radiolabelled GnRH and its analogues elute significantly later than the total column volume (V(t)) on Sephadex G25 column chromatography. However, incubation of GnRH II or GnRH agonist tracers with human placental cytosol reduced the intact tracer peak, with the concomitant appearance of a new peak eluting in the total column volume (V(t)). This peak increased with increasing cytosol concentration and duration of incubation, and probably represented degraded GnRH tracer, since (i) degradation-resistant GnRH agonist tracer, [D-Trp(6)]GnRH EtA, was inactivated more slowly than GnRH II, (ii) boiling of cytosol fractions abolished formation of this peak and (iii) peptidase inhibitors blocked its formation. A second new tracer peak eluted in the column void volume (V(o)) and was largely unaffected by peptidase inhibitor concentrations that blocked tracer degradation. The magnitude of this high molecular weight peak depended on the GnRH tracer employed, cytosol concentration, and the pH, duration and temperature of incubation. Tracer associated with this third peak appeared similar to intact GnRH tracer by TLC. Unlabelled GnRH analogues and isoforms decreased both tracer degradation and formation of the V(o) peak, but their specificity and affinity for the two processes differed. Ligand blots identified several bands that were abolished by inclusion of unlabelled agonist during incubation. Our data indicate the presence of specific GnRH binding protein(s) and GnRH peptidases that may modulate local actions of GnRH in the human placenta.

Non-genomic steroid receptors in the bovine ovary.

Over the last few years, rapid and physiologically important non-genomic actions of all classes of steroid hormones have been described in many cell types. A putative non-genomic membrane progesterone receptor (NGPR) was the first, and so far the only, non-genomic steroid receptor cloned. Two homologous NGPR proteins have been identified in the human, and a similar protein in the bovine and rat. Various detection methods have been used to identify putative NGPRs in a range of tissues: however, different methods often yield quite different molecular weights, and probably detect distinct moieties. We describe some properties of the specific cell-surface membrane binding sites for [3H]-progesterone in enriched cell membrane preparations of bovine luteal and follicular cells. Similar binding sites were also detected in cell-membranes of some (but not all) bovine tissues. Western blots of detergent extracts of bovine luteal membranes identified a protein (85kDa) that reacted with an antiserum to the N-terminal peptide of porcine NGPR. Activity was low in native non-denatured extracts, but increased dramatically in a dose-dependent manner following pretreatment with the cholesterol-complexing agent, digitonin. This protein was co-precipitated by antisera to caveolin. In contrast, a specific monoclonal antibody to the ligand binding domain of the genomic progesterone receptor (Mab C262) detected two proteins (M(r), 55 and 60kDa) in luteal membrane detergent extracts. Immunostaining of these proteins by Mab C262 was abolished by digitonin concentration-dependent manner in non-denatured extracts. However, both proteins were unaffected by digitonin in fully denatured detergent extracts, suggesting that digitonin induced a conformational change in the native protein that prevented binding of Mab C262 to its epitope. Our data suggest the presence of a complex of two or more distinct membrane-associated progesterone-binding proteins in bovine luteal membranes. Moreover, their conformations are specifically affected by removal of bound cholesterol.

Expression of mRNA encoding insulin-like growth factors I and II and the type 1 IGF receptor in the bovine corpus luteum at defined stages of the oestrous cycle.

Previous studies have implicated insulin-like growth factors I and II (IGF-I and -II), in the regulation of ovarian function. The present study investigated the localization of mRNA encoding IGF-I and -II and the type 1 IGF receptor using in situ hybridization to determine further the roles of the IGFs within the bovine corpus luteum at precise stages of the oestrous cycle. Luteal expression of mRNA encoding IGF-I and -II and the type 1 IGF receptor was detected throughout the oestrous cycle. The expression of IGF-I mRNAvaried significantly during the oestrous cycle. IGF-I mRNA concentrations were significantly higher on day 15 than on day 10, and IGF-I mRNA in the regressing corpus luteum at 48 h after administration of exogenous prostaglandin was significantly greater than in the early or mid-luteal phase (days 5 and 10). In contrast, there was no significant effect of day of the oestrous cycle on expression of mRNA for IGF-II and the type 1 IGF receptor in the corpus luteum. Expression of IGF-II mRNA was localized to a subset of steroidogenic luteal cells and was also associated with cells of the luteal vasculature. mRNA encoding the type 1 IGF receptor was widely expressed in a pattern indicative of expression in large and small luteal cells. These data demonstrate that the bovine corpus luteum is a site of IGF production and reception throughout the luteal phase. Furthermore, this study highlights the potential of IGF-II in addition to IGF-I in the autocrine and paracrine regulation of luteal function.

Human placental gonadotrophin-releasing hormone-like factors: an artefact of human placental peptidases?

Non-denatured human placental cytosol fractions displaced tracer binding in parallel with gonadotrophin-releasing hormone (GnRH) isoform and agonist peptides in GnRH-specific radioimmunoassays and radioreceptor assays. However, placental immuno- and receptor binding-GnRH-like activity was highly correlated with inactivation of GnRH tracers, suggesting that placental GnRH-like factors may be an artefact of ligand degradation during assay. The properties and inhibitor sensitivities of the major (125)I-labelled GnRH-degrading enzymes of term placental cytosol were studied using a dextran-coated charcoal (DCC) adsorption assay as a rapid screen for GnRH tracer inactivation. Three different activities were demonstrable: (i) a cathepsin D-like enzyme (M(r) 55 kDa), active against all radiolabelled GnRH isoforms and agonists tested, optimal at acid pH, and inhibited specifically by pepstatin; (ii) a metallo-thiol endopeptidase activity (M(r) 70 kDa) optimal at alkaline pH (7-9) which degraded GnRH isoforms to a greater extent than GnRH analogues, inhibited dose-dependently by low concentrations of thiol reagents (N-ethylmaleimide, thimerosal), chelating agents (o-phenanthroline, EDTA), and by tosyl-phenylalanyl-chloromethyl ketone but not by other serine protease inhibitors; and (iii) a bacitracin-sensitive enzyme optimal at physiological pH. These observations permitted the development of a robust radioreceptor assay which minimized GnRH tracer degradation. Under these assay conditions, the GnRH-like radioreceptor assay activity of human placental cytosol fractions was markedly reduced.

Human placental GnRH-like factors: parallel displacement in GnRH immuno- and receptor-binding assays can be caused by degradation of radiolabelled GnRH tracers.

Human term placental cytosol fractions decreased the specific binding of gonadotrophin-releasing hormone (GnRH) isoform tracers to placental membranes (and to rat pituitary GnRH receptors and anti-GnRH antibodies) in a dose-dependent manner, and in parallel to GnRH standard curves. However, cytosol fractions had little or no effect on the binding of two GnRH superagonist tracers. The specificity of placental binding sites for a range of GnRH-like and unrelated peptides was shown to be similar with GnRH isoforms or GnRH agonists as binding ligands, suggesting that isoforms and agonists did not bind to different forms of the GnRH-receptor. Inclusion of a cocktail of protease inhibitors during the preparation of placental cytosol significantly reduced immuno- and receptor-binding activity. Moreover, incubation of radiolabelled chicken GnRH II with placental cytosol led to marked inactivation of tracer, as assessed by radioreceptor and radioimmunoassays for GnRH, high resolution liquid chromatography, thin layer chromatography and adsorption to dextran-coated charcoal and other matrices. There was a good negative correlation between tracer degradation and apparent GnRH immuno- and receptor-binding activities. These results emphasize the important effects which proteases in un-denatured tissue extracts can have on radioreceptor and radioimmunoassays due to inactivation of peptide tracers, and suggest that previous measurements of receptor- and immuno-active GnRH-like factors may have been over-estimated due to peptidase action during the GnRH assay.

Stimulation of specific binding of [3H]-progesterone to bovine luteal cell-surface membranes: specificity of digitonin.

Non-genomic actions of progesterone have been described in the ovary, and luteal membranes of several species have been shown to possess specific binding sites for [3H]-progesterone. However, binding of radiolabelled progesterone to luteal membranes was demonstrable only in the presence of digitonin. Digitonin is a non-ionic detergent which is thought to act by forming one-to-one complexes with certain sterols. It is also a cardiotonic agent, inhibiting (Na+-K+) ATPase activity by interaction with the extracellular (ouabain/K+) binding site. We therefore investigated which properties of digitonin were responsible for its stimulatory actions on progesterone binding to bovine luteal membranes. A range of compounds with detergent, cardiotonic and or cholesterol-complexing activities were tested for their effects on [3H]-progesterone binding to bovine luteal membrane fractions, and on haemolysis of rat erythrocytes. Stimulation of progesterone binding to luteal membranes was highly specific for digitonin, and a number of ionic and non-ionic detergents, cardenolides, saponins and cholesterol-complexing reagents tested failed either to stimulate [3H]-progesterone binding to bovine luteal membranes in the absence of digitonin, or to inhibit binding specifically in the presence of digitonin. When digitonin was first reacted with excess cholesterol or pregnenolone to form the respective digitonides, stimulatory activity was greatly reduced, suggesting that the ability of digitonin to interact with (an) endogenous steroid(s) may be important in its action. High performance liquid chromatography (HPLC)-mass spectrometry of commercially available digitonin preparations indicated the presence of numerous minor impurities in most commercial digitonin preparations. Three major UV-absorbing peaks were isolated and characterised by mass spectrometry: all stimulated progesterone binding to bovine luteal membrane receptors in a dose-dependent manner, though to differing extents. Our data suggest that the unique action of digitonin on luteal membrane progesterone receptors is not related to its detergent or cardiotonic properties, but appears to be related to its ability to complex with membrane sterols.

Effects of alcohol on the human placental GnRH receptor system.

Isolation of human term placental membranes in the presence or absence of protease inhibitors indicated that protease inhibitors significantly reduced the amounts of [(125)I]-labelled gonadotrophin-releasing hormone (GnRH) binding to membrane GnRH-receptors in vitro by approximately 20%. This decrease was largely due to the ethanol used to dissolve the serine protease inhibitor, phenylmethylsulphonylfluoride (PMSF). Ethanol alone decreased the specific binding of [(125)I]-labelled GnRH isoform (IC(50), 7.9 +/- 0.8 mg/ml; n = 6) or agonist tracers (IC(50), 10.0 +/- 1.4 mg/ml; n = 6) to human placental membranes in a dose-dependent manner. Other alcohols also interfered with [(125)I]-GnRH isoform or agonist binding: inhibition increased with increasing carbon chain length and was dependent on the isomeric position of the hydroxyl group. Fractionation of term placental cytosol by gel chromatography demonstrated the presence of a high molecular weight fraction ( approximately 60-70 kDa) which inhibited [(125)I]-GnRH binding to human placental membranes. However, placental cytosol fractions did not cross-react significantly with a specific anti-GnRH antibody. Surprisingly, re-assay of cytosol fractions in the presence of a cocktail of protease inhibitors generated a factor (molecular weight approximately 40-50 kDa) which did cross-react strongly with the GnRH antibody. The generation of this factor was due to the ethanol solvent rather than to the protease inhibitors per se, as treatment of pooled 'latent' cytosol fractions with ethanol alone generated GnRH-like immunoactivity (irGnRH) which competed in parallel with GnRH standard. The amount of irGnRH generated depended on the concentration of ethanol added to the 'latent' cytosol fractions. However, ethanol had no effect on the assay in the absence of cytosol fraction, or with inactive cytosol fractions. Thus, ethanol can perturb the human placental GnRH/GnRH-receptor system in vitro in two distinct ways: by inhibition of GnRH binding to receptor, and by dissociation of complexed endogenous GnRH-like factor(s) from a GnRH-binding protein. It is postulated that high alcohol consumption in vivo may interfere with placental GnRH secretion/action and affect placental secretion of factors important to the establishment and maintenance of pregnancy.

Immune cells and cytokine production in the bovine corpus luteum throughout the oestrous cycle and after induced luteolysis.

Immune cells and their cytokine products have powerful local effects within body tissues. There has been great interest in the potential role of these cells, not only during destruction of the corpus luteum but also during its functional lifespan. In this study, lymphocytes, macrophages and major histocompatibility complex class II molecules were quantified using immunohistochemistry and the reverse transcription-polymerase chain reaction was used to detect mRNA for tumour necrosis factor alpha and interferon gamma within corpora lutea from three groups of cows: (1) corpora lutea collected at an abattoir and assessed visually into four stages (stage I (days 1-5), stage II (days 6-12), stage III (days 13-18) and stage IV (days 19-21) of the oestrous cycle); (2) corpora lutea collected around natural luteolysis (days 14-20); and (3) corpora lutea collected 6, 12 and 24 h after prostaglandin F 2 alpha-induced luteolysis. The numbers of T lymphocytes (CD5+ and CD8+) were significantly higher (P < 0.05) at stage IV and from day 16 onwards, before functional luteolysis. There were significantly higher numbers (P < 0.01) of macrophages at stages I, III and IV compared with stage II in visually staged tissue. Major histocompatibility complex class II molecules were increased (P < 0.05) at stages I and IV compared to stage II and at all times after induced luteolysis. Using reverse transcription-polymerase chain reaction, mRNA encoding tumour necrosis factor alpha and interferon gamma was detected in all luteal tissue collected around natural luteolysis and after induced luteolysis. These findings, particularly the increase in T lymphocytes before functional luteolysis, provide further evidence of a significant role for the immune system in affecting reproductive function in cows.

Bovine ovarian non-genomic progesterone binding sites: presence in follicular and luteal cell membranes.

We have shown recently that the bovine corpus luteum (CL) possesses specific luteal cell surface membrane binding sites for progesterone. We have now confirmed and extended these observations to compare the subcellular distribution of these binding sites in developing, mature and regressing CL. The median buoyant densities of luteal progesterone binding sites from early-, mid- and late-luteal phase CL were similar (though three of five density profiles for late-luteal phase CL showed association of steroid binding with a fraction with a lower density), and clearly resolved from nuclear, mitochondrial, lysosomal, peroxisomal, Golgi-endoplasmic reticulum-lysosomal and smooth endoplasmic reticulum markers. Specific binding of [3H]progesterone overlapped with the distributions of 5'-nucleotidase and luteinizing hormone receptor (luteal cell surface membrane markers) in both control and digitonin-treated gradients at all stages of the luteal phase. Since steroidogenic 'large luteal' and 'small luteal' cells of the CL are derived from the granulosa cells (GC) and theca of the preovulatory follicle, we also investigated whether similar receptors were present in the follicle, and describe for the first time specific membrane binding sites for progesterone in purified GC and thecal membranes from healthy bovine follicles of different sizes. Specific binding increased linearly with GC and thecal membrane protein concentration; however, it was detectable only when digitonin was included in the binding incubation. Binding sites were specific for progesterone; unlabelled progesterone competed for [3H]progesterone binding at low concentrations (IC50, 35 and 31 nmol/l) compared with testosterone (IC50, 905 and 870 nmol/l) and delta4-androstenedione (IC50, 1050 and 660 nmol/l) for GC and thecal receptors respectively. In contrast, oestradiol, oestrone, pregnenolone, cortisol, cholesterol, and a genomic progesterone receptor antagonist, RU486, competed poorly. Steroid binding was present in GC and thecal membranes of follicles of all sizes, but [3H]progesterone binding to GC membranes decreased significantly with increasing follicle size (P<0.02), perhaps indicating developmental regulation of GC membrane non-genomic progesterone receptors in the preovulatory bovine follicle. We suggest that these membrane steroid receptors may be involved in the autocrine/paracrine regulation of follicular function by progesterone.

Expression of monocyte chemoattractant protein-1 in the bovine corpus luteum around the time of natural luteolysis.

Monocyte chemoattractant protein (MCP-1) is a specific chemoattractant for monocytes/macrophages that could have a role in the influx of macrophages into the corpus luteum (CL) during structural luteolysis. In this study, reverse transcription-polymerase chain reaction and in situ hybridization were used to investigate MCP-1 mRNA expression in CL collected from 18 heifers between Days 15 and 20 of the estrous cycle. There was expression of mRNA encoding MCP-1 in luteal tissue from all cows; however, expression was greater in animals that had undergone luteolysis at the time of CL collection as compared to animals in which the CL was still functional. Similarly, in situ hybridization showed greater expression of mRNA encoding MCP-1 in CL after functional luteolysis. There was also evidence of increased MCP-1 mRNA expression in an animal with a functional CL where the systemic concentration of prostaglandin F2alpha metabolite was high at the time of tissue collection. T lymphocyte populations, identified by immunohistochemistry, had a distribution similar to that of cells expressing MCP-1 mRNA within the CL, but other cell types were also involved. These results demonstrate an increase in MCP-1 mRNA after functional luteolysis in the cow, which may be related to the influx of macrophages that occurs at this time.

Alternative splicing of the prolactin receptor gene generates a 1.7 kb RNA transcript that is linked to prolactin function in the red deer testis.

A cDNA encoding a putative non-membrane bound prolactin receptor was amplified by RT-PCR from red deer (Cervus elaphus) testis. Sequence analysis suggests that the testicular cDNA is generated by alternative splicing resulting in the deletion of exons 7 and 8, which code for: (a) the final 53 aa of the extracellular domain of the receptor including the fifth conserved cysteine residue and the WS x WS motif, (b) the entire transmembrane domain, (c) the first three cytoplasmic amino acid residues, and (d) two nucleotides of the fourth cytoplasmic amino acid codon. The resultant RNA would encode a putative protein of 174 aa due to a single bp frame shift and a premature stop codon. Northern blot analysis confirmed that the PCR-amplified cDNA is encoded by a specific 1.7 kb RNA transcript whereas the membrane bound receptor is encoded by transcripts of 3.5 and 2.5 kb. HPLC studies using media from 293 cells transfected with the 1.7 kb cDNA failed to detect any specific binding for prolactin. These data suggest that: (a) the deletion in the 1.7 kb transcript alters the structure of the prolactin binding domain in the putative protein encoded by the 1.7 kb transcript, and (b) alternative splicing of the prolactin receptor gene toward the 1.7 kb transcript is a means of down-regulating the expression of the full length prolactin receptor and hence may modify the role of prolactin in the testis of seasonally breeding mammals such as red deer. The sequence reported in this paper has been deposited in the Genbank/EMBL data base with accession number Y14753.

Specific non-genomic, membrane-localized binding sites for progesterone in the bovine corpus luteum.

Fractionation of bovine corpus luteum (CL) homogenates on continuous sucrose density gradients with and without preincubation with 3H-progesterone demonstrated high levels of tracer binding and high content of endogenous progesterone associated with particulate membrane fractions. Analysis of gradient fractions for a range of luteal plasma membrane and intracellular organelle marker enzyme activities indicated that endogenous progesterone content and 3H-progesterone-binding activity were associated with fractions enriched in luteal plasma membrane markers. This was confirmed by pretreatment of homogenates with the saponin, digitonin, prior to fractionation. Digitonin perturbed the buoyant density of luteal surface membrane markers and 3H-progesterone binding to a similar extent, but did not perturb the buoyant densities of other intracellular markers to the same degree. Interestingly, digitonin pretreatment also increased the proportion of progesterone tracer that entered the gradients. We consistently failed to demonstrate significant binding of 3H-progesterone to membrane fractions incubated with progesterone tracer in vitro. However, when digitonin was included in the in vitro binding assay, we observed a dramatic, dose-dependent stimulation of 3H-progesterone binding by digitonin. Other radiolabeled steroids tested (3H-cortisol, 3H-testosterone) bound poorly in the presence or absence of digitonin. 3H-Progesterone binding in the presence of optimal digitonin concentrations increased linearly with increasing luteal membrane concentration; was dependent on the pH, duration, and temperature of incubation; and low levels of progesterone (68 nM) competed for tracer binding. A range of other steroids tested (androgens, estrogens, corticosteroids, steroid precursors) competed at higher concentrations (10- to 100-fold) or did not compete at all for 3H-progesterone binding. There was no correlation between the hydrophobicity of various steroids and their ability to compete for binding. Moreover, a number of agonists and antagonists specific for the genomic progesterone receptor, agonists of peripheral benzodiazepine receptors, and inhibitors of a range of steroidogenic enzymes did not compete for 3H-progesterone binding. Western blots confirmed that detergent-solubilized progesterone-binding sites could be resolved from cytochrome P450 side-chain cleavage and 3beta-hydroxysteroid dehydrogenase. Moreover, extraction of bound steroid from the binding site and HPLC demonstrated identity to progesterone, suggesting that no metabolism of the progesterone tracer had occurred during incubation. Progesterone binding to membranes of large luteal cells was higher compared with binding to small luteal cells, and levels were similar in membranes prepared from CL at all stages of the luteal phase. We suggest that bovine luteal progesterone-binding sites may play a role either in sequestration of newly synthesized progesterone or in the mediation of autocrine and/or paracrine actions of progesterone in the CL.

Insulin-like growth factor binding protein -2 and -4 messenger ribonucleic acid expression in bovine ovarian follicles: effect of gonadotropins and developmental status.

This work is concerned with the role of insulin-like growth factor binding protein (IGFBP)-2 and -4 in the regulation of IGF bioactivity in bovine follicles during the development of dominance. We measured the expression of IGFBP-2 and -4 messenger RNA (mRNA) in small (1-4 mm) gonadotropin-sensitive follicles and medium (4-8 mm) and large (>8 mm) gonadotropin-dependent follicles using in situ hybridization. In healthy nonatretic bovine follicles, IGFBP-2 and -4 mRNA expression was confined to granulosa and theca tissue, respectively. Moreover, during the development of follicular atresia, there were distinct changes in the temporal and spatial expression of these genes. IGFBP-2 immunoactivity was localized in granulosa tissue and the basement membrane of healthy preantral follicles, whereas IGFBP-4 immunoactivity was localized in both theca and granulosa tissue. Of particular interest was the lack of IGFBP-2 mRNA expression in large (>8 mm) gonadotropin-dependent follicles, an observation that was confirmed by the lack of immunoreactive IGFBP-2 in these follicles. The regulation of IGFBP-2 and -4 mRNA expression in granulosa and theca cells was analyzed using a serum-free cell culture system. FSH inhibited the expression of IGFBP-2 mRNA in granulosa cells, whereas LH stimulated IGFBP-4 mRNA expression in theca cells. Our results provide evidence for the existence of different roles for IGFBP-2 and -4 in the developing follicle.

Ultra-structural characteristics of bovine granulosa cells associated with maintenance of oestradiol production in vitro.

We investigated whether the maintenance of oestradiol production by bovine granulosa cells (GC) in vitro was related to GC ultra-structure, and studied the effects of inclusion of serum as a cell attachment factor on oestradiol secretion, cell morphology and ultra-structure. Bovine granulosa cells from medium-sized follicles (4-8 mm diameter), in a serum-free (SF) culture system, maintained oestradiol production for 6 days, whereas oestradiol secretion by cells cultured in serum-coated (SC) wells declined rapidly with time, in culture. SF cells formed clumps consisting of two types of cells. Cells within clumps presented a phenotype similar to GC in vivo, being spherical, tightly joined by extensive gap junctions and interdigitated pseudopodia/microvilli, had abundant rough and smooth endoplasmic reticulum (ER) and mitochondria with trabecular cristae. In contrast, cells cultured in either SC wells or in the flattened base of cell clumps from SF cultures were enlarged, containing less rough ER, had fewer mitochondria (which tended to be round) and contained endosome-like structures, morphological characteristics suggestive of early luteinisation.

Co-purification of a ribonuclease and human chorionic gonadotrophin beta-core protein from human urine and displacement of 125I-human luteinizing hormone from Candida albicans binding sites by ribonucleases.

An 18 kDa pregnancy urine protein preparation, purified to apparent electrophoretic homogeneity as judged by silver-staining of polyacrylamide gels, inhibited binding of 125I-hLH (human luteinizing hormone) to Candida albicans microsomes, reacted with monoclonal and polyclonal antibodies raised against human chorionic gonadotrophin (hCG) beta-core protein and exhibited ribonuclease (RNase) activity. Eleven of the 12 amino acids at the N-terminus of a protein in this preparation were identical to those of the N-terminus of human non-secretory ribonuclease. These results indicate co-purification of hCG beta-core with a RNase. An 18 kDa RNase was also purified from a commercial hCG preparation (Chorulon). However, no RNase activity was detected in a highly purified commercial preparation (Profasi). Three commercial RNase preparations displaced 125I-hLH from C. albicans binders at extremely low concentrations (< 0.001 microg/ml RNase) whereas only slight displacement of 125I-hLH from sheep luteal binding sites was observed with very high concentrations of the RNases (100 microg/ml RNase). The co-purification of hCG beta-core and RNase from pregnancy urine and the displacement of 125I-hLH from C. albicans binding sites by RNases may be related to the close relationship that has been identified between mammalian RNase inhibitors and the extracellular domain of gonadotrophin receptors. The presence of RNase in commercial preparations of gonadotrophins should be borne in mind during any investigations that involve impure preparations of these hormones.

Protein kinase C- and Ca2+ ionophore-stimulated production of reactive oxygen species in mechanically dispersed isolated bovine luteal cells.

We measured the production of reactive oxygen species (ROS) using luminol-horseradish peroxidase-induced chemiluminescence in mechanically dispersed cell suspensions from bovine corpus luteum (CL). Since other cell types besides luteal cells were present in crude cell suspensions from CL, cell preparations were purified by centrifugation on Percoll. Only cell suspensions that gave no significant response when stimulated with formyl-methionyl-leucyl-phenylalanine, a potent stimulator of ROS production by phagocytes, were used routinely. Basal ROS production by purified bovine luteal cell preparations was low but could be stimulated rapidly and in a dose-dependent manner by nanomolar concentrations of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), though only in cells purified by fractionation on Percoll. Luteal ROS responses to PMA were quenched by returning bovine erythrocytes to purified luteal cells, or by exogenous catalase or superoxide dismutase. The magnitude of the response to PMA varied markedly from one luteal cell preparation to another but appeared to be unrelated to the stage of the luteal phase of the CL from which the cells were prepared. The luteal ROS response to PMA was blocked by staurosporine, an inhibitor of PKC. Although the inactive phorbol ester (4alpha-phorbol didecanoate; 4alphaPDD) alone had little or no effect on luteal ROS production, 4alphaPDD significantly potentiated the effects of submaximal concentrations of PMA in a dose-dependent manner. ROS production could also be stimulated by the Ca2+ ionophore A23187. This response was rapidly abolished by treatment with EDTA or EGTA. A23187 also augmented the response to submaximal PMA levels: however, pretreatment with 4alphaPDD did not significantly enhance the ROS response to A23187. In conclusion, we have shown that isolated bovine luteal cell suspensions are capable of generating a marked acute ROS response triggered by activation of PKC and/or elevation of cytosolic calcium.

The recruitment of ovarian follicles is enhanced by increased dietary intake in heifers.

The objective of this study was to determine whether dietary-induced changes in growth hormone (GH), insulin, and IGF-I alter the pattern of ovarian follicular development in heifers. Twenty-eight 2- to 3-yr-old Hereford-Friesian heifers were equally allocated (n = 7) to one of four dietary treatments: 1) Control (C) fed a maintenance diet as two meals per day; 2) twice maintenance (2M2) given as two meals per day; 3) twice maintenance (2M6) given as six meals per day; 4) feed-deprived (F) fed maintenance requirements as for C, changed to straw ad libitum for 3 d from a synchronized estrus. On d 4 (estrus = d 0) heifers were transferred to grass silage (ad libitum access) until the end of the experiment. Blood samples were collected hourly for 10 h on d 1 and 3 and daily for an additional 14 d. Follicular development was monitored daily by ultrasonography until d 14. The number of small follicles (< 4 mm) was increased (P < .05) by 37% on d 1 and 2 in 2M2 and 2M6 heifers, with no carryover effect of nutrition to the second follicular wave. Number of medium-sized (4 to 8 mm) and large (> 8 mm) follicles did not vary (P > .05) among treatments. The FSH concentrations were not different (P > .05) among treatments. Insulin concentrations were higher (P < .05) in 2M2 and 2M6 heifers than in C or F heifers, with no carryover after the diet was changed. Increase in number of small follicles was independent of changes in FSH and IGF-I, negatively associated with GH, but positively associated with circulating insulin. These results indicate that a short-term increase in nutritional plane can affect follicular recruitment in cycling heifers.