T. A. Bramley

ROLE OF GROWTH HORMONE AND INTRAFOLLICULAR PEPTIDES IN FOLLICLE DEVELOPMENT IN CATTLE

Abstract Ovarian function in cattle is controlled by complex local and systemic feedback mechanisms which ensure that in >96% of estrous cycles only one follicle will ovulate. Although there is a primary requirement for gonadotropins in stimulating follicular growth, particularly during the final stages of follicle maturation, it is now becoming apparent that locally-produced factors, such as insulin-like growth factor-I (IGF-I) and inhibin, have a modulating role. In addition to gonadotropins, other systemic agents such as metabolic hormones (GH, IGF-I and insulin), can also influence follicular growth. The ability to control the growth of the dominant follicle, together with improved understanding of the competing mechanisms controlling follicular recruitment and growth, should enable more precise control of ovarian function and superovulation in cattle.

Pretreatment with recombinant bovine somatotropin enhances the superovulatory response to FSH in heifers.

One of the primary limiting factors to superovulation and embryo transfer in cattle has been the large variability in response, both between and within animals. It appears that the primary source of this problem is the variability in the population of gonadotropin-responsive follicles present in ovaries at the time of stimulation. We have shown that treatment of heifers with recombinant bovine somatotropin (rbGH) increases the number of small antral follicles (2 to 5 mm) and, therefore, enhances the subsequent superovulatory response to eCG. To investigate further the potential of using this approach to improve superovulatory regimens in cattle, the effect of rbGH pretreatment on the response to pituitary FSH was studied. The estrous cycles of 16 heifers were synchronized using PGF2alpha. On Day 7 of the synchronized cycle, half of the animals were injected with 320 mg sustained-release formulated rbGH, while the other half received 10 ml saline. Five days later, all heifers were given a decreasing-dose regimen of twice daily injections of oFSH for 4 d, incorporating an injection of PGF2alpha with the fifth FSH treatment, to induce superovulation. All animals were artificially inseminated twice with semen from the same bull during estrus. Ova/embryos were recovered nonsurgically on Days 6 to 8 of the following estrous cycle, and the ovulation rate assessed on Day 9 by laparoscopy. Using the same animals as described above, the experiment was repeated twice, 3 and 6 mo later, with no laparoscopy in the third experiment. The animals were randomized both between experiments and for the day of ova/embryo collection. Pretreatment of heifers with rbGH significantly (P < 0.01) increased the number of ovulations, total number of ova/embryos recovered and the number of transferable embryos. The percentage of transferable embryos was significantly (P < 0.05) increased by rbGH pretreatment. In addition, the incidence (2/16) of follicular cysts with a poor ovulatory response (< 6 ovulations) for the rbGH-pretreated heifers was significantly lower (P < 0.05) when compared with the incidence (7/16) in the control animals. It is concluded that pretreatment with rbGH may provide a useful approach for improving superovulatory response in cattle.

Treatment with recombinant bovine somatotrophin enhances ovarian follicle development and increases the secretion of insulin-like growth factor-I by ovarian follicles in ewes

To investigate the effect of recombinant bovine somatotrophin (rGH) on ovarian folliculogenesis in sheep, 18 mature Scottish Blackface ewes were assigned randomly to two treatment groups. Starting from day 5 of the synchronised oestrous cycle, animals were injected daily with either vehicle (control group) or 12.5 mg rGH (rGH-treated group) for 7 days. Blood samples were collected once daily during the experimental period for the measurement of growth hormone (GH), insulin-like growth factor-I (IGF-I), insulin, follicle-stimulating hormone (FSH), luteinising hormone (LH) and progesterone. At the end of treatment animals were killed and ovaries collected. All follicles at least 1.0 mm in diameter were dissected out and diameters measured to assess follicular populations for individual animals. Five small follicles (1.0–3.4 mm in diameter) and all the large follicles (at least 3.5 mm) from each animal were incubated in 1 ml of Medium 199 for 1 h. Medium was then changed and incubation continued for a further hour. All medium samples were assayed for IGF-I, oestradiol, testosterone and progesterone. Treatment of ewes with rGH had no effect on the total number of follicles at least 1.0 mm in diameter (control, 34.4 ± 2.6; rGH-treated, 31.3 ± 1.4; P > 0.2). However, when follicles were further classified into different size categories (1.0–2.0, 2.1–3.0, 3.1–4.0, 4.1–5.0, 5.1–6.0 and over 6.0 mm in diameter), the population of follicles 2.1–3.0 mm in diameter was significantly increased by rGH treatment (control, 9.2 ± 0.7; rGH-treated, 13.8 ± 1.1; P = 0.02). The number of follicles of 3.1–4.0 mm diameter in the rGH-treated group tended to be increased (P = 0.09), whilst the population of follicles 1.0–2.0 mm in diameter was reduced (P = 0.07). Treatment of ewes with rGH significantly increased peripheral concentrations of GH (P < 0.01), IGF-I (P < 0.01), insulin (P < 0.01) and progesterone (P < 0.05). There was no effect of rGH treatment on circulating concentrations of FSH and LH. Both large and small follicles from rGH-treated ewes secreted significantly (P < 0.001) more IGF-I (37.8 ± 2.2 ng ml h−1, n = 50) than follicles from the control group (26.7 ± 1.6 ng ml−1 h−1, n = 73). However, there was no significant effect of rGH treatment on the secretion of oestradiol, testosterone and progesterone by either large or small follicles. It is concluded that treatment of mature ewes with rGH can enhance the development of ovarian follicles to the gonadotrophin-dependent stages. Furthermore, rGH appears to act through increased secretion of ovarian IGF-I, as well as increased peripheral concentrations of IGF-I and insulin.

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.

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.

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.

Subcellular fractionation of the human corpus luteum: distribution of GnRH agonist binding sites

We have investigated the subcellular localization of GnRH agonist (GnRHA) binding sites in the human corpus luteum. Corpora lutea (CL) were obtained from 13 women undergoing laparotomy during the luteal phase of the menstrual cycle. CL homogenates were subjected to subcellular fractionation by either differential rate centrifugation or by sucrose density gradient fractionation with or without digitonin perturbation. Fractions were then assayed for a number of marker activities characteristic of the major intracellular organelles and cell-surface membranes, and for specific binding of [125I]GnRHA. Specific binding of [125I]GnRH agonist was greatest in regions of the gradient (density, 1.12-1.15 g/cm3) enriched in cell-surface and endoplasmic reticulum membrane markers. Little or no GnRHA binding was associated with fractions enriched in nuclei, mitochondria or lysosomes. Digitonin treatment demonstrated that the majority of GnRHA binding sites were associated with luteal cell plasma-membranes: however, some GnRHA binding was also associated with endoplasmic reticulum. These results indicate that: (i) human luteal GnRHA binding sites were not associated with lysosomal proteases, (ii) a significant fraction (59%) of GnRHA binding sites was localised on the luteal cell-surface membrane. Specific GnRHA binding to both cell-surface and endoplasmic reticulum membranes may indicate an extensive turnover of membrane binding sites.

Particulate binding sites for steroid hormones in subcellular fractions of the ovine corpus luteum: properties and hormone specificity.

We have described previously the presence of binding sites in particulate fractions of the porcine corpus luteum (CL) which were specific for progesterone. We now demonstrate the presence of similar progesterone-specific binding sites in particulate fractions of the ovine CL. Preincubation of ovine luteal membranes with radiolabelled steroids demonstrated binding of progesterone and pregnenolone to a low-density particulate fraction (1.07-1.09 g/cm3). Preincubation with digitonin perturbed the buoyant density of this fraction (to 1.10-1.14 g/cm3) without causing release of steroid. Androgens and oestrogens did not bind appreciably to control luteal membranes, but were bound when preincubated with digitonin. In contrast, steroid conjugates (oestrone sulfate, pregnanediol glucuronide), cortisol, fatty acids (arachidonic acid, prostaglandin F2 alpha) and cholesterol ester failed to bind to ovine luteal membranes, with or without digitonin pretreatment. The effects of digitonin on steroid binding led us to examine its effects on steroid binding to ovine luteal membrane fractions in vitro. Specific progesterone binding was absent in the absence of digitonin, even at very high membrane concentrations. However, binding of 3H-labelled progesterone was stimulated 5-15-fold in a dose-dependent fashion by increasing digitonin concentrations, reaching a plateau at about 100 microM. In the presence of digitonin, [3H]progesterone binding increased linearly with luteal membrane concentration. Other detergents, saponins and cardiotonic steroids tested did not stimulate progesterone binding to ovine luteal membranes. [3H]Progesterone binding was dependent on the pH, duration and temperature of incubation. Unlabelled progesterone decreased binding of [3H]progesterone (half-maximal displacement of specific binding (IC50) at about 60 nM) whereas androgens were less potent (IC50, 500-3300 nM), whilst a wide range of other steroids and inhibitors of steroidogenic enzymes were ineffective, except at very high concentrations. Similarly, a number of progesterone receptor agonist and antagonist analogues failed to compete for progesterone binding to luteal membranes, suggesting that these binding sites were unrelated to progesterone receptors. Modifications to the 3, 4, 5 and 11 positions of progesterone, removal of the steroid side-chain or aromatization of the A-ring decreased binding potency dramatically, whereas changes to the 17 or 20 positions had relatively minor effects. Our results indicate the presence of a low density particulate fraction in ovine corpora lutea which contains specific binding sites for endogenous and exogenous progesterone.

Co-purification of a ribonuclease and human chorionic gonadotrophin β-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.

Failure to detect gonadotrophin-releasing hormone receptors in human benign and malignant breast tissue and in MCF-7 and MDA-MB-231 cancer cells

We have measured the binding of radiolabelled analogues of gonadotrophin-releasing hormone (GnRH) to homogenates of human breast cancer and benign breast tissue, and to MCF-7 and MDA-MB-231 cell lines. Although incubation of breast cancer homogenates with the 125I-labelled GnRH agonist analogues, buserelin [(D-Ser tBU6)GnRH 1-9 ethylamide] and tryptorelin [(D-Trp6) GnRH 1-9 ethylamide] appeared to show significant though low, specific GnRH agonist binding in a high proportion of breast cancers (32/42 for buserelin; 15/32 for tryptorelin) and benign breast tissues (13/16 for buserelin; 10/12 for tryptorelin), after correction for displaceable binding in control assay tubes, GnRH agonist binding to breast tissue was no longer apparent. The lack of specific binding was not due to inactivation of GnRH agonist tracers, as > 86% of the unbound tracer was still capable of rebinding to fresh placental membranes after incubation with breast cancer homogenates. GnRH agonist did not bind to MCF-7 and MDA-MB-231 cells, however GnRH agonist tracer inactivation following exposure to these cells was very high. We have shown recently that human placental receptors bound salmon GnRH and chicken GnRH II as well as GnRH agonists, but not other isoforms of GnRH. However, no isoform of GnRH bound significantly to human breast tumour tissue. In summary, we could not confirm the presence of specific GnRH binding sites in homogenates and membranes from human breast tissues in this study. Low levels of apparently specific binding of GnRH agonist tracers could be accounted for entirely by displacement of tracer from assay tubes. Inability to demonstrate specific binding was not due to extensive inactivation of GnRH tracers (although this may be a factor in the failure to demonstrate GnRH binding to MCF-7 and MDA-MB-231 cell lines).