PubMed Health⌕ Search

Biomedical subjects

M G Hunter

Publications and source records attributed to M G Hunter.

104 records · Page 6Linked to original sources
PubMed1985

The functional activity of adult mouse Leydig cells in monolayer culture: effect of lipoproteins, pregnenolone and cholera-toxin.

Further studies were carried out on purified mouse Leydig cells to determine why they lose their hormone responsiveness after several days in monolayer culture. The effects of cholera-toxin on cyclic AMP and testosterone production were examined. It was found that cyclic AMP production could still be maximally stimulated by cholera-toxin after 4 days in culture when response to luteinizing hormone (LH) has declined. Testosterone production was, however, not maintained. Because this decline in testosterone production may have been due to the lack of a suitable substrate after several days in culture, cells were cultured initially in the presence of exogenous pregnenolone and low-density lipoproteins (LDL). Both substances were found to enhance basal and LH-stimulated testosterone production and to extend responsiveness of the cells until at least day 4, but by day 7 response was lost. Cells were then cultured in the presence of rat and human LDL and HDL and in both cases LDL was found to enhance consistently testosterone production, but HDL was much less effective. Scanning and transmission electron micrographs showed that changes in cell shape occurred during culture, but indicated that the cells were not depleted of lipid droplets by the end of culture or after LH stimulation. It is concluded that the eventual decline in testosterone synthesis is not due to lack of substrate, although the addition of exogenous substrate does extend the period of responsiveness. Nor is it due to a decrease in adenylate cyclase activity. At least part of the lesion is caused by a decrease in the enzymes required for the conversion of pregnenolone to testosterone.

Animals↗
PubMed1985

Basic physiology of follicular maturation in the pig.

The pig is an excellent animal in which to study the control of folliculogenesis in a polytocous species, and particularly to examine the inter-relationships between follicles from the same animal. Follicle recruitment occurs from the proliferating pool, and various studies suggest that this recruitment occurs between Days 14 and 16 of the oestrous cycle. The growth of follicles selected for ovulation is associated with rapid atresia of smaller follicles and a block to their replacement in the proliferating pool. However, there is a considerable range in the morphological and biochemical development of the dominant follicles in the early follicular phase, suggesting that follicles are recruited at markedly different stages of development, or that recruitment continues into the follicular phase. A significant and predictable relationship has been established between follicular diameter and follicular fluid volume, and a comparison of these two characteristics demonstrates a gradual increase in follicular tissue volume as a proportion of total volume. Growth of follicles from 2 to 4 mm is associated with a proportional increase in granulosa cell numbers, but above 4 mm the relationship is very variable even in selected follicles that are steroidogenically active. Therefore, the number of granulosa cells cannot be used as an indicator of atresia in pig follicles. LH receptors are present in thecal tissue throughout development, reaching maximal levels on Day 20 of the oestrous cycle and declining on Day 21. Granulosa cells possess receptors for LH only in the later stages of maturation, and again these are maximal on Day 20. The pattern of steroidogenesis in pig follicles is consistent with the two-cell theory of steroidogenesis in that androgen produced by the theca is aromatized to oestrogen by the granulosa cells, However, in contrast to that of many other species, the theca of the pig also produces oestradiol in quantities comparable to those secreted by the granulosa. As with morphological development, the selected population of preovulatory follicles shows a considerable range of biochemical development and follicles of identical size may show great dissimilarity in follicular fluid steroid concentrations and LH binding. Androgen availability rather than aromatase activity appears to be the limiting factor for steroidogenesis. There are also several nonsteroidal factors which have been isolated from porcine tissue and play some role in follicular maturation. Although exogenous gonadotrophins are effective in promoting follicular development, other factors of extra- or intra-ovarian origin may limit follicular responsiveness to gonadotrophins.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Prolactin stimulates steroidogenesis by human luteal tissue in vitro.

Human luteal tissue recovered from varying stages of the luteal phase was minced and incubated for 3 h and the effect of human chorionic gonadotrophin (hCG), prolactin and hCG + prolactin on progesterone and oestradiol production measured. While hCG generally enhanced both progesterone and oestradiol synthesis, prolactin alone at either 20 or 200 micrograms/l had no significant effect on steroidogenesis. When prolactin was added along with hCG in four of six corpora lutea, however, progesterone production significantly increased and in three of six corpora lutea oestradiol production was increased above that induced by hCG alone. It is concluded that prolactin may play some role in the control of steroidogenesis by the human corpus luteum.

Chorionic Gonadotropin↗
PubMed1983

11. Testicular and epididymal function. Effects of isolation and purification procedures on the viability and properties of testis Leydig cells.

The effect of the isolation and purification procedures used to obtain mouse and rat Leydig cells on their viability and steroidgenic properties has been investigated. It was found that mechanical dispersion of rat testes prior to collagenase dispersion had a deleterious effect on the steroidogenic capacity of the cells. Omission of the mechanical disruption and separation of the collagenase-dispersed cells on Percoll gradients yielded 3 bands of cells. Band III (density 1.070 g/ml) contained highly pure viable Leydig cells of high steroidogenic capacity. Diaphorase histochemistry (but not trypan blue exclusion) revealed that bands I and II contained high amounts of damaged Leydig cells. These results are discussed in relation to the previously reported heterogeneity of Leydig cells. Both mouse and rat Leydig cells (band III) in monolayer culture maintained their responsiveness to LH for several days. The rat (but not mouse) Leydig cells contained LHRH receptors and responded to LHRH in a biphasic manner: initially a stimulation of both basal and LH increased steroidogenesis occurred which after 24 h became inhibitory.

Animals↗
PubMed1982

Stimulation and inhibition by LHRH analogues of cultured rat Leydig cell function and lack of effect on mouse Leydig cells.

The effect of 2 luteinizing hormone-releasing hormone (LHRH) analogues(10-8-10-6 M) on the functional activity (testosterone and cyclic AMP production and [125I]hCG binding) of purified mouse Leydig cells in culture was examined. The analogues were found to have no significant effect on the cells over a period of 3 days. No specific binding of a labelled analogue to impure or pure mouse Leydig cells could be detected. In contrast high levels of specific binding to impure rat interstitial cells occurred. Centrifugation of the rat interstitial cells on 0-90% Percoll gradients showed that the LHRH analogue bound specifically to the active lutropin-responsive Leydig cells. The purified rat Leydig cells were cultured in the presence of LHRH analogue (ICI 118630) (10-7 M) and after an initial lag period (2h) a marked stimulation of testosterone production occurred over a 32-h period (up to 400 ng/10(6) cells). The response to LH alone increased with time in culture up to 10 h, and the LHRH analogue enhanced this LH-stimulated testosterone production. When the cells were cultured for longer time periods (24 h) the LHRH analogue was found to inhibit LH-stimulated testosterone production at all concentrations of LH used (p less than 0.01). The LHRH analogue had no consistent effect on LH-stimulated cyclic AMP production, although when added alone, cyclic AMP production was increased. These results show that LHRH analogues do not bind to or have any detectable effect on mouse Leydig cells in vitro. However, LHRH analogue does bind specifically to purified rat Leydig cells. After a short lag period the analogue stimulates testosterone production which turns to inhibition after 20 h in culture.

Animals↗
PubMed1982

Catecholamine stimulation of testosterone production via cyclic AMP in mouse Leydig cells in monolayer culture.

The effects of the beta-adrenergic hormone agonist, isoproterenol, on testosterone and cyclic AMP production in mouse Leydig cells in culture have been investigated. It was found that isoproterenol increased testosterone production on days 1, 2 and 3 of culture but not in freshly cultured cells. Cyclic AMP production was however increased on all days of culture. In subsequent studies carried out on day 2 of culture the amounts of testosterone formed during incubation with isoproterenol were 20-90% of those obtained with maximum stimulating levels of luteinizing hormone. The amounts of cyclic AMP formed were extremely low compared with those obtained with luteinizing hormone (22 +/- 5.3 and 2320 +/- 100 pmoles/10(6) cells/2 h respectively). Isoproterenol (10(-8) -10(-7) M) gave a significant increase in testosterone production and reached a maximum with 10(-6) M. Similar dose-response curves for cyclic AMP production were obtained. The stimulation of cyclic AMP and testosterone by isoproterenol was highly dependent on the presence of the phosphodiesterase inhibitor, methylisobutylxanthine. Propranolol blocked, in a dose-dependent manner, both isoproterenol-stimulated cyclic AMP and testosterone production. In the presence of excess luteinizing hormone no additional effects of isoproterenol were detected. Epinephrine also stimulated testosterone production. It is concluded that catecholamines stimulate testosterone production in mouse Leydig cells in monolayer culture and that this effect if mediated by cyclic AMP.

1-Methyl-3-isobutylxanthine↗
PubMed1982

The functional activity of adult mouse Leydig cells in monolayer culture. Effect of lutropin and foetal calf serum.

Purified Leydig cells were obtained from adult mouse testes by mechanical dispersion followed by Percoll density-gradient centrifugation as described by Schumacher et al. (1978). The cells were then established in monolayer culture by maintaining them in medium and 10% serum at 32 degrees C in 95% O2, 5% CO2. The cells rapidly attached to the culture dishes, gradually flattened and became epitheloid in appearance. Testosterone production by the cells in response to maximum stimulating levels of LH (100 ng/ml) and dibutyryl cyclic AMP (1 mM) was maintained for at least 2 days (approximately 1 microgram/10(6) cells/2h) and then declined to lower levels by days 3-4. Cyclic AMP production in response to LH was higher on day 1 than day 0 and then declined to lower levels by days 3-4. Binding of [125I]hCG was similar on day 0 and day 1 (approximately 20 fmoles/10(6) cells) and then declined to lower levels by days 3-4. The functional activity of the cells cultured in 0, 1 and 10% foetal calf serum was also examined; no significant effect of the serum on LH-stimulated testosterone or cyclic AMP production was found; however, a decrease of up to 50% in the binding of [125I]hCG to the Leydig cells occurred in the presence of serum. These results demonstrate that the function of differentiated adult Leydig cells can be maintained for at least 2 days in culture.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Analysis of btuB receptor function by use of nonsense suppression.

Informational suppression of btuB nonsense mutants allows the study of the effect of known, single amino acid substitutions on receptor function. We found that ligand uptake is largely unaffected by such amino acid changes. The few instances in which certain substitutions destroyed sensitivity to the two lethal agents (phage BF23, colicin E3) without affecting vitamin B12 uptake suggest a common region on the btuB receptor involved in the binding of these proteinaceous agents.

Colicins↗
PubMed1981

Effect of hCG, cAMP and FSH on steroidogenesis by human corpora lutea in vitro.

Human corpora lutea of various ages were minced and incubated in the presence of hCG (10 i.u./ml), cAMP (10 mM) or FSH (20 mu/ml) and production of progesterone and oestradiol was measured. Cyclic AMP and hCG stimulated progesterone and oestradiol production during at least the mid- and late luteal phases, but FSH stimulated only oestradiol production during the early and mid-luteal phases and had no effect on progesterone production. This demonstrates that progesterone and oestradiol synthesis by the human corpus luteum can be independently controlled.

Chorionic Gonadotropin↗
PubMed1981

Responsiveness in vitro of porcine luteal tissue recovered at two stages of the luteal phase.

Corpora lutea (CL) were recovered from pigs at 6 1/2 or 12 1/2 days after induced ovulation and fragments of tissue and isolated cells were incubated for 3 h in vitro. Progesterone synthesis by tissue and cells from 12 1/2-day CL was stimulated by bovine LH (1 micrograms/ml), dibutyryl cyclic AMP (10 mM) and hCG (10-50 i.u./ml) but 6 1/2-day CL, although having a higher level of control synthesis, did not respond to these substances. The addition of testosterone (100 ng/ml) to the incubations was found to suppress the response to bovine LH in the 12 1/2 day tissue and cells. Separation of the dissociated luteal cells into populations of large and small sized cells demonstrated that the large cells were more synthetically active than the small cells, but cells of both sizes in the 12 1/2-day CL responded to bovine LH. These results support the concept that the porcine CL is autonomous for much of its lifespan.

Animals↗
PubMed1976

Non-identical behaviour of the subunits of rabbit muscule creatine kinase.

1. The dimeric enzyme creatine kinase (ATP: creatine N-phospotransferase, EC 2.7.3.2) from rabbit muscle was reacted with three separate reagents, each of which specifically modifies one thiol group per subunit. 2. The reactions of the enzyme with these reagents (4-chloro-7-nitrobenzofurazan, 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetate) all behave as normal second-order processes. This indicates that the thiol groups on the two subunits of the enzyme react at the same rate as each other in all three cases. 3. The effects of various ligands (Mg2+, ADP and creatine, and combinations of these) on the kinetics of the reactions were studied. In all cases the reactions behave as normal second-order processes. 4. In the presence of the ligand combination Mg2+ plus ADP plus creatine plus nitrate, which has been postulated to form a "transition state analogue" complex with the enzyme, the reactions of the thiol group show considerable deviation from second-order kinetics. This indicates that the thiol groups on the two subunits react at different rates from each other. A similar effect is also noted in the presence of the combination ADP plus creatine plus nitrate. 5. The binding of ADP to the enzyme (studied by equilibrium dialysis) is hyperbolic in the absence of other ligands or in the presence of Mg2+ or Mg2+ plus creatine. The dissociation constant is similar in all three cases. 6. In the presence of creatine plus nitrate (with or without Mg2+) the bindings of ADP to the enzyme is tightened considerably and the binding plots indicate the presence of either negative interactions between the subunits or two distinct types of binding sites. 7. Possible causes for the observed non-identical behaviour of the two subunits of the enzyme are discussed.

4-Chloro-7-nitrobenzofurazan↗
PubMedDate not supplied

The physiology of follicular maturation in the pig.

Exogenous gonadotrophin (eg. PMSG) treatment results in follicular growth in most reproductive states, and suggests that lack of FSH and LH-like activity is a likely cause of arrested-follicular development in the pig. Endogenous changes in gonadotrophins between reproductive and non-reproductive states are consistent with this concept. However, changes in the pattern of LH secretion, rather than clearly defined changes in FSH release, appear to be the effective trigger for follicular recruitment. In other situations in vivo, follicular growth is initiated in the absence of demonstrable changes in LH or FSH secretion, suggesting that other hormones interact to sensitize the ovary to gonadotrophins. Such effects have been reported for insulin, cortisol, thyroxine and IGF-1 in vitro, and the steroid environment of follicles also affects gonadotrophin receptors and receptor-mediated mechanisms. Once initiated, the regulation of follicle maturation also involves interactions between gonadotrophins and steroids, as well as other intra-ovarian regulators. Co-operation exists between theca and granulosa in the synthesis of steroids and androgen precursor availability is an important regulator of the oestrogenic activity of follicles. Considerable heterogeneity exists in the morphological and biochemical maturity of preovulatory follicles and inter-follicular regulation via endocrine and paracrine mechanisms may be an essential component of the selection process.

Animals↗