Modelling the formation of necrotic regions in avascular tumours.

The mechanisms underlying the formation of necrotic regions within avascular tumours are not well understood. In this paper, we examine the relative roles of nutrient deprivation and of cell death, from both the proliferating phase of the cell cycle via apoptosis and from the quiescent phase via necrosis, in changing the structure within multicellular tumour spheroids and particularly the accumulation of dead cell material in the centre. A mathematical model is presented and studied that accounts for nutrient diffusion, changes in cell cycling rates, the two different routes to cell death as well as active motion of cells and passive motion of the dead cell material. In studying the accumulation of dead cell matter we do not distinguish between the route by which each was formed. The resulting mathematical model is examined for a number of scenarios. Results show that in many cases the size of the necrotic core is closely correlated with low levels in nutrient concentration. However, in certain cases, particularly where the rate of necrosis is large, the resulting necrotic core can lead to regions of non-negligible nutrient concentration-dependent upon the mode of cell death.

Follicular development in the immature guinea-pig.

A method for making statistical comparisons between populations and types of antral follicles within the ovary of the immature guinea-pig is described. This was used to demonstrate that atresia in the antral follicles first appeared at around 14 days of age, but that the population of healthy antral follicles remained remarkably consistent throughout the second half of the prepubertal period, since neither the total number of healthy antral follicles nor the proportion of atretic follicles changed significantly. However, the maximum diameter of the antral follicles gradually increased with age, from 400 micron at 7 days to 1000 micron at 28 days, 4 days before the vagina first opended. Because the percentage distribution of all types of follicles was so consistent at any age, although individuals showed substantial differences in the numbers of follicles within each ovary, it is suggested that mechanisms operate which regulate the total population of antral follicles within the ovary.

Follicular development in the adult guinea-pig and responses to human chorionic gonadotrophin.

The development of antral follicles and of atretic follicles throughout the cycle of adult guinea-pigs is a continuous process, but there are two stages when atresia is most pronounced: immediately after oestrus and in the late luteal phase. New atretic antral follicles were not found in the ovaries until around day 10 of the cycle, when an injection of HCG caused atresia of the medium-sized antral follicles within the ovary and luteinization of the largest follicles but spared the smallest antral follicles. Following the induced atresia, cycle lengths were prolonged, but the population of antral follicles could be restored to normal within 10 days of gonadotrophin treatment. It is suggested that the growth rate of antral follicles is flexible and proceeds most rapidly at the end of the luteal phase. It is not clear whether ovarian steroid play an integral part in regulating follicular growth and atresia.

Characterization of transforming growth factor-alpha receptors in the avian ovary: alterations in ligand binding to granulosa cells during follicular maturation.

The presence of epidermal growth factor receptors (EGF-R) and the ligands epidermal growth factor/transforming growth factor-alpha (EGF/TGF alpha) have been reported in mammalian ovaries where they are implicated in folliculogenesis and steroidogenesis. Evidence is presented to show that authentic EGF/TGF alpha receptors are expressed by the avian granulosa cells. The TGF alpha receptors (TGF alpha-R) from chicken granulosa cells were characterized by specific binding of 125I-human TGF alpha. In this study, competition with human EGF, human TGF alpha, human IGF-I, human basic fibroblast growth factor (bFGF) and insulin for 125I-human TGF alpha binding demonstrated that the avian granulosa cell TGF alpha-R binds human EGF with 300-fold lower affinity than human TGF alpha. IGF-I, bFGF and insulin did not displace bound 125I-TGF alpha. Scatchard analysis showed that a single class of high-affinity binding sites is present on the granulosa cells (Kd 0.23 +/- 0.009 nM). However, the number of binding sites altered during follicular maturation with a significant decline in the most mature follicle. These results go some way to explaining the basis for the changing sensitivity of avian granulosa cells to EGF/TGF alpha stimulation as they mature. In addition, the gonadotrophins, LH and FSH, increased the number of receptors in cultured granulosa cells and may therefore partially influence folliculogenesis and steroidogenesis through this route.

Changes in the concentration of luteinizing hormone in plasma during development in the guinea-pig.

The concentration of LH in the plasma of guinea-pigs from day 50 of gestation to day 45 of postnatal life was assayed by radioimmunoassay utilizing a cross-reaction with anti-ovine LH antiserum. The effect of gonadectomy in infancy and in the adult upon the plasma concentration of LH was also studied. The LH concentration in the plasma of male or female foetuses was high immediately prenatally and fell at birth. High levels of LH were again detected in male, with a lesser increase in female, guinea-pigs over the first 10 days postnatally. Maternal plasma concentrations of LH remained consistently low. Removal of the gonads on days 0, 5, 10, 15, 25 or 35 of postnatal life, followed by blood collection at autopsy 10 days later, caused a significant rise in plasma LH content at all ages. The rise in plasma LH after gonadectomy in adults was less marked in male than in female guinea-pigs.

The inhibition of androstenedione production in mature thecal cells from the ovary of the domestic hen (Gallus domesticus): evidence for the involvement of progestins.

In the 24 hours before ovulation, there is an abrupt decline in the ability of theca cells from the largest chicken preovulatory follicle to produce androstenedione from all substrates except dehydroepiandrosterone. In this study, we tested the hypothesis that progesterone from granulosa cells might inhibit andostenedione production by the adjacent theca cells. Physiological concentrations of progesterone inhibited andostenedione production by dispersed thecal cells from the substrate 17 alpha-hydroxyprogesterone but not dehydroepiandrosterone in both a dose- and time-dependent manner. In contrast, the metabolites of progesterone, 17 alpha-hydroxyprogesterone, and androstenedione at a high concentration (100 nM) failed to produce such an inhibitory effect. In addition, this inhibitory effect of progesterone was reversed by the protein synthesis inhibitor cycloheximide. The results of this study seem to suggest that progesterone acts indirectly through its nuclear receptor to induce the synthesis of a protein that possibly inhibits C17,20 lyase activity and/or C17,20 lyase gene expression.

Effects of insulin-like growth factor I and interactions with transforming growth factor alpha and LH on proliferation of chicken granulosa cells and production of progesterone in culture.

The effects of insulin-like growth factor I (IGF-I), alone, and in combination with LH or transforming growth factor alpha (TGF-alpha), on replication and progesterone production by cultured avian granulosa cells obtained from the three largest (F1-F3) follicles were studied. IGF-I and TGF-alpha stimulated proliferation of granulosa cells in a dose-dependent manner, and responsiveness decreased as the cells matured. IGF-I stimulated progesterone production from granulosa cells of all the follicles with no change in ED50 value during follicular maturation; however, the maximum response was from cells derived from F1 follicles. IGF-I plus LH had an additive effect on progesterone production by cells from all follicles. In contrast, TGF-alpha inhibited basal and LH- and IGF-I-stimulated progesterone production. These data show that IGF-I and TGF-alpha may interact with each other during granulosa cell maturation, such that efficacy of IGF-I increases, while that of TGF-alpha decreases before ovulation. Furthermore, both growth factors interact with LH, either to enhance or inhibit progesterone production by granulosa cells. However, LH, IGF-I and TGF-alpha combine to stimulate proliferation of granulosa cells.

The expression of transforming growth factor alpha receptor protein and its activation in chicken ovarian granulosa cells of maturing follicles.

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) are structurally related growth factors that exert their biological actions by binding to the same cell-surface receptor, EGF receptor. However, in chicken cells, human EGF binds with approximately 100-fold lower affinity than human TGF-alpha. In a previous study, we localized EGF/TGF-alpha receptor immunohistochemically in the granulosa and theca of the developing follicles of laying hens. We have also shown that TGF-alpha binds to cell-surface receptors of the granulosa cells. The present study characterizes the nature of the EGF/TGF-alpha receptor. Immunoprecipitation of receptor proteins from cultured granulosa cells with an anti-EGF receptor antibody (12E) shows the expression of a 170-kDa receptor protein. The expression of the receptor protein decreases with follicular enlargement between the F3 and F1. Incubation of the cells with [125I]TGF-alpha followed by cross-linking with bis(sulphosuccinimidyl)suberate showed that TGF-alpha binds a similar (170 kDa) receptor protein immunoprecipitated with the 12E anti-EGF receptor antibody. The binding of TGF-alpha to granulosa cells caused receptor protein oligomerization, yielding the monomeric (170 kDa) and dimeric (340 kDa) protein forms. Oligomerization seemed to favour the formation of the dimeric rather than the monomeric form. Culturing granulosa cells with luteinizing hormone or follicle-stimulating hormone increased the expression of both monomer and dimer forms of the receptor proteins compared with the control. Western blotting analysis with anti-phosphotyrosine antibody revealed that the lysates of TGF-alpha-stimulated cells express phosphotyrosine-containing receptor proteins of 170 kDa and 340 kDa. The results show that chicken granulosa cells express the 170-kDa EGF/TGF-alpha receptor protein, which dimerizes on binding to TGF-alpha, suggesting that the receptor protein may be involved in the signal transduction of TGF-alpha actions in the chicken granulosa cells.

Inhibitory effect of PGF-2 alpha on hCG-stimulated progesterone production in vitro by luteal cells from guinea-pigs at different stages of the oestrous cycle.

Suspensions of luteal cells were prepared by collagenase dispersion of guinea-pig corpora lutea obtained at specific times during the oestrous cycle. Luteal cells incubated with hCG produced increased amounts of progesterone. For Days 3--13 of the oestrous cycle, the concentrations of hCG required for 50% of the maximum response were within the range, 1 x 10(-3) to 7 x 10(-3) i.u./ml, showing no marked loss of sensitivity to hCG with increasing luteal age. PGF-2 alpha (1 mumol/l), had no effect on basal production of progesterone but significantly inhibited hCG-stimulated progesterone production by luteal cells isolated on Days 7, 9, 10, 12 and 13 of the cycle. This concentration of PGF-2 alpha had no significant effect on progesterone production by luteal cells prepared earlier in the cycle (Days 3 and 5). It is concluded that (a) the luteolytic action of PGF-2 alpha in the guinea-pig is mediated, at least in part, by direct action on luteal cells, and (b) the cells from newly formed corpora lutea are resistant to the direct inhibitory action of PGF-2 alpha.

Steroid secretion by ovarian cells of the Japanese quail (Coturnix coturnix japonica).

Mixed cell preparations (theca plus granulosa) prepared from the hierarchy of follicles of quails ovaries were incubated under defined conditions with or without the addition of ovine luteinizing hormone (oLH), ovine follicle stimulating hormone (oFSH), theophylline, cycloheximide, or dibutyryl cyclic adenine monophosphate (db cAMP); or in the presence of androstenedione or testosterone as aromatizable substrate. Steroids secreted into the medium during the 4-hr incubation period were assayed by radioimmunoassay. Cells from the largest follicles (F1) secreted predominantly progesterone, were stimulated by LH and db cAMP, and the response was potentiated by theophylline, but FSH had no stimulatory effect. The F1 cells showed increasing basal and LH-stimulated responses between 18 and 12 hr before the next expected oviposition. Cells from the smaller follicles (F3 and F4) secreted predominantly estrogens, and were stimulated by FSH but not by db cAMP and only to a small extent by theophylline. Addition of androstenedione (10(-7) M) or testosterone (10(-7) M) enhanced estrogen secretion, which was further raised by the simultaneous addition of FSH. These results confirm previous reports on the sites of steroid secretion within quail follicles and suggest that while the action of LH on the cells from F1 follicles may be mediated in part through the adenylate cyclase system, the action of FSH on the smaller follicles may be substantially independent of cAMP.

Induction of ovulation and oviposition in female quail with luteinizing hormone, luteinizing hormone releasing hormone, or progesterone.

Regularly laying female Japanese quail were injected 12 or 18 hr before the next expected oviposition with 25 micrograms oLH, 25 micrograms luteinizing hormone releasing hormone (LHRH), or 0.05 mg progesterone, and the subsequent oviposition was recorded and ovulation determined by autopsy 9 hr after the injection. Plasma progesterone levels were measured in blood collected from a wing vein during the postinjection interval. Vehicle-injected birds served as control. All treatments resulted in premature oviposition and ovulations in 50-100% of birds when injected 12 hr before the next expected oviposition. None of the vehicle-injected birds showed any advancement in either oviposition or ovulation. Premature oviposition was generally followed by premature ovulation within 1 hr, when birds were treated 12 hr before the next expected oviposition and was preceded by a rise in plasma progesterone levels which reached values similar to those occurring during the normal preovulatory period. When birds were injected 18 hr before the next expected oviposition, the incidence of premature oviposition was very low, premature ovulation was absent, and the rise in plasma progesterone levels following treatment was substantially less than in the former group. The results suggest that oviposition and ovulation in quail may be initiated by LHRH induced LH release from the pituitary gland and that progesterone may stimulate LHRH and LH release. The timing of the ovulatory cycle may depend upon the phase of follicular maturation.

Influence of 5 alpha-reduced androgens on oestradiol secretion by granulosa cells of pregnant mare serum gonadotrophin treated immature rats.

The effect of aromatizable androgens (testosterone and androstenedione) and naturally occurring 5 alpha-androstane, 3 alpha 17 beta-diol and 5 alpha-androstane, -3 beta, 17 beta-diol on oestradiol secretion by granulosa cells isolated from preovulatory follicles of PMSG-primed immature rats was investigated. The amount of oestradiol secreted by granulosa cells in the absence of exogenous aromatizable androgen in a 4h incubation was negligible. However, the addition of testosterone or androstenedione resulted in concentration dependent increases in oestradiol secretion. The 5 alpha-reduced androgens inhibit oestradiol and oFSH-stimulated oestradiol secretion by the granulosa cells in the presence of exogenous testosterone. The least potent of the androgens tested in causing this effect being the 5 alpha-androstane-3 alpha, 17 beta-diol. This result suggests that the naturally occurring 5 alpha-reduced androgens have a direct effect on androgen-aromatizing enzymes. The effect of these androgens may have an important connotation with respect to the control of the onset of puberty and regulation of ovarian oestradiol secretion within the microenvironment of an ovarian follicle.

Calcium-dependent stimulation of estrogen secretion by FSH from theca cells of the domestic hen (Gallus domesticus).

There is little information about the stimulation of estrogen secretion from theca cells of the domestic hen by follicle-stimulating hormone (FSH), and the mechanism of action of FSH through calcium has not been considered previously. The theca interna and externa cells from the third (F3) and fourth (F4) largest ovarian follicles of hens were separated, dispersed, and incubated in M199 with FSH (0.5 micrograms ml-1) or A23187 (0.1 or 1 microgram ml-1) with or without ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) (2 mM) in calcium-replete or in calcium-free M199. Alternatively, androstenedione (10(-6) M) was added to the cells as aromatizable substrate, with or without FSH and/or EGTA. Estradiol and estrone secreted into the media during a 4-hr incubation period were measured by RIA. FSH stimulated estradiol and estrone secretion from all the cell preparations. The effect of FSH was abolished by the addition of EGTA or in calcium-free medium. A23187 stimulated estradiol and estrone secretion by the same extent as FSH, but did not do so in calcium-free medium. Androstenedione enhanced estradiol and estrone secretion, but estrogen secretion was further raised by the simultaneous addition of FSH. This action of FSH on aromatization was also abolished by EGTA. This evidence supports the hypothesis that calcium, possibly of extracellular origin, is an important mediator in the stimulation of aromatase systems in thecal cells of chickens.