Induction of adrenocortical special zone in the male possum (Trichosurus vulpecula)

The induction of adrenocortical special zone (S.Z.) by gonadotrophin administration was studied in male brush tailed possums. Castrated males injected with porcine FSH (NIH-FSH-P2) formed well developed S.Z.s, varying in sizes from 5-20% of the gland's volume. Pregnant mare serum gonadotrophin (PMSG) was ineffective. From adrenal homogenates of FSH treated possums incubated with [3H]-progesterone the major conversion products were 5 beta-reduced steroid metabolites (72%) and cortisol (4%). The conversion products from adrenals of saline and PMSG treated males were cortisol and corticosterone (65%). Of the ten untreated castrates, one had a well developed S.Z. and two had S.Z.s at an early stage of development. Significant 5 beta-reduction of [3H]progesterone was only found in one animal. The plasma FSH concentrations were in intact males 317 +/- 41 ng rat FSH ml-1 (mean +/- SEM) and in castrates 769 +/- 64. The possible reasons for the lack of spontaneous S.Z. formation in intact males are discussed.     

Follicle development in cyclic, anoestrous and FSH-treated brushtail possums (Trichosurus vulpecula)

The common brushtail possum (Trichosurus vulpecula) is a pest of considerable economic importance in New Zealand. Attempts to develop methods of suppressing reproduction in this species are currently hampered by the lack of reliable methods to synchronise oestrus and ovulation in this species. The objective of this study was to compare antral follicle populations in anoestrous and cyclic brushtail possums and to assess the efficacy of exogenous FSH to induce follicle development in anoestrous animals. Ovaries were recovered from anoestrous possums after administration of either exogenous FSH (1.0 mg/injection) or the saline vehicle alone (0.5 ml/injection) at 12-h intervals for 3 days (n = 6/group), and from cyclic animals (n = 6) that were euthanised in mid-follicular phase (5 days after removal of their pouch young). All antral follicles > or =1.0 mm in diameter were dissected free of extraneous tissue, incubated in vitro to measure oestradiol production, and then processed for histological assessment of health status. Mean weight of ovaries and vaginal cul-de-sac tissues were both significantly greater (P<0.001) in FSH-treated anoestrous females (24.2+/-5.1 mg and 6.50+/-1.34 g, respectively), but did not differ significantly between saline-treated anoestrous possums (12.4+/-3.0 mg and 1.31+/-0.27 g) and cyclic animals (13.5+/-1.6 mg and 2.62+/-0.95 g). Mean uterine weights in both cyclic (889+/-161 mg) and FSH-treated (1098+/-184 mg) animals were significantly heavier(P<0.001) than those of anoestrous possums (414+/-61 mg). The mean number of follicles (> or =1.0-mm diameter) present was significantly greater (P<0.001) in FSH-treated, than in cyclic and anoestrous possums (38.0+/-4.4, 23.2+/-3.2 and 10.7+/-3.4 follicles/animal, respectively). Cyclic animals had significantly more (P<0.01) follicles than anoestrous possums. The proportion of follicles that were classified as healthy, was significantly lower (P<0.01) in cyclic possums(38%) than in anoestrous (69%) and FSH-treated (88%) animals. The mean diameter of the largest healthy follicle present was 2.5+/-0.41, 2.1+/-0.08, and 3.1+/-0.15 mm for cyclic, anoestrous and FSH-treated animals, respectively. None of the follicles harvested from saline-treated anoestrous possums produced measurable levels of oestradiol in vitro, whereas 7% and 59% of those from cyclic and FSH-treated animals did so. In summary, cyclic possums had more antral follicles present than anoestrous animals, but a lower percentage of these follicles were healthy. Less than 10% of healthy follicles from cyclic possums, and none of those from anoestrous animals, were capable of producing oestradiol when incubated in vitro. Treatment with ovine FSH promoted follicle development in anoestrous possums, to significantly increase the number of follicles present, the proportion that were healthy and the percentage capable of producing oestradiol.     

Differences in steroid metabolism by testis, prostate and epididymis in the immature and adult possum (Trichosurus vulpecula)

Immature possums 126-195 days old and adults over 1 year old were used. Testicular homogenates from immature possums converted [3H]progesterone to nine different products, of which greater than 63% were 5 alpha-reduced androstane metabolites. The major product from adult testis was testosterone in yields greater than 60%. While metabolism of [3H]testosterone by the epididymis of immature possum was minimal, in adults 5 alpha-reduced products constituted greater than 80% of the yield. In contrast, prostatic tissue from adults converted less than 4% of [3H]testosterone to 5 alpha-reduced products, while the yields were greater than 80% from prostates of immature animals. The results showed that like in rodents, the testis of immature possum has a high 5 alpha-reductase and low 17 beta-hydroxysteroid dehydrogenase activity, which reverses in the adult state. The implications of the findings are discussed.     

Differences in steroidogenesis by the subcellular fractions of adrenocortical special zone and cortex proper of the female possum (Trichosurus vulpecula)

Steroidogenesis by subcellular fractions of adrenal cortex proper (C.P.) and special zone (S.Z.) of female possum (Trichosurus vulpecula) was studied. Mitochondrial, microsomal and cytosol cell fractions were incubated with appropriate substrates in the presence of an NADPH-generating system. The major products formed from [3H]progesterone and [3H]17 alpha-hydroxyprogesterone by the microsomal fraction of the C.P. were 11-deoxycortisol and 11-deoxycorticosterone and 3 alpha (beta)-hydroxy-5 alpha-androstan-17-one by the S.Z. The mitochondrial fraction converted [3H]11-deoxycortisol to cortisol in yields twenty times higher by the C.P. than by the S.Z. and to 17 alpha, 20 beta,21-trihydroxy-4-pregn-3-one thirty times higher by the S.Z. The conversion of [3H]androstenedione to 11 beta-hydroxyandrostenedione by the C.P. was approximately double that of the S.Z., while 18-hydroxyandrostenedione (tentatively identified) formed the highest yield in both zones. Incubation of the same substrates with cytosol formed two 5 beta-pregnane and two 5 beta-androstane derivatives in total yields less than 5% by C.P. and greater than 60% by S.Z. Aromatase activity, estimated by the release of [3H2O] from [1 beta 3H]testosterone, in the adrenals of 8 possums, was in each experiment negligibly low. Determination of total enzyme activities in the two zones revealed that 11 beta, 18 and 21-hydroxylases were higher in the C.P., while 17 alpha-hydroxylase was higher in the S.Z. Similar results were obtained when the rates of formation of hydroxylated products were estimated in the presence of saturating amounts of substrates. Active 5 alpha- and 5 beta-reductases, C17-20-lyase and 3 alpha (beta) and 20 beta-hydroxysteroid dehydrogenases were found almost exclusively in the S.Z. We conclude that the S.Z. at lower levels of activity than the C.P. could contribute to the basal secretion of corticosteroids. In addition, the S.Z. has a high capacity to form C19 steroids and 5 alpha- and 5 beta-reduced steroids. The possible role of the S.Z. in possum is discussed.     

Evidence that age-related changes in p38 MAP kinase contribute to the decreased steroid production by the adrenocortical cells from old rats

The current studies were initiated to investigate whether excessive oxidative stress exerts its antisteroidogenic action through modulation of oxidant-sensitive mitogen-activated protein kinase (MAPK) signaling pathways. Western blot analysis indicated that aging caused increased phosphorylation and activation of rat adrenal p38 MAPK, but not the ERK1/2 or JNK1/2. Lipid peroxidation measurements (an index of cellular oxidative stress) indicated that adrenal membranes from young animals contained only minimal levels of endogenous thiobarbituric acid-reactive substances (TBARS), and exposure of membranes to enzymatic and non-enzymatic pro-oxidants enhanced TBARS formation approximately 12- and 20-fold, respectively. The adrenal membranes from old animals showed much more susceptibility to lipid peroxidation and exhibited roughly 4- to 6-fold higher TBARS formation than young controls both under basal conditions and in response to pro-oxidants. Qualitatively similar results were obtained when lipid peroxide formation was measured using a sensitive FOXRS (ferrous oxidation-xylenol orange-reactive substances) technique. We next tested whether aging-induced excessive oxidative insult alters steroidogenesis through modulation of MAPK signaling pathway. Treatment of adrenocortical cells from old rats with specific p38 MAPK inhibitors restored Bt(2)cAMP-stimulated steroidogenesis approximately 60-70% of the value seen in cells of young animals. Likewise, pretreatment of cells with reactive oxygen species (ROS) scavengers MnTMPyP and N-acetyl cysteine also partially rescued age-induced loss of steroid production. In contrast, simultaneous treatment of cells with ROS scavengers and p38 MAPK inhibitor did not produce any additional effect suggesting that both types of inhibitors exert their stimulatory action through inhibition of p38 MAPK activation. Collectively, these results indicate that p38 MAPK functions as a signaling effector in oxidative stress-induced inhibition of steroidogenesis during aging.     

Involvement of 5-lipoxygenase metabolites in ACTH-stimulated corticosteroidogenesis in rat adrenal glands

Various lipoxygenase (LO) products of arachidonic acid (AA) have been found to have potent biological activities and modulate physiological processes in various cells including endocrine cells. However, no studies concerning LO products in adrenocortical cells have been reported. The present study was performed to investigate LO products in rat adrenocortical cells and its role in ACTH-stimulated adrenal steroidogenesis. LO metabolites produced in ACTH-stimulated rat adrenocortical cells prelabeled with [3H]AA was analyzed by reverse phase and straight phase HPLC and two 5-LO products, 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4) were identified. ACTH-induced 5-HETE and LTB4 production in adrenal cells was dose dependently inhibited by AA861, a specific inhibitor of 5-LO. AA861 reduced ACTH-stimulated corticosteroid production without any change in cyclic AMP formation, while indomethacin did not affect both corticosteroid and cyclic AMP production. Reduced steroidogenesis by AA861 was reversed by the addition of 5-hydroperoxyeicosatetraenoic acid (5-HPETE). Also exogenously added 5-HPETE dose dependently augmented ACTH-stimulated corticosteroid production without any concomitant change in cyclic AMP production. However, 5-HETE and LTB4 had no such effect. These results indicate that 5-LO pathway is present in rat adrenocortical cells and its metabolites, most likely 5-HPETE, may play an important role in adrenal steroidogenesis.     

Evidence for the involvement of endothelial cell products in adrenal CITED2 expression

The adrenal gland contains a well-organized network of blood vessels, and adrenocortical cells are situated in close proximity to endothelial cells. Recently, several new mechanisms have been characterized that control the release of aldosterone by adrenocortical cells, including the involvement of endothelial-cell-derived factors. Interestingly, a CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), which is necessary for adrenal development, has been linked to aldosterone synthesis. We have therefore examined the effects of endothelial-cell-conditioned medium (ECCM), as produced during the incubation of human umbilical vein endothelial cells for 24 h, on the promoter activity and mRNA and protein expression of CITED2 in adrenocortical cells as represented by the NCI-H295R cell line. We have found a dose-dependent effect of ECCM on CITED2 promoter activity; this peaks at 480%. Activation of the CITED2 promoter occurs in parallel to an increase in CITED2 messenger RNA (as quantified by real-time polymerase chain reaction) and protein. The stimulatory effect of ECCM can be reversed by blocking mitogen-activated protein kinase activity with the MEK1-inhibitor PD98059. We conclude that products secreted by endothelial cells control not only steroidogenesis, but also factors that are important for adrenocortical development, thereby highlighting the role of cellular interactions within adrenocortical development and physiology. This work was supported by a grant from the Doktor Robert Pfleger-Stiftung, Bamberg, Germany, to H.S.W.     

Alteration of follicular steroid secretion and thecal morphology after in-vitro exposure of individual pig follicles to follicle regulatory protein

Medium-sized (4-6 mm) pig follicles were incubated for 10 h and then examined via light microscopy. Treatment with pig FSH resulted in significantly increased concentrations of oestradiol, testosterone, androstenedione and progesterone in the medium. Follicle regulatory protein (FRP) alone (1 micrograms/ml) decreased follicular secretion of oestradiol (56%) and progesterone (53%) but stimulated the secretion of testosterone (226%) and androstenedione (139%). In the presence of 1 ng FSH/ml, the inhibitory effect of FRP on oestradiol secretion was enhanced (74%), progesterone values were unaffected and secretion of testosterone and androstenedione were reduced by 66% and 53%, respectively. All effects of FRP were fully overcome by 1 micrograms FSH/ml. The incidence of atresia, as defined by granulosa cell pycnosis, was similar in all treatment groups (1-3 of 10 follicles per group). The remaining follicles had intact granulosa cells. However, follicles treated with FRP (1 micrograms/ml) + FSH (1 ng/ml) had pycnotic nuclei in the theca interna cells, in the presence of an intact stratum granulosum. External exposure of follicles to FRP may not reflect physiological conditions since, in vivo, thecal pycnosis is never observed before granulosa cell pycnosis. However, the present results indicate that FRP is potentially capable of altering both follicular morphology and steroidogenesis. We suggest that FSH and FRP interact to affect follicular development.     

Factors influencing prostatic 5 alpha-reductase activity in possum (Trichosurus vulpecula)

1. There were marked differences in prostatic wts among individual possums, but no evidence of a seasonally related change in wt could be established. It was concluded that the wt differences are mainly due to the changes in secretory activities. After castration the prostate wts fell while after administration of testosterone or oestradiol partially reversed this process. 2. Seven steroid conversion products were isolated from prostatic homogenates incubated with [3H] testosterone; 5 alpha-androstane-3 beta,17 beta-diol forming the highest yield. 3. While the 5 alpha-reductase activity of prostates from intact possums was very low (approx. 8% of the total yield), it increased to over 50% after castration. 4. Administration of testosterone or oestradiol partially reversed the post-castration rise in 5 alpha-reductase, while 17 beta-hydroxy-5 alpha-androstane-3-one (DHT) was ineffective. Administration of porcine FSH-NIH-P2 to both intact or castrated possums caused a marked rise in prostatic 5 alpha-reductase activity. 5. It was concluded that in possum, FSH may have a direct stimulatory effect on prostatic 5 alpha-reductase activity. The results are discussed in relation to placental mammals.     

Effect of FSH infusion on follicle development in GnRH agonist-treated gilts

The aim was to investigate the effect of infusion of purified FSH alone on follicle development in hypogonadotrophic GnRH agonist-treated gilts. Large-White hybrid gilts (n = 12) were treated during the mid-luteal phase and again after 28 days (day 0) with a potent slow releasing GnRH agonist. On day 3, seven gilts were infused for 168 h with 1.5 S1 units oFSH h-1 (equivalent to 1.5 units of bioactivity of NIH-FSH-S1 standard) and blood samples were collected. Ovaries were then recovered and all follicles > or = 1 mm in diameter were dissected and incubated for 2 h in 1 ml Eagle's minimum essential medium. The ovaries were recovered from the remaining five GnRH agonist-treated gilts on day 10 and also from five cyclic gilts during the late follicular phase (controls). Plasma FSH concentrations in GnRH agonist-treated gilts were lower (P < 0.01) than in follicular phase controls, increased (P < 0.001) after 1 h of FSH infusion and reached a plateau similar (P > 0.1) to that of controls after 8 h. Basal LH concentrations were similar (P > 0.1) between GnRH agonist-treated and control gilts and remained unchanged (P > 0.1) throughout the infusion period. GnRH agonist treatment reduced (P < 0.01) basal oestradiol concentrations compared with control gilts. Infusion with FSH alone increased (P < 0.001) plasma oestradiol concentrations after 96 h compared with those before infusion; when the animals were killed oestradiol concentrations were higher (P < 0.01) in GnRH agonist-treated gilts infused with FSH than in controls. This was also apparent by vulval swelling and behavioural oestrus. There were more follicles > or 1 mm in diameter in the GnRH agonist-treated groups than in the controls (184, 153 and 86 per animal; P < 0.01). Infusion with FSH increased the maximum follicle diameter (GnRH agonist: < 4 mm; FSH infused: < 12 mm; controls: < 10 mm) and tended to increase (P < 0.07) the mean number of follicles > or = 6 mm diameter per animal (FSH infused: 53; controls: 21). Total oestradiol production in vitro by follicles > or = 1 mm was higher (P < 0.01) in GnRH agonist-treated gilts infused with FSH and in follicular phase controls than in animals treated with GnRH agonist alone. However, oestradiol and testosterone secretion in vitro per follicle > or = 6 mm in diameter was lower (P < 0.05) in FSH-infused animals than in controls. In summary, although infusion of FSH alone stimulated the growth of multiple follicles of preovulatory size in GnRH agonist-treated gilts, steroidogenic output by individual follicles was impaired.     

Studies on lipoprotein and adrenal steroidogenesis: I. Roles of low density lipoprotein- and high density lipoprotein-cholesterol in steroid production in cultured human adrenocortical cells

The roles of human low density lipoprotein (LDL)- cholesterol and high density lipoprotein (HDL)- cholesterol on adrenal steroidogenesis were investigated using cultured human adult and fetal adrenocortical cells and the findings were then compared to those obtained with bovine adrenocortical cells. The secretion of cortisol in both human and bovine adrenocortical cells was dose-dependently increased by the administration of LDL- or HDL-cholesterol in the presence of adrenocorticotropin (ACTH). LDL-cholesterol was utilized to a greater extent than HDL-cholesterol in both human and bovine adrenal steroidogenesis in the presence of ACTH. Exogenous lipoprotein-derived cholesterol was less utilized in human adrenal steroidogenesis than in bovine adrenal steroidogenesis, compared to the endogenous cholesterol. An increase in the secretion of cortisol and dehydroepi androsterone sulfate (DHEA-S) continued for the 5-day culture period, in the presence of lipoprotein cholesterol and ACTH in both human adult and fetal adrenocortical cells. The secretion of aldosterone increased on the first day of the culture period, then gradually decreased for the 5-day culture period in human adult adrenocortical cells, but not in human fetal adrenocortical cells in the presence of lipoprotein cholesterol and ACTH. These findings demonstrate that exogenous cholesterol utilized in the biosynthesis of steroids is mainly from LDL-cholesterol in both human adult and fetal adrenals and bovine adrenal and the proportion of cholesterol synthesized de novo is significantly larger in the human adult adrenal than in the bovine adrenal.     

Adrenal microsomal C19-steroid 5α-reductase activity in the Snell transplantable rat adrenocortical tumour 494 and the effect of oestradiol, testosterone propionate and adrenocorticotrophin in intact and gonadectomized rats

The C(19)-steroid 5alpha-reductase activity in the microsomal fraction of rat adrenal tissue under various hormonal treatments was examined. In intact control rats the activity is similar in both males and females, and after gonadectomy it is markedly increased. Treatment with oestradiol (150mug/day per animal for 7 days) or testosterone propionate (2mg/day per animal for 7 days) lowered the activity of 5alpha-reductase in castrated animals to approximately the values for intact animals in both sexes, and in intact animals the activity was also decreased by these treatments. The enzyme activity was also decreased by adrenocorticotrophin treatment but to a lesser extent than by the steroid hormones. The activity of the 5alpha-reductase enzyme in the Snell adrenocortical tumour 494 is very low when incubated as a whole homogenate, but the activity in microsomal material of the tumour was measured and unexpectedly found to be similar to that in intact controls.     

Effect of LH on steroidogenesis by hamster follicles isolated at defined stages of development

Multiple follicular growth was stimulated in groups of immature female hamsters by the administration of PMSG. The ovaries were removed 72-78 or 96-102 h later. The animals at 96-102 h were subdivided according to whether the concentration of plasma progesterone was within the range of the concentrations found at the previous period (0.45-13.18 nmol/l; Group I) or above (Group II). Thecal and granulosa cells from batches of isolated follicles (591-740 micron) were cultured together in the presence or absence of LH and the products of steroidogenesis were measured in the culture fluid. The results showed that there was a significant increase in the production of progesterone and 17 alpha-hydroxyprogesterone in follicles from Groups I and II at 96-102 h. In the presence of LH there was a significant increase in the yield of progesterone and 17 alpha-hydroxyprogesterone over the control values at all time periods. There were, however, reductions in the yields of 17 alpha-hydroxyprogesterone and androstenedione in the presence of LH at 96-102 h compared with 72-78 h and in Group II compared with Group I, although the concentrations of 17 alpha-hydroxyprogesterone were approximately 40-fold those of androstenedione. In addition, the production of oestradiol decreased significantly in follicles from both groups at 96-102 h and was reduced further in the presence of LH. It is concluded that the major rate-limiting step of steroidogenesis in hamster follicles undergoing the oestrogen-progestagen shift involves the side-chain cleavage of 17 alpha-hydroxyprogesterone.     

Spontaneous or experimentally induced formation of a special zone in the adrenal cortex of the adult brush-tailed possum (Trichosurus vulpecula)

The cytology and ultrastructure of the hypertrophied special zone, which is formed spontaneously in the adrenal cortex of adult female brush-tailed possums (Trichosurus vulpecula), was compared to the adrenocortical tissue in adult males in which the special zone, normally absent, was induced following castration alone or by additional treatment with follicle-stimulating hormone (FSH). The special zone in females was situated between the zona fasciculata and the zona reticularis, the latter being a rudimentary zone in this species. Special zone tissue extended as a broad band parallel to and on one side of the adrenal medulla. In the luteal phase of the reproductive cycle, the special zone cells showed ultrastructural features commonly associated with steroidogenic tissues, with many mitochondria and compact masses of smooth endoplasmic reticulum. Cytoplasmic lipid inclusions were rarely observed. In lactating females, however, the special zone cells exhibited cytological and ultrastructural features suggestive of a transformation in their morphology broadly divided into two types of cells: (1) cells at the periphery of the special zone (closest to the zona fasciculata) showed variable quantities of lipid inclusions, mitochondria with dispersed cristae, and segregation of the smooth endoplasmic reticulum into compact masses; (2) cells within the more central regions showed an increasing abundance of lipid inclusions which in many cells became the dominant feature of the cytoplasm. These special zone cells contained very little smooth endoplasmic reticulum and their mitochondria contained few cristae together with amorphous granular material within the matrix. In castrated males, special zone tissue developed between the zona fasciculata and the zona reticularis, appearing initially as focal islands of cells (8 months postcastration) and later (11 months postcastration) expanding into a single zone, probably via the proliferation and differentiation of adjacent cells of the zona fasciculata and longitudinal growth of the special zone. Similar focal aggregations of special zone cells were induced after 14 days of FSH treatment given to 2-month castrated males. In all castrated and FSH-treated castrated males, the ultrastructure of special zone cells was similar to that of special zone cells in luteal-phase female possums. The findings suggest that the formation and cellular composition of the special zone is associated with changes in the pituitary-gonadal axis and that FSH plays a primary role in the differentiation of this tissue.     

The effects of serum oestrogen-binding components on the unbound oestradiol-17 beta fraction in the serum of developing female rats and on inhibition of [3H]oestradiol uptake by uterine tissue in vitro

Total oestradiol concentrations in the serum of young female rats were high and decreased after about 21 days of age. High affinity serum oestradiol-binding components (EBP), however, fell steadily from 5 to 23 days of age while the unbound oestradiol-17 beta fraction, which was low early in development, increased between 21 and 28 days of age. Injection (i.v.) of immature (EBP-rich) oestradiol-free serum into 21-day-old female rats led to a decrease in the unbound oestradiol fraction and an increase in serum FSH concentrations. Incubation of uterine tissue with [3H]oestradiol, with or without the addition of diethylstilboestrol (DES), showed the rate and degree of total and DES-suppressible uptake of [3H]oestradiol to be greatest from buffer, less from adult (EBP-poor) serum and negligible from immature (EBP-rich) serum; moreover, there was a positive correlation between the degree of uptake of [3H]oestradiol and the unbound fraction of [3H]oestradiol in the incubate. It is concluded that, at least in young rats, oestradiol activity depends more on the availability of free oestradiol than on its total plasma concentrations.     

Effect of estradiol benzoate on hormonal profile and steroidogenic organs of immature and adult male rats

The effects of estradiol benzoate (E2B) at a dose of 50 micrograms/day/rat for 15 days were investigated on ascorbate metabolism, steroidogenesis, protein, cholesterol levels of testis, adrenal and serum testosterone, LH and FSH profiles of rats. The data revealed that E2B manifested a direct effect on testicular and probably adrenal steroidogenesis. But serum gonadotrophin levels remained unchanged. The treatment also brought about a decline in ascorbate metabolism, activities of 3 beta and 17 beta hydroxysteroid dehydrogenases and alteration in protein level concomitant with the accumulation of cholesterol in both steroidogenic organs. Estrogen treatment was more effective in adult male rats than in the immature ones.     

Effect of selective removal of the adrenal medulla on female sexual development

The ovary and adenohypophysis of the rat contain beta-adrenergic receptors and respond to beta-adrenergic stimulation with hormone release. To determine the importance of the adrenal medulla as a source of adrenergic influences regulating prepubertal ovarian and pituitary function, a technique was developed to remove most of the adrenal medulla without compromising adrenocortical function. Medullectomy (MED) of 24-day-old female rats depressed both spontaneous diurnal changes in plasma epinephrine (EPI), and the EPI and norepinephrine (NE) response to decapitation, without affecting corticosterone (B) levels. Vaginal opening and first ovulation were delayed in MED rats. Serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were normal in MED rats, but those of growth hormone (GH) and prolactin (Prl) were depressed. MED reduced the ovarian weight response to pregnant mare's serum gonadotropin (PMSG) and the ovarian steroidal response to human chorionic gonadotropin (hCG) in vitro, but it did not affect ovarian beta-adrenergic receptors. Cultured granulosa cells, harvested from juvenile ovaries and primed in vitro with FSH, responded to nanomolar concentrations of EPI with progesterone (P) secretion. EPI also augmented hCG- and FSH-induced P secretion. The EPI effect was reproduced by Zinterol, a beta 2-adrenergic agonist and was prevented by propranolol, a beta-adrenergic antagonist. Blockade of alpha-adrenergic receptors with phentolamine was ineffective. It is suggested that EPI of adrenomedullary origin supports female prepubertal development by a) stimulating ovarian P secretion, b) favoring Prl and GH release and c) amplifying the stimulatory effect of low gonadotropin levels on ovarian steroidogenesis. The effects of EPI on ovarian function appear to be mediated by beta-adrenergic receptors of the beta 2 type.     

The androgen receptor does not mediate progestin regulation of progesterone biosynthesis in cultured rat granulosa cells

In the present investigation the influence of androgens and progestins on the FSH modulation of progesterone biosynthesis was studied in cultured rat granulosa cells. Cells obtained from the ovaries of immature estrogen treated rats were cultured for three days in serum free medium or in medium supplemented with FSH or CPA, with or without reduced androgen DHT or the synthetic progestin R5020 alone or in combination with the anti-androgen CPA. Treatment with FSH increased pregnenolone, progesterone and 20 alpha-OHP accumulation in the culture medium 20-, 14- and 7-fold, respectively. Furthermore FSH increased the activity of the enzyme 3 beta-HSD. Concurrent treatment with DHT or R5020 augmented the FSH stimulated steroidogenesis of cultured cells. The androgen enhancement of FSH stimulated steroidogenesis of cultured granulosa cells was blocked by concomitant treatment with CPA, whereas treatment of cultures with anti-androgen did not affect the stimulatory effect of the synthetic progestin R5020.     

Follicular steroidogenesis and oocyte maturation after superovulation of goats (Capra hircus) with gonadotrophins.

Follicles were sampled at three different times after treatment with 1200 iu pregnant mares' serum gonadotrophin (PMSG) or 12 mg ovine follicle-stimulating hormone (FSH), and from untreated control animals. The meiotic status and protein synthesis of the oocyte from each follicle was determined and correlated with the intrafollicular concentration of oestradiol and progesterone. Significantly higher amounts of oestradiol were present in PMSG-treated animals at sponge withdrawal than in FSH-treated and control goats. Twenty hours later, both oestradiol and progesterone concentrations in the PMSG group were higher than those in the FSH group, and were equivalent to control animals at the onset of oestrus. At 18 h after the administration of human chorionic gonadotrophin (hCG), oestradiol decreased markedly in all three treatment groups, whereas progesterone remained significantly higher in PMSG-treated follicles. Although these high concentrations of intrafollicular steroids were associated with a higher incidence of premature condensation of chromatin in oocytes, the two events were not causally related. Moreover, cytoplasmic maturation was not prematurely activated in these oocytes and a changed pattern of protein synthesis was observed in oocytes from all three treatment groups after the hCG injection. Whereas disturbances in follicular steroidogenesis of oestradiol and progesterone occur in vivo in goats superovulated with PMSG, they do not underlie the premature activation of the initial stages of nuclear maturation in oocytes but are associated with normal cytoplasmic maturation.     

Evidence to support a follicle-stimulating hormone threshold theory for follicle selection in ewes chronically treated with gonadotrophin-releasing hormone agonist

The mean and peak concentrations of follicle-stimulating hormone (FSH) during the luteal phase of a normal cycle were measured in 8 Welsh Mountain ewes. Gonadotrophin secretion and follicle growth were then suppressed by the chronic administration of the GnRH agonist buserelin for 5 weeks. During the 6th week of agonist treatment, each ewe was given a continuous infusion of FSH to produce a peripheral concentration of FSH equal to either the mean or peak of the gonadotrophin measured for that individual in the cycle preceding agonist treatment. Treatment had no effect on the total number of follicles, the number of follicles less than or equal to 2.5 mm in diameter or the in-vitro production of oestradiol by the small follicles when compared with control animals. None of the animals infused with the mean luteal-phase FSH equivalent developed large follicles greater than 2.5 mm diameter which could be classified as preovulatory follicles (oestradiol greater than 1000 pg/follicle/h). All of the animals infused with the peak luteal-phase FSH equivalent developed large follicles, some of which were preovulatory. The results suggest that an individual threshold concentration exists for FSH above which the later stages of preovulatory follicular development are stimulated.