The role of steroids in follicular growth

The steroidogenic pathway within the ovary gives rise to progestins, androgens and oestrogens, all of which act via specific nuclear receptors to regulate reproductive function and maintain fertility. The role of progestins in follicular growth and development is limited, its action confined largely to ovulation, although direct effects on granulosa cell function have been reported. Consistent with these findings, progesterone receptor knockout mice are infertile because they cannot ovulate. Androgens have been shown to promote early follicular growth, but also to impede follicular development by stimulating atresia and apoptosis. The inability of androgens to transduce a signal in mice lacking androgen receptors culminates in reduced fertility. Oestrogens are known to exert effects on granulosa cell growth and differentiation in association with gonadotrophins. Studies with oestrogen receptor knockouts and oestrogen depleted mice have shown us that oestrogen is essential for folliculogenesis beyond the antral stage and is necessary to maintain the female phenotype of ovarian somatic cells. In summary, the action of steroids within the ovary is based on the developmental status of the follicle. In the absence of any single sex steroid, ovarian function and subsequently fertility, are compromised.     

Age-dependent changes in the hypothalamo-pituitary-ovarian axis of the laying hen.

The functional integrity of the components of the hypothalamo-pituitary-ovarian axis was examined in young and old laying hens. Ovarian function was tested by measuring the amount of progesterone released in response to an injection of LH, and pituitary function was investigated by measuring the increase in the plasma LH level after an injection of LH-RH. There were no differences between young and old birds in the response of the pituitary gland or the ovary to these stimuli. Hypothalamic function was investigated by studying the positive feedback action of a standard dose of progesterone on LH release; the positive feedback response was smaller (P less than 0.05) in old hens. It is suggested that the fall in the rate of lay in hens towards the end of their laying year is caused partly by a decrease in the response of the LH-positive feedback mechanism to progesterone.     

Decreased follicular steroids and insulin-like growth factor-I and increased atresia in diabetic gilts during follicular growth stimulated with PMSG

Four streptozotocin-diabetic gilts (maintained on exogenous insulin for 3 months) and 4 normoglycaemic gilts were treated with 600 i.u. PMSG. Diabetic gilts had insulin therapy removed at the time of PMSG administration. Plasma glucose averaged 463 +/- 5 mg/100 ml for diabetic gilts and 82 +/- 4 mg/100 ml for control gilts over the 72-h sampling period. Serum insulin was lower in diabetic than in normoglycaemic gilts (glycaemic state by time interaction; P less than 0.0001). At ovary removal 75 h after PMSG, numbers and percentages of large (greater than or equal to 7 mm) and medium (3-6 mm) non-atretic follicles were similar for diabetic and control gilts (31 vs 68%; s.e.m. = 7; P less than 0.05). Diabetic gilts had a greater percentage of atretic follicles over all size classes (50 vs 21%; s.e.m. = 7; P less than 0.03). After PMSG, LH was suppressed within 12 h in control gilts and remained similar to values in diabetic gilts until 72 h, when LH was elevated in 2 diabetic gilts (glycaemic state by time interaction; P less than 0.001). Pulsatile LH patterns during 52-55 h after PMSG were not affected by glycaemic state. Serum concentrations of IGF-I tended (P less than 0.1) to be lower in diabetic gilts. Concentrations of oestradiol and FSH in serum were similar in diabetic and control gilts. Follicular fluid concentrations of oestradiol in follicles greater than or equal to 7 mm were lower in diabetic than normoglycaemic gilts (341 vs 873 ng/ml; s.e.m. = 86; P less than 0.05). Testosterone was higher in follicles 3-6 mm in diameter in diabetic than in normoglycaemic gilts (142 vs 80 ng/ml; s.e.m. = 26; P less than 0.05). Progesterone concentrations in follicular fluid were not affected by glycaemic state. Concentrations of IGF-I in follicles greater than or equal to 7 mm were lower in diabetic than control gilts (150 vs 200 ng/ml; s.e.m. = 13; P less than 0.05). We conclude that follicles of diabetic gilts respond to external gonadotrophic stimulation with decreased hormone production and increased ovarian follicular atresia, despite an absence of effects on circulating gonadotrophin and oestradiol concentrations.     

Changes in follicular fluid concentrations of steroids, the pattern of steroid secretion and aromatization capability of incubated preovulatory follicular wall of rats

Female Wistar rats exhibiting a regular 4-day oestrous cycle were included in this study. They were killed on the day of pro-oestrus at 11.00, 14.00, 16.00, 2.00, 22.00 and 24.00 h. The isolated preovulatory follicles were placed individually on a small triple disc of filter paper. They were cut in half and the flowing out follicular fluid (FF) was allowed to soak into the underlying filter paper disc, which was subsequently placed at the bottom of incubation chamber. It was rinsed with the incubation medium and this medium, containing FF, was collected. The remaining follicular wall was incubated for 3h. Some of the incubation media were supplemented with testosterone (T). The steroid content was estimated by radioimmunoassay. The secretion of estradiol (E) by follicular wall was high until 20.00 h while that of androgens until 22.00 h, and both declined thereafter. Relatively low progesterone (P) release rose sharply at 22.00 h and this hormone became the main steroid secreted at 24.00 h. The addition of T always enhanced the release of estradiol. Follicular fluid contained very little estradiol, while prevailing steroids were androgens. Decrease of androgen concentrations was found only at 24.00 h. Progesterone level, much lower than that of androgens, started to rise at 20.00 h and P was the main steroid present in FF at 24.00 h. The present results suggest an existence of selective passage of steroids into FF and show a presence of androgenic milieu surrounding cumulus oophorus in preovulatory FF. The results also indicate that the aromatase activity is sustained until ovulation and stress the role of the P rise observed in the secretion rate of follicular wall and FF content just before ovulation.     

Formation of lipoidal steroids in follicular fluid

The presence of high levels of lipoidal pregnenolone in follicular fluid has recently been established although no evidence has been presented concerning its possible origin. The following investigation focuses on the enzymatic conversion of non-conjugated steroids into their lipoidal derivatives in preovulatory follicular fluid obtained from women undergoing in vitro fertilization. Our observations indicated that pregnenolone, an important precursor steroid, was acylated at a similar rate as cholesterol in follicular fluid. Similar studies were subsequently conducted with serum obtained from a pool of normal women and women undergoing follicular stimulation which showed little difference to the results obtained in follicular fluid. Further studies using dehydroepiandrosterone, androst-5-ene-3 beta,17 beta-diol, estradiol and dihydrotestosterone were were also performed to monitor their respective lipoidal conversion percentages in follicular fluid which revealed a marked difference of conversion rates between steroids. The indirect identification of the lipoidal pregnenolone derivatives formed in follicular fluid was also conducted by incubating radiolabelled pregnenolone in follicular fluid. The fatty acid components of the resulting lipoidal pregnenolone derivatives showed a marked resemblance to those of cholesteryl esters formed in plasma by the enzymatic activity of lecithin:cholesterol acyltransferase. The pregnenolone derivatives were comprised predominantly of unsaturated fatty acids such as linoleate, palmitoleate, oleate, linolenate and arachidonate while saturated fatty acids, namely palmitate, constituted 20% of the total lipoidal pregnenolone.     

Low temperature highlights the functional role of the cell wall integrity pathway in the regulation of growth in Saccharomyces cerevisiae

Unlike other stresses, the physiological significance and molecular mechanisms involved in the yeast cold response are largely unknown. In the present study, we show that the CWI (cell wall integrity) pathway plays an important role in the growth of Saccharomyces cerevisiae at low temperatures. Cells lacking the Wsc1p (wall integrity and stress response component 1) membrane sensor or the MAPKs (mitogen-activated protein kinases) Bck1p (bypass of C kinase 1), Mkk (Mapk kinase) 1p/Mkk2p or Slt2p (suppressor of lyt2) exhibited cold sensitivity. However, there was no evidence of either a cold-provoked perturbation of the cell wall or a differential cold expression program mediated by Slt2p. The results of the present study suggest that Slt2p is activated by different inputs in response to nutrient signals and mediates growth control through TORC1 (target of rapamycin 1 complex)-Sch9p (suppressor of cdc25) and PKA (protein kinase A) at low temperatures. We found that absence of TOR1 (target of rapamycin 1) causes cold sensitivity, whereas a ras2Δ mutant shows increased cold growth. Lack of Sch9p alleviates the phenotype of slt2Δ and bck1Δ mutant cells, as well as attenuation of PKA activity by overexpression of BCY1 (bypass of cyclase mutations 1). Interestingly, swi4Δ mutant cells display cold sensitivity, but the phenotype is neither mediated by the Slt2p-regulated induction of Swi4p (switching deficient 4)-responsive promoters nor influenced by osmotic stabilization. Hence, cold signalling through the CWI pathway has distinct features and might mediate still unknown effectors and targets.     

Possible role of prolactin in growth regulation

To study the possible role of prolactin on growth regulation pituitary grafted rats of different ages and their sham-operated controls have been used. After the transplant operation of one pituitary gland from a litter-mate donor on day 5 or on day 30 of life, a marked prolactin increase has been observed. This increase has been immediate in 30 day-old rats and delayed in 5 day-old rats in which the elevation starts being significant on day 20 for females and on day 25 for male rats. Pituitary grafting on day 5 of life, with an adult donor gland, resulted in an immediate and marked increase of prolactin values in both sexes. Litter-mate donor pituitary grafting, on day 5 of life, resulted in an increase in body weight that could be directly correlated with the increase in prolactin levels. Adult pituitary grafting resulted in an increased body weight in females with no effects being detected in males. In 30 day-old grafted male and female rats, marked body weight increases were seen, over the whole studied period, together with an increase in nose-tail length (1 cm in female and 1.5 cm in males longer than the control animals). All these changes do not seem not be related to GH modifications in pituitary grafted rats, since GH changes were very slight with a final tendency to lower values in female rats but not in males. All these data could suggest that prolactin might exert a direct effect on growth both in male and female rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oocyte-granulosa cell interactions during mouse follicular development: regulation of kit ligand expression and its role in oocyte growth

Ovarian folliculogenesis is regulated by both endocrine and intraovarian mechanisms that coordinate the processes of oocyte growth and somatic cell proliferation and differentiation. Within the follicle, paracrine interactions between the oocyte and surrounding granulosa cells are critical for normal cell development and function. This review focuses on the role of paracrine interactions during early oocyte and follicular development that ensure proper coordination of oocyte and somatic cell function. Particular emphasis is given to granulosa cell-derived Kit Ligand (KitL), whose functional importance for oocyte growth has been demonstrated by a wide range of in vivo and in vitro studies. Reported interactions between KitL and oocyte-derived growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) suggest the molecular basis of oocyte-granulosa cell interactions, but also hint at the complexity of these communications. These paracrine interactions and the structure of the oocyte-granulosa cell interface are follicle stage-specific and regulated by FSH. Elucidation of the molecular mechanisms that promote the development of healthy oocytes with good developmental competence has potential applications for improving fertility and for in vitro growth systems for oocytes from domestic animals and humans.     

Role and regulation of the fibroblast growth factor axis in human thyroid follicular cells

Thyroidal levels of fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor 1 (FGFR1) are elevated in human thyroid hyperplasia. To understand the significance of this, effects of FGFR1 activation on normal human thyrocyte growth and function in vitro and the regulation of FGF-2 and FGFR1 expression have been examined. FGF-2 stimulated cell growth, as measured by cell counting, and inhibited thyroid function as measured by 125I uptake. Sensitivity to FGF-2 disappeared after 7 days, although FGFR1 expression was maintained. Thyroid-stimulating hormone (TSH, 300 mU/l) increased FGFR1 mRNA expression within 4 h and protein expression by 8 h. Exogenous FGF-2 decreased FGFR1 protein. Endogenous FGF-2 levels were low (approximately 1-2 pg/microg protein), and TSH treatment decreased these by 50%. Protein kinase C (PKC) activation increased FGF-2 mRNA and FGF-2 secretion within 2 h. This effect was enhanced (4.4-fold) when cells were cultured in TSH. We conclude that TSH stimulates FGFR1 but not FGF-2 expression. PKC activation stimulates FGF-2 synthesis and secretion, and TSH synergizes with PKC activators. Increases in FGFR1 or FGF-2 or in both may contribute to goitrogenesis.     

In vivo and in vitro studies of the role of the adrenergic system and follicular wall contractility in the pathogenesis and resolution of bovine follicular cysts

Bovine follicular cysts (FCs) are a common cause of economic loss in modern dairy herds. Their aetiopathogenesis is not completely understood, even though an inadequate hypothalamic release of GnRH at the time of ovulation is considered to be their main cause. Much evidence, however, suggests a role for adrenergic innervation in ovarian functions, such as follicular development, steroid hormone secretion, and follicular contractility, the latter being an event important for ovulation. Moreover, in humans, polycystic ovary syndrome, a disease very similar to bovine follicular cysts, is characterised by increased density of adrenergic nerves. Given these premises, the aim of our study was to evaluate the effectiveness and mode of action of a novel strategy for the treatment of bovine follicular cysts. In the in vivo experiment, 170 Friesian cows diagnosed with follicular cysts were assigned to four groups (groups A, B, C, and D) to assess the effects of epidural administration of a β-adrenergic antagonist (carazolol) alone or in combination with a GnRH analogue (lecirelin). The four groups underwent the following treatments: Group A was administered lecirelin in combination with carazolol; Group B was administered carazolol; Group C was administered lecirelin; and Group D was administered only normal saline solution. In the in vitro experiment, strips of the walls of cystic follicles recovered post-mortem were suspended in an organ bath, connected to an isometric force transducer and exposed to increasing doses of epinephrine or to the same treatment after exposure to carazolol for 15 min (n = 10). The amplitude and frequency of the contractile activity were recorded. None of the control cows was observed in oestrus or was submitted to AI. The combination of lecirelin and carazolol induced a significant increase in the number of cows in oestrus (88%) compared to lecirelin alone or to carazolol alone (P < 0.05 and P < 0.01, respectively). The combination of lecirelin and carazolol and lecirelin alone were significantly more efficacious than carazolol alone (P < 0.01 and P < 0.05, respectively). In the in vitro experiment, epinephrine increased the amplitude of the contractions of the strips in a dose-dependent manner. This response was significantly enhanced in strips pre-treated with carazolol. The treatments had no effect on the frequency of contractions. In conclusion, our work demonstrates that the epidural administration of a GnRH analogue and a β-adrenergic blocker is an effective means of treating cows with cystic ovarian disease. Moreover, it confirms, from a clinical point of view, that alterations of the adrenergic system and of the contractility of the follicular wall can be considered aetiopathogenic factors involved in the development of FCs. The results of this study lay the basis for a new therapeutic approach to FCs.     

Distribution of follicular growth, atresia and ovulation in the ovary of the domestic hen (Gallus domesticus) at different ages

Ovaries of laying hens of 14, 18, 30, 45 and 86 weeks of age were divided transversely into two or more distinct regions. Differences in follicular populations between these regions were observed. The central segments of the ovary in birds of 14 weeks of age produced most of the visible (greater than 0.5 mg) developing follicles (P less than 0.01). This was a transient effect during ovarian maturation. In birds of 18 weeks of age, but not yet in lay, more follicles of greater than 8 mm in diameter were observed in the anterior part of the ovary than the posterior part (P less than 0.05). Follicles of this size are almost certain to ovulate. The posterior segment of the ovary of birds of 30, 45 and 86 weeks of age contained more follicles beginning the rapid growth phase, as measured by follicles of 1.4-1.8 mm in diameter, than did the anterior segment (P less than 0.01). Higher levels of atresia in the posterior segment (P less than 0.001) resulted in fewer follicles of greater than 8 mm (P less than 0.001) and fewer post-ovulatory follicles than in the anterior segment. We conclude that most of the eggs produced during the hen's laying year must be from ovulations from the anterior part of the ovary.     

Age-dependent models of population growth

P. H. Leslie (1945, Biometrika, 33, 183–212) (and others) introduced vector-matrix growth difference equations to model populations in which birth and death rates are age-dependent. We develop differential versions of these equations in both the unrestricted growth and logistic cases. We find that the vector logistic equations (difference and differential) are explicitly solvable in terms of solutions of the unrestricted equations, even when vital rates vary with time. These explicit solution formulas make it easy to determine the behavior of solutions as time goes on.     

Age-dependent alterations of c-fos and growth regulation in human fibroblasts expressing the HPV16 E6 protein.

Normal human cells in culture become senescent after a limited number of population doublings. Senescent cells display characteristic changes in gene expression, among which is a repression of the ability to induce the c-fos gene. We have proposed a two-stage model for cellular senescence in which the mortality stage 1 (M1) mechanism can be overcome by agents that bind both the product of the retinoblastoma susceptibility gene (pRB)-like pocket proteins and p53. In this study we determined whether the repression of c-fos at M1 was downstream of the p53 or pRB-like "arms" of the M1 mechanism. We examined c-fos expression during the entire lifespan of normal human fibroblasts carrying E6 (which binds p53), E7 (which binds pRB), or both E6 and E7 of human papilloma virus type 16. The results indicate a dramatic change in cellular physiology at M1. Before M1, c-fos inducibility is controlled by an E6-independent mechanism that is blocked by E7. After M1, c-fos inducibility becomes dependent on E6 whereas E7 has no effect. In addition, a novel oscillation of c-fos expression with an approximately 2-h periodicity appears in E6-expressing fibroblasts post-M1. Accompanying this shift at M1 is a dramatic change in the ability to divide in low serum. Before M1, E6-expressing fibroblasts growth arrest in 0.3% serum, although they continue dividing under those conditions post-M1. These results demonstrate the unique physiology of fibroblasts during the extended lifespan between M1 and M2 and suggest that p53 might participate in the process that represses the c-fos gene at the onset of cellular senescence.     

The role of development and adrenal steroids in the regulation of the mineralocorticoid receptor messenger RNA.

The ontogeny, adrenal-feedback regulation and regional distribution of the mineralocorticoid receptor (MR) mRNA were examined in the rat brain and kidney. In the kidney, MR mRNA levels in the adult were only 25-30% of the neonatal concentration. Adrenalectomy caused a 35% increase in total brain MR mRNA and a 94% increase in kidney MR mRNA levels. Examination of the regional distribution of the MR mRNA within the brain revealed that the hippocampus had the highest levels, and the mRNA abundance increased after adrenalectomy. The administration of dexamethasone to intact animals resulted in a significant reduction of MR mRNA in the kidney of neonatal rats but not in the brain. These data indicate that there are developmental changes in MR gene expression in kidney and that adrenal steroids can modulate MR gene expression in both the brain and kidney.