-This paper discusses the phenomenon of nutritional flushing in ewes whereby increased nutrition stimulates folliculogenesis and ovulation rate. In addition the paper reviews recent findings on the effects of increased levels of nutrition on the blood concentrations of reproductive and metabolic hormones in the ewe and some of the intraovarian changes that take place in response to nutritional stimulation. Finally, in the paper, we propose a model of the physiological mechanism for the nutritional stimulation of folliculogenesis and we review how closely the model fits recent published and unpublished evidence examining the mechanism of flushing. Nutritional stimulation alters the blood concentrations of some metabolic hormones. By using short-term models of nutritional flushing, we have shown that as the blood concentrations of insulin and leptin increase that of growth hormone decreases while that of IGF-I appears unaffected by the nutritional flushing. Nutritional flushing also alters the blood concentrations of some reproductive hormones. Again, using the same model, we have shown that there is a transient increase in FSH and a decrease in oestradiol concentrations in the blood. The changes in oestradiol are particularly evident in the follicular phase of the oestrous cycle. In the ovary, the effect of nutrition is to stimulate folliculogenesis. These changes are associated with intra-follicular alterations in the insulin-glucose, IGF and leptin metabolic systems. The stimulation of these intra-follicular systems leads to a suppression in follicular oestradiol production. The consequence of these direct actions on the follicle is a reduced negative feedback to the hypothalamic-pituitary system and increased FSH secretion that leads to a stimulation of folliculogenesis.insulin / leptin / IGF-I / glucose / FSH / oestradiol

Improved nutrition increases ovulation rate in sheep and there is evidence that intra-ovarian pathways mediate responses to nutrition. An experiment was conducted to examine the effect of dietary energy on folliculogenesis. Anoestrous Merino ewes were fed a diet of wheat straw alone (control, n = 5), or wheat straw supplemented with lupins (500 g day(-1), n = 5). Other ewes were fed wheat straw and infused with glucose (50 mmol h(-1), n = 5) or with glucosamine (3.5 mmol h(-1), n = 5). Intravaginal progestagen sponges were inserted for 12 days, and nutritional treatments were started 5 days before sponge removal. At sponge removal, the ewes were injected with a regimen of GnRH pulses (500 ng every 4 h from 0 to 12 h; 250 ng every 2 h from 14 to 24 h; and 200 ng every 1 h from 25 to 36 h) to simulate normal follicular development. Thirty-six hours after sponge removal, the animals were killed and the ovaries were collected and stored at -80 degrees C. The ovaries were sectioned serially every 10 microm. Every 20th section was stained (to estimate number and diameter of follicles) and every 17-19th section was probed by in situ hybridization for P(450) aromatase. Data were analysed using ANOVA and chi-squared tests. There was an effect of treatment (P < 0.05) on the number of follicles 2-3, 3-4 and 6-7 mm in diameter. Aromatase-positive follicles (1.6-7.9 mm) were detected in 31 follicles from 15 ewes across all four groups. In ten animals, the largest follicle was aromatase-positive. The diameters of aromatase-positive follicles were larger (P = 0.004) in lupin fed compared with glucose-infused ewes (4.9 +/- 0.5, 3.6 +/- 0.7, 5.3 +/- 0.5 and 4.2 +/- 0.5 mm for control, glucose-infused, lupin-fed and glucosamine-infused groups, respectively). Treatment did not affect the plasma concentration of FSH when compared with controls, indicating that the energy supplements were modifying recruited (2-3 mm and 3-4 mm) and selected follicles (> 6 mm) directly. In conclusion, dietary energy can directly stimulate folliculogenesis in recruited and selected follicles, and this effect may be mediated by changes in systemic leptin concentrations and the hexosamine energy-sensing pathway in the follicle.

An experiment was carried out to determine the pattern of follicular expression of mRNAs for aromatase, IGF-I receptor (IGF-IR), IGF-binding protein (IGFBP)-2, -4 and -5, leptin and the long form of the leptin receptor (Ob-Rb) in ten ewes infused with human recombinant leptin (n 5 5; 1 mg/h) or saline (n 5 5) for 72 h in the luteal phase of the oestrous cycle. At the end of infusion a follicular phase was induced with a luteolytic dose of a prostaglandin F2a analogue and the ovaries were collected 32 h later. One ovary from each ewe was serially sectioned at 10 mm using a cryostat at 220 8C. All follicles >1 mm in diameter were counted and probed with specific oligoprobes for aromatase, IGF-IR and IGFBP-2, -4 and -5 and specific riboprobes for leptin and Ob-Rb. Leptin mRNA was detected in theca and granulosa cells and Ob-Rb mRNA was detected only in granulosa cells, of some, but not all antral follicles. Leptin doubled the number of follicles with a diameter $3.5 mm (1.0 6 0.36 (S.E.M.) vs 2.4 6 0.24; control vs leptin; P < 0.02) but had no effect on the number of $1 < 3.5 mm follicles. Leptin had no effect on the number of follicles expressing aromatase mRNA but it decreased significantly the number of follicles expressing mRNA for IGF-IR (10.7 6 0.79 vs 7.4 6 0.81; control vs leptin; P < 0.05), IGFBP-2 (10.0 6 0.82 vs 5.2 6 0.87; control vs leptin; P < 0.05) and IGFBP-5 (5.2 6 1.60 vs 1.2 6 0.30; control vs leptin; P < 0.05). Leptin increased the diameter of IGFBP-2 mRNA-positive follicles (1.5 6 0.15 vs 2.2 6 0.31 mm; control vs leptin; P < 0.05) and increased follicular mRNA expression for IGFBP-2 (0.30 6 0.021 vs 0.39 6 0.027 arbitrary units; control vs leptin; P < 0.05) and IGFBP-5 (0.46 6 0.019 vs 0.25 6 0.053 arbitary units; control vs leptin; P < 0.05). The mRNA for IGFBP-4 was detected in the theca of only two follicles from the control group. Leptin increased the number of follicles expressing Ob-Rb mRNA (0.25 6 0.25 vs 1.40 6 1.17; control vs leptin; P < 0.05) but had no effect on the number expressing leptin mRNA. Leptin decreased plasma concentrations of oestradiol (P < 0.05) and increased concentrations of FSH (P < 0.001) and insulin (P < 0.001), with no effect on glucose concentrations. These data show that: (i) ovine granulosa cells express mRNA for Ob-Rb and leptin and (ii) leptin increased the number of follicles $ 3.5 mm. Furthermore, the data suggest that suppression of oestradiol production by leptin is not mediated by inhibition of aromatase gene expression. Finally, the data indicate that the action of leptin in ovarian follicles is mediated by the IGF system, because leptin increased mRNA expression of IGFBP-2 and -5. Leptin also decreased the number of follicles expressing IGF-IR and IGFBP-2 and -5. We suggest that these actions of leptin on the IGF system decrease the bioavailability of IGF-I, resulting in decreased oestradiol production.

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The IGF system is associated with ovarian folliculogenesis. The effect of the IGFs mediated through the type I receptor (IGF-IR) and IGF-binding protein-2 (IGFBP-2), is to regulate the growth and atresia of follicles. To test if the mRNAs for IGF-IR and IGFBP-2 are differentially regulated in the follicle we used nutritional treatments that stimulate folliculogenesis and measured, by in situ hybridisation, their mRNAs expression. Groups of five anoestrous Merino ewes were fed wheat straw (control) or the control diet supplemented with lupins (500 g/day). Other ewes were fed the control diet and infused with glucose (50 mmol/h) or with glucosamine (3.5 mmol/h). Intravaginal progestagen sponges were inserted for 12 days, and nutritional treatments were started 5 days before progestagen removal. Follicular development was studied after an artificial follicular phase, simulated by progestagen for 12 days and a regime of GnRH pulses given for 36 h following progestagen withdrawal, when the animals were killed. The ovaries were collected and stored at 2 80 8C until sectioning at 10 mm. Every 25-28th and 29-32nd section was probed for IGF-IR and IGFBP-2 using 35 S-labelled oligonucleotide probes. None of the nutritional treatments affected the number or size of follicles positive for IGF-IR, but glucose (P < 0.001) and lupin (P < 0.001) treatments reduced the follicular concentration of mRNA. The nutritional treatments all increased the number of follicles positive for IGFBP-2 (P < 0.05) and reduced their mean diameter (P < 0.05) and with the exception of lupin feeding, the concentration of mRNA (P < 0.05). The results show that all treatments affected the intrafollicular IGF system and suggest that IGF-IR and IGFBP-2 are nutritionally regulated in the follicle. However, the effects of treatments were variable and suggest the existence of multiple regulatory mechanisms that allow for normal variation in composition and balance of the ruminant diet.