Several studies conducted on theca and granulosa cells have shown that leptin may have direct negative effects on ovarian steroidogenesis in various mammalian species. Leptin inhibits insulin-induced progesterone and 17β-estradiol production by isolated bovine granulosa cells  and impairs the hormonally-stimulated in vitro release of 17β-estradiol by rat granulosa cells . In granulosa cells from fertile women, leptin inhibits FSH and IGF-I stimulated estradiol production [28, 29] Since leptin has a more potent inhibitory action of insulin-induced aromatase activity of granulosa cells from small than large follicles, it has been proposed that the numbers of leptin receptors in granulosa cells might decrease as follicles develop in order to make mature Graafian follicles less sensitive to the negative action of leptin [26, 30]. As shown in this study and in previous reports [14, 17], the leptin receptor was expressed in the hen ovary in both granulosa and theca cells, suggesting a direct action of leptin at the level of the ovary. It seemed that leptin might affect ovarian steroidogenesis in laying hens during fasting  but the involvement of leptin on steroidogenesis during normal follicle development remained to be determined. In this study we demonstrated that the direct action of leptin on the ovary might be modified during follicle development since the level of expression of its receptor clearly decreased during maturation of yellow follicles. This decrease was particularly evident in slow growing broiler breeder hens from the "Label" genotype and from the feed-restricted standard line. Given that fast growing chickens (ad libitum-fed standard and Experimental broiler breeder hens) have the highest reproductive problems, genetic or nutritional control of the growth rate might regulate ovarian leptin receptor gene expression and improve reproductive function. Such evolution of receptor expression in the follicular hierarchy has previously been shown for the FSH receptor. FSH-stimulated steroidogenesis declined during follicle maturation and was associated with a decrease in FSH receptor numbers . Conversely, the expression of mRNA encoding the IGF-I receptor and the related efficacy of binding of IGF-I to granulosa cells increased as the follicle matured [32, 33]. Since leptin receptor gene expression was modified during follicle development, leptin might also be involved in regulation of the follicular hierarchy and onset of preovulatory steroidogenesis, as has been proposed for gonadotrophins and growth factors including FSH and IGF-I.
Unlike mammals, progesterone in chickens is synthesized and secreted mainly by granulosa cells whereas theca cells generate estradiol . Progesterone produced by granulosa cells from mature follicles provides the positive feedback necessary to stimulate a preovulatory surge of LH . IGF-I has been involved in the regulation of ovarian steroidogenesis in both mammals and birds. IGF-I stimulates progesterone production from avian granulosa cells  whereas it up-regulates estradiol from mammalian granulosa cells [27–29]. Since leptin is considered to be an inhibitor of insulin and IGF-I action on steroidogenesis in mammals, leptin might have similar negative action in birds. Thus, the decrease in its receptor in the granulosa suggests that the inhibiting action of leptin would decrease during follicle development and consequently favours the stimulatory effect of gonadotrophins and IGF-I on follicular maturation. This hypothesis is also consistent with the weaker steroidogenic response of granulosa cell culture of ad-libitum fed standard broiler breeder hens when stimulated by IGF-I compared to granulosa cell culture from feed-restricted birds . Moreover, Onagbesan et al (2004) demonstrated in an experiment similar to that performed in the present study and using the same genotypes that plasma progesterone levels were clearly affected in SA birds. They demonstrated that plasma progesterone levels remained relatively stable between 25 and 37 weeks of age in the E, L and SA birds with a significant lower level in the SA birds (2.2 ± 0.62 ng/ml for SA birds compared to 3.9 ± 0.36 and 4.2 ± 0.54 for L and E birds respectively). In restricted standard birds, plasma progesterone levels dramatically increased and reached values (3.8 ± 0.26 ng/ml) closed to that measured in the E and L lines (Onagbesan et al., 2004, data from progesterone levels were personal communication from Dr Onagbesan, Catholic University of Leuven, Belgium).
The erratic pattern of oviposition in standard broiler breeder hens fed ad libitum has been previously demonstrated to be related to abnormal maturation of steroidogenesis, particularly in the two largest yellow follicles . Since F2 and F1 yellow follicles presented similar endocrine profiles, the preovulatory surge of LH probably triggers ovulation of the two largest follicles [21, 36]. In this study we have shown that ad libitum feeding of broiler breeder hens dramatically up-regulated expression of the leptin receptor in the granulosa cells of yellow follicles and changed the evolution of expression of this receptor with follicle development. These results suggest a strong action of leptin on the ovaries of ad libitum fed birds. Feed restriction reduced the level of expression of the leptin receptor and on the whole restored the evolution of expression of the receptor with follicle maturation. Since ad libitum feeding affects the hierarchical endocrine order of the follicles, as a potential inhibitor of hormonally induced avian steroidogenesis leptin represented a good candidate to explain the affects of follicular hierarchy. Up-regulation of the expression of the leptin receptor gene was also demonstrated in the liver. This up-regulation may be related to the control of lipogenesis. The liver plays a key role in lipid metabolism and lipogenesis in avian species [38, 39] and the standard broiler breeder hens were the fattest birds of this experiment.
Since expression of its receptor was dramatically up-regulated in SA hens, leptin probably played an important role in the increased number of large yellow follicles and abnormal follicle hierarchy. However the factors involved in regulation of the expression of the leptin receptor within the hen ovary remains to be determined. Among the plasma hormones and lipids analyzed in this study only triglycerides were found to be different between strains, with a lower level in the restricted standard broiler breeder hens that were also the leanest birds. Down regulation of the expression of the leptin receptor by homologous and heterologous signals have previously been demonstrated in both mammals [40, 41] and chickens . Leptin and insulin are able to down-regulate expression of the chicken leptin receptor in vitro. In the present study, plasma leptin and insulin levels were similar for each genotype and were not altered by feed restriction in the standard genotype. We have previously demonstrated that during the first 5 weeks of age, plasma leptin levels remained relatively stable in both broiler and layer chicken despite increased body weight . However the absence of leptin levels differences may be related to the fact plasma leptin levels were measured at 32 weeks of age. SR birds were relaxed at the start of lay, switched to breeding feeding and allowed ad libitum access as the other groups of birds. We therefore suggested that leptin and insulin are probably not involved in the regulation of ovarian leptin receptor gene expression in ad libitum or feed-restricted standard broiler breeder hens.
Evidence of the regulation of expression of the leptin receptor gene in the granulosa related to follicle maturation and nutritional state strongly suggest that leptin played an important local and sequential role in the dysfunction of the follicular hierarchy observed in standard broiler breeder hens fed ad libitum. This study suggests that the level of expression of the leptin receptor regulates the action of its ligand at the level of the ovary. This provides an interesting perspective to understanding the physiological role of leptin in the ovary.