Expression of anti-Mullerian hormone in hens selected for different ovulation rates.

In hens, the granulosa layer is the primary source of anti-Mullerian hormone (AMH), as it is in mammals. Small follicles express the greatest amount of Amh mRNA with less in the larger follicles. Laying hens have a distinct ovarian hierarchy of follicles while broiler breeder hens often have excessive follicle growth with a disrupted hierarchy. The objective of Experiment 1 was to examine Amh expression in two strains of hens differing in ovulatory efficiency. Amh expression was greater (P<0.01) in broiler breeder hens (n=6) as compared with laying hens (n=6). Experiment 2 was designed to examine whether alterations in follicular development due to diet, within the broiler breeder hens, were correlated with changes in the expression of Amh. Restricted feeding (RF) in broiler breeder hens promotes optimal follicular development. Egg production in broiler breeder hens on full feed (FF; n=8) was 78% that of hens on RF (n=9). The number of large follicles (P<0.05), total ovarian weight (P<0.01), and Amh mRNA expression were greater in FF hens as compared with RF hens (P<0.01). There was no difference in FSH receptor expression between the two groups. A direct nutritional effect was not supported because culture of granulosa cells with varying concentrations of glucose and insulin showed no effect on granulosa Amh expression. Finally, testis-conditioned medium resulted in a dose-related increase in granulosa cell proliferation, which could be inhibited by preincubation with AMH antibody. AMH may enhance granulosa cell proliferation through an autocrine or paracrine mechanism although excessive AMH may inhibit optimal follicle selection.

Expression and regulation of anti-mullerian hormone in an oviparous species, the hen.

Anti-mullerian hormone (AMH) has a critical role in regression of the mullerian duct system during development in male mammalian and avian species and in regression of the right oviduct in female avian species. AMH in adult female birds has not been investigated. Chicken-specific cDNA primers were used to isolate Amh by RT-PCR. This probe was used in Northern blot analysis to identify a 2.8-kb band with expression in total ovarian RNA and in granulosa cell RNA. Quantitative real-time PCR was used to assess Amh expression in follicles of different maturity (1, 3, 5, and 6-12 mm and the largest F1 follicle; n = 4-6 of each size). There was an increased amount of Amh mRNA in the granulosa layer of the smaller follicles and a lower amount in the granulosa layer of the larger follicles (P < 0.01). There was no difference in granulosa Amh expression between the germinal disc and non-germinal disc region of 6- to 12-mm follicles, although expression differed with follicle size (P < 0.01). To examine hormone regulation of Amh, granulosa cells (from 6- to 8-mm follicles) were cultured with various concentrations of estradiol (E(2)) and progesterone (P(4)), and Amh mRNA was assessed. Neither E(2) nor P(4) influenced Amh mRNA accumulation. Granulosa cells were also cultured in the presence of oocyte-conditioned medium (OCM), which decreased Amh mRNA expression in a dose-related manner (P < 0.05); FSH receptor expression was not affected. Heat treatment of OCM abolished the effect, but growth differentiation factor 9 antiserum did not block the suppression. Immunohistochemistry confirmed that the granulosa layer was the predominant source of AMH in the small follicles of the hen and indicated that AMH was present early in follicle development, with expression in very small follicles (approximately 150 mum).

Effect of activin A and inhibin A on expression of the inhibin/activin beta-B-subunit and gonadotropin receptors in granulosa cells of the hen.

Activin A has been shown to be abundant in the theca layer of the large pre-ovulatory follicles of the hen whereas inhibin A is produced in the granulosa layer. The purpose of this study was to investigate the effects of activin A and inhibin A on granulosa cell expression of inhibin beta-B-subunit, FSH receptor (FSHR), and LH receptor (LHR). Granulosa cells were isolated from the F1, F3+F4, and small yellow follicles (SYF; 6-12 mm diameter) of laying hens and pooled according to size. The cells were dispersed and plated in the presence of 0, 10, or 50 ng/ml recombinant human activin A (n=5 replicate cultures). RNA was subsequently extracted from the cells and Northern blots performed. Cell proliferation was determined for all treatments. An identical set of experiments was performed in which the granulosa cells were treated with recombinant human inhibin A (n=4 replicate cultures). Treatment with activin A at 50 ng/ml significantly (p<0.05) increased expression of beta-B-subunit for granulosa cells from all follicles. This dose also significantly increased expression of FSHR in granulosa cells from all follicles (p<0.05) and increased expression of LHR in cells from F1 and F3+F4 follicles (p<0.01) with no significant effect on cells from the SYF. Overall, activin A treatment significantly (p<0.05) decreased cell proliferation at the 50 ng/ml dose. Inhibin A had no significant effect on expression of beta-B-subunit, FSHR or LHR at any dose. There was a moderate stimulatory effect of inhibin A on granulosa cell proliferation. These results suggest that activin A may have an important role in regulating granulosa cell responsiveness to gonadotropins while also modulating follicle development by attenuating cell proliferation.

Expression and function of growth differentiation factor-9 in an oviparous species, Gallus domesticus.

Many studies have indicated a critical role for the oocyte growth factor, growth differentiation factor-9 (GDF9), in mammalian follicle development, but no information has been available concerning oviparous species. We cloned a cDNA for chicken GDF9 (162 base pairs) and used it in Northern blot analysis to identify a transcript at 1.7 kilobase in RNA isolated from the ovary of the hen. We also sequenced two full-length clones from a normalized chicken reproductive tract cDNA library. The cDNA clone for chicken GDF9 encodes a protein of approximately 449 amino acids and all six cysteine residues, and three of the four glycosylation sites are conserved with respect to mammalian GDF9. Chicken GDF9 is approximately 65% similar in the full-length cDNA sequence and 80% similar in amino acid sequence at the C-terminal region to GDF9 from several mammals. Quantitative polymerase chain reaction analysis (n = 5) indicated that GDF9 mRNA is greatest in follicles < 1 mm in size compared with larger follicles or granulosa layers isolated from larger follicles. Immunocytochemical analysis showed strong expression of GDF9 in hen oocytes. In yolk-filled oocytes, the GDF9 was localized at the periphery of the oocyte. Finally, oocyte-conditioned medium (from < 1-mm oocytes) resulted in a 2-fold increase in granulosa cell proliferation, which could be inhibited by preincubation of the conditioned medium with GDF9 antibody. These data suggest that GDF9 is present in the hen oocyte and that this factor is capable of enhancing granulosa cell proliferation, as has been demonstrated in mammals.