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.

Dietary aspirin decreases the stage of ovarian cancer in the hen.

OBJECTIVE: We aimed to determine the effects of dietary aspirin treatment on ovarian cancer incidence and progression in the hen as a model for the human disease. METHODS: Hens were fed a standard layer diet (control) or the same diet containing 0.1% aspirin for 1 year. Liver prostaglandin E(2) (PGE(2)) was measured using an enzyme immunoassay. Incidence and stage of ovarian cancer were determined through necropsy and immunohistochemical analysis of ovarian sections for each hen. RESULTS: Aspirin treatment decreased liver PGE(2) in treated hens as compared to control hens. Treatment with aspirin did not decrease ovarian cancer incidence. Significantly more control hens developed late stage ovarian cancer than early stage, while the same was not true for aspirin-treated hens. Hens that developed ovarian cancer, even early ovarian cancer, produced significantly fewer eggs in the year prior to diagnosis than hens without ovarian cancer. CONCLUSIONS: Aspirin treatment may inhibit the progression of ovarian cancer in the hen and egg production may be used to identify hens with early stages of the disease.

VEGF expression and the effect of NSAIDs on ascites cell proliferation in the hen model of ovarian cancer.

OBJECTIVES: We aimed to determine the expression of vascular endothelial growth factor (VEGF) and the effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on the proliferation of cells isolated from ascites in the hen model of ovarian cancer. METHODS: Ovarian tumor and normal ovary were collected from hens and ascites cells were isolated from hens with ovarian cancer. Quantitative real-time PCR was used to quantify mRNA expression. Immunohistochemical and/or Western blot analyses were used to localize protein expression in ovarian tumors, normal ovaries, and ascites cells. Cells were treated with a nonspecific, COX-1-specific, or COX-2-specific NSAID and proliferation was determined. RESULTS: VEGF mRNA was increased in ascites cells and there was a trend for a correlation between VEGF mRNA in ascites cells and ascites volume. VEGF protein was localized to theca cells of normal ovaries, in glandular areas of tumors, and to the cytoplasm of ascites cells. Aspirin and a COX-1-specific inhibitor decreased the proliferation of ascites cells, whereas a COX-2-specific inhibitor did not. CONCLUSIONS: VEGF may play a role in ovarian cancer progression in the hen and the proliferation of ascites cells can be decreased by targeting the COX-1 but not COX-2 pathway.

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).

Cyclooxygenase 1 and 2 mRNA and protein expression in the Gallus domesticus model of ovarian cancer.

OBJECTIVE: Our purpose was to determine the mRNA and protein expression of cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2) in ovarian tumors and normal ovaries of the hen, which is an excellent model for human ovarian cancer. Tissue concentrations of prostaglandin E(2) (PGE2) and PGE2 metabolites were also determined. METHODS: Tissue was obtained from ovarian tumor (n = 18) and normal ovary (n = 29) of 2- to 4-year old Single-comb White Leghorn hens. Quantitative real-time PCR with Sybr Green was used to quantify the mRNA expression of COX-1 and COX-2, using 18S expression as an internal control for COX normalization. Immunohistochemistry using antibodies for COX-1 and COX-2 was used to localize protein expression of each isoform in a subset of tumor (n = 5) and normal samples (n = 6). For determination of tissue prostaglandin concentration, tissue was obtained from ovarian tumor (n = 8) and normal ovary (n = 8). PGE2 and PGE2 metabolites were measured using competitive enzyme immunoassays (EIAs). RESULTS: Our results indicate that COX-1 mRNA expression is significantly higher (P < 0.05) in ovarian tumor samples compared to normal ovaries while there is no significant difference in expression of COX-2 between the samples. Immunohistochemistry results support this finding and show COX-1 expression only in tumor samples and COX-2 expression unchanged between normal ovary and tumor samples. PGE2 levels are significantly higher (P < 0.05) in tumor samples compared to normal ovaries, and there is no significant difference in PGE2 metabolite levels between the samples. CONCLUSION: These findings may implicate COX-1 as a suitable target for the prevention or treatment of ovarian cancer.