Anti-Mullerian hormone (AMH) is involved in the regression of the Mullerian ducts in mammalian and avian male embryos as well as the right oviduct in avian female embryos. AMH is expressed by granulosa cells of adult hens and mammals and is thought to be involved in the recruitment of follicles from the primordial pool as well as in regulating follicle-stimulating hormone (FSH) sensitivity. We have shown that AMH expression by the granulosa layer of hens is high in the small follicles but decreased in the larger hierarchical follicles. The decline in expression of AMH with increasing follicle size is associated with an increase in expression of the receptor for FSH (FSHR) in the granulosa layer, although the mechanism is not known. In this study, we tested whether vitamin D (1,25-dihydroxyvitamin D3) regulates expression of AMH mRNA in granulosa cells of the hen. Granulosa cell layers were removed from follicles 3-5 mm and 6-8 mm in size, dispersed, and cultured for 24 h in Medium 199 + 5% fetal bovine serum (n = 7). The medium was removed and replaced with Medium 199 + 0.1% bovine serum albumin and vitamin D (at doses of 0, 10, and 100 nM) and cultured for 24 h. Cells were harvested and RNA was extracted for use in quantitative PCR. Parallel 96-well plates were set up to examine cell proliferation. AMH and FSHR mRNA expressions were evaluated, and all values were standardized to 18S reactions. There was a significant (P < 0.05) dose-related decrease in the expression of AMH mRNA in granulosa cells of 3- to 5-mm and 6- to 8-mm follicles in response to vitamin D. Additionally, FSHR mRNA and cell proliferation were significantly (P < 0.05) increased by vitamin D in both groups. Western blot analysis for the vitamin D receptor (VDR) showed doublet bands at the expected sizes (58 and 60 kDa) in protein isolated from the chicken granulosa layer. Immunohistochemistry was used to identify VDR within the follicle, and it predominantly localized to the nucleus of granulosa cells. VDR mRNA expression in the granulosa layer, relative to follicle development, was increased (n = 4; P < 0.05) with follicle development, with greatest expression in the F1 follicle. There was no evidence for expression (mRNA or protein) of the calcium-binding protein, calbindin (CALB1), in the ovary or granulosa layer. Overall, these results suggest that vitamin D regulates AMH expression, and thereby may influence follicle selection in the hen.

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Iron overload disorder (IOD) can lead to organ dysfunction and may exacerbate other diseases in the critically endangered black rhinoceros ( Diceros bicornis). It is important to develop methods for monitoring the progression of iron storage (hemosiderosis), diagnosing the disease, and evaluating treatments in this species. Traditionally, an equine enzyme immunoassay (EIA) was used to measure rhinoceros ferritin, a serum protein correlated to iron stores. The goal of this study was to validate a rhinoceros-specific assay and investigate factors potentially associated with ferritin concentrations in black rhinoceros. A ferritin EIA developed for Sumatran rhinoceros was validated for black rhinoceros via Western blot analysis of liver ferritin and confirmed parallelism of serum samples to the EIA standard curve and used to analyze serum samples ( n = 943) collected from 36 black rhinoceros (<1-33 yr) at 14 U.S. institutions. Mean (±SEM) serum ferritin concentration was 6,738 ± 518 ng/ml (range: 85-168,451 ng/ml). Concentrations differed among individuals with eastern black rhinoceros (7,444 ± 1,130 ng/ml) having a higher mean ferritin than southern black rhinoceros (6,317 ± 505 ng/ml; P < 0.05) and higher mean values in wild-born (11,110 ± 1,111 ng/ml) than captive-born individuals (3,487 ± 293 ng/ml; P< 0.05). Ferritin concentrations did not differ between young rhinoceros (<5 yr old; 2,163 ± 254 ng/ml) and adults (7,623 ± 610 ng/ml) and were not correlated with age ( r = 0.143) or time in captivity ( r = 0.146, wild born; r = 0.104, all animals). Ferritin concentration was not impacted by sex (female: 2,086 ± 190 ng/ml; male: 8,684 ± 717 ng/ml), date, month, or season of collection ( P > 0.05). Data indicate ferritin concentrations are variable and not necessarily associated with IOD; ferritin is not recommended for diagnosing or monitoring IOD in black rhinoceros.