N.R. Sundaresan

High doses of dietary zinc induce cytokines, chemokines, and apoptosis in reproductive tissues during regression

In chickens, high levels of dietary zinc cause molting, and the reproductive system undergoes complete remodeling concomitant to feather replacement. In the present study, the expression profiles of cytokines and chemokines were investigated in the ovary and oviduct of control hens and of hens induced to molt by zinc feeding. The zinc-induced feed-intake suppression, the changes in corticosterone levels, the immune cell populations in the reproductive tract, and the apoptosis of reproductive tissues were analyzed. The expression of mRNAs for interleukin-6 (IL-6), interferon-γ (IFN-γ), the avian ortholog of mammalian IL-8 (chCXCLi2), and a chicken MIP-1β-like chemokine (chCCLi2) in the ovary and of mRNAs for IL-1β, IL-6, IFN-γ, transforming growth factor-β2, chCXCLi2, and chCCLi2 in the oviduct were upregulated significantly during zinc-induced molting. A simultaneous feed-intake reduction was observed with higher expression of cytokines and chemokines. The results of the present investigation also suggested that the upregulation of corticosterone was closely associated with the increased expression of cytokines and chemokines. An increase in apoptosis within reproductive tissue during tissue regression was also noted. We had previously observed the upregulation of these cytokines expression in an earlier study (molting by feed withdrawal). However, the pattern and the level of expression were different among these two methods. These findings indicate that cytokines might be a common mediator of tissue regression during molting induced by diverse methods, although the pattern of induction is different. Thus, a high dose of dietary zinc seems to induce reproductive regression via the upregulation of cytokines and chemokines, the suppression of feed intake, and the increase in serum corticosterone, resulting finally in the apoptosis of reproductive tissues.

Expression analysis of melatonin receptor subtypes in the ovary of domestic chicken

Melatonin (N-acetyl-5-methoxytryptamine), an indole hormone, regulates various biological functions through three different receptor subtypes (Mel-1a, Mel-1b, and Mel-1c). However, the distribution of different melatonin receptor subtypes in chicken reproductive tissues was not known. In the present investigation, the partial sequences of ovarian melatonin receptor subtypes (Mel-1a, Mel-1b, and Mel-1c) were characterized. Further, the expression profile of melatonin receptor subtypes in the granulosa and theca layers of different preovulatory and postovulatory follicles (POF) were studied by semi-quantitative RT-PCR. The expression of all three subtypes of melatonin receptors were observed in the ovary of domestic chicken. Analysis of partial sequences of ovarian melatonin receptors revealed that the melatonin subtypes were identical to the brain receptors. In small white ovary follicles, we observed only the expression of mel-1b receptors, but not mel-1a or mel-1c receptors. In yellow follicles, all the three subtypes of receptors expression were noticed. Interestingly, we observed the expression of mel-1a receptor only in thecal layer, but not in granulosa layer. In contrast, mel-1b and -1c receptors were expressed in both granulosa and thecal layer. During the regression of POF, we observed significant upregulation of melatonin receptors (mel-1a and 1c) expression, that downregulated in the later stages of regression. We assume that the expression of melatonin receptors might have been influenced by the atresia or apoptosis of different follicular layers in POF. Our findings suggest that the differential distribution of melatonin receptor subtypes might have distinct downstream cellular functions in the ovarian tissues.

Caspase-mediated apoptosis in chicken postovulatory follicle regression.

Chicken postovulatory follicle (POF) regression occurs via the process of apoptosis. However, the signals and initiator pathways responsible for regression of the POF are unknown. In the current study, we examined gene expression patterns of various caspases (caspase-1, -2 and -3) involved in apoptosis by semi-quantitative RT-PCR. The percentage of apoptotic cells during POF regression was also quantified by flow cytometry. Expression of caspase-3 mRNA was noted in the largest preovulatory follicle (F1). However, the initiator caspases (caspase-1 and -2) were not expressed in F1. During the regression of the POF, caspase-3 was activated during initial stages, whereas the initiator caspases were upregulated at the later stages (POF4 and POF5).The percentage of apoptotic cells was significantly higher during the regression of the POF. It might be possible that levels of caspase-3 mRNA do not necessarily reflect the cell’s potential for facilitating apoptosis, as activation of the caspase-3 by initiator caspases is required for its function. We presume that both caspase-1 and caspase-2 were key initiators in the regression of chicken POF and that the apoptosis-mediated regression of POFs might be similar to mammalian corpus luteum involution.

Spatial expression of chemokines and cytokines mRNA in the largest preovulatory follicle of chicken

In the present experiment, we studied the spatial expression profiles of chemokines and cytokines mRNA in the granulosa (F1Gr) and theca (F1Th) layers of the largest preovulatory follicle in chicken using semi-quantitative PCR. The mRNAs of IL-1β, IL-6, GM-CSF, chCXCLi2, chCCLi2, chCCLi4, chCCLi7, IFN-γ, IL-4, IL-13, IL-10 and TGF-β2 were expressed in the granulosa (F1Gr) and theca (F1Th) layers of the largest preovulatory follicle. However, the transcripts of IL-2 were not detected in any of the samples tested. Significantly higher levels of IL-6 and GM-CSF mRNA expression were noticed in F1Gr when compared to F1Th layer. Expression of chCXCLi2, a CXC chemokine, was almost similar in F1Gr and F1Th layers. However, the expression of CCL chemokines i.e. chCCLi2, chCCLi4, chCCLi7 mRNAs were almost 2 folds higher in F1Th layer in comparison to F1Gr layer. The expression of Th2 cytokines (IL-4 and IL-13) mRNA was noticed in F1Gr and F1Th layers with higher levels in the former. Expression of IFN-γ mRNA was noticed in F1Gr and F1Th layers. Significantly higher level of TGF-β2 expression was observed in F1Th in comparison to F1Gr layer. It was concluded from the present study that the mRNA expression of cytokines and chemokines are differentially regulated in the granulosa and theca layers of the largest preovulatory follicle in chicken.