Changes in the expression of gallinacins, antimicrobial peptides, in ovarian follicles during follicular growth and in response to lipopolysaccharide in laying hens (Gallus domesticus)

Abstract

The aim of this study was to identify the types of gallinacin genes (GALs) expressed in ovarian follicles and to determine the changes in their expression during follicular growth and in response to lipopolysaccharide (LPS). Follicles at different stages of growth were collected from laying hens (nZ5) and LPS-injected hens (nZ3). The expression of GALs in the theca and granulosa layers was examined by semi-quantitative RT-PCR. The expression of GAL-1, -2, -7, -8, -10, and -12 in the theca layer and GAL-1, -8, -10, and -12 in the granulosa layer was identified in white and yellow follicles. The expression of these genes was not changed in the theca and granulosa layers during follicular growth except for a decrease in that of GAL-1 in theca. The expression of GAL-1, -7, and -12 in the theca layer of the third largest follicles was increased in response to LPS at a dose of 1 mg/kg body weight and this increase was induced within 3 h and maintained until 12h postinjection. Granulosa layers did not respond to LPS until 12h injection. These results show that six and four types of GALs are expressed in the theca and granulosa layers of healthy follicles respectively, and their levels do not change with follicular growth except for GAL-1 in theca. Elevated levels of GAL-1, -7, and -12 expression in theca in response to LPS suggest that the theca cells expressing these GALs function to eliminate LPS-containing bacteria.

Changes in the Expression of Toll-Like Receptor mRNAs During Follicular Growth and in Response to Lipopolysaccharide in the Ovarian Follicles of Laying Hens

“…TLRs also mediate induction of antimicrobial peptides [19]. Expression of gallinacins (GALs), a class of antimicrobial peptide of chicken, in the theca layer is enhanced by LPS [23]. In mammals, expression of antimicrobial peptide β-defensin-2 is regulated by TLR-4 in response to LPS in intestinal epithelial cells [44].…”

“…This dose was sufficient to induce an immune response in our previous study [23]. The stock solution of LPS was prepared by dissolving LPS from E. coli 0111:B4 (Wako Pure Chemical, Osaka, Japan) in Dulbecco's phosphate buffer (Nissui Pharmaceutical, Tokyo, Japan) at 4 mg/ml concentration.…”

“…Semi-quantitative RT-PCR was conducted as described previously [23]. The RNA samples were reverse transcribed using reaction mixture (10 µl) consisting of 1 µg of total RNA, 1 × RT buffer, 1 mM of each dNTP, 20 U RNase inhibitor, 0.5 µg of oligo (dT)20 and 50 U of ReverTra Ace in a PTC-100 programmed at 42 C for 30 min, and this was followed by heat inactivation for 10 min at 99 C. The PCR was performed in a PTC-100 in a volume of 25 µl of reaction mixture containing cDNA corresponding to 1 µg of the initial total RNA, 1 × PCR buffer, 0.2 mM each dNTP, 0.4 µM each primer and 0.625 U Takara Taq (Takara Bio).…”

Expression of Antimicrobial Peptides in Cecal Tonsils of Chickens Treated with Probiotics and Infected with Salmonella enterica Serovar Typhimurium

“…Yoshimura et al (35) reported that inoculation of cell cultures of the hen oviduct with Salmonella enterica serovar Enteritidis and lipopolysaccharide (LPS) increased the expression levels of AvBD1, AvBD2, and AvBD3. Upregulation of AvBD1, AvBD7, and AvBD12 in the theca layer of ovarian follicles in LPS-injected laying hens has also been demonstrated (24). AvBD4 was expressed significantly in the liver of chickens in response to Salmonella serovar Enteritidis and serovar Typhimurium (17).…”

Three-dimensional NMR Structure of Hen Egg Gallin (Chicken Ovodefensin) Reveals a New Variation of the β-Defensin Fold

“…The sequencing of the chicken (Gallus gallus) genome revealed the presence of a cluster of 14 different genes on chromosome 3 coding for avian defensins and designated as AvBD1 to -14 (22), with specific tissue distribution (13,(22)(23)(24)(25). Proteomic analyses of the chicken egg revealed the presence of AvBDs, especially AvBD9, AvBD10, and AvBD11, identified in the egg yolk, eggshell, and eggshell/egg white/vitelline membrane, respectively (26 -29).…”