http://www.jstage.jst.go.jp/browse/jpsa doi:+* . ,+.+ /jpsa. **3*2- Copyright ῌ ,*+*, Japan Poultry Science Association. Mohamed Abdelsalam, Naoki Isobe and Yukinori Yoshimura Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima1-3ῌ 2/,2 , Japan Avian beta-defensins (avBDs) play significant roles in the innate immune system. The aim of this study was to identify immunoreactive (ir) avBDs proteins in the hen ovarian follicles and the changes in their localization with follicular growth. The ovarian follicles at di#+,- erent growth stages, namely the largest (F ), second and third largest (F and F ), prehierarchal small yellow and cortical follicles, were collected. The presence of ir-avBD-2+* , - , and - +, were examined by immunohisto- chemistry and Western blot. The three ir-avBDs showed a similar pattern of immunostainings in the follicular tissues at di# er- ent growth stages. In the granulosa cells, the ir-avBDs were identified in the cortical follicles, whereas their density was re- duced in small yellow follicles. The granulosa cells of yellow follicles (F-+ -F ) showed dense immunolabelings. The interstitial cells showed a faint immunolabeling for avBD-+, but not for avBD- 2 and - +* in the cortical follicles, whereas they were weakly stained in the small yellow follicles. Dense immunoreaction products were noticed in the theca interna cells of F- - F+ follicles. Western blot analysis showed a single band for each defensin. These results suggest that avBD-2+* , - and - +, proteins are expressed in the specific cells in the follicles, namely interstitial or theca interna cells and granulosa cells, where their amounts are likely increased with follicular growth. These avBDs may play significant roles in the host innate immune system in the follicles. Key words: avian beta-defensins, follicular growth, hen ovary, ovarian follicles J. Poult. Sci.,.1 : 11ῌ 2. , ,*+* ῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍῍ Introduction been identified in the growing follicles. In contrast, re- ports on the innate immune functions in the follicles are The hen ovary is often infected by pathogenic microor- relatively limited. Toll-like receptors (TLRs) are one of ganisms such asSalmonella bacteria, and its infection may the members able to recognize microbial agents in the cause not only the ovarian functional disorder but also innate immune system, identifiable by the type of con- contamination of eggs due to bacterial transmission to the served pathogen-associated molecular patterns in a variety yolk (Neubaueret al.,,**3 ; Gantois et al., ,**3 ). Thus, of animals (Roach et al., ,**/ ). We have identified the ovarian immunity to protect the tissue from infection is mRNA expression of TLRs, which recognize the pattern essential to maintain the normal functions of the ovary of microorganisms and induces synthesis of antimicrobial and the production of hygienic eggs safe for human con- peptide and cytokines in the theca and granulosa layers of sumption. The ovary consists of cortical follicles embedded follicles (Subediet al., ,**1 a). in the ovarian stroma, prehierarchal and hierarchal folli- Beta-defensins are antimicrobial peptides that play a cles. The follicles undergo changes in the tissue struc- significant role in the innate immune response in a variety tures, responsiveness to gonadotropins, steroidogenic ac- of animals including avians (Harwinget al., +33. ; Evans tivities and yolk absorption ability during their growthet al.,+33. ; Fogaça et al., ,**. ; Lynn et al., ,**. ; Xiao et (Johnson,,*** ). The existence of immunocompetent cells al.,,**.; Aono et al., ,**0 ). The few avian beta-defensisns involved in adaptive immunity such as antigen presenting (avBDs) that have been studied for their antimicrobial cells expressing major histocompatibility complex class I activities displayed a wide range of antimicrobial or mi- and II (Subedi and Yoshimura,,**/ a), CD .ῌ and CD 2 crobistatic activities against Gram-positive and Gram- ῌ T cells (Barua and Yoshimura,+333 ; Subedi and negative bacteria and fungi (Higgset al., ,**1 ; Milona et Yoshimura,,**/ b), and B cells (Baruaet al., +332 ) have al., ,**1 ; Akbari et al., ,**2 ; Ma et al., ,**2 ; van Dijk et al.,,**2; Soman et al., ,**3 ). The avBDs may kill the mi- Received: September3 , ,**3 , Accepted: October +3 , ,**3 Released Online Advance Publication: December+* , ,**3 croorganisms by disrupting their membranes (van Dijk et ,**2 # Correspondence: Y. Yoshimura,Graduate School of Biosphere Science, al., ). Until now, the sequences of fourteen di erent Hiroshima University, Higashi-Hiroshima1-3ῌ 2/,2 , Japan. avBDsgenes have been reported (Lynn et al., ,**1 ; van (E-mail: [email protected]) Dijket al.,,**2 ). These avBD genes are located in re- 78 J. Poult. Sci.,.1 ( + ) latively close positions, namely approximately20 Kb care Bio-Sciences AB, Uppsala, Sweden) according to the singleavBD cluster on chromosome--/-1 q . -q . (Lynn et manufacturer’s directions. The antibodies of avBD- 2 al.,,**1; van Dijk et al., ,**2 ). Moreover, we have iden- and - +* in the antiserum and normal IgG in the pre- tified gene expression of0 types ofavBDs in the theca and immune rabbit serum (for control staining) were purified . types ofavBDs in the granulosa layer of white and using Hi Trap a$ nity Protein G HP column (GE Health- yellow follicles (Subediet al.,,**1 b). The avBD-+2+* , - , - care Bio-Sciences). and-+, were expressed in both layers. Intravenous injec- Immunocytochemistry tion of birds with lipopolysaccharide (LPS) caused an The ovarian stroma containing cortical follicles, small increase in the expression ofavBD-+2 , - and - +, in the yellow follicles approximately1 mm in diameter, the third theca and a decrease inavBD-++, and - expression in the largest (F-, ), the second largest (F ) and the largest granulosa layer. We have also identified the immunoreac- follicles (F+0 ) were collected h after oviposition (n῍ . ). tive (ir) avBD-+, in the growing follicles. The ir-avBD- +, They were fixed in Bouin fixative, dehydrated with graded was negligible in the white follicles (Subediet al., ,**2 ), ascending series of alcohols, cleared with xylene and whereas it was identified in the theca and granulosa layers embedded in para$. n. Sections (m m in thickness) were of yellow follicles. Although the gene expression profiles air-dried in MAS coated pre-cleaned slides (Matsunami ofavBD s in the follicles have been shown, the localization Glass Inc. Osaka, Japan). Then, the sections were depara- of their proteins remains to be studied except for avBD- finized with xylene and rehydrated. Antigen retrieval was +,. performed by autoclaving the sections for+*+ min in . M Thus, the aim of this study was to determine the pres- citric acid, pH0* . The immunohistochemistry was per- ence of avBD proteins in the follicles and the changes in formed using Vecta Stain ABC kit (Vector Laboratories, their localization during follicular growth. The avBD- Burlingame, CA, USA). The slides were incubated with 2+*, - and - +, , whose gene expression had been identified blocking solution +/ .ῌ (vol/vol) normal goat serum in in both the theca and granulosa layers, were examined. PBS for+ h at room temperature. Sections were incubated Specific questions were addressed as follows: (+ ) which overnight with rabbit antibodies to avBD- 2 , - +* or - +, follicular cells express these avBDs, (,,* ) whether specific diluted at a concentration ofm g/mL, followed by wash- follicular cells express di#--/ erent types of avBDs, and ( ) ing with PBS (ῌ min). The sections were then in- whether the localization of avBDs changes with follicular cubated with biotinylated anti-rabbit IgG and avidin- growth, using cortical follicles, prehierarchal (small yellow biotin-peroxidase complex for+ h each, and were washed follicles) and hierarchal follicles (yellow follicles). Al- with PBS (-/ῌ min) after each step. Immunoprecipitates though the localization of ir-avBD-+, in prehierarchal and were visualized by incubating the sections with**, . ῌ hierarchal follicles has been reported (Subediet al., ,**2 ), (wt/vol) -῍῍ , - -diaminobenzidine tetrahydrochloride and it was further examined for comparison with the localiza-* . **/ῌ (vol/vol) H,, O in * . */ M Tris-HCl (pH 1 . 0 ). The tion of avBD-2+* and - . sections were counterstained with hematoxylin and dehyd- Materials and Methods rated with graded series of alcohols and mounted. They were examined under a light microscope (Nikon Eclipse Experimental Animals E, Tokyo, Japan) using a Nomarsky filter. Control stain- White Leghorn hens (approximately.** -d-old) laying / ing was carried out simultaneously in which the first or more eggs in a sequence were kept in individual cages antibody was replaced with normal rabbit IgG. Simulta- under a+. h light: +* h dark regime. They were provided neously, other sections were also stained with hematoxylin with free access to feed and water. Handling of the hens and eosin for the observation of the general tissue struc- was conducted in accordance with the regulations of ture. Hiroshima University for animal experiments. SDS-PAGE and Western Blotting for avBDs in the Follicular Preparation of Rabbit Anti-avBD-2+* , - and - +, Polyclonal Tissue Antibodies Sample Preparation Antibodies to avBD-2+* and - were raised in rabbits The F+, , F and F - follicles were collected 0 h after using synthetic peptides conjugated with keyhole limpet oviposition. The surface connective tissue on the theca haemocyanin (KLH), whereas the antibody to avBD-+, was removed. The theca