Mohamed Abdelsalam, Naoki Isobe and Yukinori Yoshimura

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http://www.jstage.jst.go.jp/browse/jpsa doi:+* . ,+.+ /jpsa. **3*2- Copyright ῌ ,*+*, Japan Poultry Science Association.

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 immunohistochemistry and Western blot. The three ir-avBDs showed a similar pattern of immunostainings in the follicular tissues at di# erent growth stages. In the granulosa cells, the ir-avBDs were identified in the cortical follicles, whereas their density was reduced 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. , ,*+*

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Introduction been identified in the growing follicles. In contrast, reports 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 was separated from the granulosa had been prepared in the previous study (Subedi et al., layer in PBS and the granulosa layer was washed in PBS to ,**2). Amino acid sequence of synthetic peptides of remove the yolk. The tissues of the three follicles were avBD-2+*+, , avBD- and avBD- used for immunization pooled and used as one sample. Each tissue was homo- were as follows: avBD-2 (NNEAQCEQAGGI), AvBD- genized in/# times volume of homogenization bu er con- +*(DTVACRTQGNF) and avBD- +, (GPDSCNHDR- sisting of,* mM Tris-HCl (pH 1 . . ), +/* mM NaCl, / mM GLCRVGNCNPGEYLAKYCFEPVILCCKP). The se- EDTA,+ῌῌ (v/v) Triton X- +** , +* (v/v) glycerol, quences of those peptides corresponded to their specific *+.ῌ (w/v) SDS and + mM phenylmethylsulfonylfluoride sequence (Xiaoet al. ,**. ). The anti-avBD- +, antibody using a polytron homogenizer (Kinematica AG, Switzer- in the antiserum was purified using HiTrap a$ nity column land). The samples were centrifuged at+, , *** X g for ,* conjugated with synthetic avBD-+, peptide (GE Health- min. The supernatant was collected and the protein con- Abdelsalamet al. : avBDs in the Ovarian Follicles 79 centration was measured using protein assay reagent (Bio- Rad Lab, Hercules, CA, USA) as described by the manufacturer. Tricine-SDS-PAGE The samples were separated by Tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Tricine SDS- PAGE;+0ῌῌ separating gel and . stacking gel) as described by Scha¨gger (,**0 ) with minor modifications. Samples containing+* m g protein were mixed with sample bu#-*/ er composed ofῌῌ (v/v) glycerol, (v/v) mer- captoethanol,.**0ῌῌ (w/v) SDS, . (w/v) bromophenol blue and+/* mM Tris-HCl, pH 1 . * , and boiled for +* min. Each+/ml of sample were loaded onto gels and run at /* V in the stacking gel and at+/* V in the separating gel. Western blotting After SDS-PAGE, the proteins in the gel were elec- trophoretically transferred onto a nitrocellulose membrane (PALL Gelman Laboratory, Ann Arbor, MI, USA). The membrane was washed briefly with western bu# er ( **, . M Tris-HCl, pH 1. . , *+/ . M NaCl, */ .ῌ (v/ v) Tween,* , and * . */ῌ (w/v) BSA) and incubated with /ῌ (w/v) casein milk (Roche, Mannheim, Germany) solution in western bu#0* er for min. The membrane was then incubated with avBD-2+*+, , - or - antibodies diluted at a concentration of+* m g/ ml in Can Get Signal immunoreaction enhancer solution+ for primary antibody (Toyobo Co., Ltd., Osaka, Japan) containing+ῌ casein milk for+ h at room temperature. The membrane was then washed in western bu#-*+*- er for min ( min X ) before incubation with alkaline phosphatase conjugated Fig.+ . Sections of hen ovarian follicles at di# erent growth goat anti-rabbit IgG (Assay Designs, Inc., Ann Arbor, stage. a) cortical follicle, b) small yellow follicle, c) the # MI, USA) diluted at+ : / , *** in Can Get Signal im- largest follicle. Note that theca layer is di erentiated into externa and interna in the small yellow and the largest munoreaction enhancer solution, for second antibody + follicle unlike the cortical follicle. Granulosa layer of the (Toyobo Co., Ltd.) for h at room temperature. The small yellow follicle shows a multi-cell layer structure. Gῌ #-*+* membrane was washed with western bu er for min ( granulosa layer; Tῌῌ theca layer, TI theca interna, TE ῌ min X- times) and the immunoprecipitates on the mem- theca externa, Yῌῌ yolk, * interstitial cells and theca interna brane were visualized by incubating in a reaction mixture cells, arrowῌῌ capillary, arrow head perivitelline membrane. composed of* . *+1ῌ (w/v) / -bromo- . -chloro- - -indolyl HE staining. Scale bars represent,* m m. phosphate disodium salt (Sigma-Aldrich, Inc., St. Louis, MO, USA) and*./ .ῌ (w/v) nitro blue tetrazolium (Nacalai Tesque, Inc. Kyoto, Japan) in substrate bu# er yellow follicles; namely, the granulosa layer consisted of a (*+ . M Tris-HCl, pH 3/ . , *+ . M NaCl and */ . M MgCl, ). single cell layer and the thickness of theca externa and The membrane was finally washed in running water to interna increased. In the theca interna, the theca interna stop the reaction. For the control staining, the first anti- cells were located near the outer border of this layer and bodies were replaced with normal rabbit IgG. The results capillary beds were well developed (Fig.+ c). were confirmed by three di# erent trials. In the granulosa layer, immunolabeling of avBD-2 was Results identified in the cytoplasm of granulosa cells facing to the theca layer in cortical follicles, whereas their density was Figure+ shows the structures of cortical, small yellow faint in the small yellow follicles (Fig., a, b). Dense and yellow follicles. The cortical follicles consisted of a immunolabeling occurred in the whole cytoplasm of granulosa layer that is single cell layered and a thin theca granulosa cells in the yellow follicles, namely F-+ῌ F (Fig. layer containing the interstitial cell islets (Fig.+, a). In the c, d). The same immunolabeling pattern as avBD- 2 was small yellow follicles, the granulosa layer had several cell observed for avBD-+* (Fig. - ) and - +, (data not shown) layers, and the theca layer was di# erentiated into the theca in the cortical, small yellow and yellow follicles. externa and interna where theca interna cells and In the theca layer, immunoreaction products of avBD-2 capillaries were observed (Fig.+, b). The yellow follicles were negligible in the cortical follicles (Fig. a), whereas (F+- -F ) showed more developed features than the small the thecal interstitial cells were faintly immunostained in 80 J. Poult. Sci.,.1 ( + )

Fig., . Sections of growing ovarian follicles immunostained for avBD-2 . a) cortical follicle, b) small yellow follicle, c) the third largest follicle, d) the largest follicle. Note that the granulosa cells are immunolabeled in all the follicles (short arrows), whereas the density is faint in the small yellow follicle. Interstitial cells of small yellow follicle show a faint immunostaining (b), and theca interna cells in the largest and third largest follicles contain dense immunoreaction products (c and d) (long arrows). *ῌ interstitial cells. See Fig.+ for other abbreviations. Scale bars represent,* m m.

Fig.- . Sections of growing ovarian follicles immunostained for avBD-+* . a) cortical follicle, b) small yellow follicle, c) the third largest follicle, d) the largest follicle. Granulosa cells contain small amount of immunoreaction products in cortical and small yellow follicles, whereas they are densely immunolabeled in the largest and third largest follicles (short arrows). Interstitial cells of small yellow follicle contain small amount of immunoreaction products and theca interna cells in the largest and third largest follicles contain dense immunoreaction products (c and d) (long arrows). See Figs.+, and for abbreviations. Scale bars represent ,*m m. small yellow follicles (Fig., b). In the yellow follicles, the not shown) in the theca layer showed the same proﬁle as theca interna cells near the outer boarder of the theca avBD-2 , except that some immunoreaction products of interna showed immunolabeling for avBD-2, (Fig. c, d). avBD- +, were identiﬁed within interstitial cells in the The immunolabeling of avBD-+* (Fig. - ) and - +, (data cortical follicles. Abdelsalamet al. : avBDs in the Ovarian Follicles 81

Fig./ . Western blot of avBD-2+* , - and - +, in the ovarian follicular tissues. Lanes Aῌῌ avBD-2+* , Lanes B avBD- , Lanes Cῌῌ avBD-+, , Lanes D control staining using normal IgG antibody. The bands of avBD-2+* , - and - +, exist at,+ , ++ and ,2 KDa (arrow heads). Gῌ granulosa layer, Tῌ theca tissue. Fig.. . Sections of growing ovarian follicles immunostained using normal rabbit IgG for control. a) cortical follicle, b) small yellow follicle, c) the largest follicle. No staining is follicles, but then increased in yellow follicles. The im- observed in all sections. See Figs.+, and for abbreviations. munoreaction products in the thecal cells increased with Scale bars represent,* m m. follicular growth with their first appearance in the small yellow follicles (avBD-2+* and - ) and cortical follicle (avBD-+, ). Table+ . Summary of the immunolabeling profiles in Western blot analysis showed a single immunoreactive # the ovarian follicles at di erent growth stages band for each of avBD-2,+ ( KDa), - +*++ ( KDa) and - +, ,2 / Tissues Follicles avBD-2+*+, avBD- avBD- ( KDa) in both the granulosa and theca layers (Fig. ). Granulosa Cortical follicle ῌῌ ῌ Discussion layer SYF ῌ῍ῌῌ ῌ῍ ῌ῍ ῌ We report that ir-avBD-2+* , - and - +, proteins were F- ῌῌ ῌῌ ῌῌ F, ῌῌ ῌῌ ῌῌ localized in the theca and granulosa layers of hen ovarian + F+ ῌῌ ῌῌ ῌῌ follicles. Significant findings were ( ) the three types of ir-avBDs appeared in the interstitial cells or theca interna Theca layer Cortical follicle ῍῍ῌ῍ῌ cells as well as granulosa cells, (, ) hierarchal yellow SYF ῌ῍ῌῌ ῌ῍ ῌ - ῌῌ ῌ follicles contained more amount of ir-avBDs than cortical F - F, ῌῌ ῌ and small yellow follicles, and ( ) Western blot analysis F+ ῌῌ ῌ showed a single band for each avBD in both granulosa and theca layers. It is suggested that each antibody to avBD- ῍῎no immunoreaction products, ῌῌ ῍῎ faintly immunolabeled, 2+*, - and - +, specifically recognized the antigens because ῌ῎moderately immunolabeled, ῌῌ῎ densely immunolabled. SYF ῎῎small yellow follicle, F+- -F the largest to third largest follicles Western blot analysis revealed single bands for each avBD and control staining did not show the bands. The sizes of the bands appeared in Western blot were,+ , ++ and ,2 Immunoreaction products were not observed in the KDa for avBD-2+* , - and - +, , respectively. In contrast, control sections examined using normal rabbit IgG in the calculated molecular weights based on the amino acid place of the first antibodies (Fig.. ). sequences (total of signal peptide, propiece and mature Table+ summarizes the results of immunostaining of peptide) reported by Xiaoet al. (,**. ) are 1.-+ , 1+00 and avBDs in the follicles. The immunostaining pattern was1,*/ KDa. Although the exact reason why the bands of the same in avBD -2+* , - and - +, . The density of im- avBDs appeared at larger sizes than expected sizes is not munoreaction products in the granulosa layer was initially known, we assume that the molecules might have formed reduced in small yellow follicles compared with cortical some complexes with other residues. Yudinet al. (,**/ ) 82 J. Poult. Sci.,.1 ( + ) reported that DEFB+,0 , a macaque beta-defensin, pos- in structures, cell proliferation, yolk uptake and endocrine sessed a polypeptide backbone of about+* kDa, but functions (Johnson, ,*** ), but also in immune functions exhibited an electrophoretic mobility of-.ῌ -0 kDa with during the recruitment process from prehierarchal to hier- SDS-PAGE, indicating that a major portion of the molec- archal phase. The granulosa cells show a high prolifera- ular mass resided in the O-linked oligosaccharides. Isobe tive activity before entering the hierarchal stage (Yoshimura et al.(,**3 ) described that the lingual antimicrobial pep- et al., +330 ). Compared with the prehierarchal follicles, tide, a bovine beta-defensin, in the milk was glycosylated the hierarchal yellow follicles receive more blood flow and showed a larger molecular size than predicted size of (Scaneset al., +32, ), suggesting that more circulating an- matured peptide. From these reports, it may be possible tigens may migrate into the larger follicles. More macro- that avBDs synthesized by the chicken granulosa and phages and more developed phagocytotic activity of the theca cells were also glycosylated and showed a larger theca interna cells were observed in the hierarchal yellow molecular size than predicted size. The molecular weight follicles (Baruaet al., +332 ; Yoshimura and Okamoto, of avBD-+, in chicken oviduct was -. KDa in our recent +332 ). The current study showed that the density of study (Abdel Mageedet al., ,**3 ), whereas that in the ir-avBDs in the interstitial cells of the cortical follicles and follicular cells was,2 kDa in the current study. Thus, the small yellow follicles was negligible or faint, whereas that components involved in glycosylation of avBDs may be in the theca interna cells of the yellow follicles were high. di# erent among the tissues. Their density in the granulosa cells was higher in the We have reported that the theca layer expressed yellow follicles than the cortical and small yellow follicles. mRNAs ofavBD-,-,-,-,-+,12+* and - +, , whereas These results suggest that host defense system mediated by granulosa layers expressed those ofavBD-,-+2+* ,- and - +, avBDs may develop with the follicular growth in associa- (Subediet al., ,**1 b). The current study using Western tion with immunocompetent cell members including mac- blot and immunohistochemical analysis showed the pres- rophages. Such development of the host immunity in ence of avBD -2+* , - and - +, in both layers. The expres- larger follicles may be necessary to protect the tissues sion of ir-avBD-+, in the theca interna and granulosa from infection because opportunities of influx of the layers has been also reported by Subediet al. (,**1 a). circulating pathogens may have increased with follicular These results suggest that the theca and granulosa layers growth. It is reported that the smaller follicles are more express not only mRNA but also the protein products of susceptible toSalmonella invasion than large yellow folli- theavBD -2+* , - and - +, genes. cles (Howard et al., ,**/ ). The ir-avBD-2 , - +* and - +, were observed in the cells In conclusion, we suggest that avBD- 2 , - +* and - +, near the outer border of the theca interna and the proteins are expressed in specific cells in the follicles, granulosa cells in the hierarchal yellow follicles. They namely interstitial or theca interna cells and granulosa were also localized in the interstitial cells and granulosa cells. Their amounts in the cells are likely to increase cells in the small yellow follicles. Thus, it is likely that the during follicular growth from prehierarchal to hierarchal cells synthesizing them are common among the three phase. These avBDs may play significant roles in the host di## erent avBDs. Secretion of di erent types of avBDs in innate immune system in the follicles. a tissue may enable to kill a wider spectrum of micro- Acknowledgments organisms if the antimicrobial activities di# er among the di# erent types of avBDs. It is reported that the innate We wish to thank Prof. Dr. Lawrence M. Liao, Gradu- immune system via avBDs responds di# erently to Salmo- ate School of Biosphere Science, Hiroshima University for nellaand Campylobacter infection in chickens; namely critical editing of this manuscript. This work was sup- avBD--+* , - and - +, were significantly increased in reported by a Grant-in-Aid for Scientific Research from sponse toSalmonella, whereas avBD--.2+- , - , - , - and - +. Japan Society for the Promotion of Science. ,**3 were reduced byCampylobacter (Meade et al., ). References Circulating microorganisms may infiltrate and colonize first in the theca interna because capillary beds are well Abdel Mageed AM, Isobe N and Yoshimura Y. Immunolocaliza- developed there (Perryet al., +312 ). Then, the invasive tion of avianb -defensins in the hen oviduct and their changes +-2 microorganisms may migrate into the theca externa and in the uterus during eggshell formation. Reproduction, : 31+ῌ 312 ,**3 granulosa layers (Gantoiset al.,,**3 ). Salmonella enter- .. Akbari MR, Haghighi HR, Chambers JR, Brisbin J, Read LR itidis experimentally injected in birds was identified in the ,**- and Sharif S. Expression of antimicrobial peptides in cecal theca interna and granulosa layers (Takata et al, ). tonsils of chickens treated with probiotics and infected with In vitroexperiments also showed Salmonella bacteria Salmonella enterica serovar typhimurium. Clinical and Vac- attached to the surface of the granulosa cells followed by cine Immunology,+/ : +023ῌ +03- . ,**2 . cytoplasm invasion (Thiagarajanet al., +330 ). The avBDs Aono S, Li C, Zhang G, Kemppainen RJ, Gard J, Lu W, Hu X, in theca interna and granulosa layers may play roles in Schwartz DD, Morrison EE, Dykstra C and Shi J. Molecular host defense against the pathogens migrating into these and functional characterization of bovineb -defensin-+ . Vet- tissues. erinary Immunology and Immunopathology,++- : +2+ῌ +3* . The follicular tissues undergo marked changes not only ,**0. Abdelsalamet al. : avBDs in the Ovarian Follicles 83

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