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Poult Sci 2008. 87:979-984. doi:10.3382/ps.2007-00283
© 2008 Poultry Science Association
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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION

Expression of Avian β-Defensins in the Oviduct and Effects of Lipopolysaccharide on Their Expression in the Vagina of Hens

A. M. Abdel Mageed, N. Isobe and Y. Yoshimura1

Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan

1 Corresponding author: yyosimu{at}hiroshima-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aims of this study were to (i) determine the types of avian β-defensin genes (AvβD) expressed in the hen oviduct and (ii) to examine the effects of lipopolysaccharide (LPS) treatment in vivo on their expression in the vagina. Birds were i.v. treated with LPS (1 mg/kg of BW), and subsequently the oviducts were analyzed 0, 3, 6, 12, or 24 h after LPS administration. The mRNA expression for AvβD was examined by reverse transcription-PCR using RNA preparations from the mucosal tissues of all the oviductal segments. Furthermore, changes in their mRNA expression profiles in the vagina were analyzed by semiquantitative reverse transcription-PCR. The AvβD-1, -2, -3, -4, -5, -7, -8, -9, -10, -11, and -12 were identified in each oviductal segment from infundibulum to vagina. Among these AvβD, the expression of AvβD-3, -5, -10, -11, and -12 in the vagina were significantly increased in response to LPS treatment, whereas the others did not show significant changes. These results suggest that all 11 types of AvβD are expressed in the hen oviduct and at least 5 of them in the vagina show increased expression in response to LPS.

Key Words: avian β-defensin • lipopolysaccharide • hen oviduct • vagina


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The hen oviduct consists of the infundibulum, magnum, isthmus, uterus, and vagina. Egg formation, including the secretion of albumen, egg-shell membrane, and egg-shell components, is completed during the passage of the yolk through the oviduct. Immune functions in the oviduct play essential roles in protection of the oviductal tissues from infections and production of pathogen-free eggs. The oviduct itself may be infected by various microorganisms such as Salmonella enteritidis (Barnhart et al., 1993) and Mycoplasma meleagridis (Yamamoto and Herrad, 1966). An adaptive immune response through immuno-competent cells in the oviduct including antigen presenting cells expressing MHC class II as well as T and B cells has been well described (Yoshimura et al., 1997; Zheng et al., 1997, 1998, 2001; Zheng and Yoshimura, 1999). Although the innate immunity plays an essential role in the first line of defense against infection, there is only limited information available as what extend on the hen oviduct.

Defensins are antimicrobial peptides that may trigger an innate immune response and are divided into 3 groups, namely {alpha}-, β-, and {Theta}-defensins. The β-defensins are characterized by 6 cysteine residues, and have been found in many animal species such as bovine (Luenser and Ludwig, 2005), ovine (Luenser et al., 2005), and porcine and human (Yongming et al., 2006). These peptides are potentially kill a wide range of microorganisms including gram-positive and gram-negative bacteria, fungi, and yeast (Haryadi and Pak, 2004). Avian antimicrobial peptides classified as β-defensins had been previously called gallinacins. However, it has now been agreed to use their gene names [i.e., avian β-defensin (Lynn et al., 2007)]. We therefore decided to use the new terminology for this study rather than previous reports. So far, 13 avian β-defensin genes (AvβD) have been identified. Zhao et al. (2001) reported that gallinacin gene (Gal)-1 and -2 were expressed in bone marrow and lung, whereas Gal-3 was more preferentially expressed in bone marrow, tongue, trachea, and the bursa of Fabricii. Xiao et al. (2004) described that Gal-1 to -7 are predominantly expressed in the bone marrow and the respiratory tract, whereas Gal-8 to -13 were restricted to the liver and urogenital tract. The theca and granulosa layers of ovarian follicles expressed 6 types (theca) or 4 types (granulosa layer) of Gals, respectively, whereas the ovarian stroma expressed 12 types including Gal-1 to -12 (Subedi et al., 2007). Milona et al. (2007), who studied the antimicrobial activity of Gal-4, -7, and -9 in the intestine of the chicken, suggested that gallinacins act as antimicrobial agent constituting an integral part of the avian host innate defense system.

Ohashi et al. (2005) identified the expression of Gal-1, -2, and -3 in all the oviductal segments with greater expression in the infundibulum and vagina than in the other segments. The vagina is one of the sites that may be contaminated by microorganisms because it opens to the cloaca. Recently, Yoshimura et al. (2006) reported that the expression of Gal-1, -2 and -3 was enhanced in response to Salmonella enteritidis infection or in response to purified lipopolysaccharide (LPS) in the cultured vaginal cells. The LPS is a gram-negative bacterial cell wall component (Sunwoo et al., 1996), which mimics the effects of a bacterial infection (Leshchinsky and Klasing, 2003). Although expression of 3 types of AvβD have been shown in the oviduct, that of the other types of AvβD in the different segments of oviduct have not yet been reported. If the synthesized avian β-defensins play roles in the host immunity to eliminate microorganisms, their expressions are expected to be enhanced in response to bacterial components.

The aim of this study was to determine the types of AvβD expressed in the oviduct and the effects of in vivo treatment with LPS on the expression of AvβD in the vagina. The types of AvβD expressed in the oviduct were examined using birds treated with or without LPS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Birds

White Leghorn hens (approximately 400 d old) regularly laying 5 or more eggs in a sequence were used. Hens were kept in individual cages under a daily light regimen of 14L:10D and provided with feed and water ad libitum. They were i.v. treated with LPS preparations from E. coli (Wako Pure Chem., Osaka, Japan) dissolved in PBS at a dose of 1 mg/kg of BW. This dose of LPS had been confirmed to increase AvβD expression in the ovarian theca tissue in our previous report (Subedi et al., 2007). Birds were euthanized under anesthesia with sodium pentobarbital (Abbott Lab., Chicago, IL) after 0, 3, 6, 12, or 24 h of LPS treatment to collect the oviduct. Handling of birds was done in accordance with the regulations of Hiroshima University for animal experiments.

RNA Extraction

Total RNA was extracted from the mucosal tissues of the infundibulum, magnum, isthmus, uterus and vagina, and liver tissue using Sepazol RNA I super (Nacalai Tesque, Kyoto, Japan) according to the manufacturer’s directions. The obtained RNA pellet was then dissolved in Tris-EDTA buffer and kept at –80°C until use. A mixture (10 µL) of the RNA sample, 2 U DNase I (RNase free; TaKaRa Shuzo, Shiga, Japan), and 1x DNase buffer was subjected to 37°C for 1 h, at 80°C for 10 min, then at 4°C using Programmable Thermal Controller PTC-100 (MJ Research, Waltham, MA). The concentration of the purified total RNA was then determined using Gene Quant Pro (Amersham Pharmacia Biotec, Cambridge, UK).

Semiquantitative Reverse Transcription-PCR

Semiquantitative reverse transcription-PCR was performed as described by Subedi et al. (2007). The RNA samples were reverse-transcribed using ReverTra Ace (Toyobo Co. Ltd., Osaka, Japan) according to the manufacturer’s instructions. The reaction mixture (10 —L) consisted of 1 µg of the total RNA, 1x RT buffer, 1 mM dNTP mixture, 20 U of RNase inhibitor, 0.5 µg of oligo (dT)20, and 50 U of Rever Tra Ace. The reverse transcription was performed at 42°C for 30 min, followed by heat inactivation for 10 min at 99°C using the PTC-100 programmable thermal controller (MJ Research, Waltham, MA). The PCR was performed in a reaction mixture of 25 µL containing 0.5 µL of cDNA, 1 x PCR buffer, 0.2 mM dNTP mixture, 0.4 µM each primer, and 0.625 U of Takara Taq (Takara Bio. Inc., Shiga, Japan). Table 1Go shows the primers used for PCR, which were the similar primers as have been used in a previous study (Subedi et al., 2007). To determine the types of AvβD expressed in the oviduct, the oviducts collected pre or at 3 h after LPS treatment were used. The cDNA samples from each oviductal segment were amplified using the primers of all types of AvβD at different annealing temperature ranging from 52 to 60°C and 40 PCR cycles. For semiquantitative reverse transcription-PCR analysis of AvβD expression in the vagina, different PCR cycles (30, 35, 40, and 45 cycles) were examined to optimize the amplification. A linear response for the 30 through 45 cycles was observed, and 35 cycles for AvβD-12 and 40 cycles for the other AvβD and β-actin were considered optimal. Each observed AvβD was amplified using an optimal number of cycles to examine the changes in their expression in response to LPS in the vaginal mucosa and liver tissue. The cycle parameters were denaturation at 94°C for 30 s, 35 cycles (for AvβD-12), or 40 cycles (for AvβD-1 to -11 and β-actin), annealing at 58°C (for AvβD-1, -2, -4, -5, -7, -9, and -10, and β-actin) or 60°C (for AvβD-3, -8, -11, and -12) for 30 s, and extension at 72°C for 1 min, followed by the final extension at 72°C for 6 min. The PCR products were separated by electrophoresis on 2% (wt/vol) agarose gels containing 0.5 mg/mL of ethidium bromide and photographed under UV illumination. Densitometry for the PCR product bands was performed using UN-SCAN-IT gel TM, ver. 6.1, (Silk Scientific Corporation, Orem, UT), and then the ratio of AvβD to β-actin densities was obtained.


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  		Table 1. The PCR primers and their accession numbers used for profiling of avian β-defensins (AvβD) and β-actin
 
Statistical Analysis

The values of each AvβD expression were obtained as the mean ± SEM of the ratio of AvβD/β-actin. The significance of differences in the expression of AvβD after LPS treatment was examined by 1-way ANOVA, followed by Duncan’s multiple range test. Differences with P value of < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Figure 1Go shows the pattern of PCR products of AvβD obtained from the oviduct after 3 h of LPS treatment. The PCR products of AvβD-1, -2, -3, -4, -5, -8, -9, -10, -11, and -12 were observed in all the oviductal segments, and AvβD-7 products showed only faint bands in the isthmus, uterus, and vagina. No AvβD-6 and -13 expression was observed in any segments of the oviduct. In the oviduct of nontreated birds, some of the bands that could be identified in the treated birds were faint or undetectable (data not shown).


Figure 1
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  		Figure 1. Pattern of reverse transcription-PCR products for avian β-defensins mRNA (AvβD) in the mucosal tissues of hen oviduct treated with lipopolysaccharide (LPS). The hens were i.v. treated with 1 mg of LPS/kg of BW before 3 h of examination. The PCR products were electrophoresed on 2% agarose gel containing ethidium bromide. M = marker.

 
Effects of LPS treatment on the expression of 11 types of AvβD in the vagina are shown in Figure 2Go. The expression of AvβD-3, -5, -10, and -11 increased by 3 to 6 h of treatment. Then, the expression of AvβD-5 and -10 were kept higher until 24 h of treatment, whereas those of AvβD-3 and -11 showed a tendency to decline after the peak at 3 h and 6 h of posttreatment. The AvβD-12 expression was also increased by LPS treatment although the significance was found only at 24 h of posttreatment. The average values at the peak of AvβD-1, -2, -4, -7, -8, and -9 showed approximately 2- to 5-fold in the LPS-treated groups compared with pretreatment; however, the differences were not statistically significant.


Figure 2
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  		Figure 2. Changes in the expression of avian β-defensins mRNA (AvβD) in the vagina and AvβD-3 in the liver after i.v. treatment with lipopolysaccharide (LPS). Birds were i.v. treated with 1 mg of LPS/kg of BW, and AvβD expression in the vaginal mucosa was examined by semiquantitative reverse transcription-PCR at different periods after LPS treatment. (a) – (k): AvβD expression in the vagina. (l) AvβD-3 expression in the liver. Values are mean ± SEM of the AvβD/β-actin ratio (n = 4). a–cValues with different letters are significantly different (P < 0.05).

 
The effect of LPS treatment on the expression of AvβD in the liver was also examined. The liver also expressed 11 types of AvβD (except for AvβD-6 and -13). The expression of AvβD-3 was decreased by 3 h of posttreatment and kept lower until 24 h (Figure 2Go-l), whereas the other AvβD did not show significant changes in their expression (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We are reporting the types of AvβD expressed in hen oviduct and the changes in their expression in response to i.v. treatment with LPS. Significant findings were (1) 11 types of AvβD were expressed in all segments of oviduct, (2) expression of 5 types of AvβD was increased in the vagina in response to LPS treatment.

Ohashi et al. (2005) reported that all the oviductal segments expressed Gal-1, -2, and -3 with a higher expression in the infundibulum and vagina. The current study provided further findings that 11 types out of 13 reported AvβD were expressed, but the expression of AvβD-6 and -13 was not observed in any segments of the oviduct. Previous reports suggested that avian β-defensins play significant role in the host innate immunity of mucosal tissues; namely, the expression of Gal-4, -6, -7, and -9 were expressed in the intestine and Gal-3, -4, -5, -6, and -7 were expressed in the trachea (Albert et al., 2007; Milona et al., 2007). Although the healthy mucosal tissues of oviduct and other organs express AvβD, the types of the AvβD are likely to be different among the organs. However, the current results suggest that the types of AvβD are same among each oviductal segment; namely, 11 types of AvβD could be expressed in all the segments.

We found that the expression of AvβD-3, -5, -10, -11, and -12 in the vagina was increased by the i.v. treatment with LPS. Yoshimura et al. (2006) reported that the expression of Gal-1, -2, and -3 was increased in the cultured vaginal cells by stimulation with Salmonella enteritidis or LPS. The current study confirmed that AvβD-3 expression is enhanced by LPS even in vivo. Furthermore, the current results suggested that expression of AvβD-5, 10, -11, and -12 was also increased by LPS stimulation. However, there were temporal differences for the increase of expression; namely, that of AvβD-3 and -11 was increased only from 3 to 6 h of posttreatment with LPS, AvβD-5 and -10 were elevated from 6 to 24 h, and AvβD-12 was increased only after 24 h. These temporal differences in the increase may enable the oviductal mucosa to express AvβD for a longer time to attack the microorganisms by the combination of different types of AvβD. Although the reason why such differences in the increase of expression occur still unknown, we assume that the types of cells expressing AvβD may not be single and the time to recognize circulating LPS, the time of intracellular response to express AvβD, or both, may be different among these cells. Ohashi et al. (2005) showed AvβD-1, -2, and -3 expression in the basal cells of the vaginal mucosal epithelium. Leukocytes that distribute in the vaginal mucosa may also contribute to synthesize AvβD (Evans et al., 1994; Harwig et al., 1994; Brockus et al., 1998).

The expression of Gal-1 and -2 of cultured vaginal cells was significantly enhanced by the LPS stimuli (Yoshimura et al., 2006), whereas their expression did not show statistically significant changes under in vivo treatment with LPS in the current study. Although the reason why these differences exist between the 2 studies is not known, we assume that the intensity of the LPS stimulation to the vaginal cells was different between the studies. Furthermore, the expression level of not only AvβD-1 and -2 but also AvβD-4, -7, -8, -9 did not change significantly in response to LPS, although the average value differed 2- to 5-fold between pre- and posttreatment. It is assumed that a proper stimulation by LPS may also cause a significant increase of those AvβD.

Unlike in the oviduct, AvβD-3 expression in the liver was reduced by LPS treatment, whereas no change was observed in the expression of the other AvβD. Thus, the effect of LPS on the AvβD expression may be different among the different tissues and cells.

In conclusion, we suggest that 11 types of AvβD were expressed in all the segments of oviduct and at least 5 types were enhanced in the vagina in response to LPS.


   ACKNOWLEDGMENTS
 
We wish to thank W. Schwaeble, Department of Infection, Immunity and Infection, University of Leicester, UK, and Animesh Barua, Rush University Medical Center, Rush University, Chicago, IL, for critical reviewing of this manuscript. This work was supported by Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

Received for publication July 12, 2007. Accepted for publication February 10, 2008.


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 REFERENCES
 
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