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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION |
Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan
1 Corresponding author: yyosimu{at}hiroshima-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: avian beta-defensin • sperm • testis • male reproductive tract • oviduct
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-, β-, and 
-defensins (Klotman and Chang, 2006)]. The β-defensin family in chicken (avian β-defensin: AvBD) was previously called gallinacins, which contains 3 intermolecular cysteine disulfide bonds (Harwig et al., 1994; Lynn et al., 2007). Currently, genes of 13 different AvBD (AvBD 1 to 13; identical to gallinacin 1 to 13) have been reported (Xiao et al., 2004; Lynn et al., 2007). The ovary and oviduct expressed several types of AvBD, which were changed in response to Salmonella enteritidis or lipopolysaccharide (Yoshimura et al., 2006; Subedi et al., 2007, 2008; Abdel Mageed et al., 2008). Zhao et al. (2001) characterized AvBD-3 gene structures and showed the epithelial expression in several organs. They showed a significant increase of the mRNA expression of AvBD-3 in the trachea after experimental infection of chickens with Hemophilus paragallinarum. These reports strongly suggest that AvBD play significant role in the innate immunity for the defense against microorganisms. Avian sperm produced in the seminiferous tubules enter the epididymal tracts and ductus deferens. In hen oviducts, sperm are stored in the sperm storage tubules (SST) in the utero-vaginal junction (UVJ) for a prolonged period and participate in fertilization that occurs in the infundibulum. The reproductive organs may be invaded by microorganisms in both males and females (Donoghue et al., 2004; Boltz et al., 2006; Cole et al., 2006; Jackson et al., 2006), and if they attach to the sperm, their survivability and fertility may decline. Expression of β-defensin by various cells in the male reproductive tract of different species and on sperm surface has been reported. These defensins may play roles not only in innate immunity but also in male fertility, sperm survival in male reproductive tract, and sperm-egg interaction in mammals (Com et al., 2003; Palladino et al., 2003; Yudin et al., 2003; Zhou et al., 2004). Sperm associated antigen (SPAG11), known as epididymal protein 2 (EP2) in monkey, human epididymis 2 (HE2), and rat spag 11, are antimicrobial peptides attached on the sperm surface (Hamil et al., 2000; Yenugu et al., 2004, 2006a, b; Avellar et al., 2007). It has not been reported whether avian sperm express antimicrobial peptides. Recently, we have raised antibody to AvBD-3 using its synthetic peptide, which could be used for immunohistochemical identification of AvBD-3 (identical to gallinacin-3) in the epithelial cells of trachea, oviduct, cloacal gland, and uropygialis (Yoshimura et al., 2007). If the chicken sperm express AvBD-3, it is expected to be identified using the antibody.
The goal of this study was to determine whether the chicken sperm synthesize AvBD-3 in the testis and retain it during the storage in the male and female reproductive tract. We have performed immunohistochemistry for AvBD-3 in the testis and the male reproductive tract to identify the stage when the AvBD-3 appears on the sperm. Then, the AvBD-3 in oviductal UVJ of inseminated hens was also examined to show whether the molecule is kept during the storage of sperm in SST. The analysis by reverse transcription (RT)-PCR was performed to confirm the synthesis of AvBD-3 by testicular cells and ejaculated sperm.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Barred Plymouth Rock roosters (approximately 22 mo old) and White Leghorn hens (approximately 18 mo old) laying 5 or more eggs in a sequence were used. All the birds were kept in individual cages under a 14L:10D lighting regimen and were provided with feed and water ad libitum. Ejaculates were taken from the roosters using the abdominal massage technique and used for artificial insemination of hens and for analysis of AvBD-3 protein and mRNA expression in the ejaculated sperm. Artificial insemination was performed with 0.05 mL of undiluted fresh semen 1 h before examination. The testes and oviducts were collected after euthanization of birds under anesthesia with sodium pentobarbital. Handling of chickens was done in accordance with the regulations of Hiroshima University for animal experiments.
Tissue Preparations for Immunostaining
The testes, epididymis, and ductus deferens of roosters (n = 3) and oviducts of inseminated hens (n = 3) were fixed with Bouin’s solution and processed for paraffin sections. The sections (4-µm thickness) were air-dried on slides. After deparaffinization, the sections were autoclaved in 2 mM citric acid (pH 6.0) for 1 min to activate antigen and then washed with PBS. To identify the AvBD-3 in the ejaculated sperm, fresh semen was air-dried on slides and fixed with 10% formal-in in PBS. The semen was collected from 3 roosters and examined separately. The sections and sperm on the slides were incubated with 1% blocking reagent in PBS (Roche Diagnostics GmbH, Mannheim, Germany) for 30 min, and then rabbit anti-AvBD-3 antibody diluted at a concentration of 13 µg/mL in PBS containing 1% blocking reagent for 4 h at room temperature. The anti-AvBD-3 antibody was an affinity purified polyclonal antibody, which was raised in rabbit using a synthetic peptide (Arg-Phe-Pro-His-Ile-Ala-Ile-Gly-Lys-Cys-COOH) in our previous study (Yoshimura et al., 2007). Then, the sections were washed with PBS for 15 min (5 min x 3 times). The immunoreaction products were identified using Vectastain ABC kit (Vector Lab. Inc., Burlingame, CA) according to manufacturer’s instructions. Briefly, sections were incubated with biotin-conjugated anti-rabbit IgG and avidin-biotin-peroxidase complex for 1 h each, followed by washing with PBS for 15 min (5 min x 3 times). The immunoreaction products were visualized by incubating the sections with a reaction mixture consisting of 0.02% (wt/vol) 3, 3'-diaminobenzidine tetrahydrochloride, and 0.005% (vol/vol) H2O2 in 0.05 M Tris-HCl buffer, pH 7.6. The sections were dehydrated and covered. For the control staining, the first antibody was replaced with 0.1% normal rabbit serum or normal rabbit IgG (13 µg/mL) in PBS-BSA. Staining was not observed in the control sections except for staining in the epithelium of the efferent ductules. Immunostained sections were observed under a light microscope (Nikon Eclipse E600, Nikon, Tokyo, Japan) using a Nomarsky filter.
RT-PCR Analysis for AvBD-3 mRNA Expressions
Total RNA was extracted from testes and ejaculated sperm using Sepasol RNA I super (Nacalai Tesque, Kyoto, Japan) according to the manufacturer’s directions. The RNA sample was resuspended in TE buffer (10 mM Tris, pH 8.0, with 1 mM EDTA) and treated with 10 U of DNase I (Roche Diagnostic GmbH) at 37°C for 1 h, 80°C for 30 min, and 4°C for 5 min. Then the concentration of total RNA was determined using Gene Quant pro (Amersham Pharmacia Biotech, Cambridge, UK) and the RNA stored at –80°C until use.
The RNA samples were reverse transcribed using Rever Tra Ace (Toyobo Co. Ltd., Osaka, Japan). The reaction mixture (10 µL) consisted of 1 µg of total RNA, 1 x RT buffer, 1 mM of each dNTP, 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 a Programmable Thermal Controller, the PTC-100 (MJ Research Inc., Waltham, MA). The PCR was performed in the PTC-100 in a reaction mixture of 25 µL containing 0.5 µL cDNA, 1 x PCR buffer, 0.2 mM of each dNTP, 0.4 µM of each primer, and 0.625 U of Takara Taq (Takara Bio Inc., Shiga, Japan). The PCR cycle parameters were denaturation at 94°C for 30 s, 40 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min, and a final extension at 72°C for 6 min. The expression of β-actin was also examined to confirm the presence of mRNA in samples. The PCR products were separated by electrophoresis on 3% (wt/vol) agarose gels containing ethidium bromide (0.5 mg/mL) and photographed under UV illumination. The primers used in this study were as follows: for the AvBD-3, F-CTGCCGCTTCCCACACATAG and R-GCAATGCCAAACTGCACGCCTTTA (accession number: NM_204650 [GenBank] ), and for β-actin, F-TTCCAGCCATCTTTCTTG, and R-TCCTTCTGCATCCTGTCA (accession number: X00182 [GenBank] ). These primers had been used in our previous studies (Ohashi et al., 2005; Subedi et al., 2007).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The immunoreaction products for AvBD-3 were identified in the middle part of the seminiferous tubules, whereas the other parts of the seminiferous epithelium and interstitial tissues were negative in the immunolabeling (Figure 1b). The immunoreaction products were localized in the elongated spermatids attached to the seminiferous epithelium and in the sperm in the tubular lumen (Figure 1c). No staining was observed in the control sections stained using normal rabbit serum or IgG in place of the first antibody (Figure 1d).
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). Positive staining for AvBD-3 was found in the sperm in the epididymal ducts (Figure 2b). The epithelial cells of efferent ductules and epididymal ducts were faintly stained in the current immunostaining; however, it was considered nonspecific staining because same staining was observed occasionally in the epithelial cells in the control staining (data not shown). Immunoreaction products were also found in the sperm in the ductus deferens (Figure 2c). No staining was observed in the sperm in reproductive tracts in control sections (data not shown).
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). The SST was observed in the UVJ of hen oviduct, where sperm were stored in the inseminated birds (Figure 3b). The immunoreaction products of AvBD-3 were identified in those sperm stored in the SST (Figure 3c). The RT-PCR analysis showed the induction of PCR products (approximately 280 bp) derived from AvBD-3 mRNA in the testicular tissue, but the products did not show a clear band in the ejaculated sperm (Figure 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the testes, immunoreactive AvBD-3 was expressed only in the elongated spermatids and luminal sperm. The other cells in the seminiferous epithelium and interstitial tissue were not immunostained by the anti-AvBD-3 antibody. The RT-PCR analysis confirmed AvBD-3 mRNA expression in the testes. These results suggest that the elongated spermatids and sperm in the testes synthesize AvBD-3. The mammalian testes are reported to produce β-defensins that exert antimicrobial activity (Com et al., 2003; Palladino et al., 2003). In human testes, immunoreactive β-defensin-1 was localized in the Leydig cells and early and elongated spermatids, whereas discrete immunolabeling occurred in pachytene spermatids (Com et al., 2003). Sperm associated antigen II (SPAG II), the gene of which is included in the β-defensin cluster, was immunolocalized in late stage spermatids and in Sertoli cells in cattle testes (Avellar et al., 2007). Therefore, it is suggested that late stage of spermatids produce AvBD-3 in the chicken testes as some of the mammalian spermatids. Although the other testicular cells were not immunolabeled by anti-AvBD-3 antibody, it remains unknown whether they produce other types of AvBD.
The sperm in the epididymis and ductus deferens were immunolabeled by AvBD-3 antibody, and no staining was observed in control staining. Thus, sperm are likely to retain AvBD-3 produced in the testes during the passage and storage in those ducts. Mammalian sperm undergo maturation in the epididymis. During the storage in the epididymis, sperm are coated with sperm binding protein produced by the epithelial cells of epididymis (Hamil et al., 2000; Avellar et al., 2007). Those proteins synthesized in the epididymis involve β-defensin members that may play roles not only in host immunity of reproductive tract but in the regulation of sperm motility, sperm maturation, and anti-sperm immunity (Zhou et al., 2004; Yudin et al., 2005). In the epididymis of birds, sperm density is condensed by reabsorption of seminal fluid, unejaculated sperm are removed by phagocytosis, and some substances are secreted from the epithelium into the fluid (Tingari and Lake, 1972; Clulow and Jones, 1988). In the current study, the epididymal epithelial cells showed occasionally nonspecific staining in which the first antibody was replaced with normal rabbit serum or IgG. Thus, it remains unknown whether the epithelial cells in the epididymis secrete AvBD-3 for a sperm-associated protein.
In the ejaculated sperm, immunolabeling for AvBD-3 occurred in the midpiece and upper part of principal region of the tail, whereas RT-PCR analysis failed to identify a clear band to show mRNA expression of AvBD-3. Therefore, it is suggested that the ejaculated sperm retain AvBD-3 synthesized by themselves in the testis, but may not synthesize further AvBD-3 after ejaculation. In mammals, it is reported that sperm are coated by β-defensin. Some type of those β-defensin may be synthesized by the epididymal epithelium under a regulation of androgen (Yenugu et al., 2004). However, the others may be synthesized by sperm themselves not only in the testis but also even after ejaculation because ejaculated sperm expressed mRNA for human neutrophil peptide and β-defensin (Com et al., 2003).
In the oviduct of inseminated birds, sperm were stored in the SST and were stained positive for immunoreactive AvBD-3. The immunocompetent cells, including antigen presenting cells expressing major histocompatibility complex class II, T, and B cells, distribute in the hen oviduct (Yoshimura et al., 1997; Zheng et al., 1997, 1998). If anti-sperm immunoresponse occurs in the oviduct, it may affect the sperm survivability in the oviduct (Zheng et al., 2001). Recently we reported that the immunoreaction to the sperm stored in the SST may be suppressed because the expression of transforming growth factor-β, which is believed to suppress immune response, was enhanced there after insemination (Das et al., 2006). Yudin et al. (2005) suggested that β-defensin 126, which covers the entire primate sperm surface, plays a role in protecting the sperm from immune recognition. They showed that when β-defensin 126 of sperm was released after capacitation or sialic acid of that defensin was removed, those sperm became immunogenic. Thus, AvBD-3 of chicken sperm may also play roles in part in the regulation of immunorecognition against them in the oviduct.
In conclusion, we suggest that AvBD-3 is synthesized by late stage of spermatids in the testis, and this molecule is retained by the sperm during the passage through epididymis and ductus deferens and even during the storage in the oviduct. The sperm AvBD-3 is assumed to play roles in protection of the sperm from the microbial infection and sperm survival in the oviduct.
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ACKNOWLEDGMENTS |
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Received for publication May 26, 2008. Accepted for publication July 29, 2008.
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