Prolactin Inhibits Annexin 5 Expression and Apoptosis in the Corpus ...

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Endocrinology 144(8):3625–3631 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2003-0118

Prolactin Inhibits Annexin 5 Expression and Apoptosis in the Corpus Luteum of Pseudopregnant Rats: Involvement of Local Gonadotropin-Releasing Hormone MITSUMORI KAWAMINAMI, YUTAKA SHIBATA, AKIKO YAJI, SHIRO KURUSU, INORU HASHIMOTO

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Veterinary Physiology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan We investigated a specific relationship between the expression of annexin 5 and prolactin in the corpus luteum of pseudopregnant rats, with particular interest in GnRH and apoptosis of luteal cells. The expression of ovarian annexin 5 mRNA was significantly decreased at mid-pseudopregnancy and recovered at the end, whereas it remained low on the corresponding day of pregnancy. The dopamine agonist CB154, administered at mid-pseudopregnancy (d 5), increased ovarian annexin 5 mRNA, whereas prolactin, given daily for 3 d to cycling rats, decreased it. An immunocytochemical study also showed that annexin 5 increased in the corpus luteum on d 6 and 7 of pseudopregnancy after treatment with CB-154 on d 5. The distribution of annexin 5-positive cells was not uniform in the corpus luteum and matched that of termi-

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NNEXIN 5, A MEMBER of a novel calcium-phospholipid binding protein family, distributes preferentially to the corpus luteum in the ovary (1, 2). The synthesis of annexin 5 is thought to begin after luteinization of the follicular cells, because the granulosa cells do not express annexin 5. Annexin 5 is therefore postulated to be involved in luteal functions. Although various functions have been reported for the annexin 5 protein, their relevance in a cellular context is not known. One function reported for annexin 5 is inhibition of protein kinase C (3). Because this inhibition has been seen in vivo (4) and it has been suggested that protein kinase C of the corpus luteum is involved in various functions of luteal cells (5–7), we hypothesize that luteal annexin 5 is involved in regulating such corpus luteum functions as progesterone production. Because annexin 5, like other annexins, also inhibits the activity of phospholipase A2 (8), luteal annexin 5 could also affect luteolysis, which is promoted by prostaglandin F2␣ (9). Other reported functions for annexin 5 include forming calcium channels and inhibiting blood coagulation (10, 11). Although annexin 5 may have many different physiological roles, we are specifically interested in the relationship between ovarian annexin 5 expression and corpus luteum function. We have already shown, in a preliminary report, that the ovarian expression of annexin 5 is reduced during pseudopregnancy and is increased by the suppression of prolactin release with a dopamine agonist, CB-154 (12). It was subsequently shown by others, with DNA Abbreviations: DIG, Digoxigenin; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.

nal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells. Because GnRH stimulates annexin 5 mRNA expression in the gonadotropes, involvement of the GnRH receptor was examined. Local administration of a GnRH antagonist, Cetrorelix, to hemilateral ovarian bursa of pseudopregnant rats simultaneously receiving CB-154 abrogated both the expression of annexin 5 and the TUNEL reaction. The present results clearly demonstrate that prolactin decreases annexin 5 mRNA in the luteal cells during pseudopregnancy. Prolactin is suggested to suppress the local action of GnRH, which stimulates annexin 5 synthesis and apoptosis of functional luteal cells during pseudopregnancy. (Endocrinology 144: 3625–3631, 2003)

microarray screening, that luteal annexin 5 expression is suppressed by prolactin in hypophysectomized rats (13). Annexin 5 is widely distributed in many tissues but is cell type specific (1, 14, 15). Hence, the signals for regulation of annexin 5 synthesis are assumed to be tissue specific, which may dictate its biological functions in the various tissues. We hypothesize, however, that the regulation is achieved by the same mechanism in different tissues. Recently, we revealed that annexin 5 expression in the pituitary gonadotropes is augmented by GnRH (16, 17). These results suggest that a similar cellular mechanism might exist in the luteal cells, as well. We conducted the present study to determine the ovarian expression of annexin 5 in relation to luteal activity in rats and to study the effects of the major luteotropic hormone, prolactin, on the expression of annexin 5 mRNA. Furthermore, because both GnRH and its receptor are reported to be expressed in the ovary and GnRH was shown to inhibit progesterone production (18 –20), we also examined the mediating effects of GnRH between prolactin and annexin 5. Materials and Methods Animals Adult female Wistar-Imamichi rats (weighing 250 –350 g) bred in our animal facility were housed in groups, in air-conditioned rooms, with lights on from 0500 –1900 h. They were given free access to standard laboratory chow and tap water. The animals were treated according to the protocols for animal use approved by the institutional committee that follows the National Institutes of Health Guide. Daily vaginal smears were taken, and rats showing regular 4-d estrous cycles were used. Pseudopregnancy was induced by mating with a vasectomized male rat in the evening of the proestrous day. Pregnancy was effected

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by moving proestrous rats into the cages of male rats in the evening of proestrus and leaving them until the next morning. Pregnancy was verified by the presence of sperm in the vaginal smear upon separation from the males. The estrous day was designated as d 0 of pregnancy and pseudopregnancy.

Kawaminami et al. • Prolactin Suppression of Annexin 5

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) Apoptotic cells were detected by TUNEL using the In situ Cell Death Detection Kit, Fluorescein (Roche Molecular Biochemicals) following the manufacturer’s instructions. The specimens were observed with a fluorescence microscope.

Northern blot analysis of annexin 5 mRNA in the ovary Ovarian expression of annexin 5 mRNA was examined by Northern blotting for both pseudopregnant and pregnant rats. Each experimental group consisted of three rats. Some pseudopregnant rats were given the dopamine agonist CB-154 (2-Br-␣-ergocriptine, 300 ␮g/0.2 ml, 0.3% tartaric acid, Novartis Pharma, Tokyo, Japan) at 17:00 h on d 5, and the ovaries were harvested on d 6. To see the effect of prolactin, ovine prolactin (33 IU/mg, Sigma, St. Louis, MO) was given to cycling rats twice daily (1000 and 1700 h) starting on the estrous morning. The administration of prolactin (10 IU/0.2 ml saline) was continued for 3 d, and the ovary was collected on the diestrous day (1700 h). The ovaries were swiftly removed after decapitation of the rats, and total RNA was extracted by the acid-guanidinium-thiocyanate-phenol-chloroform method using TRIzol (Invitrogen Japan, Tokyo, Japan). Northern blot analysis was performed using standard procedures with a digoxigenin (DIG)-labeled PCR probe and visualized with a chemiluminescent substrate. Briefly, 10 ␮g of total RNA were electrophoresed in an agarose gel (1%) and blotted on a nylon membrane (Roche Molecular Biochemicals, Mannheim, Germany). The mRNA for rat annexin 5 was detected with a PCR probe synthesized with DIG-11-UTP (Roche Molecular Biochemicals). The primers for the PCR probe, 5⬘-ATG GCT CTC AGA GGC ACC GT-3⬘ and 5⬘-GTC ATC CTC GCC TCC ACA GA-3⬘, amplify the entire 957-bp reading sequence of annexin 5 cDNA (8). A cloning vector, pUC119, containing an annexin 5 cDNA insert, was used as the template (21). The PCR was performed with EX Taq polymerase (Takara, Tokyo, Japan) and deoxynucleotide triphosphates, including DIG-11-UTP, for 40 cycles of 0.75 min at 95 C, 1 min at 48 C, and 2 min at 72 C. The hybridized probe was detected with alkaline phosphatase-conjugated anti-DIG serum (Roche Molecular Biochemicals) and CSPD (disodium 3-(4-methoxyspiro{1,2-dioxetane-3, 2⬘-(5⬘-chloro)tricyclo[3.3.1.13,7] decan}-4-yl)phenyl phosphate; Roche Molecular Biochemicals) as substrate. Chemiluminescence was detected by x-ray film exposure (Kodak XAR-5, Kodak, Tokyo, Japan) for 1–3 h. Film was scanned with an FLA2000 fluoro-scanner (Fuji Film Co., Tokyo, Japan), and the intensity of each band was measured using Image Gauge software (Fuji Film Co.). Band intensity was normalized to the intensity of 18S rRNA measured by scanning the ethidium bromide-stained gels with FLA2000 and Image Gauge software before the hybridization step.

Tissue preparation Ovarian tissue of pseudopregnant rats was subjected to immunocytochemistry. Some of the pseudopregnant rats were given CB-154 (300 ␮g/0.2 ml, ip) to suppress prolactin secretion on d 5 of pseudopregnancy. Each rat was deeply anesthetized with Somnopentyl (pentobarbital sodium, 25 mg/kg body weight, Kyoritsu Shoji Co., Tokyo, Japan), then perfused through its ventricle with PBS (0.1 m, pH 7.4), followed by 4% paraformaldehyde/PBS at a rate of 200 ml/h for 15 min. Ovaries were dissected and further fixed in ice-cold paraformaldehyde/ PBS overnight. The tissue was then incubated in PBS for another night in an ice bath. Dehydration and paraffin embedding were performed per standard procedures.

Immunocytochemistry Paraffin-embedded ovary samples were cut to 4-␮m thickness. Paraffin was removed from the sections by a series of xylene and ethanol rinses. Immunocytochemistry was performed with the antirat annexin 5 rabbit antiserum raised in our laboratory (21). Antibody was diluted 1:1000 in the antiserum binding buffer (50 mm Tris, 150 mm NaCl, 5 mm EDTA, 0.25% gelatin, 0.05% Nonidet P-40, pH 7.4) and the signal was detected with Vectastain ABC AP kit and Vector Red substrate (Funakoshi, Tokyo, Japan).

Local administration of GnRH antagonist The GnRH antagonist Cetrorelix was kindly provided by Zentaris GmbH (Frankfurt, Germany). Cetrorelix was administered locally by an Alzet Osmotic Pump (Model 1003D, DURECT Co. Cupertino, CA) at a rate of 50 ng/␮l䡠h into a hemilateral ovarian bursa of a CB-154-administered pseudopregnant rat. On d 5 of pseudopregnancy, CB-154 (300 ␮g/0.2 ml) was administered sc. Simultaneously, an Alzet Osmotic Pump filled with Cetrorelix (50 ␮g/ml) was implanted under light ether anesthesia. A polyethylene tube (PE 60, Becton Dickinson, Tokyo, Japan) was attached to the pump and the other end was inserted into the ovarian bursa. The canula was fixed with a suture to the surrounding fat of the mesometrium.

Statistics ANOVA and the Bonferroni method were used for comparison of the means. P ⬍ 0.05 was considered to be significant.

Results Changes in the expression of ovarian annexin 5 mRNA during pseudopregnancy

Northern blot analysis revealed that annexin 5 mRNA decreased in the ovarian tissue during pseudopregnancy (Fig. 1A) but recovered at the end of pseudopregnancy (d 12). To see whether the observed variation is related to the age of the corpus luteum or to its functional status, we compared the level of annexin 5 mRNA expression between ovaries of the same age from pseudopregnant and pregnant rats. On d 12, the level of annexin 5 mRNA in the ovary was significantly lower in the pregnant rats than in the pseudopregnant rats (Fig. 1B). Effect of prolactin on annexin 5 expression in the ovary

The secretion of prolactin is enhanced during both pseudopregnancy and early pregnancy (22, 23). To see whether prolactin inhibits the level of ovarian annexin 5 mRNA during pseudopregnancy, the dopamine agonist CB-154, which suppresses prolactin release, was administered to pseudopregnant rats at 1700 h on d 5. We previously observed that CB-154 precipitously reduces plasma prolactin levels in pseudopregnant rats and stops pseudopregnancy (our unpublished data). The ovaries were harvested at 1000 h on d 6, total RNA was extracted, and Northern analysis was performed. CB-154 clearly increased annexin 5 mRNA expression in these ovaries (Fig. 2A). Conversely, we also examined the effect of prolactin on the expression of ovarian annexin 5 mRNA in estrous cycling rats. Twice-daily administration of prolactin (10 IU each) beginning the morning of estrus, which generates the functional corpus luteum, significantly reduced the expression of annexin 5 on diestrous 2 (Fig. 2B). Immunohistochemistry and TUNEL

The distribution of annexin 5 in the corpus luteum was observed by immunocytochemistry in the pseudopregnant


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FIG. 1. Northern blot analysis of annexin 5 mRNA expression in rat ovarian tissues. A, Ovaries were harvested from pseudopregnant rats on d 0, 3, 6, 9 and 12. B, Ovaries were harvested from pseudopregnant and pregnant rats on d 12. Each experimental group consisted of three rats. Total RNA was subjected to Northern blot analysis with a DIG-labeled PCR probe. The signal was visualized with the chemiluminescent substrate, CSPD. The upper panels show the ethidium bromide-stained gels of control 28S and 18S rRNA after electrophoresis, with samples from three animals in each column. The middle panels show Northern blots of annexin 5 mRNA, and the lower panels are graphical representations of the densitometric measurements of the blots. The relative levels of annexin 5 mRNA were normalized to the corresponding level of 18S rRNA. The data shown are the mean plus SE of three samples. The asterisk reveals a statistically significant difference from the value of d 0 (P ⬍ 0.05).

rats treated with CB-154 on d 5 of pseudopregnancy. The levels of annexin 5 expression gradually increased during the 2 d following treatment. The annexin 5-immunopositive cells did not distribute uniformly; rather, the intensity of immunostaining varied within each corpus luteum, as indicated in Fig. 3, C and D). To see whether apoptosis, a known function of the corpus luteum, is affected by annexin 5 or CB-154, we used the TUNEL assay, which detects apoptotic cells, to observe the effect of CB-154 on the ovarian tissue. CB-154 increased TUNEL-positive cells in the corpus luteum, and the distribution of TUNEL-positive cells completely matched that of the annexin 5-immunopositive cells (Fig. 4, B and D). Local administration of GnRH antagonist

Because we previously found that GnRH stimulates annexin 5 gene expression in pituitary gonadotropes, we were interested in studying the effects of GnRH on annexin 5 expression in the corpus luteum. When CB-154 was administered to the pseudopregnant rat on d 5, an osmotic pump filled with the GnRH antagonist Cetrorelix was implanted simultaneously. A short canula was connected to the pump to deliver Cetrorelix into a hemilateral ovarian bursa, whereas the other ovary was left untreated. Two days later, both ovaries were subjected to immunocytochemistry with antiannexin 5 antibody and to the TUNEL assay. Surprisingly, local administration of Cetrorelix completely inhibited both annexin 5 expression and TUNEL staining (Fig. 5, A vs. B and C vs. D).

Discussion

The present study is the first to clearly demonstrate that the ovarian expression of annexin 5 mRNA is decreased during the pseudopregnancy of rats and that prolactin suppresses annexin 5 expression in the corpus luteum. Annexin 5 is a member of the annexin family of proteins categorized by their structural similarity and the common property of calcium-dependent binding to phospholipids (24, 25). It has been postulated that this protein family is involved in some important physiological processes, but little has been clarified (26, 27). Although the genomic sequences of annexins suggest that they are housekeeping genes (25), it has been repeatedly demonstrated that their expression is regulated under various circumstances (28 –30). Thus, it was not surprising to find that ovarian annexin 5 expression differed under various conditions in the present study. We previously reported that the expression of annexin 5 in the pituitary gonadotropes was augmented in ovariectomized rats and that annexin 5 stimulates gonadotropin release (1, 17). We then demonstrated that GnRH augments annexin 5 mRNA expression in primary pituitary cell culture (16). These findings suggest that GnRH stimulation of annexin 5 gene expression is a necessary step to augment gonadotropin secretion in the gonadotropes. It is then also possible that annexin 5 synthesis is specifically regulated for its physiological functions in different cell types that express annexin 5. Although this regulation may be achieved by the same mechanism in different tissues, namely GnRH receptor signaling, it is perhaps initiated in response to different physiological

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FIG. 2. Effect of prolactin on the expression of annexin 5 mRNA in the ovary. A, On d 5 of pseudopregnancy, 300 ␮g of CB-154 was administered sc at 1700 h, and the ovaries were harvested in the morning (1000 h) of the next day. B, Prolactin (10 IU each, ip) was administered twice daily starting the morning of estrus, and the ovaries were harvested in the afternoon of diestrus. Each experimental group consisted of three rats. The upper panels show the control ethidium bromide-stained gels, the middle panels show the Northern blots for annexin 5, and the lower panels are graphical representations of the densitometric measurements of the blots. The relative levels of annexin 5 mRNA were normalized as in Fig. 1. The data shown are the mean plus SE of three samples. The asterisk shows statistical significance (P ⬍ 0.05).

FIG. 3. Immunocytochemistry of annexin 5 in the ovaries of pseudopregnant rats treated with dopamine agonist. Pseudopregnant rats were administered CB-154, or vehicle for controls, in the afternoon of d 5 of pseudopregnancy. The ovaries were collected on the two following days (d 6 and 7), fixed, stained with annexin 5 antibody (red reaction product), and counterstained with hematoxylin. Panels show vehicle control samples (A and B) and CB-154-administered samples (C and D), from d 6 (A and C) and d 7 (B and D). Highly immunopositive cells are indicated with arrows. Scale bar, 100 ␮m.

events, although all of these events are not clear. It is, thus, important to understand the mechanisms by which expression of ovarian annexin 5 is down-regulated during pseudopregnancy. The expression of annexin 5 mRNA in the pregnant ovary on d 12 was lower than that of the pseudopregnant ovary. The pregnant corpus luteum of d 12 secretes abundant progesterone, whereas the pseudopregnant corpus luteum is

already regressed. The reciprocal relationship between annexin 5 and progesterone secretion could indicate that progesterone down-regulates annexin 5 expression, or that the suppression of luteal annexin 5 expression is necessary for luteal progesterone secretion. The latter point raises the possibility that the increase in annexin 5 expression at the end of pseudopregnancy is a necessary step for the cessation of progesterone secretion and/or luteolysis.



FIG. 4. Immunocytochemistry of annexin 5 and histochemical assessment of apoptosis (TUNEL) in the ovaries of pseudopregnant rats treated with a dopamine agonist. Pseudopregnant rats were administered CB-154 (B and D) or vehicle (A and C) in the afternoon of d 5 of pseudopregnancy, and ovaries were collected on d 7. The ovaries were then stained with annexin 5 antibody (red reaction product) and counterstained with hematoxylin (A and B) or subjected to the TUNEL assay (C and D; TUNEL-positive is seen as green fluorescence). Panels A and C are adjacent sections, as are B and D. Arrows point out the identical regions in the sections. Scale bar, 100 ␮m.

FIG. 5. Immunocytochemistry of annexin 5 and histochemical assessment of apoptosis (TUNEL) in the ovaries of pseudopregnant rats treated with a dopamine agonist, followed by local administration of a GnRH antagonist to the hemilateral ovarian bursa. Pseudopregnant rats were administered CB-154 in the afternoon of d 5 of pseudopregnancy. The ovaries were either not treated further (A and C) or treated with Cetrorelix administered by osmotic pump (B and D). Ovaries were collected 2 d later (d 7). Sections were stained with annexin 5 antibody (red reaction product) and counterstained with hematoxylin (A and B) or subjected to the TUNEL assay (C and D). Panels A and C are the adjacent sections, as are B and D. Arrows indicate the matched regions of immunopositive and TUNEL-positive cells in adjacent sections. Scale bar, 100 ␮m.

Although it is possible that progesterone is a direct suppressant of annexin 5, the data presented here favor prolactin in this role. CB-154, a dopamine agonist that inhibits prolactin secretion, increased annexin 5 expression in the ovary at mid-pseudopregnancy. In contrast, the administration of prolactin to cycling rats, in which plasma progesterone levels are low, significantly reduced annexin 5 expression. These data indicate that the expression of luteal annexin 5 is physiologically down-regulated by prolactin. Stocco et al. (13) have reported, using a cDNA expression array, that prolactin evidently down-regulates the expression of luteal annexin 5 in the corpus luteum of hypophysectomized pregnant rats. This observation supports our data demonstrating reduction in the luteal annexin 5 mRNA induced by prolactin during pseudopregnancy. Interestingly, the expression of annexin 5 was low on d 12 of pregnancy in the present study even though prolactin surges are known to cease on d 10 of preg-

nancy (31). Placental lactogen (PL-1), which has been shown to bind to the prolactin receptor, may take the place of prolactin after d 11 of pregnancy when the secretion of PL-1 increases (32, 33). Therefore, the expression of annexin 5 in the luteal cells may still be under the effect of activated prolactin receptor even on d 12 of pregnancy (13). Prolactin is the hormone necessary to initiate the sequence of events in the ovary that facilitates progesterone synthesis (34, 35). Mechanical stimulation of the uterine cervix in the evening of proestrus induces twice-daily prolactin surges and causes a pivotal change in the corpus luteum that starts progesterone secretion (22, 23). Because prolactin is a primary luteotropic hormone in rats, the reduction of annexin 5 expression during pseudopregnancy may be necessary for the augmented progesterone secretion. It is demonstrated by the present study that the expression of annexin 5 and TUNEL-positive apoptosis are concomi-

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tantly induced in the same population of luteal cells shortly after inhibiting prolactin secretion of the pseudopregnant rat. The cessation of prolactin secretion during the functional luteal phase has been known to induce luteal regression (36, 37). Although annexin 5 is often used for detecting apoptotic cells, the results obtained here may not relate to this practical application of this protein. Detection of apoptotic cells with annexin 5 is based on its high affinity to phosphatidylserine, which is usually located inside the plasma membrane and is moved to the outer surface at a very early step of apoptosis (38). The present immunocytochemistry suggests the cytosolic distribution of annexin 5. It appears that annexin 5 is synthesized in the luteal cells during apoptosis to play an as yet unidentified role. The present results show that local administration of a GnRH antagonist specifically suppresses both annexin 5 expression and TUNEL staining in the corpus luteum. Previously, we found that GnRH specifically stimulates annexin 5 mRNA expression in the pituitary gonadotropes. Annexin 5 also stimulates gonadotropin secretion in a primary pituitary cell culture (17). It is hypothesized that annexin 5 mediates GnRH receptor signaling in the gonadotropes. As GnRH receptor is also found in the corpus luteum (39), it is possible that annexin 5 lies between the GnRH receptor and downstream cell functions, such as apoptosis, in the luteal cells. Several reports published recently suggest a role for annexins in signal transduction. Overexpression of annexins 1 and 5 was shown to down-regulate the MAPK/ERK pathway (4, 40), and the phosphorylation of annexin 1 was revealed to block ligand-induced association of Grb2, p21(ras), and Raf in the A549 human adenocarcinoma cell line (41). Furthermore, the intracellular domain of the vascular endothelial growth factor receptor, Flk-1, was revealed to interact with annexin 5 (42). Annexin 5 also inhibits the activity of protein kinase C (3). Although there is as yet no explanation to accommodate all of these pieces of evidence, annexin 5 is hypothesized to play a role in the MAPK pathway. If annexin 5 functions in the signal transduction of luteal cells, there also may be a mechanism to regulate it, using prolactin to change the expression of annexin 5. The present result, to our knowledge, is the first demonstration of the antagonizing effects of prolactin on GnRH action in the ovary. In summary, this report demonstrates the specific expression of annexin 5 in the corpus luteum and physiological suppression of luteal annexin 5 expression during pseudopregnancy by prolactin. Prolactin is suggested to restrain GnRH action in the corpus luteum and GnRH may facilitate luteal regression and apoptosis through annexin 5 synthesis. Acknowledgments The authors are grateful to Ms. Miyoko Nakata for her assistance in preparing the manuscript. Received January 24, 2003. Accepted April 29, 2003. Address all correspondence and requests for reprints to: Dr. Mitsumori Kawaminami, Veterinary Physiology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 0348628, Japan. E-mail: [email protected]. This work was supported partly by Grants-in-Aid for Scientific Research (No. 1436018 to M.K.) from Japan Society for the Promotion of


Science and by a Grant for Scientific Research from Kitasato University, School of Veterinary Medicine and Animal Sciences (S-0201).

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