Effects of sexual maturation and gonadal steroids on the localization of IgG-, IgM- and IgA-positive cells in the chicken oviduct

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1 Effects f sexual maturatin and gnadal sterids n the lcalizatin f IgG-, IgM- and IgA-psitive cells in the chicken viduct W. M. Zheng, Y. Yshimura and T. Tamura Graduate Schl fr Internatinal Develpment and Cperatin, and Faculty f Applied Bilgical Science, Hirshima University, Higashi-Hirshima 739, Japan The effects f sexual maturatin and gnadal sterids n the lcalizatin f immunglbulin-psitive cells in chicken viducts were studied. Oviductal tissues were cllected frm laying hens and chicks treated with stilbestrl (DES, an analgue f estrgen) r prgesterne. Paraffin wax sectins f the tissues were immunstained fr IgG, IgM and IgA, and the frequency f cells staining psitive was examined using an image analysis system. Sme f the cells in the mucsal epithelium and plasma cell-like cells in the strma f the viduct stained psitive fr IgG, IgM r IgA. In the mucsal epithelium f laying hens, there was a significantly greater number f IgG-psitive (IgG) cells in the shell gland than in the infundibulum, magnum and isthmus, mre IgM cells in the magnum than in the infundibulum, and mre IgA cells in the magnum than in the ther segments f the viduct with the exceptin f the vagina. The frequency f IgG and IgM cells in the mucsal epithelium f all viductal segments and IgA cells in the magnum, isthmus and vagina was significantly higher in laying hens than in immature birds. In the subepithelial strma f laying hens, there was a significantly greater ppulatin f IgG cells in the infundibulum and vagina than in the magnum and isthmus, mre IgM cells in the infundibulum than in the magnum, and mre IgA cells in the utervaginal junctin and vagina than in the magnum and isthmus. The frequency f IgG, IgM and IgA cells in the subepithelium f infundibulum, utervaginal junctin and vagina was significantly greater in laying hens than in immature birds. The number f IgM cells in all viductal segments and f IgA cells in the magnum f the mucsal epithelium f the chicks treated with DES increased significantly cmpared with thse f cntrl chicks. In additin, the number f IgG cells in the shell gland and vagina and f IgM cells in the vagina f the strma f DES-treated birds were increased. Treatment f immature birds with prgesterne had n effect n the lcalizatin f Ig cells in the viduct except fr a decrease in the number f IgM cells in the shell gland. These results suggest that the lcal immunity in the viduct develps during sexual maturatin, pssibly under the cntrl f estrgen. Intrductin The chicken viduct cnsists f the infundibulum, magnum, isthmus, shell gland, utervaginal junctin (UVJ) and the vagina. In the viduct, egg cmpnents that surrund the ylk, including albumin, the shell membrane and the egg shell are frmed. In additin, spermatza entering via the vagina are stred in the sperm-strage tubules in the UVJ (Fujii, 1963; Fujii and Tamura, 1963) befre taking part in fertilizatin which ccurs in the infundibulum. The lcal immune system in the chicken viduct may play an imprtant rle in preventing infectin as the vagina pens t the claca which cntains numerus exgenus pathgens (Shivaprasad et al, 1990; Pppe et al, 1992). The viductal immune system may als affect the selectin, survival and the fertilizing ability f ""Crrespndence. Received 3 March spermatza (Steele and Wishart, 1992; Bakst et al, 1994). It was reprted that incubatin f spermatza with anti-sperm antisera in turkeys drastically reduces the fertilizing ability f the sperm cells (Burke and Rieser, 1972; Burke and Yu, 1979). In the viduct f laying hens, plasma cells (Van Krey et al, 1987) and Ig-cntaining cells (Lebacq-Verheyden et al, 1972; Kirk et al, 1989; Kimijima et al, 1990) that may have a rle in lcal immunity have been demnstrated. Yshimura et al (1996) reprted that immuncmpetent cells, including majr histcmpatibility cmplex class II psitive cells, and cells and Ig psitive (Ig ) cells, are numerus in the mucsal surface f the viduct in laying hens, whereas, in multing hens, they are reduced. Previus studies in mammals have indicated that estrgen enhances humral immune respnses (Trawick and Bahr, 1986; Erbach and Bahr, 1988, 1991) and that the lcal immune system in the reprductive tract is cntrlled by gnadal sterids (Grssman, 1984). Therefre, it is pssible that the immune

2 system f the hen's viduct is affected by the reprductive cycle and influenced by gnadal sterids. Hwever, it is nt knwn whether the presence f Ig cells in the viductal wall is under the cntrl f gnadal sterids in hens. The aim f the present study was t determine the changes in the lcalizatin f IgG, IgM and IgA cells in the viduct f the hen during sexual maturatin and the effects f estrgen and prgesterne n these Ig cells. Treatment f birds Materials and Methds Immature and laying White Leghrn hens were kept in individual cages under a light regimen f 14 h light and 10 h dark, and prvided with feed and water ad libitum. The chicks (50 days ld, n 5 birds) and mature hens (apprxi mately 260 days ld, = 5 birds) that were laying mre than five eggs in a sequence were used in Expt 1. The laying hens were used 6 h after vipsitin, that is, when there was an egg in the shell gland. In Expt 2, immature chickens (36 days ld) were injected i.m. daily with 1 mg stilbestrl (DES, Nacalai Tesque. Inc., Kyt,) r 1 mg prgesterne (Sigma C., St Luis, MO) fr 7 days. These sterids were disslved in sesame il (vehicle) at a cncentratin f 10 mg ml. Cntrl ~ birds received 0.1 ml vehicle. Six birds were used in each grup. All birds were killed by decapitatin. The viduct was pre-fixed in 10% (v/v) frmalin slutin fr apprximately 15 h, and small pieces (apprximately 3-5 mm lng) were cut frm the middle part f the infundibulum, magnum, isthmus, shell gland, UVJ and vagina, and re-fixed fr anther 36 h in the same fixative. Sectins were embedded in paraffin wax (6 µ thick) and immunstained fr Igs. Sme f the sectins were stained with haematxylin and esin fr histlgical studies. Immuncytchemistry Antibdies. The first antibdies used fr the staining f, IgG IgM and IgA cells were rabbit anti-chicken IgG (... Cncepts Inc., Tmas River, NJ), gat anti-chicken IgM (Bethyl Lab. Inc., Mntgmery, TX) and gat anti-chicken IgA (Bethyl Lab. Inc., Mntgmery, TX), respectively. These first antibdies were diluted t a cncentratin f 1:500 with PBS cntaining 0.05% (w/v) BSA. Vectr-staining ABC-PO kits (Vectr Lab. Inc., Burlingame, CA) fr the detectin f rabbit IgG (in the case f detectin f IgG cells) and gat IgG (fr detectin f IgM and IgA cells) were used t detect the immunreactin prducts f the first antibdies. Immunstaining. Sectins were deparaffinized and washed with PBS fr 15 min (5 min 3 times). They were then incubated with nrmal hrse serum (dilutin 1:150) fr 15 min fllwed by incubatin with the first antibdy fr 2 h. After washing in PBS (5 min 3 times), they were incubated with the secnd antibdy fr 1 h. Sectins were then washed in PBS (5 min 3 times) and incubated with avidin bitin-perxidase cmplex fr 1 h. After washing with PBS (5 min 3 times), the immunreactin prducts were revealed by incubating with a mixture f 0.02% (w/v) 3,3 '-diaminbenzidine tetrahydrchlride and 0.005% (v/v) H202 in 0.05 ml Tris-HCl l"1 buffer, ph 7.6. Sectins were then rinsed with water and cunterstained with haematxylin. Cntrl sectins were pre pared by the same prcedure except that the first antibdy with nrmal rabbit (in case f staining fr IgG cells) r gat (in case f staining fr IgM and IgA cells ) serum, and n psitive staining was bserved in these sectins. All steps were carried ut at rm temperature. Observatins and analysis f data Sectins were examined under a light micrscpe. Ppulatins f cells psitive fr immunreactins in the mucsal epithelium and in the strma were analysed by an image analysis cmputer system, MacAspect (Mitani C., Fukui). The subepithelial strma f 50 µ depth frm the basal bundary f the mucsal epithelium cvered the whle strmal tissue f each viductal segment in immature chickens regard less f treatment. Hwever, in laying hens, it did nt cver the middle part f strma because the tubular glands had devel ped there. Therefre, the frequency f psitive cells in the strma was examined in the subepithelial strma (the area 50 µ in depth frm the bundary f the mucsal epithelium) f immature hens and in the subepithelial and middle part f the strma f laying hens. The number f psitive cells was cunted in tw different areas (apprximately µ each) in ne viductal segment f a bird and the values were calculated t be the number f cells in 5000 µ 2. The average f the tw cunts was then used as the number f cells in ne segment f a f difference bird. The significance in the number f cells amng treatments r segments was examined by ne-way ANOVA fllwed by Duncan's multiple I test. Results Experiment 1: changes in the Ig cell ppulatin befre and after sexual maturatin in the viduct The mucsal tissue f the viduct cnsisted f the mucsal epithelium and strma in bth immature and laying hens. Tubular glands were well develped in the magnum, isthmus and shell gland in laying hens. Plasma cell-like cells in the strmal cnnective tissues in each viductal segment stained psitive fr IgG (Fig. la f), IgM and IgA in bth immature and laying hens. Sme mucsal epithelial cells als stained psitive fr IgG (Fig. lb, d, f), IgM and IgA in laying hens, whereas few f these cells stained in immature hens. The frequency f Ig cells in the viductal mucsal epi thelium in laying and immature hens is shwn (Fig. 2). In the mucsal epithelium f laying hens, the ppulatin f IgG cells in the shell gland was significantly higher than that in the infundibulum, magnum r isthmus. The frequency f IgM cells in the magnum was significantly higher than that in the infundibulum, and the frequency f IgA cells in the magnum

3 1. Sectins f the viducts f immature and laying hens immunstained fr IgG. Arrws and arrwheads indicate examples f psitively stained cells in the strma and mucsal epithelium, respectively. E: mucsal epithelium; S: strma; : tubular gland in the strma; L: lumen f viduct. Scale bars represent 20 µ (a) The magnum f an immature hen. There are very few IgG psitive (IgG ) cells in the strma and nne in the epithelium, (b) The magnum f a laying hen. Tubular glands have develped in the strma and psitive cells are bserved in the mucsal epithelium and strmal cnnective tissue, (c) The shell gland f an immature hen. There are very few IgG cells in the strma and nne in the epithelium, (d) The shell gland f a laying hen. There are many cells in the mucsal epithelium and in the strmal cnnective tissue, (e) The vagina f an immature hen. There are a few psitive IgG cells in the strma and nne in the epithelium, (f) The vagina f a laying hen. Many IgG cells can be seen in the mucsal epithelium and in the strma. Fig. significantly higher than that f the ther viductal with the segments exceptin f the vagina. In immature hens, there was n significant difference in the frequency f each kind was Ig cells in the mucsal epithelium amng the viductal segments. The frequency f IgG and IgM cells in the mucsal epithelium f all viductal segments and IgA cells in f

4 20 (a) 15 r SD E 20 (C) Ma Is Sg Uv Va Fig. 2. Frequency f the cells psitive fr immunglbulins (a) IgG, (b) IgM and (c) IgA in the mucsal epithelium f the viduct in (D) immature and ( I ) laying hens. Each bar represents the mean ± sem f the number f psitive cells in 5000 µ tissue (n = 5 birds fr each bar). Bars with different superscripts are significantly different (P < 0.05) fr laying hens. There were n significant differences amng the segments frm immature hens. An asterisk indicates a significant difference between laying and immature birds (P < 0.05). In: infundibu lum; Ma: magnum; Is: isthmus; Sg: shell gland; Uv: utervaginal junctin; Va: vagina. the magnum, isthmus and vagina was significantly higher in laying hens than in immature birds. The frequencies f Ig cells in the strma f viductal tissues are shwn (Figs 3 and 4). The ppulatins f IgG cells in the subepithelial strma in the infundibulum and in the vagina were significantly higher than thse in the magnum and the isthmus in the case f laying hens. In additin, the frequency f IgM cells in the infundibulum was significantly higher than that in the magnum, and the frequencies f IgA Is Sg Uv Fig. 3. Frequency f the cells psitive fr immunglbulins (a) IgG, (b) IgM and (c) IgA in the subepithelial strma f the viduct in ( ) immature and ( ) laying hens. Each bar represents the mean ± sem f the number f psitive cells in 5000 µ 2 tissue (n = 5 birds fr each bar). Fr each type f immunglbulin, bars with different superscripts are significantly different fr laying hens (P<0.05). There was n significant difference amng the segments frm immature chickens. An asterisk indicates that the difference between laying and immature birds is significant (P<0.05). In: infundibulum; Ma: magnum; Is: isthmus; Sg: shell gland; Uv: utervaginal junctin; Va: vagina. cells in the UVJ and vagina were significantly higher than thse in the magnum and isthmus (Fig. 3). In the middle part f the strma f laying hens, there were greater numbers f IgG and IgM cells in the infundibulum and vagina and a greater number f IgA cells in the vagina than in the magnum and isthmus (Fig. 4). In immature chickens, n significant differences were detected in the frequency f each kind f Ig cells in the subepithelial strma amng viductal segments (Fig. 3). The frequencies f IgG, IgM and IgA cells in the subepithelial strma f infundibulum, UVJ and vagina and als that f IgG cells in the shell gland were significantly greater in laying hens than in immature birds (Fig. 3).

5 1 SD O E Is Sg Uv Va Fig. 4. Frequency f the cells psitive fr immunglbulins (D) IgG, (0) IgM and ( ) IgA in the middle part f viductal strma f laying hens. Each bar represents the mean ± sem f the number f psitive cells in 5000 µ 2 tissue (n = 5 birds fr each bar). Within each type f immunglbulin, bars with different gruped superscripts ( fr IgG, fr IgM, "' fr IgA) are significantly different (P< 0.05). In: infundibulum; Ma: magnum; Is: isthmus; Sg: shell gland; Uv: utervaginal junctin; Va: vagina. Experiment 2: effects f gnadal sterids n the lcalizatin f viductal Ig cells in immature birds The mucsal tissue f the viduct in cntrl and prgesterne-treated chicks shwed an undifferentiated feature, which was similar t that seen in nrmal immature chickens, whereas in DES-treated chicks, the mucsal tissue shwed a develped structure including the frmatin f tubular glands in the magnum and shell gland (Fig. 5). Plasma cell-like cells psitive fr IgG, IgM and IgA were bserved in the strmal cnnective tissue f all viductal segments in chickens treated with il, DES r prgesterne (Fig. 5a f), and a few f the mucsal epithelial cells als stained psitive fr IgG, IgM and IgA. The frequencies f IgG, IgM and IgA cells in the viductal mucsal epithelium and strma f chickens treated with il, DES r prgesterne are shwn (Figs 6 and 7). In the mucsal epithelium, the effect f treatment with DES n the ppulatin f IgG cells was nt significant. Hwever, the frequency f IgM cells in the mucsal epithelium f all viductal segments and that f IgA cells f the magnum were significantly increased by DES treatment. Prgesterne treatment had n significant effect n the frequency f Ig cells in the mucsal epithelium. The ppulatin f IgA cells in the mucsal epithelium f the DES-treated grup was significantly greater in the magnum than in the shell gland and vagina (Fig. 6). A significant increase in the ppulatin f IgG cells was bserved in the viductal strma f the shell gland and vagina, and f IgM cells in the vagina as a result f treatment with DES (Fig. 7). Treatment f chickens with prgesterne had n significant effect n the ppulatins f Ig cells in the viductal strma with the exceptin f the ppulatin f IgM cells in the shell gland which was significantly decreased by treatment. In DES-treated birds, the ppulatin f IgG cells was apprximately 2 4 times higher than thse f IgM and IgA cells in the magnum, shell gland and vagina. In additin, the ppulatins f IgG cells were nt significantly different amng the magnum, shell gland and vagina, whereas the ppulatin f IgM cells in the vagina was significantly higher than in the magnum and shell gland, and that f IgA cells was significantly higher in the shell gland and vagina than in the magnum. Discussin The significant findings f the present study are that there are significantly mre Ig cells in bth the mucsal epithelium and strma f the viducts f laying hens than in immature chickens, and that stilbestrl, an analgue f estrgen, significantly increases the ppulatin f viductal Ig cells in immature hens, while prgesterne has n such effect. The Ig cells are lcalized in the cells f mucsal epithelium and plasma cell-like cells in bth the subepithelial and the middle part f strma in laying hens. This bservatin supprts previus reprts which lcalized Ig cells in the viductal strma (Lebacq-Verheyden et al, 1972; Van Krey et al, 1987; Kirk et al, 1989; Kimijima et al, 1990) and mucsal epithelium (Kimijima et al, 1990) in laying hens. It is nt knwn whether the mucsal epithelial cells prduce immunglbulins r if they absrb them frm bld r plasma cell-like cells lcalized in the subepithelial strma. Immunglbulins secreted frm the epi thelium int the viductal lumen may act lcally. It has been reprted that in laying hens infected with Salmvnella enteritidis the level f infectin was much lwer in the viduct than in the intestine, even thugh the vagina pens t the claca, and als that cntaminatin f the egg cmpnents was much less than that n the surface f the eggshell (Shivaprasad el al, 1990; Pppe et al, 1992). The presence f many Ig cells in the strma f bth ends f the viduct may prtect the viduct Barr and Parr frm infectin by bacteria and ther pathgens. (1985) demnstrated that, in mammals, IgA, IgG and pssibly IgM bind t many f the bacteria in the luminal cntents f the

6 5. Sectins f the magnum, shell gland and vagina f immature chickens treated with stilbestrl and cntrl sectins immunstained fr IgG. Arrws indicate examples f psitive cells in the strma. E: mucsal epithelium; S: strma; : tubular gland in the strma; L: lumen f the viduct. Scale bars represent 20 µ. (a) The magnum f a cntrl chick. There are very few IgG psitive (IgG ) cells and n tubular glands have frmed in the strma, (b) The magnum f immature bird treated with stilbestrl. Nte the presence f many psitive cells and the frmatin f tubular glands in the strma, (c) The shell gland f a cntrl bird. Only a few psitive cells are present, (d) The shell gland f the immature bird treated with stilbestrl. There are mre psitive cells cmpared with the cntrl sectin and tubular glands have develped in the strma, (e) The vagina f a cntrl bird. Only a few psitive cells are bserved in the strma, (f) The vagina f an immature bird treated with stilbestrl. Nte the increased number f IgG cells cmpared with the cntrl sectin. Fig. uterus. in the They als suggested that the secretry immune system reprductive tract f female mice may be invlved in returning the uterus t an aseptic state by at least three mechanisms, namely the blcking f the bacteria-binding sites f the mucsal epithelium, the agglutinatin f bacteria and the psnizatin f bacteria in preparatin fr phagcytsis. The

7 10 r > > 10 - (b) un 6-4 E 10 8 (c) 20 - _, hüí Magnum Shell gland Vagina Fig. 6. Frequency f the cells psitive fr immunglbulins (a) IgG, (b) IgM and (c) IgA in the mucsal epithelium f the viduct in immature chickens treated with (0) stilbestrl (DES) r ( ) prgesterne. (D) Cntrl. Each bar represents the mean ± sem f the number f psitive cells in 5000 µ 2 tissue (n 6 birds fr each bar). Within each = viductal segment, bars with different superscripts are significantly different (P < 0.05). (c) In the DES-treated birds, bars with superscripts m n are significantly different fr the frequency f IgA cells (P<0.05). infundibulum, UVJ and vagina are clsely related t fertiliz atin, strage and selectin f spermatza in hens (Fujii, 1963; Fujii and Tamura, 1963; Bakst et al, 1994). Recent reprts have indicated that there is an increase in the number f leukcytes in the lumen f the vagina just after inseminatin and that phagcytsis f spermatza by viductal cells ccurs in UVJ (Higaki et al, 1995), suggesting that an anti-sperm immunreactin ccurs in the viduct f hens. Steele and Wishart (1992) reprted that spermatza recvered frm the vagina carried Igs, suggesting that Igs may affect the survival, r are invlved in the immunlgical selectin, f spermatza in the Fig. 7. Frequency f the cells psitive fr immunglbulins (a) IgG, (b) IgM and (c) IgA in the subepithelial strma f the viduct in immature chickens treated with (0) stilbestrl (DES) r ( ) prgesterne. (D) Cntrl. Each bar represents the mean sem f the number f psitive cells in 5000 µ 2 tissue (n = 6 birds fr each bar). Within each viductal segment, bars with different superscripts are significantly different (P<0.05). In birds treated with stilbestrl, bars with superscripts x/y are significantly different in the frequency f (b) IgM r (c) IgA psitive cells (P < 0.05). viduct. Immunglbulins in the vagina, UVJ and infundibulum may participate in these anti-sperm immunreactins as mst spermatza remain in these parts f the reprductive tract just after inseminatin (Bakst et al, 1994; Higaki et al, 1995). Therefre, we suggest that the infundibulum, UVJ and vagina cntain greater numbers f Ig cells, and that these segments play an imprtant rle in the immune respnses t bth pathgenic agents and spermatza. cells in The present results shw that there were mre Ig the infundibulum, shell gland, UVJ and vagina f laying hens than in these areas f immature birds. Treatment f immature

8 birds with DES increased the number f IgM cells in the mucsal epithelium in all viductal segments, f IgA cells in the mucsal epithelium f the magnum, f IgG cells in the strma f the shell gland and vagina, and f IgM cells in the strma f the vagina. Prgesterne had n effect. We pre viusly reprted that the ppulatin f IgG cells, as well as cells (CD3 psitive cells) and cells (Bu-lb psitive cells) was greater in laying hens than in multing hens (Yshimura et al, 1996). These results indicate that the increase in Ig cells during sexual maturatin is clsely assciated with viductal grwth which is pssibly under the cntrl f estrgen. Leitner et al. (1996) demnstrated that the administratin f estrgen t hens significantly enhanced the humral immune respnse t Escherichia clt and sheep erythrcytes, whereas treatment with an anti-estrgen strngly inhibited these immune respnses. Therefre, estrgen may increase the humral immune respnse and als lcal immunity in the specific rgans that are the targets fr estrgen, like the viduct. The ppulatin f IgM cells in the shell gland was lwer in prgesterne-treated birds than in cntrl birds. Althugh we did nt examine the effects f prgesterne n the Ig cell ppulatin in the viducts f laying hens r DES-treated birds, prgesterne may suppress the infiltratin f Ig cells int the viduct. Studies in mammals have als prvided evidence that gnadal sterids might play signifi a cant rle in immune functin (Trawick and Bahr, 1986; Erbach and Bahr, 1988, 1991). Wira and Sande (1980) reprted that estrgen increased uterine IgA and IgG cncentratins in variectmized r hypphysectmized rats and that the spntaneus increase f Ig cncentratins during the estrus cycle resulted frm the actin f estrgen in the uterus. Wang et al (1996) reprted that the transprt f plymer IgA frm the bld int female muse genital tissues decreased signifi cantly after variectmy, and that this decline was rectified by estradil but nt by prgesterne. The effects f prgesterne n the Ig further. cells in the hen's viduct need t be examined In cnclusin, the present results suggest that the lcal immunity in the viduct develps during sexual maturatin and is under the cntrl f estrgen. The presence f relatively greater numbers f Ig cells in bth ends f the viduct suggests that the immune system in these tissues plays an imprtant rle in the immune respnse t pathgenic and agents spermatza. This wrk was supprted by Grant-in-Aid fr Scientific Research frm the Ministry f Educatin, Science and Culture f Japan (N ) t Y. Yshimura. 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