DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. I0, pp. 597-602, 1986. 0145-305X86 $3.00 + .00 Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.

ASPECTS OF THE IMMUNOLOGY OF THE FEMALE GENITAL TRACT OF THE ELASMOBRANCH, SCYLIORHINUS CANICULA L.

S. Hart, A. B. Wrathmell, T. A. Doggett and J. E. Harris. Department of Biological Sciences, Plymouth Polytechnic, Plymouth, U.K.

INTRODUCTION

Recently, Ogra et al. (i) reviewed the growing literature on the immunology of the reproductive tract of higher vertebrates. It has been established that in a number of species a local immune response may develop during natural infections, to experimentally administered antigens and to spermato- zoan and seminal antigens.

The morphology of the reproductive system in fishes is well documented. (2). However, reproductive immunology has received limited attention other than an investigation of immunoglobulin transfer from mother to young in oviparous and viviparous teleosts (3) and immune relations between mother and foetus in viviparous poecilid fish Xiphorphorus helleri Haeckel (4, 5, 6, 7). Extensive interest in the reproduction of chondrichthyan fishes, reviewed by Wourms (8), has established that they exhibit a variety of reproductive strategies. The common dogfish Scyliorhinus canicula L. exhi- bits oviparity; it produces a fertilised egg sealed in a protective egg case which develops outside of the reproductive tract. The mucosa of this reproductive tract is potentially vulnerable to challenge from pathogens, and paternal or zygotic antigens. As part of a broader study of mucosal immunology in elasmobranches (9, I0), a preliminary investigation of the histology and ultrastructure of the mucosa, and an immunological analysis of the mucus of the reproductive tract was undertaken.

MATERIALS AND METHODS

Fish. Female dogfish (0.75 - 1.5 kg) were supplied by the Marine Biological Association (Plymouth, U.K.). Fish were anaesthetised in MS222 (Sandoz, Basel, Switzerland), pithed and the reproductive tract immediately excised and perfused with chilled elasmobranch saline (ll).

Histology. Material was fixed in 4% paraformaldehyde at 0 °C for i h, dehydrated through 30-70% alcohol at 0 C, then through 70% to absolute at -18 "C and infiltrated with TAAB Lowocryl K 4m resin at the same tempera-

597

598 IMMUNOLOGY OF ELASMOBRANCH GENITAL TRACT Vol. i0, No. 4

ture (TAAB Lowocryl K 4m Handbook, 1982). One micron sections were cut with glass knives on a Beichert Jung Autocut and stained with Giemsa or methyl green pyronin (12).

Ultrastructure. Material was fixed for 1 h in 2% glutaraldehyde in elasmo- brance saline at 4 °C, post-fixed in 1% osmium tetroxide, dehydrated in graded alcohols and embedded in TAAB Spurr resin according to the manufac- turer's instructions.

Immunological analysis of mucus. Mucus was collected from Zone 1 (see Fig. i) with a glass pipette, care being taken to avoid any contamination with blood.

Preparation of antiserum. A specific antiserum to dogfish IgM was raised in rabbits by a modification of the technique described by Ellis (13). Briefly, an antiserum to sheep red blood cells (SRBC) was first raised in adult dogfish to a titre of i:i000. This was then used to agglutinate SRBC which were then washed exhaustively and injected subcutaneously into rabbits at 1 month intervals. A rabbit anti-whole dogfish serum was also prepared (14).

Immunoelectrophoresis. This was performed using a Shandon 600 x i00 mm tank and Vokam 400 Power Pack (Shandon, London U.K.). The proteins were separated in 1% ionagar dissolved in barbitone buffer (0.8 M, pH 8.2) at 30 mA for 2 h at room temperature. Gels were incubated with antiserum overnight, exhaustively washed in saline, blotted dry, stained with Coo- massie brilliant blue and destained in an aqueous solution containing 10% acetic acid and 10% methanol.

RESULTS

For the purposes of this study the female reproductive tract was arbitrarily divided into 6 zones (Fig. i) and the major histological characteristics of these zones are outlined in Table i.

The genital tract of the female dogfish (Fig. i) contained leucocytes occu- pying two niches: the connective tissue and the epithelium. Plasma cells, which were basophilic with Giemsa, exhibited pyroninophilia and showed characteristic endoplasmic reticular elements (Fig. 2, 3), were identified only in the subepithelial connective tissue and predominantly in the upper regions of the tract (Table i). Intraepithelial cells, apparently in vacuo- lar spaces (Fig. 4), were observed throughout the tract occupying a basal position in the epithelium and, like the plasma cells, were found in highest concentrations in the proximal end of the tract. Additionally intralaminal leucocytes were observed in the nidament~l gland (Fig. 5).

Mucus was most abundant and hence most easily sampled in the distal region of Zone 1. On immunoelectrophoretie examination with rabbit antiserum to whole dogfish serum two precipitin arcs were revealed (Fig. 6a). One of the componenets was positively identified as IgM using a rabbit anti- dogfish IgM serum (Fig. 6b). A second arc, barely visible on this plate, was also observed and may represent IgM in another form, this faint arc is also present in serum and bile (15).

Vol. i0, No. 4 IMMUNOLOGY OF ELASMOBRANCH GENITAL TRACT 599

i ! i i ~ i! ~ i

! i ~ ii i

Fig. i. Reproductive tract of a female common dogfish ScFliorhinus canicula L. Liver (L), Spleen (S), Spiral intestine (SP), Reproductive tract (R), Epigonal (E), Nidamental gland (N). 2. Collection of plasma cells. Lumen of blood vessel (L), Plasma cell (P). 3. Pair of plasma cells (P), Collagen (C). 4. Mucosa of zone 2. Goblet cell (G), Cilia (C), Intraepithelial leucocyte (I), Basement membrane (BM), Sinus (S). 5. Nidamental gland. Glandular Tissue. Accumulation of leucocytes (A). 6a. IEL of rabbit anti whole dogfish serum run against reproductive tract mucus. 6b. Immunoelec- trophoresis (IEL) of rabbit anti dogfisk IgM run against reproductive tract m U C U S .

600 IMMUNOLOGY OF ELASMOBRANCH GENITAL TRACT Vol. I0, No. 4

TABLE l

H i s t o l o g y o f the Female Reproduct ive Trac t

ZONE ] 2 3 4 5 6

NATURE OF S t r a t i f i e d , f o l d e d as zone as zone fo lded h i g h l y

EPITHELIUM 672-900um co lumnar , 2 2 c i l l e t e d f o l ded ,

many g o b l e t 40-50um 20-40um c l I i e ted c e l l s 20-40um

NATURE OF muscular musculor, es zone as zone accumulat ions large

SUBEPITHELIUN numerous sub- 2 2 of Intreleminel prop-

e p i t h e l i a l leucocytes in ortlon

sinuses in the connec t ive tsken up

c l o s e p r o x i m i t y t i s s u e of the by b lood

to the base~N3nt n idamenta l s inuses

membrane orgon

DISTRIBUTION OF - /+ - as zone as zone

SUBEPITHELIAL 2 2

PLASMA CELLS

-/4 ÷~+

DISTRIBUTION OF -/+ + as zone ms zone -/+

INTRAEPITHE L IAL 2 2

LEUCOCYTES

DISCUSSION

In ScFliorhinus the right ovary is functional; eggs are collected in the top of the oviduct, albumen and egg case added in the nidamental gland and the encased egg is extruded through the vent (8).

The mucosa of the reproductive tract in Female dogfish has a substantial number of cells which may have a specific immunological potential. Plasma cells were identified in the lamina propria, and as is the case in the dogfish gut (9), they occurred predominantly in the proximal zones. The antigen and hence the degree to which the epithelium is penetrated by anti- gen. The thick stratified epithelium of the distal tract may be less ac- cessible to antigen than the relatively thin proximal epithelium.

Immunoglobulin (IgM) was detected in the lumen of the distal region of the tract (Fig. 6b); the converse of the plasma cell distribution. It may, however, have been secreted from the proximal mucosa, directed down- wards by mucus flow and collected in the expanded distal zone prior to discharge via the cloaca. It is not known whether the immunoglobulin detec- ted in the lumen is derived from the local plasma cells or from direct transudation from the serum or both. A second protein was detected by immunoelectrophoresis with the rabbit anti-whole dogfish serum (Fig. 6a), the identity of which is as y~t unknown. A variety of non-specific Factors,

Vol. i0, No. 4 IMMUNOLOGY OF ELASMOBRANCH GENITAL TRACT 601

which aid resistance to infection in the urinogenital tract of female mam- mals, exists (i). The eleetrophoretie mobility of the second precipitin arc is comparable to that of serum transferrin which is included amongst these factors at least in mammals (16). Intraepithelial leucocytes were observed throughout the genital tract but specific cells were not identified in this study. It would be interesting to determine if a common mucosal leucocyte population exists in the respiratory, gastrointestinal and repro- ductive mucosae. Histological work undertaken to date on these tissues suggests there are broad similarities in plasma cell distribution between the gut and urinogenital tract.

Besides the work on the materno-foetal relations (3, 4, 5, 6, 7) and a brief mention by Wourms (17) on immunity in viviporous fish, little informa- tion exists on local immunity in the reproductive tract of fishes. Similar- ly the immunity of other mueosal surfaces; gill and gut have also received little attention (18).

In the absence of any definitive information on local immunity of the repro- ductive tract of fishes we feel that internal fertilization mechanisms and the development of viviparity may have created a necessity for immuno- competence at this mucosa as a result of (a) infection of this mucosa by transfer of micro-organisms or other pathogens on the claspers of the male during mating or by passive entry at other times, (b) sperm or seminal antigens stored in Scyliorhinus and other elasmobranchs (8), which have the potential to act immunogenically and (c) fertilisation, the zygote being allogeneic (although this may have greater significance in placental sharks than in Sc~liorhinus where the zygote is coated by albumen and an egg case both of maternal origin).

Although this is only a preliminary study we feel that the reproductive immunology of elasmobranchs, which exhibit a spectrum of gestatory strate- gies from oviparity to true placental viviparity offers an exciting model for the examination of the development of local immunity, tolerance mechan- ism to spermatazoan and seminal antigens, and of materno-foetal immunologi- cal relationships.

REFERENCES

1 OGRA, P.L., YAMANAKA, T. and LOSONSKY, G.A. Local immunologic defences in the genital tract. In: Reproductive Immunology. N. Gleischer (Ed.) New York: Alan R. Liss, Inc., 1981 p 381.

2 HOAR, W.S. Reproduction. In: Fish physiology. Vol. 3. W.S. Hoar and D.J. Randall (Eds.) New York: Academic Press, 1969, p i.

3 BLY, J.E. The ontogeny of immunity in teleost fishes with particular reference to foeto-maternal relationships. PhD Thesis. University College of North Wales, Bangor. 1984.

4 HOGARTH, P.J. Immunological aspects of foeto-maternal relations in lower vertebrates. J. Reprod. Cert. Suppl. 3, 15, 1968.

5 HOGARTH, P.J. Immune relations between mother and foetus in the vivi- parous poeciliid fish Xiphophorus hellerii Haeckel. I. Antigenieity of the foetus. J. Fish Biol. 4, 265, 1972.

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6 HOGARTH, P.J. Immune relations between mother and foetus in the vivi- parous poeciliid fish Xiphorphorus hellerii Haeckel. II. Lack of status of the ovary as a favourable site for allograft survival. J. Fish Biol. 4, 271, 1972.

7 HOGARTH, P.J. Immune relations between mother and foetus in the vivi- parous poeciliid fish Xiphophorus hellerii Haeckel. III. survival of embryos after ectopic transplantation. J. Fish Biol. 5, 109, 1973.

8 WOURMS, J.P. Reproduction and Development in Chondrichthyan fishes. Am. Zool. 17, 379, 1977.

9 HART, S., WRATHMELL, A.B. and HARRIS, J.E. Gut associated lymphoid tissue (GALT) in the dogfish ScFliorhinus canicula L.: a light micro- scopic study. J. Mar. Biol. Assoc. U.K. 66, 721, 1986.

i0 HART, S., WRATHMELL, A.B. and HARRIS, J.E. Ontogeny of gut associated lymphoid tissue in the dogfish, SeFliorhinus eanicula L. Vet. Immunol. Immunopathol. 12, 107, 1986.

ii HALE, L.J. Biological Laboratory Data. London: Methuen, 1965.

12 PEARSE, A.G.E. Ltd.

Histochemistry. 3rd edition, vol. 1 and 2, Churchill

13 ELLIS, A.E. Leucocytes and related cells in the plaice Pleuronectes platessa. J. Fish Biol. 8, 143, 1976.

14 MORROW, W.J.W., HARRIS, J.E., DAVIES, D.H. and PULSFORD, A. Isolation and partial characterisation of dogfish (ScFliorhinus canicula L.) anti- body. J. Mar. Biol. Assoc. U.K. 63, 409, 1983.

15 HART, S., WRATHMELL, A.B., HARRIS, J.E. and DOGGETT, T.A. bulins in the gut of the dogfish Sc~liorhinus canieula L. tion).

Immunoglo- (In prepara-

16 INGRAM, G.A. Substances involved in the natural resistance of fish to infection - a review. J. Fish Biol. 16, 23, 1980.

17 WOURMS, J.P. Viviparity: the Maternal-Foetal relationship in Fishes. Am. Zool. 21, 473, 1981.

18 LAMERS, C. Reaction of the immune system of fish to vaccination. thesis. University of Wageningen, Netherlands. 1985.

Received: May, 1986 Accepted: June, 1986

PhD