Immunoglobulin synthesis in fetal sharks

Camp. B&hem.

Physiol.,

1973, Vol. 45A, pp. 247 to 256. Pergamon Press. Printed in Great Britain

IMMUNOGLOBULIN DAVID

GITLIN,

ANITA

SYNTHESIS PERRICELLI

IN FETAL

SHARKS*

and JONATHAN

D. GITLIN

Department of Pediatrics, University of Pittsburgh School of Medicine and the Children’s Hospital, Pittsburgh, Pennsylvania 15213, U.S.A. (Received 7 October 1972)

Abstract-l.

Fetal serum immunoglobulin levels in seven viviparous and one ovoviviparous species of shark ranged from less than 0.3 to 4.8 per cent of the maternal levels. 2. Apparently little or no maternofetal transfer of immunoglobulins occurs in sharks either via the placenta as it does in some mammals or via the yolk as it does in birds. 3. Immunoglobulins can be synthesized by fetal shark spleen and in the adult shark by spleen, liver, stomach and the anal organ. 4. The low serum levels of immunoglobulin in the spiny dogfish fetus may be due in part to a relative unresponsiveness to antigens before birth.

INTRODUCTION

THE MAMMALIAN fetus can synthesize immunoglobulin M (IgM) as early as the end of the first trimester of gestation and immunoglobulin G (IgG) soon thereafter (van Furth et al., 1965; Tyan & Herzenberg, 1968; Gitlin & Biasucci, 1969). Yet, relatively little of either immunoglobulin is normally produced antenatally (Orlandini et al., 1955 ; Hitzig, 1961; Gitlin et al., 1963 ; M&-tensson & Fudenberg, 1965). This paucity of immunoglobulin synthesis by the fetus has been attributed to a lack of antigenic stimulation in utero, since the fetus can respond to both injected antigens and to infection with the formation of specific antibodies and elevated serum concentrations of IgM (Stiehm et al., 1966 ; Remington et al., 1968 ; Silverstein et al., 1970). In some mammals, the placenta is selectively permeable to IgG, and IgG is transferred from the mother to the fetus during the last half of gestation (Bangham et al., 1958; Gitlin et al., 1964; Koch et al., 1967). In other mammals, the placenta is impermeable to both IgM and IgG, and maternal IgG is transferred to the neonate via colostrum (Sterzl et al., 1966; Brambell, 1970). In birds, there is little or no significant synthesis of immunoglobulins by the embryo, but maternal IgG is stored within the yolk and reaches the embryo via the vitelline circulation during development (Ryle, 1957; Brambell, 1958; Mitchison, 1962). * Supported by a grant (No. HD-01031) from the National Institutes of Health, U.S. Public Health Service. Presented in part at the Second Symposium on Elasmobranch Biology held in Bar Harbor, Maine, 21 June 1971. 247

248

DAVIDGITLIN, ANITA PBRRICELLIANDJONATHAND. GITLIN

In the present report, the maternofetal transfer of immunoglobulins and the synthesis of immunoglobulins by the fetus were studied in ovoviviparous and viviparous sharks. In the ovoviviparous shark, the developing young is at first enclosed in an egg-case membrane, and the fetus is later released to grow freely within the uterine lumen without the formation of a placenta. Fidler et al. (1969) have found that the nurse shark, an ovoviviparous species, possesses only small amounts of immunoglobulin at birth. In viviparous sharks, the fetus becomes attached to the uterine wall by means of a placenta which develops from the yolk sac, and in some species the young develop within individual uterine compartments which separate the fetuses from each other as well as from the sea during the last half of gestation. MATERIALS

AND METHODS

Sharks studied Gravid females of the eight species listed in Table 1 were captured, and serum was obtained from both the gravid females and their fetuses. All of the galeoid sharks were viviparous; the squaloid shark was ovoviviparous. Of the galeoid fetuses studied, all but those of the smooth dogfish and the bonnethead shark were in the last trimester of gestation, and consequently had well-developed placentas; in addition, the fetuses of a given litter were already separated from each other by uterine membranes which completely enclosed each fetus within a given volume of uterine fluid (Schlernitzauer & Gilbert, 1966). Aliquots of uterine fluid were obtained from individual fetal compartments by transuterine puncture. The fetuses of the smooth dogfish and those of the bonnethead shark were obtained at the end of their first trimester of gestation, still encased in egg membranes ; there was no uterine compartmentalization at this stage of development in either species and placental formation was not yet evident. Young of the spiny dogfish were obtained at two stages of development : (a) in the candle stage, or during the first third of gestation, in which there were two to four embryonated eggs enclosed within a single egg membrane (Hisaw & Albert, 1947), and (b) in the last third of gestation, with the fetuses lying free within the uterine cavity but still possessing well-vascularized yolk sacs (Hisaw & Albert, 1947). Aliquots of uterine fluid bathing the spiny dogfish pups were obtained by transuterine puncture. Antisera to shark immunoglobulins Rabbit antiserum specific for lemon shark immunoglobulins (anti-LIg) and rabbit antiserum specific for nurse shark immunoglobulins (anti-NIg) were generously contributed by Dr. L. William Clem of the University of Florida, Gainesville, Florida; the antisera had been prepared using purified immunoglobulins of the respective shark species as antigens (Clem et al., 1967; Clem & Small, 1967). Rabbit antisera specific for shark immunoglobulins were also prepared as described elsewhere (Gitlin et aZ., 1972): briefly, rabbits were immunized with serum from adult sharks of a given species, and the resulting antisera were then adsorbed with 0.5 vol. of homologous fetal shark serum. Adsorbed antisera were prepared against the immunoglobulins of the lemon shark, or anti-L (Ig), blacktip shark, or anti-Bt (Ig), bull shark, or anti-B (Ig), great hammerhead shark, or anti-GrH (Ig), scalloped hammerhead shark, or anti-ScH (Ig), the smooth dogfish, or anti-Sm (Ig), and the spiny dog&h, or anti-Sq (Ig). Antiserum prepared against the immunoglobulins of one shark species cross-reacted intensely with the immunoglobulins of each of the other shark species studied, and the results obtained with the adsorbed antisera were virtually identical to those obtained with the unadsorbed antisera prepared against purified shark immunoglobulins. In addition to the immunoglobulins, some of the adsorbed antisera precipitated an or-globulin in homologous adult and fetal shark sera but not in heterologous sera; this precipitation was easily distinguished from that of the immunoglobulins (Fig. 1).

IMMUNOGLOBULIN

SYNTHESIS IN FETAL SHARKS

249

Immunochemical methods Immunoelectrophoresis was performed essentially as described by Scheidegger (1955) using O-1 M borate buffer at pH 8.6. Uterine fluids were concentrated approximately twentyfold before immunoelectrophoresis by dialysis against distilled water followed by lyophilization and resolution of the lyophilized proteins in 0.1 M borate buffer at pH 8.6. Immunoglobulin concentrations were determined by radial immunodiffusion (Mancini etaZ., 1965) using serial dilutions of pooled homologous adult shark serum as relative concentration standards. Adult shark serum contains both 19 S and 7 S immunoglobulins (Marchalonis & Edelman, 1965, 1966; Frommel et al., 1971; Clem et al., 1967; Clem & Small, 1967 ; Suran et al., 1967) ; since 19 S molecules diffuse through agar more slowly than 7 S molecules, estimations of immunoglobulin concentrations in a serum which may contain mostly 19 S molecules, as has been found in the neonatal nurse shark (Fidler et aZ., 1969), may be approximately 10 per cent less than the actual concentration when adult shark serum is used as the concentration standard (Fidler et al., 1969). In addition, since shark serum contains from two to four immunochemically distinguishable immunoglobulins (Gitlin et al., 1972), at least two precipitin rings were discernible on radial immunodiffision; the relative concentrations of maternal and fetal immunoglobulins were derived from measurements of the outer precipitin ring only. The limit of sensitivity of this method as used in this study was approximately 0.3 per cent of the immunoglobulin concentration present in the maternal serum. Tissue cultures Tissues were obtained from fetal blacktip sharks and fetal great hammerhead sharks as well as from their mothers. Tissues were also obtained from adult non-gravid bonnethead sharks. All tissues were collected within minutes after the animals were sacrificed. The individual tissues were minced with scissors, and up to 250 mg of tissue were placed in either flasks or roller tubes together with 2-3 ml of tissue culture medium. The medium used for these cultures was Eagle’s basal medium in Hanks’ solution but modified to contain 250 m-equiv. of Na, 240 m-equiv. of Cl, 350 m-mole of urea, 90 m-mole of trimethylamine oxide and 50 ml of heat-decomplemented horse serum per 1. To each culture was then added O-1 ml of culture medium containing from 4 to 7.5 &i of 14C-labeled amino acid, either as uniformly labeled L-leucine or as a mixture of uniformly labeled amino acids obtained from “C-labeled algal hydrolysates. The cultures were kept in the dark at ambient temperatures between 20 and 23°C for l-3 weeks, dialyzed against distilled water for 3 days, lyophilized, then reconstituted to 0.2-0.3 ml with 0.1 M borate buffer at pH 8.6 and centrifuged. An aliquot of either homologous or heterologous adult shark serum was added to an aliquot of culture fluid concentrate to provide carrier serum immunoglobulins, and immunoelectrophoresis of the mixture was performed. The slides were developed for 3-4 days with specific antiserum, then washed in four to five changes of 0.1 M NaCl over a period of 3 days, dried and inverted on Tri-X film (Eastman Kodak Co., Rochester, N.Y.) for 6-10 weeks for radioautography. Egg-case membrane permeability

studies

The permeability of the egg-case membrane of the spiny dogfish to human serum albumin was studied by immersing intact candles, each containing two to four embryos, in 7 1. of sea water to which had been added 30 &i of lr61-labeled human serum albumin. The baths were kept at 10°C. At irregular intervals up to 5 days, during which period the embryos remained alive and active, two candles were removed from the bath, rinsed in two consecutive baths of fresh sea water and carefully wiped with a soft dry towel. Aliquots of clear fluid overlying the embryos were obtained by puncturing the candles with a needle. The embryos from a given candle were removed, pooled and homogenized. Aliquots of the internal egg fluid, aliquots of the labeled bath and the pooled embryos were assayed for 12sIlabeled protein by adding equal volumes of 20% trichloroacetic acid (TCA), washing the

250

DAVIDGITLIN, ANITA F%RRICELLIANDJONATHAND. GITLIN

resulting precipitates twice with at least 10 vol. of 10% TCA and counting the precipitates in a well-type NaI crystal coupled to a 400 channel spectrometer. The permeability of the egg-case membrane of the smooth dogfish to serum albumin was examined by loosely holding the wet membrane across one open end of a plastic tube 2 cm in diameter and securing the membrane to the tube with rubber bands. The end of the tube covered by the membrane was placed 4 cm beneath the surface of a 180~ml bath of sea water which contained 6 &i of 1Z61-labeled human serum albumin. The upper surface of the membrane, or that enclosed within the tube, was covered with a thin layer of sea water. After 29 hr at 13°C all of the fluid within the tube, approximately 1 ml in each case, was removed and assayed for TCA-precipitable la61 together with aliquots of the bath. Spiny dogfish pups obtained during the last trimester of their gestation were placed in 7 1. of sea water containing 30 &i of Y-labeled human serum albumin and kept at 910°C. At irregular intervals up to 5 days, during which period the pups remained quite active, two pups were exsanguinated by caudal vein puncture; their sera and aliquots of the labeled bath were assayed for lzaI precipitable in 10% TCA. RESULTS

Maternal serum immunoglobulins developed relatively intense bands of precipitation on immunoelectrophoresis using either homologous (Fig. 1) or heterologous (Fig. 2) anti-immunoglobulin antisera, and at least two antigenically different immunoglobulins could be distinguished as has been reported earlier (Gitlin et al., 1972). On the other hand, the immunoglobulin precipitation bands that formed with fetal serum were faint and diffuse at best (Figs. 1 and 2). On radial immunodiffusion the concentration of immunoglobulins in third trimester fetal serum was found to be approximately l-5 per cent of that in the mother’s serum (Table 1); the serum concentration of immunoglobulins in the first trimester embryos of the smooth dogfish and the bonnethead shark was below O-3 per cent of that in the maternal serum, the limit of sensitivity of the method. Immunoglobulins could not be detected in the yolk of any of the fetuses. Cultures of spleen and liver from adult bonnethead sharks in media containing i*C-labeled amino acids produced radio active immunoglobulins (Fig. 3A and B) as did cultures of spleen and liver from the maternal blacktip shark (Fig. 3C and D) and from the maternal great hammerhead shark. Traces of radioactive immunoglobulin were detected in some of the cultures of the stomach and in cultures of the anal organ from these sharks, but no radioactive immunoglobulin could be found in cultures of pancreas, kidney, heart, skeletal muscle or blood. Small amounts of radioactive immunoglobulin were found in fetal spleen cultures of the blacktip (Fig. 3E) and great hammerhead sharks, but not in any of the other fetal tissues cultured including the blood, liver, stomach, intestine, pancreas, kidney, heart, skeletal muscle, yolk sac and placenta. Cultures of adult and fetal tissues in media which did not contain radioactive amino acids did not produce any lines on radioautography. In addition, radioactive immunoglobulin was not formed when fetal and adult shark sera were incubated alone with media containing radiolabeled amino acids. Immunoglobulins were not detected in any of the uterine fluids even when the fluids were concentrated twentyfold. Since the lower limit of sensitivity of the

Maternal shark sera were placed in FIG. 1. Immunoelectrophoretic patterns. the antigen wells marked “m” and fetal shark sera in the larger antigen wells marked “f”; the precipitin lines were developed with antisera against the immunoglobulins of the homologous shark species. A. Lemon shark sera developed with anti-L (Ig). B. Great hammerhead sera developed with anti-GrH (Ig). C. Scalloped hammerhead sera developed with anti-ScH (Ig). D. Bull shark sera developed with anti-B (Ig). E. Blacktip shark sera developed with Bt (Ig). Arrows indicate the precipitin bands formed with the immunoglobulins in fetal sera.

FIG. 2. I~nmunoelectrophoresis of maternal shark sera (smaller wells) and fetal shark sera (larger wells). A. Spiny dogfish sera developed with anti-Sq (Ig), arrow indicating immunoglobulins in fetal serum. B. Smooth dogfish sera developed with anti-Sq (Ig). C. Atlantic sharpnose shark sera developed with antiSq (Ig). D. Atlantic sharpnose shark sera developed with anti-L (Ig).

FIG. 3. Negatives of radioautographs; immunoelectrophoresis of shark tissue culture fluids with added unlabeled adult blacktip shark serum as sourl:e of carrier immunoglobulins. A through D developed with anti-Bt (Ig) and E developed with unadsorbed antiserum against adult blacktip shark serum. A. Adult bonnet-head shark spleen culture. B. Adult bonnethead shark liver culture. C. Maternal blacktip shark spleen culture. D. Maternal blackti:: shark liver culture. E. Fetal blacktip shark spleen culture (arrows indicate immunoglobulin precipitin line).

Negaprion brevirostris Carcharhinus limbatus C. leucas Sphyrna tiburo S. mokarran S. bxuini Mustelus canis Squalus acanthias

Carcharhinidae

Triakidae Squalidae

Sphyrnidae

Genus and species

Family

THAT

Lemon shark Blacktip shark Bull shark Bonnethead shark Great hammerhead Scalloped hammerhead Smooth dog&h Spiny dogfish

3.2-3.7 (3) 2.6-2.9 (3)
0*90-1.5-t (2) 26-3.4 (6) 4*0-M (2)
Immunoglobulins in fetal serum*

IN MATERNALSERUM

Common name

RELATIVETO

* Given as a percentage of maternal concentration; maternal concentration = 100 per cent. Numbers in parentheses indicate number of fetal litters: serum from fetuses of a given litter were pooled; number of fetuses per litter varied from as few as four to eight in the spiny dogfish to as many as twenty-eight in the great hammerhead. t Range. 3 First trimester embryos: rest are third trimester fetuses. Lower limit of sensitivity of method = 0.3 per cent of maternal concentration.

SQUALOIDEA

GALEOIDEA

Suborder

TABLE ~-CONCENTRATION OF IMMUNOGLOBULINS INFETALSERUM

m

P 5?

3

E 5! z: s z E: z v)

DAVIDGITLIN, ANITA PERRICJXLLI ANDJONATHAND. GITLIN

252

method was less than O-3 per cent of the maternal serum immunoglobulin level, the uterine fluid concentrations of immunoglobulins, if present, were less than 0.02 per cent of that in maternal serum. Among the galeoid sharks, the concentration of urea in the fluids obtained from the individual uterine compartments was the same as that in the mother. In the spiny dogfish, however, the concentration of urea in the uterine fluid obtained during the candle stage of development was only two-thirds of that in maternal serum (Table 2), and by the third trimester, the urea level of spiny dogfish uterine fluid was less than one-hundredth of the maternal serum level; the maternal serum urea level was the same at both stages of pregnancy. In addition, the protein concentration in uterine fluid from the spiny dog&h fell and the concentrations of Na, Cl and Ca increased dramatically between the first and third trimesters of gestation until the levels of these substances were the same as those in the surrounding sea water (Table 2). TABLE

2--COMPOSITION

OF UTERINE

FLUIDS

OF SPINY DOGFISH COMPARED WITH MATERNAL

AND FRTAL SERA AND SEA WATER

Serum

Protein (g/100 ml) Urea (m-mole/l .) Na (m-equiv/l.) Cl (m-equiv/l.) K (m-equiv/l.) Ca (mg/lOO ml)

Maternal

Fetal

Early

Late

Sea water in area of capture

wvt

(5)

(3) o-25 254 344 378 18 20

(5) O-08 2.5 432 524 9.6 36

(3) 0.04 0.03 445 510 9.5 36

2.7$ 360 250 245 2.7 14

Uterine fluids*

3-o 325 260 220 4.0 12

* Early, first trimester; late, third trimester. t Number of maternal sharks in parentheses; each fetal serum represents serum pooled from entire litter. 1 Averages given; ranges were less than f 5 per cent of the given averages.

When the candles of the spiny dogfish were immersed in sea water containing radioactive human serum albumin, small amounts of labeled protein did seem to pass through the candle membrane. After 1.7 days, the concentration of labeled protein in the clear fluid overlying the embryos was approximately 0.5 per cent of that in the sea water and at 4.6 days, it was as high as 5 per cent. On the other hand, little or no radiolabeled albumin was found within the embryos in these candles. The egg-case membrane of the smooth dogfish also seemed to be slightly permeable to radiolabeled human albumin: when stretched gently over a tube and immersed in sea water for 29 hr with a positive pressure of approximately 4 cm of water being exerted on the outside of the membrane, the concentration of labeled albumin in the fluid found inside the tube was about 1.5 per cent of that in the sea water. When ten third trimester fetuses of the spiny dogfish were kept for

IMMUNOGLOBULIN

SYNTHESIS IN FETAL SHARKS

253

periods of 2-4 days in sea water containing labeled human serum albumin, eight of the fetuses did not have detectable amounts of labeled albumin in their sera, but two had serum levels of 0.5 per cent of that in the sea water bath. DISCUSSION

It is apparent from the data in this report that the near term fetuses of viviparous sharks have relatively low serum concentrations of immunoglobulins: the fetal serum levels found during the third trimester in five viviparous species ranged from 0.9 to 4.8 per cent of the serum immunoglobulin concentrations in their mothers. The serum immunoglobulin concentration in the third trimester fetus of the ovoviviparous spiny dogfish ranged from 1.6 to 1.9 per cent of the maternal level, and Fidler et al. (1969) have reported that the immunoglobulin concentration in the ovoviviparous nurse shark at birth is about 5 per cent of that in the adult. Since the fetal serum immunoglobulin levels relative to those in the mother are similar in both viviparous and ovoviviparous sharks, and since immunoglobulins were not detected in the yolk, there would not seem to be: (a) much, if any, transfer of immunoglobulins in viviparous sharks from mother to fetus across the placenta as occurs in many mammals, or (b) much, if any, transfer of maternal immunoglobulins to the fetus via the yolk as occurs in birds (Ryle, 1957; Brambell, 1958; Mitchison, 1962). The immunoglobulins found in the fetus are at least in part synthesized endogenously, since such synthesis was demonstrable in cultures of spleen from fetal blacktip and fetal great hammerhead sharks. Histologic examination of the placentas of each of the viviparous sharks in the present study revealed, as was emphasized by Gilbert (Schlernitzauer & Gilbert, 1966; Gilbert & Schlernitzauer, 1966), that the egg-case membrane is interposed between the maternal uterine epithelium and the fetal placental epithelium. The egg-case membrane of the viviparous smooth dogfish, as well as that of the spiny dogfish, proved only slightly permeable to labeled human serum albumin. Hence the egg-case membrane layer in the shark placenta could serve as an additional semipermeable barrier to protein transfer. It should also be noted, however, that immunoglobulins were not readily detected in the fluids obtained from individual uterine compartments, indicating that the non-placental uterine epithelium was relatively impermeable to immunoglobulins in the direction of mother to uterine fluid. While the morphology of the uterine compartments and the chemical composition of the uterine fluid in the viviparous sharks suggest that there is little direct contact of the near term fetus with the external environment, this is certainly not the case in the spiny dogfish. Sea water has been shown to be taken into the uterus during late pregnancy in the spiny dogfish (Burger & Loo, 1959) ; in the present study, the chemical composition of the uterine fluid in the third trimester was found to be the same as that of sea water and quite unlike that of either maternal or fetal serum. The composition of the uterine fluids even during the first trimester in the spiny dogfish suggests that sea water intermittently enters the spiny dogfish uterus early in gestation. Thus, the spiny dogfish fetus is directly exposed to

254

DAVIDGITLIN, ANITA PERRICELLI AND JONATHAN D. GITLIN

antigens in the external environment at least during the third trimester of development, and perhaps even indirectly during the candle stage, since the candle membrane is not entirely impermeable to protein molecules. Yet, the spiny dogfish fetus maintains a relatively low level of serum immunoglobulins. In the neonatal nurse shark, serum immunoglobulin levels begin to rise soon after birth. (Fidler et al., 1969). It would seem then that the low level of immunoglobulin synthesis in the spiny dogfish fetus during late gestation might be attributable to either the immaturity of those tissues which synthesize immunoglobulins, or, less likely, interference in utero to antigenic stimulation of the fetus. It may be noted that the injection of antigens into avian embryos does not readily elicit humoral antibody synthesis (Beveridge & Burnet, 1946; Burnet et al., 1950; Buxton, 1954), although cells presumably capable of producing immunoglobulins have been found in the avian embryo as early as 13-14 days of incubation (Cooper et al., 1971). In the sense that antigenic exposure may not necessarily result in enhanced immunoglobulin production, the ovoviviparous shark is similar to the bird. Higher on the evolutionary ladder, the low levels of immunoglobulin synthesis in the normal mammalian fetus are attributed to a lack of antigenic stimulation of the fetus in utero (Silverstein et al., 1970), since stimulation of the fetus in utero can result in brisk antibody responses similar to those seen in adults. Hence, with evolution, the immunoglobulin synthesizing system has apparently developed the capacity for antigenic response during fetal development even if not always called upon to do so. SUMMARY

Using rabbit antisera against shark immunoglobulins, it was found that the serum immunoglobulin concentrations in third trimester shark fetuses of five viviparous species were from 0.9 to 4.8 per cent of those in the mother, and in an ovoviviparous shark, the concentrations ranged from 1.6 to 1.9 per cent of the maternal levels. In two other species of viviparous sharks, the serum immunoglobulin levels in the first trimester young were less than 0.3 per cent of the mothers’ levels. That the third trimester fetus can synthesize immunoglobulins was demonstrated in cultures of fetal spleen in 14C-labeled amino acids; in the adult shark, immunoglobulins are synthesized primarily by spleen and liver and to a lesser extent by the stomach and anal organ. There seems to be little or no maternofetal transfer of immunoglobulins in the shark fetus either through the placenta or via the yolk. The spiny dogfish fetus is bathed in sea water taken into the uterus principally in the third trimester; it is suggested that the low levels of immunoglobulin in this species before birth may be attributable in part to an unresponsiveness of the fetal immunoglobulin-producing cells to antigenic stimuli. Acknowledgements-The authors are deeply indebted to Dr. L. William Clem of the University of Florida, Gainesville, Florida, for his gift of the antisera against purified lemon and nurse shark immunoglobulins, and to Mr. William Fite and the Captains of the Islamorada Charter Boat Association for their help in obtaining many of the sharks used in this study.

IMMUNOGLOBULIN SYNTHESISIN PETALSHARKS

255

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DAVIDGITLIN, ANITA PERRICELLIANDJONATHAND. GITLIN

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shark fetus;

fetal immunoglobulin

syn-