ffects of Oestrogen Treatment Q onstituents Associated with Vitell Eiasmobranch Scyi~ort-dnus c J. C. A. CRAIK’ Department

of Zoology,



of North





Accepted April 17. 1978 Seventeen days after intramuscular (im) implantation of oestradiol pellets into adult female 5cyliorhinus cur&&, plasma phosphoprotein was significantly elevated by 0.40 mg of protein phosphorus/l00 ml above controls, corresponding to an increase in the levei of plasma vitellogenin by a factor of 1.7. Plasma phospholipid was not significantly affected. Oestradiol uptake was < 1 pg/lOO g body weight/day. Plasma oestradiol levels 17 days after implantation were (mean ? SE) 48 * 5 rig/ml, a value which is close to values found in freshly caught fish in midwinter (the seasonal maximum), while in controls they fell to 6 2 3 rig/ml. The im injection of oestradiol benzoate (3 mg/kg body weight) caused elevation of plasma phosphoprotein by 9.5 mg of protein phosphorus/l00 ml after 25 days, corresponding to a 17-fold increase in plasma vitellogenin level above mat found in freshly cat&t fish. Tt also caused significant elevation in plasma phospholipid, total lipid, calcium, and protein. Plasma oestradiol levels after 14 days were 103 2 8 rig/ml, while in controls they were 17 2 14 rig/ml. Male dogfish injected with oestradiol benzoate (3 m&kg) were fmmd to give a response which was qualitatively similar to that of females, but its magnitude was not assessed. The response of this species to administered oestrogen is thus qualitatively similar to that seen in other oviparous vertebrates, suggesting mat elasmobranchs do not differ fundamentally from other vertebrates either in their mechanism of vitellogenesis or in its immediate endocrine control. However, the largest change seen (that of injected female dogfish after 25 days) was considerably smaller than changes which have been reported in some other lower vertebrates after oestrogen treatment.

Evidence from representative species suggests that the yolk precursor, vitellogenin, a complex calcium-binding lipophosphoprotein, is found generally in the blood plasma of vitellogenic females of oviparous vertebrates (Laskowski, 1936; Craik, 1978). The vitehogenin may be detected and/or estimated either directly by immunological methods (e.g., Hosoda et al., 1955; Amirante, 1972; Goedmakers and Verboom, 1974; Redshaw and Follett, 1976; Craik, 1978) or indirectly by measuring the elevation which its presence in large amounts causes in plasma levels of phosphoprotein (Dessauer and Fox, 1959). It is 1 Present address: Department of Oceanography, The University. Southampton, U.K.

very likely that the presence of vite~~og~~i~ incipaJ cause of elevations w described n-r plasma Jeve oodhead, 1968)? total hpid and Woodhead, 1966), ph and Pritchard, 1968), an (?3ooke, 1964). There is muc confined to relatively few species, that the vitehogenin is synthesised and secreted by the hver under the influence of ~es~r~g~~~c

out oviparous vertebrate classes, of the administration of oestrogens. IIn viteflogemc femaies, this treatment raises levels of plasma vitehogenin above those found



naturally or in control animals; this is presumably because such treatment produces plasma oestrogen levels considerably above those of untreated animals. Vitellogenin does not occur in detectable levels in plasma of males, immatures, or nonvitellogemc females, but its appearance may be induced in all these groups by oestrogen administration (e.g., Amirante, 1972; Aida et al., 1973a; Yu and Marquardt, 1973) In males and ovariectomised females, the normal destination of the vitellogenin is absent, and consequently the response tends to be greater and more long-lasting than in intact females (Wallace and Jared, 1968; Pickering, 1976). In both sexes, oestrogen treatment induces increase in size and changes in the fine structure of the liver which are closely associated with the hepatic role in vitellogenin synthesis (Egami, 195.5; Oguro, 1956; Follett et uZ., 1968; Aida et al., 1973b). This vitellogenic response to administered oestrogen has been described in birds (e.g., Vanstone et al., 1957), reptiles (Clark, 1967; Hahn, 1967; Suzuki and Prosser, 1968; Gerstle and Callard, 1972), amphibia (e.g., Follett and Redshaw, 196Q teleosts (Bailey, 1957; Chung-Wai Ho and Vanstone, 1961; Fleming et aZ., 1964; Plack and Pritchard, 1968; Woodhead 1969a; Plack et al., 1971; Amirante, 1972; Aida et al., 1973a; Campbell and Idler, 1976), and lampreys (Pickering, 1976). Experiments on elasmobranchs have provided contradictory results. Urist and Schjeide (1961) failed to produce any demonstrable effect in two shark genera (Triakis and Heterodontus) after treatment with oestrone at a dose which caused a teleost, an amphibian, a reptile, and a bird species to respond. However, Woodhead (1969b) working with the lesser spotted dogfish, Scyliorhinus canicula, found that injected oestradiol benzoate elevated plasma calcium. Since calcium is a component of vitellogenin, he concluded that this elasmobranch resembled other vertebrates in its response to oestrogens.

This paper sets out to resolve this apparent contradiction by examining in greater detail the effects of administered oestrogen on the oviparous elasmobranch, s. canicula. MATERIALS


Dogfish were caught by trawling in Caernarvon Bay. Blood plasma samples were collected, and determinations of plasma levels of protein phosphorus, lipid phosphorus, calcium, total lipid, and total protein were made as previously described (Craik, 1978). The fish were maintained in outdoor tanks (approximate dimensions, 1.3 x 0.7 x 0.3 m) of circulating seawater at ambient temperatures. Since this species does not feed spontaneously in captivity in tanks of this size, the fish were force-fed by the method of Dodd et ul. (1959). Blood samples ‘(1.5 ml per fish at each collection) were always taken at 1500-1700 hr. Immunoelectrophoresis was conducted in 1% agar gel in Verona1 buffer (pH 7). Statistical comparisons were made by Student’s t test, Injection of oestradiol benzoate. Adult females caught within the previous 6 days (early September) were used; body weights ranged from 700 to 900 g. Six were injected im dorsally above the pectoral fin with a solution of oestradiol benzoate in arachis oil (B.D.H.: 5 mg/ml; 0.5 ml per fish). A control group of six fish was similarly injected with arachis oil. The water temperatures during the experiment were within the range lo-12O. The fish were force-fed 7 and 19 days after injection, and blood samples were taken immediately before and 7, 11, 14, 17, and 25 days after injection. Implantation of oestradiol pellets. Adult females caught 2 days previously (early November) were used: body weights ranged from 900 to 1100 g. They were anaesthetised with MS 222 (Sandoz). Six fish were each implanted with three weighed pellets of fused oestradiol (ca. 50 mg per pellet; Organon). Three 5-mm incisions were made in the dorsal musculature of each fish, a pellet was placed in each incision at a depth of 3-5 mm, and each incision was closed with three stitches. In each of six control fish three incisions were made and stitched without implantation. The wounds did not heal during the 22 days of the experiment. Water temperatures were 7-9O, and the fish were force-fed 11 and 16 days after operation. Blood samples were taken at operation and after 17 days, and the pellets were removed after 22 days. The initial and final pellet dry weights were measured to the nearest 20 pg on a Cahn electrobalance. Determination of plasma levels of oestradiol. These were determined by radioimmunoassay, details of which are given eIsewhere (Sumpter and Dodd, in preparation).


The effects of injection of oestradiol benzoate on five plasma components are shown in Fig, I. Leveis of protein phosphorus, hpid phosphorus, total protein, total lipid, and cahAum m treated fish were all elevated above those of control fish 2S days after injection at the levels of significance shown. 0f these five components, phosphoprotein owed the most profound change and, e level of nonvitellogenin protein rus is very low, the course of its may be taken as that which most closely represents the increase m plasma vitellogenm. This increase appears to be sigmoid rather than linear, increasing relatively ?.essduring the first 7 and last 8 days than during the intervening period. The vaiue in mjected fish after 25 days was 9.5 in phosphorus/~00 ml above 8.9 above freshly caught adult is assumed that zero vitellogemn is represented in this species by 0.6Q mg of protein phosphorus/l00 ml and that the value m freshly caught adult females is 1.16 gllO0 ml (Craik, 1978), then 25 days after i~~jection plasma vitellogenin levels were about 17 times greater than in freshly caught adult females; after 17 days the factor was 15~ If it is also assumed that the vitehogenm, hke the yolk granules, contams I. 1% protein phosphorus (CraikY then vitellogenin levels may be calcuFor example, 25 days after injection ormone, the protein phosphorus level j mg/lOO ml corresponds to about f vite~~oge~in/~OO rnl~ In the vehicleinjected controls, plasma phosphoprotein had declined almost to the zero vitellogenin level after 25 days, possibly mdicating that the endogenous hormonal system controihng vitehogenesis had been adversely atfected by the period of captivity. Plasma oestradiol levels 14 days after injection were (means 5 SE): injected fish (n = 4), 103 & 8 rig/ml; controls (H = 3), 17 2 14 nglml .

The effects of oestradioi ~m~~a~ta~~0~~on plasma levels of o§~~Q~rQt~~~ and $-K6phohpid are shown in Table 1 in phosphoprotem levels of i durmg the 17 days was (mea 25 0.093 mg of protein phos which was sign~~ca~t~y gre O.Q76. The difference



trols? the plasma change s~gn~~ca~t~y during the 17 days the same assumptions a,s above are ma lanted fish 17 days afkr ~p.erat,~on had about I.7 times as much vite COIlh-Qb or freddy canght a&&s. of im ~rnp~a~ltat~o~was therefore about :-Gne injecthi, a$ a Vitel~Qge~j~ levels l’? days after treatment and assuming th interval between experiintroduce a seasonal va~iation in sensitivity to h~rmme. In the in-k

ean tiptake of oestradiol body weight per day of =CI pg. than this was possible e limits of sensitivity of the asma oestradiol levels 17

s of imm~~oe~ect plasma of adult females inject tradiol benzoate and fresmy caugh: adult females and males are shown in Fig 2. In six plasma samples the vi~e~~~~e~i~appears as a single arc which has slightly from the origin t~war There is cons more viiehgenin in twi3 fi§h which ktad tradid benzoate tha



FIG. 1. Effects of injection of oestradiol benzoate (3 mgkg) on five plasma constituents in adult female s. cunzkulu. In each of the five graphs the predominantly upper curve represents the treated fish (?I = 6) and the lower curve represents the controls (/t = 6). Means k SE. Significance of difference between treated and control groups 25 days after injection is denoted as follows: * P < 0.05, ** P < 0.01, *** p < 0.001. TABLE EFFECT OF IMPLANTATION PROTEIN PHOSPHORUS



Protein phosphorus (mg/lOO ml)

Implanted fish (n = 6) Controls (n = 6)



canicda Lipid phosphorus (mg/lOO ml)

Day 0

Day 17

Day 0

Day 17

1.08 2 0.08 0.99 k 0.08

1.55 ? 0.12 1.15 k o&x3

4.17 Zk 0.35 3.22 k 0.22

4.33 k 0.46 3.35 2 0.32

FE, 2. Immunoelectrophoresis of plasma of oestrogen-injected adult females and freshly caught adult females and males. The round wells were filled with plasma and subjected to electrophoresis (20 mA for 1 hr: catbode at top, anode at bottom). Then the long troughs were filled with anti-dogfish yolk granule serum (Craik, 1978). Precipitated vitellogenin appears as symmetrical arcs between each well and the adjacent two noughs; it has migrated slightly from the well toward the anode during electropboresis. From the left, first two wells: two adult females injected with oestradiol benzoate (3 mgikg) 14 days previously; next four wells: four adult females caught 8 hr previously; next two wells: two adult males caught 8 hr previously. More vitellogenin is present in the plasma of oestrogen-treated females than in normal females (as judged by the lengths of the arcs). No vitellogenin is detectable in the plasma of the males.

Immunoelectrophoresis of plasma samples from two adult male dogfish which had been injected as described with oestradiol benzoate 10 days previously showed the presence of vitellogenin in concentrations igher than those of normal or control females. No other comparisons or measurements were made with plasma from oestrogen-treated males. It appears, therefore3 that both sexes show a vitellogenic response to oestrogen treatment, as is the case in other oviparous vertebrates. Implantation of oestradiol pellets intraperitoneally (ip) was attempted with female dogfish, but the perforation of the abdominal wall did not heal and unacceptably high mortality (four out of four fish) ensued within 2 ays of operation.

In Table 2 the effects describe workers of oestrogen treat composition of three other lower vertebrates (a lamprey? a teleost, and an ~rn~bi~ wi ths results of the bian) are compared present study of an elasrnQb~a~c1~~ The figures are the mean increases above control values induced in plasma co S by the treatments shown. (P&ho e phosphoprotein levels of ~~~~~~§ were measured by a different method from used for the other species~ it is reason to assume that the differences caused by oestrogen in all four species are direct1 comparable.) The table s thouglr im injection is much more effective than im implantation in dogfish7 im ~~j~c~~~~

The effects of oestrogen treatment on certain plasma constituents associated with vitellogenesis in the elasmobranch Scyliorhinus canicula L.

GENERAL AND CQMPARATIVE 35,455-464 ENDOCRINOLOGY (1978) ffects of Oestrogen Treatment Q onstituents Associated with Vitell Eiasmobranch Scyi~ort...
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