Comp. Biochem. Physiol. Vol. 73B. No. 4, pp. 965 to 970, 1982 0305-0491/82/120965-06503.00/0 Printed in Great Britain. © 1982 Pergamon Press Ltd

EFFECTS OF OESTRADIOL-17fl ON THE SYNTHESIS OF RNA, PROTEINS AND LIPIDS IN THE PYLORIC CAECA

OF THE FEMALE STARFISH ASTERIAS RUBENS

A. J. VAN DER PLAS, A. H. L. KOENDERMAN, G. J. DEIBEL-VAN SCHIJNDEL and P. A. VOOOT

Laboratory of Chemical Animal Physiology, State University of Utrecht, 8 Padualaan, 3508 TB Utrecht, The Netherlands

(Received 19 April 1982)

Abstract---1. The effects of oestradiol-17/~ on processes of synthesis in the pyloric caeca of female Asterias rubens were studied.

2. In vitro treatment with 2.5 × 10 7 M oestradiol-17,8 resulted in significantly higher RNA levels, 3. In vivo treatment with oestradiol-17/~ resulted in higher lipid levels in the pyloric caeca, but RNA

levels, protein levels and the incorporation of 6-[~4C]orotic acid, L-leucine-[~4C] and sodium 1-[~'tC] acetate into RNA, proteins and lipids, respectively were not affected, neither were the indices of the gonads and pyloric caeca.

INTRODUCTION

The size of the pyloric caeca of several asteroids changes in inverse relationship to the size of the gonads. This has been demonstra ted by investigators who determined organ indices throughout the year (Farmanfarmaian etal . , 1958; Giese, 1966; Rao, 1966; Crump, 1971; Jangoux & Vloebergh, 1973; Lawrence, 1973; Ferguson, 1974; Lowe, 1978; Barker, 1979; Oudejans e ta l . , 1979; Van der Plas & Voogt, 1982). During the gonadal rest period the pyloric caeca store reserve nutrients, which are transferred later on to the gonads for use in gametogenesis. In Asterias rubens a peak protein level in the pyloric caeca was followed by a peak protein level in the ovaries (Schoenmakers & Dieleman, 1981). Biochemical studies of Asterias rubens, carried out th roughout the year, have shown that all biochemical consti tuents of the pyloric caeca decrease simultaneously with the pyloric caeca index (Van der Plas & Voogt, 1982) and that all yolk com- ponents in the ovaries increase rectilinearly with the gonad index (Oudejans & Van der Sluis, 1979). These results have led to the hypothesis that vitellogenin- like substances are t ransported from the pyloric caeca to the gonads (Oudejans & Van der Sluis, 1979). The maximum protein level in the pyloric caeca coincided with a peak level of the steroid oestrone in the ovaries (Schoenmakers & Dieleman, 1981). Since the decrease in protein content in the pyloric caeca is too small to account for the increase in protein in the ovaries, Schoenmakers & Dieleman (1981) have supposed that in addi t ion to the release of proteins from the pyloric caeca there must be biosynthesis of proteins in this organ.

When female specimens of Asterias rubens in stage 1 or stage 2 of the reproductive cycle were treated with oestradiol-17/3 there was a decrease in the weight of the pyloric caeca, rapid growth of the ovaries, ac- celerated oogenesis and active vitellogenesis (Schoen- makers et al., 1981).

F rom the foregoing it can be assumed that pro- cesses of synthesis occurring in the pyloric caeca and

subsequently promot ing gametogenesis are affected by oestrogens. Since yolk material consists mainly of proteins and lipids, the effects of oestradiol-17/~ on the synthesis of RNA, proteins and lipids in the pyloric caeca of female Asterias rubens were studied. The results are reported in this paper.

MATERIALS AND METHODS

Animals

Adult specimens of the starfish Asterias rubens were col- lected east of the island of Texel (Wadden Sea, The Nether- lands). The animals were kept in a flow-through sea-water system at 12C and acclimatized for at least three days.

Chemicals

All chemicals were of reagent grade. Sodium 1-1-~'~C]ace - tate (spec. act. 2.0GBq/mmole) and L-leucine-[~'~C]UL (spec. act. 12.62GBq/mmole) were purchased from New England Nuclear Co. (Boston, USA), while 6-p4C]orotic acid (spec. act. 2.2 GBq/mmole) was supplied by the Radio- chemical Centre (Amersham, UK).

Conditions under which the animals were kept

Animals, irrespective of sex, were divided at random into an experimental group and a control group. The animals were kept in two identical aquaria containing 2501. of fil- tered and continuously aerated sea-water at a temperature of 12'C, were exposed to a daily light period of 12 hr and fed ad libitum with sea mussels (Mytilus edulis).

Oestr adiol-17 fl treatment in vitro Pieces ( _+ 500 rag) of the pyloric caeca were incubated for

4hr in various concentrations of oestradiol-17/3 in sea- water.

Oestradiol-17[~ treatment in vivo The animals of the experimental group were injected

intra-coelomically with 10/A per gram fresh weight of a 2.5 x 10-VM oestradiol-17,8 solution in sea-water on day 1 and day 7. The dosage was equally divided over the five arms. The animals of the control group were treated in the same way with sham (sea-water) injections. The oestra- diol-17r~ solution and the sea-water were sterilized with a

965

966 A. J. VAN I)ER PLAS et al.

Falcon filter (B D; Dickenson and Co.). Sterile syringes of l ml were used for the injections. On day 3 and day 8 three or five female starfishes from each group were sacrificed for incubation with radio-isotopes. The indices of the gonads and pyloric caeca were determined for each animal. These indices were defined as the ratio between the fresh weight of the organ concerned and that of the total starfish multi- plied by 100'~,,

Incubation with 6-[14C]-orotic aeid Three pieces ( ± 500 rag) of the pyloric caeca of each ani-

mal were incubated in 2 ml Hanks' solution (Castle et al., 1979) containing 37 kBq 6-[l'~C]-orotic acid. The incuba- tions were carried out in a continuously shaking water- bath at 18C in an air atmosphere. After 2 hr the incuba- tion was terminated by adding 2 ml 20':~, trichloroacetic acid containing 0.04 M sodium pyrophosphate.

Incubation with sodium l-[laC]-acetate Three pieces (_+ 200 rag) of the pyloric caeca of each ani-

mal were incubated in 2 ml Tris -HCI buffer (0.8 M, pH 7.4l containing 168.2kBq sodium 1-[a~C]-acetate. The incuba- tions were carried out in a continuously shaking water- bath at 18C in an air atmosphere. After 4hr the incuba- tion was terminated by adding 5 ml chloroform methanol (1:2: v:v).

Incubation witll ~.-leucine-[ 14C]-UL Two pieces (_+400 rag) of the pyloric caeca of each ani-

mal were incubated in 2 ml of minimal essential medium with Earle salts, containing a final concentration of 20 mM N-2-hydroxyethylpiperazine- N'-2-ethanesulphonic acid (HEPES), 50 Units/ml of penicillin, 50 #g/ml of streptomy- cin and 37 kBq L-leucine-[14C]-UL. The incubations were carried out in a continuously shaking water-bath at 18~C in an air atmosphere for 4 hr.

The incorporation o/" 6-[laC]orotk a~id int¢~ ribonuch:ic odds

All samples were homogenized with a Potter Elvehjem homogenizer after the addition of 2 ml 20"o trichloroacetic acid {TCA} containing 0.04 M sodium pyrophosphate. The homogenate was centrifuged at 15,000g for 10 min at 4'C. The pellet was washed twice with 2 ml 107,, TCA contain- ing 0.02 M sodium pyrophosphate and dissolved overnight in 5 ml 0.3 N NaOH. Perchloric acid was added to a con- centration of 0.5 N and after being allowed to stand for 20min at 0 C , the solution was centrifuged at 15,000g for 10 min at 4 C. The amount of RNA in the supernatant was measured with the orcinol reagent {Almog & Shirey, 1978) using yeast RNA as standard. DNA in the pellet was measured with the diphenylamine reagent (Burton, 1956) with calf thymus DNA as standard. The amount of radio- activity in both the supernatant and the pellet was deter- mined.

The incorporation of sodium l-[~4C]-aeetate into total lipids All samples were homogenized with a Potter Elvehjem

homogenizer after adding chloroform-methanol to the incubation media. The total lipids were extracted accord- ing to the biphasic chloroform-methanol method of Bligh & Dyer (1959).

Thin-layer chromatography (TLC) O#total lipids Thin-layer chromatography was applied to the total

lipids without prior separation of total lipids into neutral and polar lipids. Neutral lipids were separated by TLC (ready-to-use plates, Bakerflex IB 2) using the solvent sys- tem: hexane diethylether acetic acid {90: 10:1, v/v/v. Bj6rkman et al., 19721. For identification spots were visua- lized after spraying the plates with a solution of y3;, cupric acetate in 8°~i aqueous phosphoric acid (Fewster et al., 1969) and charring at 180'C for 20 min. Polar lipids were

separated by TLC using the solvent system: methyl acet- ate-n propanol chloroform methanol 0.05')~i potassium chloride 125:25:25:10:9, v:'v:wv'v, Vitiello & Zanetta, 1978).

The incorporation ol" L-leucine-[ ~'~C]- U L into proteins The incubation media containing pyloric caeca tissue

were centrifuged at 2000 ,q for 5 min at 4 C. The tissue was washed twice with 1 ml 25 mM Tris HCI buffer (pH 7.0) containing 100raM NH4HCO3 and 5raM NaHSO~. It was homogenized with a Potter Elvehjem homogenizer in 2 ml of the same buffer containing in addition 0.1% 2-mer- captoethanol and 150/tg/ml phenylfluoride. After aliquots had been taken to measure protein content (Coomassie brilliant blue G250 method: Bradford. 1976) and the amount of radioactivity. 7.5 N perchloric acid I PCA) was added to a tinal concentration of 0.5 N. After 30 min at 0 C the homogenate was centrifuged at 15.000.q for 10 min at 0' C. The sediment was washed twice with 2 ml 0.5 N PCA. The radioactivity and the amount of free amino acids in the supernatants were measured (Moore & Stein, 1948). The sediment was resuspended in 1 ml Tris HCI buffer (pH 7.0) and after the addition of 2 ml 5'!, TCA, was heated at 100'C for 10 min and filtered through Whatman M3 paper. The filter was washed successively with 2 ml cold TCA {5!!~,). 2 ml diethylether:ethanol (1:1 : vw) and 1 ml diethyl- ether. The amounts of free amino acids in the filtrate and radioactivity in both the filter and the filtrate were deter- mined.

Measurement of' radioactivity All samples were dissolved in Packard Emulsifier (Scin-

tillator 299 TMI. Radioactivity was measured with a Pack- ard Liquid Scintillation Counter equipped with an external standard channel ratio for quench correction.

Statistics The data were statistically analysed by the Student's

t-test or by nested analysis of variance.

RESULTS

OestradioI-17# treatment in vitro

The effect of in vitro t r ea tmen t wi th oestradiol-17# on processes of synthesis was s tudied only for RNA. For each exper iment the pyloric caeca of one animal was used. The results o f exper iments on 22 Sep- tember , 6 O c t o b e r and 20 Oc t obe r 1980 are presented in Table 1. In none of these exper iments was the in- co rpo ra t ion of 6[14C]-orot ic acid into R N A signifi- cant ly affected by t rea tment with oestradiol-17fl. However , the fresh weight level of RNA was signifi- cantly increased by t rea tment with 2.5 × 10 7 M oes- t radiol-17# on 6 and 20 October , but the cellular level of R N A was increased only on 6 Oc t obe r 1980.

Oestradiol-17# treatment in vivo

The mean values and s t andard deviat ions of the organ indices of the exper imenta l and cont ro l g roup in September , N o v e m b e r and December 1980 are shown in Table 2. The indices of the exper imenta l g roups never differed significantly from the cont ro l groups.

The oestradiol-17fi t rea tment affected nei ther the R N A con ten t of the pyloric caeca nor the incorpor- a t ion of 6 - [ ~ C ] - o r o t i c acid into R N A (Table 3).

The results of the exper iments on lipid synthesis with and wi thout oes t radiol -17# t rea tment are presented in Table 4 (September, N o v e m b e r and

Effects of oestradiol on synthesis in A. rubens

Table 1. Effect of 4hr oestradiol-17fl treatment in vitro on the incorporation of 6114C]orotic acid into RNA in pyloric caeca tissue

Concentration mg oestradiol-17fl mg RNA/g pc* RNA/mg DNA dpm/mg RNA dpm/g pc

Date: 22 September 1980; Animal: GI t 1.2; PCI:~ 20.0: n = 2 0 2.72 + 0.11 2.34 + 0.09 2312 + 385 6300 + 1292

2.5 x 10 9 M 2.56 ± 0.40 2.74 + 0.18 4090 ± 2325 9981 + 429 2.5 x 10 8 M 2.61 ± 0.30 2.35 + 0.18 3905 ± 557 10,110 _ 293 2.5 × 10 7 M 4.74 ± 0.73 2.32 ± 0.24 1406 ± 480 6501 + 1250

Date: 6 October 1980; Animal: GI 0.6; PCI 22.l; n = 4 0 4.07 ± 1.16 6.44 ± 2.10 5935 ± 864 23,876 __+ 5927

2.5 x 10 9M 4.39 ± 1.17 6.55 ± 1.82 4388 + 613 19,372 + 6336 2.5 x 10 8M 4.19 ± 0.64 7.15 ± 1.49 4985 ± 1300 21,381 __+ 8717 2.5 x 10 ~M 7.88§± 1.11 14.40§± 2.29 4702±2277 36,876 ± 17,862

Date: 20 October 1980; Animal: PCI 16.0; n = 6 0 7.92 ± 1.41 26.87 ±_ 9.54 3043 ± 1018 25,092 ± 8894

2.5 x 10 9 M 7.36 ± 1.18 20.78 ± 5.46 2648 ± 1241 19,531 ± 9553 2.5 x 10-SM 8.58 ± 2.76 19.93 ± 5.06 2600 ± 309 21,717 ± 4356 2.5 x 10 v M 9.56§ ± 1.36 23.75 ± 3.70 2718 ± 1452 26,999 __+ 17,628

* pc, pyloric caeca: t GI, gonad index; + PCI, pyloric caeca index. § The difference from the control value is statistically significant (P < 0.05). Values are means ± SD.

967

December). The incorporat ion of sodium l-[14C]-ace- tate into lipids was not significantly affected by oes- tradiol-17,8 treatment, whereas the lipid content of the pyloric caeca was increased. In the November and December experiment the increase in lipid content was significant.

The propor t ional dis tr ibut ion of the radioactivity over the lipid classes was not affected by oestra- diol-17fl t reatment, neither was the total radioactivity in each lipid class (Table 5).

Neither the protein levels of the pyloric caeca nor the incorporat ion of e-leucine-[14C]-UL into proteins were affected by t reatment with oestradiol-17/~ during September or November 1980 (Table 6).

DISCUSSION

In vitro t reatment of pyloric caeca tissue with oes- tradiol-17/~ before incubat ion with 6-[14C]-orotic acid resulted in higher levels of RNA (Table 1). In the October experiments these levels differed significantly from those in the control tissue. The RNA was also increased at cellular level. Only the highest concen- t ra t ion ofoestradiol-17fl used, 2.5 x 10 v M, had this effect on RNA levels, whereas the incorporat ion of orotic acid into RNA was not affected.

Treatment with oestradiol-17/~ in vivo did not affect the levels of RNA and proteins or the incorporat ion of orotic acid, acetate and leucine into RNA, lipids and proteins, respectively. The organ indices of the animals treated with oestradiol-17/~ were not signifi- cantly different from the organ indices of the animals treated with sea-water. Only the lipid content of the pyloric caeca was significantly increased after in vivo t reatment with oestradiol-17[/ in November and December.

Schoenmakers et al. (1981) have shown that daily injection with oestradiol-17/? for a period of 16 days

yielded a higher matura t ion index of the treated ani- mals than of the control animals. In our experiments the starfishes were injected once a week with a higher dosage, to prevent mortal i ty due to daily treatment. Since Schoenmakers et al. (1981) did observe higher gonad and lower pyloric caeca indices after 16 days of oestradiol-17,6 treatment, substances must have been t ransported during these 16 days from the pyloric caeca to the ovaries. Biochemical studies of the ovaries and pyloric caeca of Aster±as rubens led to the hypothesis that a vitellogenin-like substance is trans- ported from the pyloric caeca to the ovaries (Oudejans & Van der Sluis, 1979; Oudejans et al.,

Table 2. Indices of gonads (GI) and pyloric caeca (PCI) of animals from experimental and control groups

G1 PCI

Date: 25 September 1980; 8 days after the first and 1 day after the second injection with oestradiol-17/~; n = 3. Control group 1.63 + 1.15 19.81 -t- 3.53 Experimental group 1.99 + 1.13 15.78 ± 3.26

Date: 14 November 1980; 2 days after the injection with oestradiol-17fl; n = 5. Control group 2.52 + 0.34 17.21 + 1.16 Experimental group 3.32 + 1.55 21.39 + 3.69

Date: 20 November 1980; 8 days after the first and 1 day after the second injection with oestradiol-17/~; n = 5. Control group 3.39 + 2.41 21.06 __+ 3.56 Experimental group 3.80 + 1.05 20.66 __+ 4.24

Date: 3 December 1980; 8 days after the first and 1 day after the second injection with oestradiol-17fl; n = 3. Control group 2.53 + 0.24 18.82 + 2.00 Experimental group 3.58 ___ 0.72 19.70 + 3.80

Values are means ± SD.

968 A. J. VAN DER PLAS eta[.

Table 3. Effect of oestradiol-17]$ treatment in vivo on the incorporation of 6-[~'*C]-orotic acid into RNA in pyloric caeca tissue

mg RNA/g pc* mg RNA:mg DNA dpm/mg RNA dpm,,g pc

Date: 25 September 1980:8 days after the first and I day after the second injection with oestradiol-17/:t; n = 3 Control group 3.25 i 0.78 1.11 ± 0.36 3622 ± 1908 11,953 ± 6314 Experimental group 3.43 + 1.00 1.55 ± 0.62 6267 + 3092 19,701 ± 9799

Date: 14 November 1980:2 days after the injection with oestradiol-17/:/: n - 5 Control group 7.73 ± 1.45 6.21 ± 1.49 1494 ± 347 11,188 ± 2485 Experimental group 7.84 ± 2.27 7.63 ± 1.62 1065 ± 343 9,013 ± 5806 Date: 20 November 1980:8 days after the first and l day after the second injection with oestradiol-17fl: n = 5. Control group 7.26 ± 0.39 6.31 ± 0.80 972 ± 323 6,999 ± 2467 Experimental group 7.26 i 1.16 5.62 ± 0.77 842 ± 299 6.097 ± 2199

* pc: pyloric caeca. Values are means + SD.

1979t. If more yolk material is t ransported to the ovaries during oestradiol-17/3 treatment, this may be at t r ibuted either to the increased synthesis of a vitel- logenin-like substance in the pyloric caeca, followed by increased t ranspor t to the ovaries or to increased t ranspor t of a vitellogenin-like substance that has been synthesized earlier in the reproductive cycle.

It is generally accepted that in oviparous verte- brates the synthesis of vitellogenin, which is a glycoli- poprotein, in the liver is regulated by oestradiol-17,B (Clemens, 1974; Tata, 1976; Tata & Smith, 1979; Knowland, 1980). If the synthesis of the vitellogeniv~- like substance in Aster±as rubens is also regulated by oestradiol-17/3, it will be synthesized during the period when oestradiol-17/3 is administered and not earlier in the reproductive cycle. However, oestra- dio1-17/3 did not stimulate the incorporat ion of pre- cursors of RNA, protein and lipid in Aster±as rubens. This may be because the animals were insensitive to oestradiol-17/3, or because the t reatment was too short or the dosage too low.

Our in vivo experiments were performed in Sep- tember and in November, whereas Schoenmakers et

al. (198t) carried out their experiment in September. If it is only the gametogenesis of animals in stage 1 or stage 2 of the reproductive cycle which can be stimu- lated with oestradiol-17/3, experiments carried out in November may be too late in the reproductive cycle. The total dosage used in our experiments is the same as the total dosage used by Schoenmakers et al. (1981). In vitro t reatment with oestradiol-17/3, 2.5 x 10 ; M, caused an increase in RNA levels. To obtain the same concentrat ion of oestradiol-17/3 in the coelomic fluid in vivo a dosis of 10pl per gram fresh weight of a 62.5 x 10 _7 M oestradiol-17;q sol- ution has to be used (+_ 17 ng/g fresh weightl. This dosage would still be very low compared with vitello- genin inductive doses of oestradiol-17/3 in for example Salmo ,qairdneri (Van Bohemen, 1981). Schoenmakers et al. (1981) have shown that oestradiol-17/3 affects gametogenesis in Aster±as rubens. Fur ther studies are needed to prove that oestradiol-17/3 also stimulates processes of synthesis in the pyloric caeca. In these studies it would be better to use animals in stage 1 of the reproductive cycle and higher doses of oestra- diol- 17/3.

Table 4. Effect of oestradiol-17/3: treatment in vivo on the incorporation ot sodium 1-[14C]acetate into lipids in pyloric caeca tissue

mg lipid/g pc* dpm/mg lipid 103 dpm/g pc

Date: 25 September 1980; 8 days after the first and 1 day after the second injection with oestradiol-17/~; n - 3. Control group 87.9 ± 3.7 31,469 ± 12,484 2769 ± 1095 Experimental group 106.2 + 18.9 36,435 ± 18,403 3653 ± 1742

Date: 14 November 1980; 2 days after the injection with oestradiol-17/£ t~ - - 5 .

Control group 75.9 ± 6.4 38,460 + 39,278 2674 ± 2515 Experimental group 84.9+ ± 6.3 33.075 ± 10,115 2683 ± 745

Date: 3 December 1980:8 days after the first and 1 day after the second injection with oestradiol-17/~; n - 3. Control group 88.2 + 13.0 1748 ± 1189 143 ± 77 Experimental group 106.3+ + _+ 1.0 2376 ± 710 250 ± 76

* pc, pyloric caeca: ?P < 0.05; ++P < 0.01. Values are means _+ SD.

Effects of oestradiol on synthesis in A. rubens

Table 5. Proportional distribution of the radioactivity over the lipid classes and the total radioactivity in each lipid class from animals from 25 September (Table 4)

969

Proportional Distribution Radioactivity/g pyloric caeca Control group Experimental group Control group Experimental group

Neutral lipids TLC PL + MG 39.5 + 5.1 49.9 ± 6.2 DG 7.2 ± 0.6 8.6 ± 2.4 ST 4.6 + 0.8 5.4 ± 2.4 FAlc 4.6 ± 1.3 5.2 ± 1.6 FFA 1.0 + 0.0 1.I ± 0.4 TG 24.4 _+ 4.2 17.6 ± 2.7 ELI 2.2 ± 0.5 1.2 ± 0.2 EL2 6.9 ± 0.9 4.8 ± 2.8 EL3 8.0 _+ 2.8 5.3 ± 3.2 EL4-5 1.0 ± 0.2 0.6 ± 0.1 EL6 0.1 ± 0.1 0.1 ___ 0.1 SE 0.2 _+ 0.0 0.2 ± 0.1 HC 0.4 _+ 0.2 0.2 ± 0.1

Polar lipids TLC Start 1.7 ± 1.6 1.6 ± 0.7 Lyso + SPH 7.7 ± 3.1 9.5 ± 1.6 PC 11.8 ± 4.2 9.4 ± 2.0 LysoPE 3.5 ± 2.0 3.5 ± 1.6 PS 2.0 _+ 0.6 2.6 ± 0.3 PI + PA 7.9 ± 3.2 4.5 ± 0.3 PE 1.1 ± 0.7 1.6 ± 0.8 SULPH 3.1 ± 1.0 5.9 ± 1.2 CER 1.0 ± 0.6 1.0 ± 0.2 9 1.6 ± 0.2 1.5 ___ 0.3 MG 9.2 + 2.2 3.4 ± 0.8 NL 49.1 + 3.8 55.4 ± 5.0

140,937 ± 52,981 204,974 ± 81,755 25,408 ± 8883 37,361 ± 19,710 16,125 ± 5696 23,535 ± 14,034 16,080 ± 7278 20,617 ± 8729

3588 ± 1357 5058 ± 3540 87,978 ± 39,728 78,880± 48,182

7877 ± 4015 5059 ± 2945 24,432 ± 8782 21,996 ± 21,989 30,754 ± 19,174 25,480± 26,666

3593 ± 1281 2708 ± 1564 510 ± 359 356 ± 245 718 ± 272 642 ± 214

1461 ± 1165 499 ± 247

4711 ± 3310 6344 ± 2845 26,313 ± 12,999 41,823 ± 26,104 45,357± 28,622 38,070± 14,896 12,(152 ± 9316 12,628 ± 1354

7257 ± 3749 11,545 ± 6602 31,276 ± 19,996 19,527 ± 10,852

3877 ± 3222 5853 ± 1245 11,403 ± 6283 23,535 ± 7541

4148 ± 3183 3923 ± 1574 5653 ± 1570 6484 ± 3893

32,256 ± 13,195 14,120 ± 6490 174,050± 61,827 243,383 ± 133,885

PL: polar lipids; MG: monoacyl glycerols; DG: diacyl glycerols; ST: free sterols; FAIc: fatty alco- hols; FFA: free fatty acids; TG: triacyl glycerols; ELl to EL6: ether lipid classes; SE: steryl esters; HC: hydrocarbons.

SPH : sphingomyelin; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PS : phosphatidylser- ine; PI: phosphatidylinositol; PA: phosphatidic acid; lysoPE: lysophosphatidylethanolamine; SULPH: sulphatides; CER: cerebrosides; M G: monoacylglycerols; NL: neutral lipids.

Table 6. Effect of oestradiol-17/3 treatment in vivo on the incorporation of L-leucine-[~4C]-UL into proteins in pyloric caeca tissue

mg protein/g pc* mg FAa¢/g pc dpm/mg protein dpm/g pc

Date: 25 September 1980; 8 days after the first and 1 day after the second injection with oestradiol-17fl; n = 3. Control group 86.2 ± 11.7 23.2 _+ 3.7 13.4 _+ 2.5 1148 _+ 247 Experimental group 66.8 ± 13.2 25.9 _+ 0.9 20.6 _+ 7.6 1355 _+ 478 Date: 14 November 1980; 2 days after the injection with oestradiol-17fl; n = 5. Control group 31.4 ± 6.5 21.3 _+ 2.4 28.7 _+ 8.5 1487 ± 1256 Experimental group 35.4 ± 4.6 25.6 + 2.8 26.3 _+ 9.8 947 ± 475

Date: 20 November 1980; 8 days after the first and I day after the second injection with oestradiol-17fl; n = 5. Control group 56.5 + 6.8 17.5 ± 1.5 10.9 ± 4.3 600 _+ 250 Experimental group 51.8 + 6.2 19.2 _+ 1.4 7.7 ± 3.6 408 _+ 231

* pc, pyloric caeca; t FAA, free amino acids.

Acknowledyements--The authors wish to thank Mr J. M. de Ree for his technical assistance during preliminary ex- periments and Miss A. M. E. R. de Lange for typing the manuscript.

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