An in vitro bioassay for a molluscan gonadotropin

Octopus vulgaris Cuvier can be caused to mature precociously by cutting the nerve supply to one or both of its optic glands; this causes the glands to secrete a gonadotropin that is normally released only in small quantities before the onset of sexual maturity. The operation is done most conveniently by removal of an area including the subpedunculate lobe from the back of the supra-oesophageal part of the brain. Within about two weeks (at 25 °C), females develop enlarged ovaries and the eggs begin to accumulate yolk rapidly. Ovary weight increases until it comprises 10–20% of the total body weight. The octopuses will then lay eggs, which may be fertile and which they brood in a normal manner (Wells & Wells, 1959).

If [¹⁴C]leucine is injected into the bloodstream of maturing females, it is rapidly taken up by the ovary; within 5 or 6 h nearly 40% of the counts injected have accumulated in protein there. This process ceases within 5 days if the optic glands are removed. Cephalopods are unlike arthropods and vertebrates in that most and perhaps all of the yolk protein is synthesized within the ovary itself (by the follicle cells as we shall show below) rather than being assembled elsewhere and transported to the eggs via the bloodstream (O‘Dor & Wells, 1973). This means that the system should be peculiarly suitable for an in vitro study of the hormonal control of yolk synthesis in addition to its use as an assay for cephalopod, and possibly other molluscan gonadotropins. The paper that follows shows that in vitro synthesis does indeed occur in excised octopus eggs and that it can be controlled with extracts taken from the optic glands of octopuses of either sex. The rate of incorporation of [¹⁴C]leucine in vitro is a function both of the rate of amino acid uptake and of protein synthesis. The relationship between uptake and incorporation is examined, and the merits of both parameters as measures of hormonal activity are discussed.

Octopus vulgaris Cuvier from the Bay of Naples were used. After collection the animals were kept in individual tanks with circulating sea water and fed upon crabs and anchovies. Approximately 10 g of food per day was given to animals weighing 200–300 g, rather more to octopuses heavier than 300 g. This is less than the animals will eat at 25 °C if fed ad lib. (Nixon, 1966), but sufficient to ensure a modest weight gain in controls.

After a few days, animals that were feeding regularly were anaesthetized in 3% urethane in sea water, and two operations were carried out. In the first of these the central supra-oesophageal part of the brain was exposed and a vertical transverse cut made about half way along the vertical lobe. This cut extended downwards through the vertical and subvertical lobes into the dorsal part of the posterior basal lobes (for the anatomy of the brain of Octopus see Young, 1971). A horizontal cut from behind completed the operation and allowed a region of tissue to be removed, including the posterior half of the vertical lobe, much of the dorsal basal and the whole of the subpedunculate lobe on each side of the brain. This last lobe is the source of nerves that run to the optic glands and apparently control their secretion (Wells & Wells, 1959). Control animals had the brain exposed but no tissue removed. At the same time as the removal of tissue from the brain, a second operation was performed on the mantle cavity of all the octopuses. The vertical pair of muscles connecting the roof and floor of the mantle cavity at its anterior end was cut. This allows the mantle of anaesthetized animals to be turned inside out, so that the condition of the gonads can be examined and biopsies and injections performed. There were no deaths as a result of these operations.

The condition of the ovary was examined after about 10 days. By 8–10 days after excision of the subpedunculate lobes the ovary, oviducts and oviducal glands were generally considerably enlarged. The ovary was typically white and opaque. At a slightly more advanced stage (10–12 days) the ovary became yellowish, an appearance that corresponded with the development of extensive infoldings by the follicle cells surrounding the ova. By now the eggs were accumulating yolk very rapidly, and growth was very fast. At about 14 days (depending on the state of each individual ovary) we carried out a further operation on the animals. At this operation some of the animals had their optic glands removed (OG⁻ animals). Others had the optic glands examined but left in (OG⁺ animals). The optic glands of the animals with brain lesions were always enlarged and bright yellow or orange in colour; controls had minute pale yellow or transparent glands.

For the m vitro experiments the animals were killed and their eggs taken from 1 to 3 (usually 2) days after removal or examination of their optic glands. Each ‘egg’ at this stage consisted of an ovum surrounded by an epithelium of several hundred follicle cells. A typical sample of eggs as used for the in vitro experiments would consist of 50–100 such complexes, each 1–2 mm long, together with numerous smaller eggs in earlier stages of development. Some 90 % of the total volume, and nearly all of the yolk, would be found in the most advanced eggs.

For in vivo experiments, OG⁺ and OG⁻ animals were injected with [¹⁴C] leucine (for details of radiometric techniques see below), placed in the bloodstream through the left branchial heart; if urethane anaesthesia is carried out appropriately, animals with their mantle musculature relaxed still have the hearts beating, so that the material is at once circulated through the bloodstream.

Details of dosages, and precautions taken to check on possible leakage in the in vivo experiments, are given below.

In vitro experiments. For these, eggs were removed from the animals and incubated in sea water or in a medium made up as follows: 0·2 g NaHCO₃, 27 ml distilled water, 73 ml seawater; with 0·96 g Powder Medium 199, 2 ml penicillin/strepto-mycin at 5000 units/ml and 1 ml 1·0 M HEPES buffer (all from Biocult Laboratories, Glasgow). This gives a solution of pH 7·4.

Uniformly labelled [¹⁴C] leucine (50 μCi/ml, 342 μCi/mMol, from the Radiochemical Centre, Amersham) was added at the rate of 0·25 ml to 100 ml of medium unless otherwise specified.

As a standard procedure approximately 100 mg samples of eggs were separated from the ovary, blotted dry and placed in 1·0 ml of sea water in weighed tubes. In some cases the sea water included optic gland extracts. Reweighing established the weight of each sample. Typically the samples were then pre-incubated for 3 h at 25 °C, before the addition of [¹⁴C] leucine in 1·0 ml of the nutritive medium. Throughout incubation the samples, usually six replicates for each treatment, were kept in sloping test tubes on a moving platform shaker. After a further 3 h incubation 100 μl samples of the medium were taken, added to 0·4 ml of distilled water in vials and counted in 5 ml of Instagel scintillation fluid (Packard Instrument Co.). Uptake is calculated as the difference between the initial counts per minute (cpm) in the medium and the cpm remaining after incubation. In the kinetics experiments suitable corrections for quenching were made to convert cpm to disintegrations per minute (dpm) to permit calculation of molar ratios.

To determine incorporation into protein, eggs were rinsed in sea water and then homogenized in 0·5 ml of distilled water. Protein was precipitated with 1·0ml of 0·1 M-Ieucine in 10% (w/v) aqueous trichloracetic acid (TCA). The precipitates were treated in the tubes with solutions as described by Mans & Novelli (1961) to remove non-protein counts. The ether-washed powder which remains at the end of this procedure was resuspended in 1·0 ml of distilled water and 10 ml of Instagel was added. The resulting clear suspension was transferred to vials for counting in a Packard Tri-Carb scintillation spectrophotometer.

For these [¹⁴C]leucine as above was diluted 1:3 with sea water and 0·2 ml of this solution injected. The animals were revived by passing sea water over the gills, and a sample of this water taken to check possible losses through leakage. In earlier experiments (O‘Dor & Wells, 1973) the animals were then kept in a closed circulation and a further check made by sampling this after 5 h. This later check never showed any significant escape of counts from the animals, and the precaution was abandoned in the present series.

Five h after injection, animals used for the in vivo experiments were sacrificed. They were anaesthetized in urethane, bled for about 10 min through a cannula inserted into the dorsal aorta, and the eggs removed. Duplicate weighed samples of eggs (approximately 200 mg each) were treated in the manner previously described to determine incorporation into protein.

The purpose of this was to discover whether eggs for in vitro studies were best used ‘fed’ by pre-incubation in a rich culture medium, or ‘starved’ by pre-incubation in sea water.

The procedure described above was used to prepare four sets of six samples of eggs in sea water. Two of the sets had 1·0 ml of nutritive medium added to each sample. One of these and one of the sea-water-only sets had an extract of optic glands added. The extract was prepared by grinding up the optic glands from the egg donor, from a similar female of 270 g, and from an 820 g male which had had the subpedunculate lobe removed 24 days earlier. The 4 sets were then incubated on the shaker for 3 h.

At the end of this period 1·0 ml of labelled medium was added to each of the sea water sets. Those already in unlabelled medium had a quantity of [¹⁴C] leucine in sea water added to bring them up to the same level of radioactivity.

After a further 3 h incubation, uptake and synthesis rates were determined as described above.

The results are shown in Fig. 1. When pre-incubation was in medium, added extract caused a large increase in uptake and incorporation; but the amino acid incorporated into protein was only a small percentage of the total uptake. When eggs were pre-incubated in sea water, the increase in uptake and synthesis with extract added was disappointingly small, but a much larger proportion of the uptake was made into protein. Both results show a definite effect of the extract on protein synthesis, though control levels were quite high. Comparison of the two indicates that the hormone may have an effect on uptake that is independent of synthesis. This question is examined in detail in a later section.

In the preliminary experiment the rather small differences in uptake and synthesis brought about by addition of hormone extract could have been due to persistence of hormone from the egg donor, since her optic glands were intact and active when the eggs were removed immediately before pre-incubation. The next section describes studies carried out to determine how long the effects of optic gland hormone continue in vivo after removal of the glands. They provide a basis for lowering control levels and improving the sensitivity of the eggs to hormone in the in vitro experiments described subsequently.

At the beginning of the present series of experiments it was known that octopuses would not synthesize significant quantities of labelled protein from injected [¹⁴C]-leucine if their optic glands had been removed 5 days previously (O‘Dor & Wells, 1973). The preliminary experiment shown in Fig. 1 suggests that effects of optic gland hormone were still present 3–6 h after removal of the eggs from an animal with its glands active until the operation. To examine further the persistence of hormonal effects, a series of precociously maturing animals was prepared in the usual manner. The active optic glands from these were removed and from 1 to 5 days later the animals were injected with 0·2 ml of [¹⁴C] leucine in seawater (see Methods, above). Five h after injection the octopuses were killed. Fig. 2 shows the relative incorporation of [¹⁴C] leucine into protein in their eggs. Protein synthesis in the ovary is clearly related to the time elapsed since optic gland removal. As found previously, there is almost no synthesis after 5 days; but there is considerable activity (incorporation at about 50% of control values) after 24 h. This persistence could account for the rather small differences between controls and experimentáis found when optic gland hormone was added to eggs taken from octopuses with ripe glands in the tests summarized in Fig. 1.

Two days after optic gland removal, protein synthesis has declined to about 30% of its original level, and it was decided to use animals deprived of their optic glands 2 days previously as the source of eggs in all the subsequent experiments of the present series. Greater differences between controls and experimentáis might have been expected from the use of 5-day OG^- octopuses. But it was felt unwise to use such ‘old’ eggs in the absence of detailed histological information; eggs in animals deprived of their optic glands break down eventually (in a matter of 2–3 weeks), and we are not yet in a position to estimate the point at which any changes become irreversible.

The amount of [¹⁴C] leucine incorporated depends on the synthesis rate and on the ratio of labelled to unlabelled leucine at the site of synthesis. This internal ratio is influenced by the free leucine present in the eggs, as well as the ratio of labelled to unlabelled leucine in the medium. The cold leucine in the eggs will decrease the internal ratio by diluting the label as it is taken up, but if the amino acid moves in both directions across cell membranes internal leucine will also increase uptake of label by exchange. If the cells lose leucine the internal concentration would decrease in sea-water pre-incubation, decreasing dilution and uptake during incubation but increasing the proportion of label in any protein that is made. Pre-incubation in a rich medium would have the opposite effects. We know from the preliminary experiment (Fig. 1) that the amount of label taken up is considerably in excess of the small amount incorporated into protein.

Fig. 3 shows the disappearance of label into eight egg samples of about too mg each (for this experiment eggs were weighed into tubes directly from the ovary of an OG⁺ animal and labelled medium added immediately at 1·00 ml per 100 mg). The solid curve is calculated from an equation (see caption to Fig. 3) developed by Sheppard & Beyl (1951) to describe the disappearance of ²⁴Na into irradiated red blood cells; they found rapid exchange superimposed on a steady net uptake. The Sheppard & Beyl formula describes our results so well that we can assume that we are dealing with a similar system and can calculate S₂₍₀₎ (the free leucine in the eggs at the start of the experiment) by extrapolation (see Fig. 3). The free leucine in the eggs calculated on this basis is 0·04 μmol/100 mg. This value is near the lower end of the range of nonprotein leucine (0·024–0·290μmol/100 mg) reported in O. vulgaris muscle by Florkin (1966), and appears reasonable for a tissue actively synthesizing protein. The net inward flow (▵) and exchange rate (ρ) for this experiment were 0·009 and 0·093μmol/h/100 mg, respectively.

In Fig. 4 a similar analysis is used to compare net inward flow and exchange, separately, in control and extract-stimulated eggs from a 2-day OG^- animal. Two samples of approximately 700 mg were pre-incubated for 3 h in 6μ0 ml of sea water or sea water with homogenized optic glands prior to the addition of 6μ0 ml of ¹⁴C medium. Uptake from the medium was followed during the next 3 h and samples of about 300 mg were taken from the eggs for protein determination at the end of this period. The remaining eggs in each sample were blotted dry and transferred to unlabelled medium diluted 1:1 with sea water or sea water plus optic gland extract. The release of label into this fresh medium was followed, and after 3 h protein determinations were made. For convenience the results shown in Fig. 4 are expressed as dpm/mg sample on the assumption that all label not present in the medium or protein is free leucine inside the eggs. This is not the graphical form used for calculation but allows all of the data to be displayed in a single figure. The solid lines are reconstructions of curves of the type shown in Fig. 3 based on analysis of the system as two compartments under non-steady-state conditions as described above.

The curves during the uptake period are essentially linear, with no evidence of a rapid exchange component, implying that the exchangeable leucine after pre-incubation in sea water is negligible. This means that the specific activity throughout the system is constant during this period, and that the cold leucine taken up and incorporated is proportional to the labelled leucine. It also allows calculation of A from the rate of disappearance of label and the initial specific activity. Thus the data from the uptake period provide an estimate of the rate of net inward movement of leucine and of the quantity of free leucine (incorporated leucine is subtracted) inside the sample at the start of the exchange period. Assuming ▵ remains constant it is then possible to calculate leucine levels in both the medium and the eggs during the exchange period. With this information and the R₁/R₁₍₀₎, values for the disappearance of label from the eggs can be calculated for each observed R1/R₁₍₀₎ (see legend, Fig. 3). In this analysis the label is inside the eggs, which must now be considered as compartment 1.

It should be noted that the movement of label due to exchange will be in the opposite direction to that due to uptake, resulting in a reversal of slope at about 2 h. As shown in Fig. 4 the equation for the 2-compartment model using the calculated values of ▵ and ρ fits the observations fairly closely, including the suggestion of a reversal. The deviation in each case is in the direction expected due to removal of free leucine by formation of protein inside the sample.

These results suggest an active transport of leucine (the estimated internal concentration at 6 h is 0·125 μmol/100 mg-within Florkin ‘s (1966) range, but 15 times the outside concentration) which is directly stimulated by addition of optic gland extract. The increased incorporation cannot account for the increased uptake; nor can the uptake account for the increased incorporation, since the specific activity of the leucine is the same in each case. Two separate processes appear to be involved in the response to the hormone.

Quite consistent results can be obtained if animals of similar size with ovaries of similar size are used. Fig. 5 A, B and C shows the results of experiments carried out with 2-day OG⁻ animals weighing 265–275 g, having ovaries weighing between 4·40 and 7·15 g. In each case there was substantial, nearly uniform, uptake and synthesis by controls and an approximate doubling of protein synthesis following the addition of extracts from optic glands.

These experiments also show that the hormone derived from male optic glands will induce yolk protein synthesis, apparently as readily as that from female glands. Experiment A used four glands, two from a ripe female weighing 295 g, and two from a ripe male weighing 285 g. The fresh extract in experiment B used two glands only, from a rather larger ripe male weighing 400 g. The males had been operated upon in the same manner as the females (subpedunculate lobes removed 14 days before death) and the glandular homogenate was diluted to approximately the same extent. Experiment C used a mixture of male and female glands, and D female glands only.

The effectiveness of male glands is consistent with the findings from transplantation experiments. Male glands implanted into the orbital sinus of female octopuses will secrete and induce precocious maturation of the ovary as readily as optic glands taken from other females (Wells & Wells, in preparation).

Freezing the glands is an effective means of preserving hormonal activity. The glands used in A were from animals killed within an hour of the beginning of the experiment. B used both fresh and frozen glands, the latter derived from octopuses killed 2 days before and stored at – 20 °C. C and D used frozen glands.

Experiment C also included a group of six samples incubated in 5 × 10⁻⁴ M cyclic adenosine monophosphate (cAMP). There was no significant effect on either uptake or synthesis. This test at a single dose does not, of course, rule out a role for cAMP in mediating the effect of the hormone.

In some (and perhaps all) other animals, gonadotropins are produced cyclically, following circadian rhythms (Mills, 1973). For our experiments we tended to use optic glands derived from animals killed in the morning or late afternoon. Since this was a possible source of variability, samples of fresh optic glands from female donors were collected a.m. and p.m. and tested on eggs from the same 11·3 g, 2-day OG⁻ ovary. Care was taken to match the optic gland samples as closely as possible, thus:

• (1) The a.m. sample (ca. 09·00 h) - one gland each from animals weighing 320 g and 490 g, two glands from an animal weighing 375 g, deep frozen for 45 min.

• (2) The p.m. sample (ca. 19·00 h) - one gland each from animals weighing 330 g and 455 g, two from an animal weighing 375 g, deep frozen overnight.

All the glands were ‘large’ and orange in colour, and all came from females with ovaries at approximately the same stage of development (enlarged, but not yet yellow in colour; that is, just beginning to lay down yolk in quantity).

Both a.m. and p.m. glands caused a large increase in uptake and synthesis as shown in Fig. 5 D, and differences between them were not significant. This experiment does not, of course, prove the absence of a secretion cycle; measurement of activity at two times a day only could not possibly do so. It is in any case probable that our dose rate was high compared with the naturally occurring titre of optic gland hormone, so that the responses may have been maximal. What experiment D does show is that the time of day of collection of the glands was not a limiting factor under the conditions of our experiments.

The second group of samples in Fig. 5D was inadvertently pre-incubated in an 8 % alcohol sea water solution and the eggs are presumed ‘dead’. The data are included as further evidence that considerable active uptake and synthesis occurs even in controls. The ‘uptake’ by these ‘dead’ eggs is about 5% of the total counts in the medium and merely represents the dilution of 2·00 ml of medium-sea water mixture by too mg of eggs.

The results summarized above show that bunches of eggs take up amino acids and make proteins. A large fraction of the protein made is water-soluble, as would be expected for a cephalopod yolk protein (see O‘Dor & Wells, in preparation). This material could be made by the ovum itself or by the follicle cells. The follicle cells are the most likely site, and light-microscope studies of other cephalopods (Lankester, 1875; Yung Ko Ching, 1930) have indicated export of materials from the follicle cells to the ovum. This has been confirmed by studies of Octopus eggs at electronmicroscope level, and Fig. 6, Plate 1 and Fig. 7, Plate 2 show, respectively, sections of follicle cells from OG⁺ and OG⁻ animals. The former, in full flush of uptake and synthesis, are packed with rough endoplasmic reticulum (ER) and active Golgi apparatus. Electron-dense vesicles are being formed by the Golgi and appear to be proceeding toward the finger-like processes that join the follicle cells and the ovum through the developing chorion; it is not yet clear whether these interdigitate or form continuous bridges.

In contrast, the follicle cells from 2-day OG⁻ eggs (Fig. 7, Plate 2) show rough ER that is tending to round off into vesicles. Golgi complexes are still present, but their saccules appear empty.

The follicle cells of the OG⁺ animals clearly show all the characteristics normally associated with active synthesis and export of protein (Droz, Pisam & Chrétien, 1973). Compared with these the ovum itself at this stage is devoid of ER and has few other cell organelles. It is packed with yolk. In the OG⁻ egg, protein synthesis in vivo has been cut to about 30% of its value in OG⁺ eggs (Fig. 2) and this is reflected in the intracellular appearance of the follicle cells. There would seem to be no doubt that we are studying the effects of optic gland hormone on these rather than on the ovum itself.

Removal of the optic glands in vivo reduces and eventually stops yolk protein synthesis by the follicle cells in Octopus (O‘Dor & Wells, 1973 and Fig. 2). The present series of experiments show clearly that this effect can be at least partially reversed in vitro by the addition of aqueous extracts of male or female optic glands. The minimum dose tested was one-third of an optic gland per 2 ml test sample. This quantity (experiment B) produced a highly significant (P < 0·001 by t-test) 62% increase in synthesis over the control, so the minimum effective dose would be considerably lower. Measurement of in vitro synthesis in the system described is a fairly rapid and sensitive means of detecting the gonadotropic principle in extracts of optic gland.

There are several possible means of improving sensitivity. The simplest would be a five- to tenfold reduction of test sample size. This could also improve reproducibility between samples, since the major cause of variation seems to be the presence of connective tissue or damaged eggs. These could be selected out in smaller samples.

Another is suggested by comparison of Fig. 1 (B, pre-incubation in sea water) and Fig. 5, which show a decrease in control levels resulting from the removal of the optic glands in vivo 2 days before using the eggs in vitro. The delay approximately halves protein synthesis by controls; synthesis by eggs with hormone added is reduced, though to a lesser extent. It should be noted, however, that these are probably minimal figures for the difference produced by two days without hormone, since the ovary used in the Fig. 1 experiment (3·6 g from a 280 g octopus) was at a somewhat earlier stage in development than those used in the Fig. 5 series (which ranged from 4·4 to 11·3 g out of animals weighing 265–310 g). Provided the effect of hormone withdrawal remains reversible, a longer waiting period should further decrease control levels and increase sensitivity.

A further means of increasing sensitivity is shown in Fig. 1. Pre-incubation in medium containing cold leucine results in a markedly higher uptake by eggs treated with hormone, which is, however, achieved at the cost of reduced incorporation into protein. The kinetic studies in Figs 3 and 4 indicate that uptake as measured in Fig. 1 has two components. One is a result of exchange and depends on the level of cold leucine in the eggs when [¹⁴C] leucine is added. The second depends on the rate of net inward movement of leucine. The hormone appears to increase this rate by a mechanism independent of its effect on synthesis. In eggs pre-incubated in sea water nearly all of the uptake is due to the net inward movement, but in eggs pre-incubated in medium, uptake is increased by a large exchange component. The magnitude of this exchange component depends on the amount of leucine inside the eggs when the [¹⁴C] leucine is added to the medium, which in turn depends on the rate of net inward movement during pre-incubation. It also depends on the ratio of P⁴C]leucine to total leucine in the medium, which is increased by movement of leucine from the medium to the eggs during pre-incubation. Thus pre-incubation in medium amplifies any change in net inward movement caused by the hormone.

We have then two means of assaying the optic gland hormone. One is to measure uptake, which has the advantage of being fast and very simple. The other is to measure protein synthesis, more tedious to carry out but showing a greater percentage change when the hormone is added. Given these two techniques, it should be possible to develop both qualitative and quantitative assays useful in the characterization not only of the optic gland hormone, but also perhaps of other molluscan gonadotropins.