Comp. Biochem. Physiol. Vol. 106B, No. 2, pp. 321-329, 1993 0305-0491/93 $6.00 + 0.00 Printed in Great Britain © 1993 Pergamon Press Ltd

ELECTROPHORETIC PATTERNS OF YOLK PROTEINS THROUGHOUT OVARIAN DEVELOPMENT A N D THEIR RELATIONSHIP TO VITELLOGENIN IN THE RAINBOW

TROUT, ONCORHYNCHUS MYKISS

CHARLES TYLER

Department of Biology and Biochemistry, Brunel University, Uxbridge, Middlesex, UB8 3PH, U.K.

(Received 15 February 1993; accepted 19 March 1993)

Abstraet--l. Electrophoretic patterns of yolk proteins were investigated throughout ovarian development and their relationship to vitellogenin determined in a pulse-chase experiment with 3H-vitellogenin.

2. Using a radioimmunoassay for viteUogenin, viteUogenin/yolk protein products of vitellogenin were detected in follicles throughout ovarian development and in ovulated eggs.

3. The majority of yolk proteins in follicles measuring less than 1.0 ram in diameter appeared to be derived from sources other than viteilogenin. In contrast, in the larger follicles all of the major yolk proteins detected were derived from vitellogenin.

4. Pulse-chase with 3H-viteUogenin revealed that all of the major yolk proteins in 3.0 nun follicles were derived from vitellogenin. The major peptides eluted with molecular masses of 110 and 30 kDa under non-reducing conditions (these are very likely to represent lipovitellin 1 and lipovitellin 2), and 88, 22, 16, and 12 kDa under reducing conditions.

5. There were no apparent differences in the major yolk proteins in ovulated eggs compared to those in vitellogenic follicles, indicating that no extensive proteolysis of these proteins had occurred during maturation and/or ovulation.

INTRODUCTION

Oocyte growth in oviparous vertebrates and invert- ebrates occurs principally during vitellogenesis through the sequestration of extra-ovarian proteins. In the South African clawed toad, Xenopus laevis, yolk proteins have been shown to account for over 80% of the egg's dry weight (Callen et al., 1980). The predominant groups of yolk proteins in this species are the lipovitellins, phosvitins and phosvettes, all of which have been shown to be derived from the hepatically synthesised glycolipophosphoprotein, vitellogenin (VTG; Wallace, 1985).

Similarly, studies on fish have also shown that VTG is a major yolk protein precursor (Wallace and Begovac, 1985; Tyler et al., 1988a,b; 1990a,b; Ka- nungo et al., 1990). Uptake of VTG is selective and recently a follicle receptor for VTG has been demon- strated in a number of fish species, including the tilapia (Oreochromis niloticus; Chan et al., 1991), coho salmon (Oncorhynchus kisutch; Stifani et al., 1990) and rainbow trout (Oncorhynchus mykiss; Le Menn and Rodriguez, 1991; Tyler and Lancaster, 1993).

Yolk proteins have been isolated and/or partially characterized by gel electrophoresis and chromato- graphic procedures in several species of fish (reviewed in Selman and Wallace, 1989). In fish, both lipovitellin-like and phosvitin-like proteins have been isolated; however, molecular weight estimates of these proteins vary widely, both within and between

species: molecular weight estimates for lipovitellin(s) and phosvitin(s) in salmonids alone range between 130 and 390 kDa (Campbell and Idler, 1980; Tyler et al., 1988a,b) and from 19.4 to 45 kDa (Ando, 1965; Campbell and Idler, 1980; Tyler et al., 1988a,b), respectively, depending, at least in part, on the tech- nique employed. Phosvettes also appear to be a constituent of teleost yolk (Wallace and Begovac, 1985). It should be pointed out, however, that only in very few cases have any of these yolk proteins been shown to be directly derived from VTG (Wallace and Begovac, 1985; Tyler et al., 1988b; Nagler and Idler, 1989). Interestingly, there are several studies on fish claiming that VTG is not processed into smaller yolk polypeptides after being taken up into the oocyte (Skinner and Rogie, 1978; Hori et al., 1979; Hara et al., 1980).

Another yolk protein, the//-component, that has neither the characteristics of lipovitellin or phosvitin, has been isolated from vitellogenic oocytes of several Oncorhynchus species (Markert and Vanstone, 1968). Originally it was thought that the fl-component was derived from a plasma protein distinct from VTG, however, other studies have indicated that the //- component is related immunologically to VTG, suggesting that it is derived from VTG (Campbell and Idler, 1980). Clearly, the present picture on VTG's processing in teleost oocytes, largely due to the complexity of the molecule, is unclear.

In the winter flounder, Pseudopleuronectes ameri- canus, apparently there are yolk proteins that consti-

321

322 CHARLES TYLER

tute a significant proportion of the oocyte contents, yet are derived from a precursor distinct from VTG (Nagler and Idler, 1989). Evidence for yolk proteins derived from precursors distinct from VTG in salmo- raids has not been forthcoming, but vitellogenic follicles of the rainbow trout can sequester serum proteins other than VTG (Campbell and Jalabert, 1979), as well as heterologous proteins (Tyler et al., 1988b). Indeed, it is likely that a whole series of other components vital for the production of viable eggs are selectively sequestered, albeit in varying amounts, by the developing oocyte.

Although VTG has often been assumed to be the major yolk protein precursor in teleosts, there have been very few studies that actually show this. Which yolk proteins are derived directly from VTG and quantifications of VTG's contribution to oocyte growth have yet to be fully determined in any fish species. This study investigated changes in the elec- trophoretic patterns of yolk proteins during oocyte development and, by including a pulse-chase exper- iment with radiolabelled VTG, assessed which of these yolk proteins were directly derived from VTG in the rainbow trout.

MATERIALS AND METHODS

Electrophoretic analysis of yolk proteins in oocytes during ovarian development

Preparation of yolk. The first part of this study set out to determine which yolk proteins constituted the major components of rainbow trout follicles during ovarian development. Ovarian follicles were collected from rainbow trout throughout the reproductive season. The study was initiated when the largest follicles within a developing ovary were less than 0.3 mm in diameter and, at subsequent intervals during ovarian development, when populations of developing follicles measured 0.3, 0.5, 0.8, 1.0, 2.0 and 3.0 mm in diameter. Finally, at the end of the season, eggs (4.5 mm in diameter) were stripped from fully mature females. Developing follicles were dis- sected out from the ovary and divested of their connective tissues and surface epithelium using watchmaker forceps (this removed any blood vessels that would contain contaminating VTG). This pro- cedure was not necessary for the preparation of yolk extracts from eggs because they are composed of yolk surrounded only by the oolemma and chorion. Yolk proteins were collected by puncturing the oocytes (or eggs) and extruding their contents into ice-cold physiological saline containing the enzyme inhibitors aprotinin (1000 kallikrien units/ml) and phenyl- methylsulphonylftuoride (PMSF; 2 mM). The only exception to this protocol was in the collection of yolk from the follicles measuring less than 0.3 mm in diameter. Here, due to the difficulty of handling the small follicles, whole lamellae containing the follicles, after removal of the major blood vessels, were ma- scerated. The yolk contents were spun at 2500 g for

10 min. The resulting centrifugate consisted of an opaque fluid with a surface layer of lipid; there was little or no preciptate, indicating that all of the yolk proteins were contained in the soluble fraction. The protein extract was withdrawn and frozen at -20°C until required.

Electrophoresis. One-dimensional gel electrophor- esis was carried out according to Laemmli (1970) on 3-17% linear gradient mini-polyacrylamide gels (Hoeffer Scientific Instruments, Newcastle, Stafford- shire, U.K.) Gels were run under both reducing and non-reducing conditions and proteins stained with Coomassie Brilliant Blue stain, Silver Stain or Stains- All. All preparations were diluted 1:4 with sample buffer (1% SDS, 0.1M Tris-HC1 (pH6.8), 10% glycerol, 0.02% Bromophenol Blue). Samples that were reduced were treated with 1% B-mecapto- ethanol and heated at 90°C for 4min. Yolk preparations were loaded at similar protein concen- trations to provide a comparision of the quantitiative changes in the different yolk proteins during ovarian development. The amounts of protein loaded are given in the figure legends. Protein concentrations of both yolk extracts and plasma samples were deter- mined using bicinchoninic acid (protein assay kit, Sigma Chemical Co., Poole, Dorset, U.K.). Molecu- lar mass estimates of the separated yolk and plasma proteins were determined from logarithmic plots of the migration coefficients vs molecular mass of stan- dard molecular weight markers (Lambin, 1978).

Vitellogenin radioimmunoassay. Yolk (and egg) preparations were radioimmunoassayed for VTG, using an homologous VTG radioimmunoassay (Sumpter, 1985), to determine both which sized fol- licles contained VTG/VTG's cleavage products and to gain an idea of what proportion of the total yolk protein was made up by the products of VTG at the different stages of development. (Previous studies have shown that certain yolk protein products (lipovitellins) crossreact well with antibodies raised to VTG; Tyler et al., 1988a.)

Pulse-chase with 3 H-vitellogenin

A vitellogenic female trout was injected with 3H- VTG and the yolk protein processing subsequently traced on electrophoretic gels.

VTG was radiolabelled in vitro with a mixture of 3H amino acids, according to Tyler et al. (1991b). Briefly, a vitellogenic female rainbow trout was in- jected with 17fl-oestradiol to induce the transcription of the VTG gene(s) and 1 week later the fish was killed and slices of liver incubated with a mixture of tritiated amino acids. De novo synthesis of VTG occurred in culture and the radiolabelled amino acids became incorporated into the polypeptide backbone. The 3H-VTG was subsequently purified from the culture medium using a combination of gel-filtration and ion-exchange chromatography. A maturing female rainbow trout was injected via the intraperi- toneal cavity with 25 p Ci of 3H-VTG. One week later,

Rainbow trout yolk proteins 323

a blood sample was collected from the Cuverian sinus into a chilled heparinized syringe containing 2 mM PMSF. The blood was spun at 2500g for 10 rain and the plasma extracted and frozen at - 2 0 ° C until required. The fish was then killed and 20 follicles (measuring 3.0 + 0.1 mm in diameter) were dissected out of the ovary, divested of their surface epithelium, and counted for radioactivity. Each follicle was placed in a separate glass vial with 1 ml of hydrogen peroxide and the vials incubated at 37°C overnight to solubilize the tissue. The digests were then allowed to cool before adding 10 ml of Liquiscint scintillation fluid (Canberra Packard, Pangbourne, Berkshire, U.K.). Samples were counted for 15 rain in a Tri- Carb 2000CA scintillation counter which had a counting efficiency of 35%.

Yolk protein extracts were prepared as described above and run on large (16 x 18 crn) 3-17% gradient gels under both reducing and non-reducing con- ditions. (Large gels were run so that sufficient amounts of yolk extract could be applied to give detectable counting signals in the separated proteins.) A plasma sample was diluted in sample buffer and run under reducing conditions. The amounts of pro- tein loaded are given in the figure legends. Both the reduced and non-reduced gels were stained with Coomassie Brilliant Blue stain and then pho-

tographed before preparing the gels for counting of radioactivity by cutting out the separate running lanes, sectioning them into 4mm wide horizontal strips, and treating each strip for scintillation count- ing, as described above for the follicles.

RESULTS

Figures 1 and 2 show the electrophoretic separ- ations of yolk proteins from follicles at various stages of ovarian development, under non-reducing and reducing conditions, respectively, stained with Coomassie Blue.

Yolk extracts from follicles measuring less than 1.0 mm in diameter contained an array of proteins of different sizes; under non-reducing conditions the major bands (or groups of bands) migrated with molecular masses of 200, 74 and 61 kDa, between 30 and 40, 15 and 11 kDa. The proteins present at the highest concentrations eluted with molecular masses of 200 and 61 kDa. Similarly, under reducing con- ditions, there was a whole series of different-sized proteins, migrating with molecular masses ranging between 200 and 15 kDa. There were no obvious differences in the electrophoretic patterns of proteins in follicles measuring between 0.3 and 0.8 mm in diameter. In 1.0 mm diameter follicles, a similar array

Molecular weight (Oaltons) :

340k ~,

170k =,

975k t,

55Ak

365k I.

20k

Lane 1 2 3 4 5 6 7 8

Fig. 1. SDS-polyacrylamide gradient gel electrophoresis of yolk extracts from follicles at various stages of ovarian development. Lane 1: a homegenate of lamellae which contained follicles less than 0.3 mm in diameter; lanes 2-8: yolk extracts from 0.3, 0.5, 0.8, 1.0, 2.0 and 3.0 mm follicles and ovulated eggs, respectively. Protein loadents were between 15 and 30 #g per lane. Samples were not reduced before application. Proteins were stained with Coomassie Brilliant Blue stain. The migration positions of the

molecular weight standards are indicated.

324 CHARLES TYLER

Molecular weight (Daltons)

170k ~,

97.4k . _ ~

55.4k.._~

365k

20k . _ ~

Lane

~/ii! iliill iiiiil/~ ¸̧ i~i ~i ̧

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Fig. 2. SDS-polyacrylamide gradient gel electrophoresis of yolk extracts from follicles at various stages of ovarian development. Lane h a homegenate of lamellae which contained follicles less than 0.3 mm in diameter; lanes 24: yolk extracts from 0.3, 0.5, 0.8, 1.0, 2.0 and 3.0 mm follicles and ovulated eggs, respectively. Protein loadings were between 15 and 30 gg per lane. Samples were reduced with 1% fl-mecaptoethanol before application. Proteins were stained with Coomassie Brilliant Blue stain. The

migration positions of the molecular weight standards are indicated.

of proteins was observed to that seen in the smaller follicles with the exception of the appearance (or at least considerable increase in concentration) of two major peptides, eluting with molecular masses of around 88-115 and 30 kDa under non-reducing con- ditions, and around 88-90 and 16 kDa under reduc- ing conditions. Another protein seen in follicles 1.0 mm and above in diameter, under reducing con- ditions, eluted with a molecular mass of 12 kDa. In the larger vitellogenic follicles examined (2.0 and 3.0 mm) and in ovulated eggs, a small number of yolk proteins became by far the greatest protein constitu- ents. These major yolk proteins eluted with molecular masses of 110, 88 and 30 kDa, under non-reducing conditions and 88, 22, 16 and 12 kDa under reducing conditions. In 2.0 and 3.0 mm follicles, and ovulated eggs, the peptides eluting at 22, 16 and 12 kDa in reduced samples were also seen as very faint bands (present at very much lower concentrations) in the non-reduced samples. Many proteins of the array present in the smaller follicles either appeared as very faint bands or were absent in the larger vitellogenic follicles and ovulated eggs. In gels that were stained with Silver Stain or Stains-All, there were no other major protein bands to those shown in the gels stained with Coomassie Blue (there were additional faint bands detected by the more sensitive Silver- Staining technique, data not shown).

Products of VTG (lipovitellins) were detected by radioimmunoassay in all the follicles examined. The proportion of the total yolk protein constituted by lipovitellin(s), however, varied considerably in the different-sized follicles. In the smallest follicles (<0.3 mm) only 0.05% of the total protein could be linked to VTG. Similarly, in follicles 0.3mm in diameter, less than 0.1% of the total protein could be linked to VTG. In follicles measuring 0.5 and 0.8 mm in diameter, the contribution of VTG increased from 0.5 to 3%, respectively. In 1.0 mm diameter follicles, there was a dramatic change and at least 50% of the total protein was derived from VTG. Similarly, in 2.0 and 3.0 mm follicles, and in ovulated eggs, over 60% of the protein present was shown to be derived from VTG.

Figure 3 shows the electrophoretic separation of plasma proteins from a vitellogenic female 1 week after it was injected with 3H-VTG. Of the array of proteins in the plasma, those present at the highest concentrations eluted with molecular massess of 170, 60, 30 and 12 kDa. Only the protein band eluting at 170 kDa contained detectable amounts of radiolabel.

One week after injection of the 3H-VTG, the follicles (mean diameter 3.0 _+0.1 mm) each con- tained 10000 _ 1200 dpm (N = 20). When extrapo- lated to the whole ovary (see Tyler et al., 1988b)

Rainbow trout

approximately 34% of the injected 3H-VTG had been sequestered by the developing follicles.

Figure 4(a) and (b) shows electrophorectic separ- ations of yolk proteins, run under non-reducing and reducing conditions, respectively, 1 week after the fish had been injected with 3H-VTG. The major yolk proteins eluted with molecular masses of 110, 88, and 30 kDa, under non-reducing conditions and 88, 22, 16 and 12 kDa, under reducing conditions, identical to that seen previously on the mini-gels. In the non-re- duced yolk protein extract, the radioactivity was associated with the proteins eluting at 110 and 30 kDa. On the reduced gel, all four of the protein bands contained tritium. Most of the label (approxi- mately 80%) was associated with the largest protein, eluting with a molecular mass of around 88 kDa.

D I S C U S S I O N

It has generally been accepted that the uptake and proteolytic processing of VTG in fish accounts for a considerable proportion of an oocyte's yolk protein reserves; however, direct evidence for this has not been forthcoming. Indeed, there have been very few studies that show even an association between VTG and yolk proteins. This study illustrates that during early ovarian development, rainbow trout follicles contained a whole series of yolk proteins, presently of undetermined origin. However, when follicles

yolk proteins 325

reached between 0.8 and 1.0 mm in diameter, proteins proteolytically derived from VTG constituted by far the greatest proportion of the yolk reserves. In large follicles (2.0 mm and above), no other proteins were detected on gels in significant quantities using three different protein-staining procedures, suggesting that autosynthesis of yolk protein and derivation from other sources provides little of the protein in these larger oocytes.

Follicles measuring 0.8 mm and below in diameter contained a whole series of yolk proteins (of un- known origin). Two major proteins in these smaller follicles eluted with molecular masses of around 200 and 61 kDa. Selman et al. (1986) and Inoue and Inoue (1986) working on the killifish (Fundulus hete- roclitus) and rainbow trout, respectively, have simi- larly shown that a 200 kDa protein is a predominant component of oocytes during early ovarian develop- ment. Their studies showed the 200 kDa compound to be a glycoprotein conjugate found within the cortical alveoli. In this study, because protein(s) contained in the major bands in 2.0 and 3.0 mm follicles and eggs became such a large proportion of the total yolk protein constituents, most of the other proteins seen in the extracts from the smaller follicles (including the 200 kDa protein) were diluted such that they comprised only a small proportion of the total protein content. When gels were run consider- ably overloaded with protein from these follicle/egg

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Molecular weight (Daltons)

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Fig. 3. SDS-polyacrylamide gradient gel electrophoresis of plasma (3/zl; 120 #g total protein) from a female rainbow trout 1 week after injection with 3H-VTG showing the pattern of eluting plasma proteins and associated radioactivity. The sample was reduced with 1% fl-mecaptoethanol before application, Proteins were stained with Coomassie Brilliant Blue stain. The migration positions of the molecular weight

standards are indicated.

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100

326 CHARLES TYLER

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Molecular weight (Daltons) _~ J.e _~ ,Ig

n o n r e d u c e d

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Molecular weight (Daltons) .M . i

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700

E 600 Q.

500

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Fig. 4. SDS-polyacrylamide gradient gel electrophoresis of yolk ( l l0gg total protein) from follicles (3.0+0.1 mm in diameter) taken from a maturing female 1 week after it had been injected i.p. with 3H-VTG. The pattern of during plasma proteins and associated radioactivity are illustrated. Samples were run under both non-reducing (4a) and reducing (4b) conditions. Proteins were stained with Coomassie

Brilliant Blue stain. The migration positions of the molecular weight standards are indicated.

extracts, however, the 200 kDa protein, as well as a whole array of other proteins present in the smaller follicles, could be seen clearly (data not shown), indicating that the 200 kDa protein, and many of the

other "minor" protein components, persist through- out ovarian development into the ovulated egg. Inoue and co-workers have demonstrated that the 200 kDa protein depolymerizes to 9 kDa subunits after fertil-

Rainbow trout

ization when the contents of the cortical alveoli are released into the perivitelline space.

Radioimmunoassay of the contents of the yolk extracts showed that VTG/products of VTG were present in the ooplasm of all the follicles examined. The proportion of yolk protein derived from VTG increased 10-fold, from <0.05% in follicles measur- ing less than 0.3 mm in diameter, to 0.5%, in 0.5 mm follicles and further, to 3% in 0.8 mm follicles. In follicles measuring 1.0 mm in diameter, there was a dramatic change in the proportion of yolk protein that was derived from VTG. Using the VTG radio- immunoassay, over 50% of the yolk protein reserves were shown to be derived from VTG and, further, over 60% in the 2.0 and 3.0 mm follicles and ovulated eggs. Studies on the polychaete worm, Perinereis cultrifera, have shown that a similar high percentage (60%) of total protein in mature oocytes is accounted for by vitellin (a yolk protein derived from VTG: Baert and Slomianny, 1987). It should be pointed out, however, that the percentages given in this study are only estimates of VTG's contribution to total yolk protein as the antibody used in the radioimmuno- assay was raised to VTG that was probably intact, and hence the antibody may not detect all of the degradation products of VTG, or detect them all quantitatively. Nevertheless, the results do give an idea of the relative contribution VTG makes to oocyte growth at the various stages of ovarian devel- opment. It is likely, looking at the gels run of whole yolk extracts from the larger follicles and eggs, where all of the major protein bands were shown to be derived from VTG, that VTG accounts for more than 60% of the total protein in mature oocytes.

Studies on the rainbow trout have suggested that follicles have to reach a certain size before they are able to sequester VTG (Tyler et al., 1991a). In that study, follicles measuring less than 0.6 nun in diam- eter did not sequester 3H-VTG, whereas those of a greater size did. Similarly, histological studies on ovarian development in the rainbow trout have shown that yolk granules (which store the products of VTG) appear when the follicles are around 0.6 mm in diameter (Upadhyay et al., 1978; Sumpter et al., 1984). The results in this study, however, show that VTG or products of VTG were present in follicles below 0.3 mm in diameter, indicating that uptake of VTG occurs throughout ovarian development rather than being confined to specific stages of oocyte growth. This apparent anomaly could well be ex- plained by the fact that in the studies mentioned above the detection systems employed for assessing VTG uptake were less sensitive than the radio- immunoassay for VTG used here. Our observations concur with that recently demonstrated in Perinereis diversicolor, where uptake of VTG into oocytes was shown to occur outside the realms of the so-called "vitellogenlc phase" of development (Baert and Slo- mianny, 1992). In female rainbow trout, VTG is present in the blood and therefore available for

yolk proteins 327

sequestration by the developing ovary throughout the life of the fish, increasing from levels (in ng/ml) in fish around 50 g to levels as high as 50 mg/ml in mature female fish (Copeland et al., 1986; Scott and Sumpter, 1983) Together, these observations question the ap- propriateness of the present stageing system used for fish oocytes that has been based mainly on cytological observations.

Proteins derived from VTG were not seen in extracts run on gels from the smaller follicles (< 0.8 ram) possibly because they represented a small percentage only of the total yolk protein. It is not possible, therefore, to determine whether the specific intra-oocytic processing of VTG seen in the larger follicles has been activated in the smaller follicles.

When follicles reached between 0.8 and 1.0 mm in diameter there was a dramatic increase in the amount of VTG sequestered; in this study, products of VTG represented only 3% of the total protein in 0.8 nun follicles but over 50% in 1.0 nun follicles. This dra- matic increase in VTG uptake is not a reflection of any considerable increase in the levels of VTG in the blood (these increase gradually throughout this period of ovarian development, see Copeland et aL, 1986). It is more likely that the dramatic increase in VTG sequestration at this time results from the activation of, and/or enhanced functioning of, the receptor system for VTG (receptors for VTG are present in follicles as small as 0.4 mm in diameter-- our own unpublished observations).

In 1.0, 2.0 and 3.0 mm follicles, and also in ovu- lated eggs, three and four proteins comprised the major yolk constituents on gels run under non-reduc- ing and reducing conditions, respectively. Figure 4(a) and (b) show that all of these peptides contained radiolabel and were therefore derived from the pro- teolytic processing of 3H-VTG. The peptides eluting at 110 and 30 kDa on gels run under non-reducing conditions are very likely to represent lipovitellin l(Lv 1) and lipovitellin 2 (Lv 2). Previous studies, which have assumed these proteins are derived from VTG, give molecular mass estimates for lipovitellin 1 (under non-reducing conditions) of between 105 and 112 kDa, in the goldfish, Carassius auratus (de Vlam- ing et al., 1980) and rainbow trout (Babin, 1987), respectively. For lipovitellin 2, molecular mass esti- mates range from 25 kDa in the goldfish (de Vlaming et aL, 1980) to between 30 and 35 kDa in the rainbow trout (Babin, 1987; Ando and Hatano, 1991).

Most electrophoretic separations of fish yolk pro- teins under reducing conditions indicate that there are between four and seven major peptides (de Vlam- ing et al., 1980; Wallace and Selman, 1985; Babin, 1987; Ando and Hatano, 1991; Carnevali et al., 1992). Comparing the molecular mass estimates of the proteins run on gels under reducing conditions in this study with the work conducted by the above authors suggests that the 88 and 22 kDa peptides are derived from the l l 0 k D a Lvl and the 16, and 12 kDa peptides from the 30 kDa Lv2.

328 CHARLES TYLER

The phosphoprotein components of oocytes, the phosvitins and phosvettes, which are known to be derived from VTG in Xenopus (Wallace, 1985) and Fundulus (Wallace and Begovac, 1985; Wallace and Selman, 1985) were not seen on gels in this study (even when the gels were stained with Stains-All rather than Coomassie Blue, a dye that preferentially stains phosphoproteins; data not shown). Phospho- proteins have been shown to be present in rainbow trout oocytes (Tyler et al., 1988a,b); however, they represent a low percentage of ovarian yolk proteins (less than 3%, Campbell and Idler, 1980) and there- fore it is likely that they had been diluted such that the small amounts loaded on the gels were undetect- able.

Work on a number of marine species, including the halibut, Hippoglossus hippoglossus, cod, Gadus morhua, plaice, Plueronectes platessa, killifish, Fun- dulus heteroclitus and gilthead sea bream, Sparus aurata, suggests that a secondary proteolysis of yolk proteins occurs during oocyte maturation (Greeley et al., 1986; Norberg, 1987; Carnevali et al., 1992). One suggestion to account for the purpose of this process is to generate the osmotic gradient required for water uptake during the hydration phase, a process necessary to generate pelagic eggs in many marine fish species. Although it is not possible to say whether there were changes in the minor yolk protein components of the ovarian extracts, there were no apparent gross changes in the "lipovitellins" in ovu- lated eggs compared to those in vitellogenic follicles, indicating that no extensive proteolysis had occurred during maturation and/or ovulation.

Acknowledgement--I would like to thank Professor John Sumpter for his constructive criticism of the manuscript,

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Copeland P., Sumpter, J. P., Walker, T. K. and Croft M. (1986) Vitellogenin levels in male and female rainbow trout (Salmo gairdneri Richardson) at various stages of the reproductive cycle. Comp. Biochem. Physiol, 83B, 487-493.

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