Regulation of germinal vesicle breakdown in starfish oocytes

624 REGULATION

OF GERMINAL STARFISH A. W. SCHUETZZ

Marine

Biological

BREAKDOWN

IN

OOCYTES and J. D. BIGGERSB

Laboratory,

Received

VESICLE

Woods Hole, Mass.,

December

U.S.A.

8, 1966

A polypeptide, which causes shedding of ova when injected into ripe females, can be extracted from the radial nerves of starfish [3, 91. Concurrently the germinal vesicles break down and the oocytes undergo meiotic maturation. These effects can also be induced in vitro by the addition of the radial nerve factor (RNF) to ovaries and ovarian fragments immersed in artificial sea water [I, 2, 6, 81. Recently evidence has been obtained which suggests that in Aster&x forbesi Desor the RNF induces germinal vesicle breakdown by stimulating the production of a low molecular weight intermediary compound. The evidence obtained in the summer of 1965 and 1966 is summarized in this paper. Partially purified preparations of RNF were prepared by first extracting isolated radial nerves with acetone and ether. The dried powders were extracted overnight at 4°C in distilled water. The supernatants were centrifuged at 12,000 g, and then desalted by passage through a column of Sephadex G-25. In each preparation the active fractions were pooled and lyophilized. The first indication that the RNF stimulates the production of a substance mediating germinal vesicle breakdown was obtained in studies on isolated oocytes and ovarian fragments maintained in calcium-free sea water (CaFSW). Ovarian tissue was freed of almost all oocytes and placed in a dish of CaFSW containing oocytes with intact germinal vesicles. When RNF was added the germinal vesicles first broke down in those oocytes nearest the fragment of ovarian tissue (Fig. I). Later, the oocytes further away from the ovarian fragment also broke down. These initial observations suggested that a substance, hereafter called the ovarian factor, diffused from the ovarian tissue and caused the breakdown of the germinal vesicles. Crude extracts of the ovarian factor were prepared from batches of RNF-stimulated ovaries which were freed of as many oocytes as possible in order to prevent utilization of the ovarian factor. During the first season the ovarian factor was extracted in CaFSW, while in the second season some preparations were made with normal sea water (NSW). In both methods the procedure was otherwise the same. Ovaries were washed in NSW or CaFSW for l-5 hr. As many oocytes as possible were removed by squashing the ovaries with a scalpel blade and rinsing with sea water. The remaining ovarian tissue was minced with scissors and suspended in sea water. RNF was then added to the suspension. After 2-8 hr the suspension was homogenized and centrifuged. The activities of the crude ovarian extracts were examined by measuring their ability to cause germinal vesicle breakdown in groups of oocytes with intact germinal 1 Research aided by Training Grant 5TI-HD-26-04 from the National Institute of Child Health and Human Development, U.S. Public Health Service and by the Lalor Foundation. 2 Present address: Johns Hopkins School of Hygiene & Public Health, Baltimore, Md, U.S.A. Experimental

Cell Research 46

Oocyte maturation

in starfish

625

Fig. l.-Response of oocytes to ovarian tissue exposed to radial nerve shedding factor. Ovarian tissue prev,rashecl in CaFSW was placed lvith oocytes with intact germinal vesicles. Following addition of 50 ,ug of nerve factor germinal vesicle breakdown was seen first in the immediate area of the ovary. s 20.

vesicles. The results are expressed as the percentage of oocytes which have lost their germinal vesicles after one hour’s exposure to the extracts. During the first season these tests used oocytes which had been isolated in CaFSW, thereby preventing them from undergoing spontaneous maturation. Ripe ovaries, or ovarian fragments, were placed in CaFSW which had been made isotonic with magnesium chloride, for 0.5-Z hr. The CaFSW was changed 4-5 times during this period. Small pieces of the ovaries were then transferred to 10 ml CaFSW and the oocytes released either by light pressure or by tearing the ovarian walls. During the second season tests were also done with oocytes similarly isolated in normal sea water (NSW), and those oocytes in which germinal vesicle breakdown did not spontaneously occur within an hour after being released into NSW were used for assays. Batches of 25 or 50 oocytes, with intact germinal vesicles and no surrounding follicle cells, were transferred into small preparation dishes containing 0.5 ml CaFSW or NSW. Various quantities of the crude ovarian factor preparation were then added to these dishes. Since this involved the addition of different volumes, the total volume of fluid in each dish was equalized by adding extra CaFSW or NSW respectively. Table I shows the results of a test of activity of one ovarian extract on oocytes prepared in CaFSW. The replicate tests are not significantly different, and indicate that

the percentage

of germinal

vesicle

breakdown

is highly

dependent Eqxrimental

on the amount Cell Research 46

A. TV. Schuetz

626

and J. D. Biggers

of ovarian extract added to the dishes. These results other ovarian extracts tested on oocytes prepared

are typical

of those

obtained

with

in CaFSW. The addition of RNF alone to immature oocytes has no effect on the germinal vesicles. Attempts to characterize the ovarian factor were made by molecular sieving on columns of Sephadex G-25 or Sephadex G-IO. The supernatants of ovarian extracts prepared in NSW and CaFSW were passed through G-25 equilibrated with the appropriate

sea water,

and

samples

were

tested

for

biological

activity.

The

ovarian

factor was markedly retarded on the column, in contrast to the RNF, indicating TABLE

I. Effect of an extract from ovaries treated with radial nerve shedding (RNF) on germinal vesicle breakdown of starfish oocytes.

a

factor

Ovarian tissue (1.65 g) was exposed to 300 ,ug of RNF in 25 cc of calcium-free sea water. Two replicates were done. Fifty oocytes were exposed to each dose of the ovarian extract. The total volume of fluid in each dish was 1.5 ml. 50 pg RNF alone failed to break down any germinal vesicles. Percentage germinal vesicle breakdown

Amount of ovarian extract (ml)

Replicate

0 0.1 0.2 0.3 0.4 0.5

TABLE

1

Replicate

0 0 6 34 80 100

2

0 6 16 62 78 80

II. Effects of ovarian extracts, prepared from normal ovaries or ovaries prewashed in CaFSW and exposed to RNF, on oocyte germinal vesicle brealcdown.

Twenty-five oocytes isolated in NSW, with intact germinal vesicles, were exposed to each dose, and all preparations were tested at the same time. The total volume of fluid in each dish was equalized to 5 ml with normal sea water. Five ml of ovarian extract was incubated with 5 mg pronase for 0.5 hr prior to being tested. Percentage Crude extract prepared in NSW

germinal

vesicle breakdown

G-10 (CaFSW)

G-25 (CaFSW)

Volume extract tested (ml)

1

2

1

2

1

2

Control Pronase treated

100 92

100 100

100 100

100 100

100 100

100 100

RNF

(100 pg) 0 76. Control

Experimental Cell Research 46

0 7.6.

Oocyte maturation

in starfish

621

molecular weight considerably less than 5000 [7]. The active material from G-25 columns eluted with CaFSW was then passed through a G-10 column equilibrated with CaFSW. The biologically active material was also retarded on this column, indicating its molecular weight is less than 700. The biological activity of crude ovarian factor prepared in NSW, and partially purified ovarian extracts prepared in CaFSW, was tested with oocytes prepared in NSW. The effect of the proteolytic enzyme pronase was examined simultaneously. The results, shown in Table II, demonstrate that oocytes prepared in NSW respond to ovarian extracts prepared in both NSW and CaFSW, and that the biological activity of the ovarian factor is not affected by pronase. The results presented thus far only prove that a factor causing germinal vesicle breakdown can be extracted from starfish ovaries. The following experiment shows that RNF stimulates the production of this ovarian factor. Ovarian tissue, free of oocytes, was isolated in CaFSW and subsequently divided into two batches. One batch was treated with RNF, and the other was left untreated as a control. Crude extracts were prepared from each of these batches and tested in different concentrations for activity. The results are shown in Table III. Clearly the ovarian factor is only produced in significant amounts in ovaries treated with RNF. A similar result was obtained with ovarian factor isolated from ovaries exposed to RNF in NSW, The nature of the ovarian factor is unknown. However, the results demonstrate that it is a much smaller molecule than RNF, and that it is produced in ovarian tissue under the influence of RNF. Other studies have shown it to be heat stable, dialyzable, TABLE

III.

Effect of radial nerve shedding factor (RNF) on the capacity of ovarian extracts to produce germinal vesicle breakdown in starfish oocytes.

Eggs were dissected from mature ovaries washed in CaFSW and 125 mg of the remaining tissue was exposed to 200 ,ug RNF for 5 hr. 125 mg of control ovarian tissue was left untreated. The tissues were homogenized in 9 ml of CaFSW and centrifuged. The supernatant was used for assay purposes. Percentage

Amount of ovarian extract (ml) RNF

Replicate

treated ovarian 0.25 0.5 1.0

Untreated ovarian 0.25 0.5 1.0 Assay controls RNF (50 /LB) Untreated

1

breakdown Replicate

2

tissue 44 96 100

4 88 100

2 0 0

0 0 0

0 0

0 0

tissue

Experimentul

Cell Research 46

628

G. L. Tritsch

and insoluble in acetone and ether. The latter results, together with its insensitivity to pronase indicate it is not protein in nature. Further investigation of the ovarian factor is important for several reasons. There is ample evidence that cytoplasmic maturation of oocytes in the starfish [4] and toad [5] is dependent on changes in the germinal vesicle. The discovery of a substance which closely regulates the breakdown of the germinal vesicle may yield systems for the detailed biochemical analysis of the changes involved in the maturation of oocytes of starfish. Furthermore, the changes involved in the maturation of mammalian ova may be controlled by similar mechanisms. If so, new light may be shed on the perplexing problem as to why a few mammalian ova mature within the individual female while the majority undergo atresia. Summary.-A small molecular weight factor, which causes breakdown of the germinal vesicle of isolated immature oocytes maintained in normal or CaFSW, was separated from the ovaries of starfish. Addition of RNF to ovarian tissue increased the amount of this substance. REFERENCES 1. CHAET,

2. __

A. B., Biol.

Bull.

126, 8 (1964).

ibid. 130, 1 (1966).

3. 4. 5. 6.

CHAET, A. B. and MCCONNAUGHY, R. A., Biol. Bull. 117, 407 (1959). DELAGE, Y., Arch. Zool. ezpfl gen. (Ser. 3) 9, 234 (1901). DETTLAEF, T. A., NIKITIN.I, L. A. and STROEVA, 0. G., J. Embryol. expfl Morph. 12, 851 (1964). KANATXNI, H., Science 146, 1177 (1964). 7. K.~N.%T~NI, H. and NOUMUR.&, T., Dobufsugaku Zasshi 43, 65 (196-i). 8. MOORE, B. D. and BIGGERS, J. D., BioZ. BUZZ. 127, 381 (1964). 9. NOUIIIURA, T. and KANATANI, H., J. gac. Sci. Uniu. Tokyo Sec. 4, 9, 397 (1962).

THE SELECTIVE

UTILIZATION CELL

OF SERUM PROTEINS

LINES

GROWING

IN

BY DIFFERENT

VITRO

G. L. TRITSCH Clinical Biochemistry Section, Department of Medicine Roswell Park Memorial Institute, New York State Department Buffalo, NY. 14203, U.S.A.

(C), of Health,

Received December 9, 1966

THE basis

of the protein requirement of cells cultured in uifro is not understood. Although there is evidence that actual utilization of protein takes place [9], the precise fate and function of the protein has remained elusive. The data herein presented will demonstrate that different cell lines in culture may have different qualitative and quantitative protein requirements for growth. Experimental.-Two cell lines were used in this study: RPM1 No. 5205, established in 1965 from human peripheral leukemic leukocytes [6] and RPM1 No. 2402, established in 1962 from a small bowel carcinoma of the Syrian hamster [7]. They were Experimental

Cell Research 46