(Foulerton Student of the Royal Society).

Transcription

1 6I2.62I:6I2.OI8 STUDIES ON OVULATION. VI. Relative importance of concentration and absolute amount of the ovulation-producing hormone. BY F. W. ROGERS BRAMBELL AD A. S. PARKES (Foulerton Student of the Royal Society). (From the Department of Zoology, University College, Bangor.) I. INTRODUCTION. IT is now reasonably certain that ovulation follows copulation in the rabbit as the result of reflex stimulation of the hypophysis causing secretion of the ovulation-producing hormone by the anterior lobe. This combination of nervous and endocrine events suggested that instructive results might be obtained from cross-circulation experiments, i.e. from the anastomosis of a normal copulated animal with a hypophysectomized unmated oestrous one'. Before such experiments could be carried out, however, it was necessary to find an anti-coagulant with no effect on ovulation, and to investigate the effect of lowering the concentration of the ovulation-producing hormone by dispersing it into a double circulation. The first problem has been dealt with in the preceding paper [Brambell and Parkes, 1932]: the second is considered in this one. It was evident from earlier work [Fee and Parkes, 1929] that the whole amount of the hormone required to produce ovulation is secreted by the anterior pituitary body within 1 hour after copulation; it was also a reasonable assumption that the major part of the hormone is in circulation during the following 30 min. It seemed, therefore, that the concentration of the hormone could be altered by replacing a proportion of the blood with gum saline. Such a technique, however, would also lower the absolute amount of the hormone and introduce a second variable. To eliminate this complication, i.e. to lower the concentration without altering the absolute amount per follicle, it was necessary to remove a corresponding proportion of the follicles. If a greater proportion of follicles than of blood was removed, the absolute amount of hormone 1 This idea was originally elaborated by the late Dr A. R. Fee, who actually carried out one experiment before his death.

2 174 F. W. ROGERS BRAMBELL AND A. S. PARKES. available per follicle could be raised above normal, and vice versa. A long series of experiments was carried out on these lines and, as might be expected from their nature, the details of the results are not very coherent. In their main outline, however, the experiments are quite decisive, and give the somewhat surprising result that the concentration of the hormone can be lowered very considerably without necessarily inhibiting ovulation. Our experiments on hormone concentration and on the use of Chicago blue as an anti-coagulant, suggest that it will be possible to carry out cross-circulations for the required time. A variety of purely technical difficulties have arisen, especially diffuse haemorrhage from cut surfaces, but preparations have already been maintained for more than 9 hours. The present paper is concerned solely with the effects of altering the concentration of the ovulation-producing hormone in single rabbits. II. METHODS AND MATERIAL. CEstrous rabbits were obtained as previously described and a preliminary laparotomy performed to ascertain the number of ripe follicles. Anaesthesia was induced as before. Calculation of the total blood volume. Before a definite proportion of the blood could be removed it was necessary to calculate the total blood volume of the animal. So far as could be ascertained the most complete data on blood volumes are those of Dreyer and Ray [1910] who give a curve for the blood volume of rabbits according to weight. This curve was used throughout our experiments. Calculation of the amount of blood removed. It was clearly undesirable that an appreciable percentage of the total blood of an animal should be removed at once. It was decided, therefore, to obtain the required amount in steps; fluid equal to 5 p.c. of the original blood volume being removed at a time and a similar quantity of gum saline replaced immediately. In an animal with a blood volume of 100 c.c., therefore, 5 c.c. would be removed each time and 5 c.c. gum saline replaced. The first 5 c.c. removed would be undiluted blood, but the second 5 c.c. would be 5 x c.c. blood plus 5 x c.c. gum saline. Thus, after the second removal, c.c. original blood would remain in the animal. The third 5 c.c. would only contain 5 x x c.c. blood, leaving c.c. still in the animal. The amount of blood remaining after each removal and replacement by gum saline is shown in Fig. 1. To remove 50 p.c. of the blood it was therefore necessary to remove

3 OVULATION-PRODUCING HORMONE. 5 p.c. of the circulating fluid and replace with gum saline thirteen and a half times; to remove 40 p.c. to make ten removals, etc. Bleeding technique. The ripe follicles were counted and one ovary or the other extirpated, according to the proportion of follicles it was desired to remove. Bleeding was then carried out from a carotid, gum saline being run into the opposite jugular immediately after each removal of blood. No cannula was put into the carotid: an inch or more k 80\ 0 70.i Number of removals Amount of original blood remaining after successive removals Fig. 1. of 5 p.c. of the circulating fluid and replacement with gum saline. of the artery was dissected out and severed, so that the stream of blood could easily be directed into the measuring cylinder. Control experiment. As a control on the possibility of hsmorrhage affecting the occurrence of ovulation, an animal was given an injection of the ovulation-producing substance (3 c.c. urine of pregnancy) immediately after the replacement of 50 p.c. of the blood by gum saline, both ovaries being left in. Ovulation took place at about the normal time, showing that this amount of bleeding is not incompatible with ovulation provided that an adequate amount of hormone is available.

4 176 F. W. ROGERS BRAMBBLL AND A. S. PARKES. III. EXPERIMENTAL RESULTS. It is evident that the technique described above is subject to a number of variable factors. Chief among these would appear to be individual variation in (a) the amount of surplus hormone, (b) the general reaction to the bleeding process, (c) the exact time after copulation at which the ovulation-producing hormone reaches its maximum concentration in the blood, and (d) the proportion of ripe follicles which would have ovulated without treatment. Also, difficulty was experienced in some animals in determining the number of mature follicles. In these circumstances no great coherence could be expected in the results, but it is possible to arrive at certain conclusions from the thirty satisfactory TABLE I. Effect of altering the concentration and the absolute amount available per follicle of the ovulation-producing hormone after copulation in the rabbit. Percentage Absolute blood left Ripe follicles amount in, i.e. con-, of hor- centration No. of Total Number mone per of hormone animal number left in follicle p.c. Result CCB follicles ovulated CCB , CCB No ovulation CCB No ovulation CCB follicle ovulated CCB follicles ovulated CCB No ovulation CCB ,, CCB ,, CCB follicle ovulated CCB follicle ovulated CCB No ovulation CCB No ovulation CCB Pt CCB ,, CCB follicles ovulated CCB No ovulation CCB CCB CCB follicle ovulated CCB No ovulation CCB CCB CCB CCB CCB CCB follicle ovulated CCB follicles ovulated CCB No ovulation CCB follicle ovulated experiments performed. The details are recorded in Table I and a summary in Table II. The following conclusions may be drawn:

5 OVULATION-PRODUCING HORMONE. (a) Reduction of the concentration of the hormone down to 70 p.c. does not necessarily inhibit ovulation, even when the absolute amount of hormone per follicle is also reduced. CCB 22, for instance, ovulated eleven follicles with both the concentration and the absolute amount of the hormone lowered to 0-7 normal. (b) Reduction of the concentration to 60 p.c. of the normal or less does, however, seem to inhibit ovulation under the conditions obtaining, unless the absolute amount of hormone available per follicle is raised by the removal of a large proportion of the original mature follicles. The second of these conclusions might be explained on the grounds that: (a) the rate at which a follicle could withdraw the hormone from the blood would be proportional to its concentration; (b) the more follicles present, the more rapidly would the initial concentration sink to a level at which withdrawal would be too slow to permit of ovulation in the required time. Therefore, with a low initial concentration, the absolute amoant available per follicle must be greater than normal so that the concentration does not sink prematurely below the effective point. TABLz II. Concentration of hormone 70 p.c. normal or more 60 p.c. normal or less Absolute,, amount per No. of No. No. of No. follicle experiments ovulating experiments ovulating Less than normal Normal More than normal Total As regards cross-circulation experiments, the non-copulated rabbit must be supposed to have a certain amount of the hormone in the blood, and therefore anastomosis will not result in a concentration as low as one-half that originally present in the copulated animal. Nevertheless, it is probable that care will have to be taken to reduce the number of follicles present. IV. SUMMARY. 1. The concentration of the ovulation-producing hormone in the circulation of the rabbit after copulation was altered by the removal of varying amounts of blood and replacement with gum saline. The absolute amount of hormone available per follicle was adjusted by the removal of a varying proportion of the ripe follicles. 2. Reduction of the concentration by up to 30 p.c. does not necessarily

6 178 F. W. ROGERS BRAMBELL AND A. S. PARKES. inhibit ovulation, even when the absolute amount per follicle is also allowed to decrease coincidently. 3. Reduction of the concentration by 40 p.c. or more inhibits ovulation under the conditions obtaining, unless the absolute amount per follicle is raised by the removal of a large proportion of the original ripe follicles. The expenses of the work were defrayed from a grant to F.W.R.B. from the Government Grants Committee of the Royal Society. REFERENCES. Brambell, F. W. R. and Parkes, A. S. (1932). J. Physiol. 74, 65. Dreyer, G. and Ray, W. (1910). Phil. Tran8. B, 201, 133. Fee, A. R. and Parkes, A. S. (1929). J. Physiol. 67, 383.