University College, London.)

Transcription

1 6I2.62I:6I2.492 STUDIES ON OVULATION. I. The relation of the anterior pituitary body to ovulation in the rabbit. BY A. R. FEE AND A. S. PARKES (Beit Memorial Research Fellows). (From the Department of Physiology and Biochemistry, University College, London.) I. INTRODUCTION. The relation between the anterior pituitary and the ovary. While working on the growth promoting factor of the anterior pituitary body Evans and co-workers (1, 2) found that sodium hydroxide extracts of ox anterior lobe brought about complete cessation of the cestrous cycle in the rat. This effect was due to the luteinization of the epithelium of all the larger follicles without the intermediate act of ovulation. As a result of this the ovaries of treated animals came to consist almost exclusively of vast numbers of atretic corpora lutea. The presence of this luteal tissue was apparently responsible for the inhibition of oestrus. Subsequently Smith and Engle6 and Zondek and Ascheim(s) demonstrated an entirely opposite effect of anterior pituitary substance. These authors were able to produce the normal maturation and ovulation of large numbers of follicles in both the immature and adult mouse by daily intramuscular implantations. In the former the precocious occurrence of cestrous symptoms coincided with the premature ovulation. Both these types of reaction have been obtained by other workers, but it is uncertain as yet whether one or more anterior pituitary substances are concerned in the regulation of the ovarian cycle. It is quite evident, however, that ovarian periodicity is to some extent dependent upon the anterior pituitary body. The only observations on the effect of hypophysectomy on the cestrous cycle appear to be those of Smith (5) who found ancestrus in the rat after the operation. Most of the work on this problem has been done on the rat and mouse, but peculiarities in the reproductive processes of the rabbit seem to make this animal very suitable for such work.

2 384 A. R. FEE AND A. S. PARKES. Ovulation in the rabbit. It was long ago shown by Heap e (4) that ovulation in the rabbit only takes place after copulation, and this observation has been frequently confirmed. In the absence of the male cestrus persists through the entire breeding season and the only cycle found is the alternation of cestrus and ancestrus. The exact mechanism whereby the act of copulation causes ovulation to occur within 10 to 14 hours (2) has never been adequately worked out, but in view of the work mentioned above it would seem that the pituitary body must be concerned, and that the effect of copulation is not exerted directly upon the ovary by nervous or other means. It is still necessary, however, to suppose that the immediate effect of copulation is nervous (artificial insemination does not result in ovulation (3)) and the action is presumably exerted upon the anterior pituitary body. On such a view ovulation in the rabbit depends upon two consecutive events, the first of which is nervous and the second endocrine. The experiments which are recorded here deal with the effect on ovulation of removing the anterior pituitary body at different intervals after copulation. II. METHOD AND MATERIALS. Animals. Mature rabbits were bought direct from dealers and isolated for at least a month before any attempt was made to use them. By this means the difficulty of their previous history being unknown was overcome and a good supply of animals in cestrus obtained. Nevertheless, a few animals which later proved to be immature or pseudopregnant were used. These, however, were always detectable from examination of the ovaries, uterus and mammary glands and are omitted from the experimental records. Operative technique. The fact that the final maturation of the follicle is comparatively rapid and that the time of ovulation can be calculated to within an hour or two suggested that the problem could be treated by means of acute experiments. The method finally adopted to remove the pituitary body was as follows. The animal was placed under deep chloroform anbesthesia, the carotid arteries and external jugular veins ligatured and a tracheal cannula inserted. The skin of the head was then removed in the region of the thyroid cartilage around to the base of the ear on both sides and the anterior part of the head removed with a hacksaw; the cut being made immediately behind the temporo-mandibular articulation to a point just posterior to the eyes. Hemorrhage was arrested during the operation by compression on the vertebral arteries and later by the application of hot saline swabs. Such a cut transects

3 STUDIES ON OVULATION. 385 the brain immediately posterior to the optic chiasma and anterior to the pituitary fossa, removing all of the frontal lobes and part of the temporal and parietal regions. After practice this operation can be done with little or no hemorrhage and the level at which the brain is transected determined quite accurately. Such animals will live for upwards of 24 hours provided that an anesthetic dose of urethane is given every 4 hours to inhibit the reflex movements characteristic of the decerebrate rabbit. Temperature must also be regulated to within to C. The ultimate cause of death in undisturbed preparations was either circulatory failure resulting from sepsis or else secondary haemorrhage. This technique has, for our purpose, several distinct advantages over the usual trephine method employed in brain operations. In the first place owing to the small size of the rabbit's skull it is very difficult to ensure both a rapid and complete removal of the pituitary body when approached in the usual manner; the partial decerebration method was only used because it afforded an easy route to the pituitary body and made the severity of operation practically the same in the control and hypophysectomized animals. To effect hypophysectomy the anterior wall of the pituitary fossa was broken down and the pituitary body shelled out. Usually it was possible to remove all of the gland in a single piece. In earlier experiments the ovaries were exposed before the calculated time of ovulation in order that continuous observation could be maintained, and in a few animals the ovaries were removed at different times to provide histological material at two stages from the same animal. The ovaries, together with portions of the uterus and vagina, were fixed in Bouin's fluid and the sections stained with hwemotoxylin and eosin. In the earlier experiments the ovaries were fixed and imbedded whole, but later, to avoid the labour of cutting complete serial sections, only the essential portions of ovaries were imbedded. Complete serial sections were cut through the selected tissue from critical experiments. III. EXPERIMENTAL RESULTS. Unoperated animals. A number of initial observations were made on- normal animals anesthetized about the time of ovulation. The ovulation time found in these was essentially the same as that recorded by Walton and Hammond(7) (see Table I). Although copulation will sometimes take place during pregnancy or pseudopregnancy we have failed, in common with other workers, to find ovulation at this time.

4 386 A. R. FEE AND A. S. PARKES. TABLE I. Ovulation after copulation in the cestrous rabbit. Time of killing after copulation No. of animal hr. mi. Time of ovulation State of ovary at death APO Large follicles. No ovulation APO ? Ovulated APO : 00 to 11: 45 Young corpora lutea Operated animals with anterior pituitary intact. Seven animals are available which survived partial decerebration for 12 or more hours after copulation. The results are summarized in Table II. TABLE II. Ovulation with anterior pituitary intact. Time of decerebration after Duration of No. of copulation Time of experiment State of ovaries at animal hr. min. ovulation hr. min. death APO Follicles hemorrhagic APO : 05-17: Young corpora lutea APO : 00-11: Pt.. APO : ,... (or earlier) APO :35-12: APO :20-12: APO : 10-11: From these results the following conclusions may be drawn: (a) No operative effect can be observed on the occurrence or the time of ovulation. (b) The involution of the ruptured follicle and the formation of the young corpus luteum is normal. (c) Histological observations failed to reveal any abnormality in the final maturation and rupture of the follicle (see Plate I, figs. a, b, and d). The secretion of secondary liquor folliculi, the separation of the discus proligerus and the hiemorrhage in the follicle wall just previous to its rupture, all proceeded normally. Operated animals with the anterior pituitary removed. Ten animals are available which lived beyond the normal ovulation time with the anterior pituitary removed. The results are summarized in Table III. The following conclusions may be drawn from these experiments: (a) Hypophysectomy within the first hour after copulation inhibits rupture of the follicle for at least 16 hours beyond the normal ovulation time and probably permanently. (b) Hypophysectomy later than the first hour after copulation does not appear to influence the occurrence or time of ovulation.

5 STUDIES ON OVULATION. 387 TABLE III. Ovulation with anterior pituitary removed. Time of decerebration after Duration of copulation experiment State of ovaries No. of animal hr. min. hr. min. at death APO No ovulation APO VP APO ,, APO APO APO APO Ovulated APO ,, (at 12 hr. 05mmin.) APO APO (c) Histological examination suggests that where ovulation occurred the corpora lutea (presumably owing to the absence of the anterior pituitary body) did not show normal development. We hope to investigate this aspect of the problem in more detail later. (d) Histological examination of those follicles which did not ovulate showed that atretic changes occur after the time when ovulation should have taken place (see Plate I, fig. c). These changes consist of general shrinkage, resulting in crumpliug of the follicular wall, and in disintegration of the membrana granulosa and of the corona radiata, together with vacuolar degeneration in the ovum. The changes in the follicular epithelium are of the same nature as those which precede normal ovulation, but they are carried much further. We have satisfied ourselves that the extensive degeneration observed in these experiments is neither of post-mortem origin nor due to inferior histological technique. The ultimate fate of the degenerating follicles has not been observed owing to the limitations of our operative technique. IV. DIscussION. It will be noticed that although both the anterior and posterior lobes of the pituitary body were removed, it has been assumed that the anterior lobe was responsible for the differences observed between the control and experimental animals. This could be objected to on the grounds that removal of the posterior lobe might cause circulatory changes sufficient to inhibit ovulation. No such changes, however, could be detected by observation of the heart rate and general arterial blood-pressure; nor was there any appreciable difference in the appearance of the ovaries and viscera of the control and hypophysectomized animals. It is too early to discuss the bearing of these experiments on the

6 388 A. R. FEE AND A. S. PARKES. problem of whether one or more substances are secreted by the anterior pituitary body for the regulation of the ovarian cycle, but it may be pointed out that copulation must immediately cause either an increased secretion of the substance concerned in the initial maturation of the follicle or the secretion of another substance concerned solely with the final stages of maturation and the luteinization of the follicular epithelium. V. SUMMARY. 1. Hypophysectomy in the rabbit within 1 hour after copulation inhibits ovulation, which would normally take place 10 to 12 hours later. 2. Hypophysectomy later than 1 hour after copulation has no such inhibitory effect, although the subsequent development of the corpora lutea appears to be abnormal. 3. Mature follicles which fail to ovulate owing to early hypophysectomy begin to undergo atresia after the normal ovulation time. 4. In view of previous work on the relation between the pituitary body and the ovary these effects may be attributed to the removal of the anterior lobe. Our best thanks are due to Prof. J. P. Hill for allowing us histological facilities. The expenses of this research were defrayed by a grant to one of us (A.S.P.) from the Medical Research Council. REFERENCES. 1. Evans. Harvey Lectures, 19. p Evans and Long. Anat. Rec. 21. p Hammond. Reproduction in the Rabbit. Edinburgh Heape. Proc. Roy. Soc. B, 76. p Smith. Anat. Rec. 32. p Smith and Engle. Amer. Journ. Anat. 40. p Walton and Hammond. Journ. Exp. Biol. 6. p Zondek and Ascheim. Arch. f. Gyn p

7 THE JOURNAL OF PHYSIOLOGY, VOL. LXVII, No. 4 PLATE I I W, i, t.t T.I. Guide letters: C.R. Corona radiata; D.P. Discus proligerus; G.E. Germinal epithelium; H. Haemorrhage; M.G. Membrana granulosa; 0. Ovum; T. E. Theca externa; T.I Theca intema. Fig. a. Mature follicle (APO 28) at time of copulation, showing relation to surface of ovary. Fig. b. Follicle about to rupture (APO 22) 12 hours after copulation, showing hiemorrhage at point where rupture will take place. Pituitary body intact. Fig. c. Follicle 22 hours after copulation (APO 42). Ovulation inhibited by removal of pituitary body. Note degenerative changes. % Fig. d. Youngf corpus luteum 16i hours after copulation (APO 17). Pituitary body intact.