(6, 7, 8, 9). cycle, a result in agreement with the experiments of PAPANICOLAOU and

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1 THE NATURE OF THE ANCESTROUS CONDITION RESULTING FROM VITAMIN B DEFICIENCY. By A. S. PARKES, Beit Memorial Research Fellow. From the Department of Physiology and Biochemistry, University College, London. (Received for publication 19th March 1928.) THE systematic investigation of the effects of dietary deficiencies on mammalian reproduction, and particularly on the cestrous cycle, was initiated in 1922 by EVANS and BISHOP (1) with the aid of the vaginal smear technique. They showed that under-feeding of rats with an otherwise complete diet resulted in general aberration of the ovulation cycle, a result in agreement with the experiments of PAPANICOLAOU and STOCKARD (2); they further found disturbance of the cycle on unbalanced diets, and on diets deficient in salts. On diets deficient in vitamin A, rats showed abnormally prolonged cestrous symptoms, whereas the absence of vitamin B resulted in a complete cessation of the cestrous cycle. Vitamin B deficiency has since been shown (PARKES and DRUMMOND (3)) to produce degenerative changes in the testis. These effects of dietary deficiency are of interest in relation to another group of facts, which seem to suggest that the onset of the cestrous cycle at puberty is brought about by certain substances in the soma, hitherto absent or used elsewhere, becoming available for the use of the ovaries. In other words, the somatic and not the ovarian age determines the appearance of puberty. Two well-established facts point to this conclusion: 1. Ovaries from young, immature animals when grafted into adult males or females become mature (4, 5, 6) long before they would have done in their original environment. 2. The number of follicles maturing at each ovulation is approximately constant whether the animal possesses (experimentally) three ovaries, two ovaries, one ovary, or only a fragment of ovarian tissue (6, 7, 8, 9). The ancestrous condition observed in vitamin B deficiency may be due, therefore, to the disappearance or non-availability of these somatic substances necessary for ovarian function. It is possible, however, that though the cestrous stimulus may become operative, the reproductive organs are incapable of responding. An obvious method for differentiating between these alternatives would be the injection of cestrin VOL. XVIII., NO

2 398 Parkes during the deficiency-ancestrus to ascertain if it is possible to induce cestrus in the vitamin-b-deficient animal. Such experiments are described below. Method8 aind Material.-As in previous experiments variations in the degree of deficiency were obtained by varying the percentage of a yeast extract added to a basal, vitamin-b-free diet. This basal diet consisted of: Extracted caseinogen per cent. Extracted rice starch Salt mixture Cod liver oil Hardened vegetable oil..,10 This basal diet was converted into graduated B-deficiency diets by the addition of 0 per cent., 0 5 per cent., 1 per cent., 2 per cent., 3 per cent., or 4 per cent. of yeast extract, the latter being sufficient for normal growth and reproduction. In order to serve as a guide to the degree of deficiency required to stop the cycle, a batch of fourteen rats of known cestrous history was put on diet for an initial test, and as a result of experimenting with various yeast extract percentages, conclusions were arrived at which enabled a further batch of animals to be reduced to a condition of ancestrus in a very short time. Vaginal smears were taken daily, and an accurate check was kept on the cestrous histories of the animals. Where it was desired to follow the ovarian cycle, vital staining by subcutaneous injection of trypan blue (1.0 c.c. of 1 per cent. aqueous solution) was resorted to. Lack of stained corpora lutea after injection was considered to be evidence of ovarian inactivity after the injection. Serial sections were, however, made of all ovaries. Experimental Results.-The first fourteen animals were examined for fifty days, and their normality was adequately demonstrated; they were then put on to basal diet+1 per cent. of yeast extract (B+1). After twenty-two days on this dietary the weight had not appreciably changed, and the cestrous cycle was unaltered. They were then put on to the basal diet only, with the result that the cestrous cycle soon stopped in all of them. The stoppage of the cycle occurred so quickly after the change that it appeared possible that it was not due to it. The animals were therefore put back on to B +0 5 diet, but still there was no spontaneous cestrus. After this induced ancestrus had lasted for some time, certain of the animals (OB 1, 3, 6, 8, 10, 14) were injected with cestrin (ten mouse-units). Of these six animals, two (OB 6 and 10) died before any reaction could have appeared, but the remaining four showed all the vaginal symptoms of cestrus. Two of these (OB 1 and 14) were subsequently injected a second time and showed a similar reaction.

3 Ancestrous Condition Resulting from Vitamin B Deficiency The histories of these animals are summarised in the following table: Average length of cycle. Length of Total length No. of No. of On deficiency ancestrus of ano2strus induced animal. On complete animal diet dficiecy before beore before iducedbefore induced autopsy death csru pestrous dietary. onset of estrus. (days). periods. ancestrus. 399 OB OB Not injected 21 OB OB *4 Not injected 19 OB Not injected 23 OB OB Not injected 20 OB 8 4* OB Not injected 21 OB OB Not injected 34 OB 12 3*9 11*0 Not injected 35 OB Not injected 28 OB ' We may conclude that: (a) The cestrous cycle sooner or later stops when rats are subjected to vitamin B deficiency represented by the addition of not more than 1 per cent. of yeast extract to the basic diet. Vital staining and histological examination of the ovaries show that the ovarian cycle also ceases. Only old corpora lutea and atretic follicles were present. (b) The cessation is abrupt-that is to say, the cycle is normal until it stops altogether, no sign of a gradually lengthening dicestrus being found on the deficiency diets. (c) Adjustment of the degree of B deficiency failed to produce a state where ancestrus was found, but where life was prolonged indefinitely. (d) The ancestrus was therefore normally terminated by death. (e) The time elapsing between the disappearance of spontaneous cestrus and death (col. 5) was, however, very variable. (f ) Injection of cestrin during the ancestrous period resulted in the appearance of cestrous symptoms in the accessory organs of reproduction. The ovaries of injected animals were not detectably different from those of the uninjected animals, so that no stimulating effect appears to have been exerted. A further batch of eleven animals was next put on a deficiency diet with a view to investigating further (a) the reaction of the animals in artificial ancestrus to cestrin injections, and (b) the relation of the onset

4 400 Parkes of ancestrus to the fatal decline in weight. They were put straight on to B +1 diet, and left on this so as to maintain a constant degree of deficiency. After a series of normal cycles ancestrus appeared, and after this had lasted for some time, 10 m.u. of cestrin was injected into each. All the animals except one responded by showing cornification of the vagina, but it was shown by vital staining immediately before injection and also by subsequent histological examination that ovula- /6 U) I$.,t' XD, D/A-us c Norrna/zAr.us /i,dwcaa' 2s/ra,s t/t '1Clb"n of.xsfr#, Q Deofh Isl /Od0oys 0or/io,17/0/scICl =N: tion did not take place at the induced oestrous periods. The growth curves of eight of these animals are shown in the accompanying diagram, from the time when dieting commenced to death or killing. Incorporated with these curves are the cestrous histories. From this diagram it will be seen that the cessation of the cestrous cycle occurs in most cases immediately before the fatal decline in weight begins. Thus (a) the induction of cestrous symptoms in the accessory organs of reproduction is not paralleled by the appearance of ovarian cestrous changes, i.e. by ovulation, and (b) the cessation of spontaneous cestrus occurs immediately before the decline in body weight sets in. My thanks are due to Professor J. C. DRUMMOND for advice regarding

5 Ancestrous Condition Resulting from Vitamin B Deficiency the diets, and to the Medical Research Council for grants for material and apparatus. SUMMARY A deficiency of vitamin B, represented by 1 per cent. or less of yeast extract added to a basal diet, brings about an abrupt cessation of the cestrous cycle in rats in about four weeks. 2. The ancestrus so produced, which begins just before the characteristic decline in weight appears, applies both to ovarian and extraovarian phenomena of cestrus, and is terminated at any time up to two months later by the death of the animal. 3. Injection of cestrin (10 m.u.) during the ancestrous state res-ults in the induction of cestrous symptoms in the accessory organs, without, however, causing the ovary, now much atrophied, to ovulate. 4. It is concluded that ancestrus due to this deficiency results not from inability of the animal to respond to the cestrus-producing stimulus, but from failure to produce cestrin. BIBLIOGRAPHY. (1) EVANS and BISHOP, Journ. Metab. Res., 1922, i (2) PAPANICOLAOU and STOCKARD, Proc. Soc. Exper. Biol. Med., 1920, xvii (3) PARKES and DRUMMOND, Proc. Roy. Soc., 1925, B, xiii. 98, 147. (4) LIPsCHtTZ, Compt. rend. soc. biol., 1925, xciii (5) LONG and EVANS, Mem. Univ. Calif., 1922, No. 5. (6) HAMMOND, " Reproduction in the Rabbit," Edinburgh, (7) ARAI, Amer. Journ. Anat., 1920, xxviii. 59. (8) CARMICHAEL and MARSHALL, Journ. Phys., 1908, xxxvi (9) LIPSCHIUTZ, Brit. Journ. Exper. Biol., 1925, ii PRINTED IN GREAT BRITAIN BY NEILL AND CO., LTD., EDINBURGH.