successfully induced an increase in size of the ovaries, due to an markedly in other species investigated. There has nevertheless been

Transcription

1 : THE REACTION OF FEMALE MONKEYS TO VARIOUS TYPES OF GONADOTROPIC EXTRACTS. By P. DE FREMERY. From N.V. Organon Laboratories, Oss. (Received for publication 4th January 1939.) HISTORICAL. IN 1921, Houssay pointed out that one of the difficulties in the investigation of pituitary extracts was the variation in the response from species to species. Since that time, our great increase of knowledge about the pituitary gland has chiefly been derived from the observation of common laboratory rodents, rats and mice, and the responses have often differed markedly in other species investigated. There has nevertheless been a tendency to regard these reactions of mice or rats as a standard, valid for all mammals, including man, and to make tentative clinical prognoses accordingly. Clearly, attention should be concentrated on species more closely related to man. Already most of the experiments on monkeys have shown that their reaction to gonadotropic hormones is widely different from that of the mouse or rat. With pituitary gonadotropic extracts in immature Macaca mulatta, Hisaw, Fevold, and Leonard [1931] successfully induced an increase in size of the ovaries, due to an enormous enlargement of follicles with no trace of luteinisation, withdrawal of treatment being followed by menstruation; these observations have been confirmed [Saiki, 1932; Engle, 1933]. The gonadotropic principle of human pregnancy urine is inactive in the immature female of this species [Engle, 1932, 1934; Johnson, 1935; Hamburger, 1936], though active in the male since it induces descent of the testes and growth of the accessory sex organs, more especially the seminal vesicles [Engle, 1932 b]. But in Macaca ecaudatus Courrier and Gros found that urinary extracts were active in the immature female as well as in the male. The activity of the urinary hormone in humans is fully established for the male, as shown by descent of the testes in numerous case-series of unilateral or bilateral cryptorchidism. But it remains doubtful whether and to what extent it can induce ripening and luteinisation of the follicles in women: the literature is contradictory [Westman, 1935; Stockl, 1934; Rock, 1935; Anselmino and Hoffman, 1935; Hamblen and Ross, 1936; Ross, 1937]. 203

2 204 de Fremery EXPERIMENTAL. In most of our experiments we worked with immature Macaca mulatta of less than 2 kg. body-weight, in a few with immature Macaca irus. Injection in every instance was performed intramuscularly every day for 8 days, the animals being killed 24 hours after the last injection of the series. Gonadotropic extracts from the following sources were tested:- 1. Human pregnancy urine. 2. Hog anterior pituitary. 3. Human anterior pituitary. 4. Human placenta. 5. Human menopausal urine. (a) Gonadotropic Hormone from Pregnancy Urine. The inactivity of this material in the immature female Macaca mulatta was confirmed. The highest dose used was 1000 Janssen- Fic. 1. Ovary of monkey No. 8, receiving 13,000 R.U. of pregnancy urine extract (Pregnyl) in 8 days. Loeser rat units,1 daily during 13 days (Pregnyl, Organon Laboratories). No external phenomena due to increased ovarian activity were observed; at autopsy the sexual tract was infantile (No. 8 in table; fig. 1), presenting no histological indication of increased hormone 1 The preparations were standardised in our laboratory. The minimal amount of material, injected subcutaneously in infantile female rats and producing cestrus in 50 per cent. of the animals, was considered as a unit. Six injections were given divided over three days, and vaginal smears taken 84, 100, and 108 hours after the first injection.

3 The Reaction of Female Monkeys to Gonadotropic Extracts 205 production, and the ovary was comparable in every respect to that of an untreated control. Repetition of the experiment (8 injections of 1000 R.U.) showed a similar absence of response in Macaca irus (No. 19, see table). An immature male Macaca mulatta responded to the same dosage and conditions by testicular enlargement and descent, and enlargement of the accessories, especially the seminal vesicles. Histologically, the interstitial tissue appeared to have been actively stimulated, but there was no sign of spermatogenesis. (b) Gonadotropic Hormone from Hog Pituitaries. The activity of mammalian pituitary extracts recorded by others was confirmed. An extract of hog pituitary was used (Ambinon, Organon Laboratories) containing the thyrotropic hormone and both gonadotropic principles (follicle-stimulating and luteinising); 0-1 ml. of this preparation gave ovaries of an average weight of 20 mg. in immature rats. An immature Macaca mulatta receiving 1.0 ml. daily, developed faint reddening of the sexual skin on the 3rd day and complete external FiG. 2.-Ovary of monkey No. 22, receiving 8 c.c. of hog pituitary extract (Ambinon) in 8 days. sign of cestrus on the 5th, with simultaneous enlargement of the mammary glands and nipples. Autopsy showed an enormous development of the genital tract (XNo. 2 in table) and ovaries which contained numerous follicular cysts, some filled with blood, but no detectable trace of luteinisation or ovulation (fig. 2). Repetition of the experiment

4 206 de Fremery showed an exactly similar response in Macaca irus (No. 20 in table). Another animal, treated in the same way with the same pituitary preparation, came to autopsy 5 days after the last injection was given. During this time menstruation occurred, but no traces of luteinisation were found in the ovaries. Since it induced follicle ripening exclusively in these monkeys, the influence of hog pituitary extract appeared to be similar to that of pure follicle-stimulating hormone, though our standardisation of the extract in rats had suggested that luteinising factor was present as well. Histological examination of the uterus, tubes, vagina, and mammary glands confirmed the existence of changes due to cestrogenic hormone and the absence of any due to progesterone. Clearly, therefore, the stimulated ovaries produced cestrogenic hormone exclusively. The thyrotropic component of the extract had induced an intense hyperplasia of the thyroid gland; but the thyroid activity so stimulated is scarcely likely to account for the difference in reaction between the hypophyseal and the urinary extract. Hamburger states that gonadotropic extracts of pregnant mare's serum, containing no trace of thyrotropic hormone, have produced ovarian changes in monkeys precisely similar to those induced by pituitary extracts. It might be contended that the response to hog pituitary extract was incomplete because of insufficient dosage. The technique of administration may also influence the result, as can be seen in the immature rat: in this species the response to amounts which produce exclusively follicle ripening may be augmented and altered by subdivision of the dose or by the addition of substances such as ZnSO4, which retard hormonic absorption [Maxwell, 1934]. The activity of hog's pituitary extract in rats is as much as 25 to 40 times greater after the addition of 5 mg. of ZnSO4. An attempt was made to raise the activity of this extract in the female monkey by applying the Maxwell reaction, but the result was negative, since the response of Macaca mulatta to 1 c.c. hog pituitary extract plus 10 mg. ZnSO4 (No. 4 in table) was exactly similar to that obtained by injection of the extract alone. (c) Combination of the Hormones present in Human Urine of Pregnancy and Hog Anterior Pituitary Gland. Several investigators who claim to have separated the follicleripening and luteinising factors of pituitary gonadotropic extracts have stated that the activity of the luteinisor in the rat depends entirely on the preliminary activity of the follicle stimulator. We therefore considered the possibility that the luteinising factor of the urinary gonadotropic extract which is active in rats might also be active in monkeys, provided the follicle stimulator were present in adequate quantity.

5 The Reaction of Female Monkeys to Gonadotropic Extracts 207 An immature female Macaca mulatta received 1 c.c. of hog's pituitary extract and 200 R.U. of urinary extract daily for 8 days. At autopsy on the 9th day, the sexual organs showed an exactly similar degree of stimulation to that obtained by hog pituitary extract alone. The synergistic reaction of Evans, whereby the activity of hog's pituitary extract in the immature rat becomes about 50 times greater, is thus absent in the infantile Macaca mulatta (No. 3 in table). In a further experiment, 2 animals received 1 c.c. of hog pituitary extract daily for 8 days, and the development of external cestrous phenomena proved that both subjects had reacted well to the hormone and that enlarged follicles were present; conditions for the artificial induction of luteinisation therefore seemed favourable. After this introductory treatment, one of the animals was kept as a control and the other received 1000 R.U. of the urinary extract for 5 days. In each instance swelling of the sexual skin regressed rapidly, and bleeding occurred from the vagina. At autopsy, on the day after the last injection of urinary extract, no significant difference was found between the animals; both showed regression of the enlarged ovarian follicles with absence of any trace of luteinisation, but many young secondary follicles, of which a large number contained several eggs (maximum 8), were present. The bleeding was of pseudo-menstrual type, occurring from an endometrium in the proliferative phase. It was thus not possible to demonstrate any activity of urinary gonadotropic principle in immature Macaca mulatta in these combined experiments. (d) Gonadotropic Extracts from Human Pituitary Gland. Numerous observations have established the non-identity of the gonadotropic substance in the anterior pituitary with that in the urine of pregnancy, and there are also differences between the extracts of the pituitary glands of various species. de Fremery and Scheygrond [1937] found that the reaction of the immature female rat to extracts of human pituitary differs considerably from that induced by pituitary extracts of animal origin: these authors obtained neither the Maxwell reaction nor a synergistic effect with human material. Evidently gonadotropic extracts of human pituitary and human pregnancy urine are more similar than those of human and hog pituitary. It therefore seemed of interest to determine the activity of the human pituitary hormone in monkeys. An immature Macaca mulatta was given 14 mg. of desiccated gland daily for 8 days, and whereas urinary gonadotropic extract had proved inactive in much higher unitage, this daily dose (75 Janssen- Loeser R.U.) of human pituitary extract caused reddening of the sexual skin after 3 days and development of the external phenomena of full cestrus by the 5th day. At autopsy, on the day after the last VOL. XXIX., NO

6 208 de Fremery injection, all the genital organs were found to have undergone a marked increase in size, especially the ovaries (fig. 3; No. 16 in table), and histological examination showed that the changes were exactly similar I, ). f FiG. 3. Ovary of rnonkey No. 16, receixving 600 R.U. of hium-ani pituitary extract in 5 days. to those produced by hog's pituitary extract, namely, cystic follicles with no trace of luteinisation. This experiment shows clearly that the gonadotropic principles of the human pituitary and of human pregnancy urine are not identical. When the experiment was repeated with an immature female Macaca irus instead of ]llacaca mulatta, an exactly similar response was obtained (No. 20 in table). (e) Gfonadotropic Hormone from Human Placenta. In the rat, similar reactions are produced by the gonadotropic hormones of human placenta and of human pregnancy urine; but pituitary extracts of human origin fail to s aowthe Maxwell and the synergistic reactions, which typically occur with similar extracts of mammalian origin. We thought it would be interesting to see the effect of the human placental hormone in the monkey. This experiment was performed exclusively on Macaca irurs owing to a scarcity of Macaca mulatta. Two immature females. therefore. received 450 Janssen-Loeser R.U. daily for 8 days. At autopsy, no reaction was found (Nos. 20 and 25 in table). This negative result points to an identity of the gonadotropic principles present in the human placenta and in human urine.

7 The Reaction of Female Monkeys to Gonadotropic Extracts 209 In an older specimen of the same species, which menstruated during treatment (daily dose 125 R.U.), one ovary contained a corpus luteum, and the bleeding was found to have occurred from a fully developed endometrium, indicating true menstruation. Apparently the gonadotropic placental extract had not disturbed the normal cycle (No. 18 in table). (f) Gonadotropic Hormone from Human Menopausal Urine. A gonadotropic substance is detectable in the urine of women at the menopause, and preparations made from such material induce ripening and luteinisation of the ovarian follicles in the immature rat. The characteristic effects of this substance should, according to several authors, correspond with those of pituitary extracts and differ from those of extracts of human pregnancy urine. One immature female Macaca mulatta received the highest tolerated dosage of a moderately purified preparation of this kind, 100 R.U. daily for 8 days (No. 24 in table). The response was positive, as might be expected from previous experience with the rat; but weak, in accordance with the low unitage (fig. 4). FIG. 4.-Ovary of monkey No. 24, receiving 800 R.U. of menopausal urine extract in 8 days. Thus, non-identity of the gonadotropic substance excreted in small amounts at the menopause with that excreted in large amounts in pregnancy was easily demonstrated by using the immature monkey as the test animal. (g) Effect of Antihormones in Monkeys. Bachman, Collip, and Selye [1934] found that when an animal had been subjected to gonadotropic stimulation continuously for several

8 210 de Fremery months a refractory condition set in, and the blood serum acquired the capacity to inhibit the activity of gonadotropic hormone of similar origin in other animals. Rowlands and Parkes [1937] treated a goat for a long time with urinary gonadotropic extract (pregnyl) and obtained an antigonadotropic serum. They then injected immature female rats with the serum and an extract of human pituitary gland simultaneously. There was a total inhibition of the gonadotropic activity of the human pituitary extract. Extracts from the pituitary glands of other species were not affected. Sera prepared by Wolfe and Meyer [1937] and by Gustus, Meyer, and Dingle [1935] from monkeys that had been receiving injections of pregnant mares' serum for several weeks, inhibited the gonadotropic activity of pregnant mares' serum in the rat; Fevold [1936] records a similar result after the use of a gonadotropic extract from cattle pituitary. Pregnant monkeys as well as pregnant women excrete gonadotropic substances in the urine-the chimpanzee for the first few months [Zuckerman, 1935], the macacque for only a few weeks [Hamlett, 1937]. Theoretically, an insensitivity to these substances might be acquired during foetal life, spent by this species in a milieu very rich in gonadotropic substances; Laroche and Simonnet [1936] have noted instances where the simultaneous injection of human serum has inhibited the gonadotropic effect of extract of human pregnancy urine in the male mouse. Accordingly the serum of an untreated immature female Macaca mulatta was tested for antigonadotropic activity. Three c.c. of the serum divided into 6 injections given in the course of 3 days was administered simultaneously with 20 R.U. of the human urinary extract to a series of immature rats. The serum had no detectable inhibitory influence, for cestrus and ovarian luteinisation appeared in every member of the series. We may therefore conclude that the insensitivity of the female Macaca mulatta to gonadotropic urinary extracts is not due to an antigonadotropic property of its serum. Histological examination of the hypophysis of several members of this series showed the presence of all the normal cell types, and no striking difference from the gland of males of similar age. Moreover, female pituitaries must contain gonadotropic material, since their implantation into the hind leg of immature rats, one pituitary to each animal, led to follicle ripening and luteinisation of the ovaries examined 100 hours later. Clearly, therefore, the cause of the refractoriness of the female monkey at this early age is not due to inadequate development of the pituitary gland. It remains possible that the ovary itself cannot react to substances of chorionic origin. Since Rowlands and Parkes [1937] have proved that an antigonadotropic goat serum will neutralise the activity of human pituitary extract in the rat, it seemed useful to find out whether such a serum, inhibitory to a gonadotropin to which female monkeys do not react,

9 The Reaction of Female Monkeys to Gonadotropic Extracts 211 could neutralise the activity of human pituitary extract in these. An immature female Macaca mulatta was injected with human pituitary extract, 14 mg. into the left side, and antipregnyl serum 1 from goats, 2 c.c. into the right, daily for 8 days; no external signs whatsoever of cestrus appeared during treatment, and at autopsy on the 9th day the genitalia were found to be infantile, without the slightest trace of stimulation (No. 17 in table). Indeed, the lack of development throughout the whole genital tract was more marked than in any other animal examined during the investigation. The possibility that the high dosage of antipregnyl serum injected had caused atrophy of the genital tract will be verified on adult monkeys. On the other hand, two immature Macaca mulatta, treated with 1 c.c. of hog pituitary extract and 2 c.c. of the goat antiserum daily for 8 days, both responded very well to the gonadotropic preparation, and no inhibition was observed. Thus we confirmed for Macaca mulatta the observation of Rowlands and Parkes, that antiserum to human pregnancy urine does not inhibit the activity of non-human pituitary extracts. DIscuSSION. That women produce more than one gonadotropic substance has been shown by many workers: the distinctions are based on the quantitative reaction of the immature rat ovary and on the persistence or loss of reactivity in the hypophysectomised animal. In the immature female monkey, as in the immature hypophysectomised rat, the gonadotropic principles of human placenta and pregnancy urine are relatively inactive. Hence change of character during renal excretion cannot account for the difference of behaviour between the pituitary hormones and those found in pregnancy urine. The reactivity of the ovary to substances of pituitary origin differs considerably in the monkey and the rat: two characteristic responses, ovulation and luteinisation, fail in the former, though growth of the follicles will always occur and some of them be filled with blood [Hisaw, Fevold, and Leonard, 1931; Saiki, 1932; Engle, Hamburger, 1936; and others]. Two pituitary preparations were tested with identical results: a hog extract which is strongly luteinising but only weakly cestrusproducing in the immature rat; and a human extract which, conversely, is only weakly luteinising but strongly cestrus-producing in these animals. In neither instance did the immature monkey exhibit any sensitivity to the luteinising substance. In the rat, the reactions to human pituitary extract resemble 1 The anti-serum was tested as follows on the rat: 0 03 c.c. of serum injected simultaneously with pregnyl, 20 R.U., into immature female rats, led to a total inhibition of the gonadotropic response throughout the entire series.

10 212 de Fremery those to urinary extract [de Fremery and Scheygrond, 1937] in many respects, such as the absence in either instance of the synergistic and Maxwell reactions which are seen so easily when hog pituitary extract is used. Monkeys, however, show no augmentation of potency of any of the three types of extract. Casida, Meyer, and McShan [1937] augmented the activity of pituitary extract in the rat by adding heme, but were similarly unable to do so in the monkey. It is likely that both the synergistic and Maxwell reactions depend on the luteinising activity of the extract used, and gonadotropic extracts of pituitary origin contain no luteinising substance that is active in the immature female monkey-a sufficient reason why the reactions do not appear. A gonadotropic extract of human menopausal urine induced in the immature female monkey a positive response similar in every way to that induced by pituitary gonadotropic extracts. Such activity proves that the principle excreted at the menopause is different from that of pregnancy urine. The possibility that the blood serum of the young animal may have antigonadotropic properties, acquired in foatal life by exposure to a high concentration of chorionic gonadotropins, might account for the later refractoriness to gonadotropins from human placenta and pregnancy urine, assuming that the chorionic gonadotrope of the pregnant monkey is identical with that excreted by the pregnant woman. The serum of our monkeys, however, in no way inhibited the gonadotropic activity of an extract of human pregnancy urine injected simultaneously into the immature female rat. The response of the infantile female rat, to gonadotropic hormones of the chorionic type, depends largely on the presence of the pituitary gland. It is therefore of interest that the pituitaries of infantile monkeys used in these experiments contained gonadotropic hormone. An antiserum from another species, of known activity against our preparation of human pregnancy urine, completely inhibited the response of the immature female monkey to pituitary gonadotropin of human, though not of hog, origin. Rowlands and Parkes [1937] have demonstrated the same reaction in the immature rat. For discrimination between the two types of gonadotropic hormone which the human female may excrete in the urine, the immature female monkey is an excellent experimental animal. It would appear desirable to determine the nature of the gonadotropic hyperexeretion occurring in certain clinical pathological states, and we hope to extend the work to an enquiry of this kind. It seems doubtful whether the reactions of the monkey are identical with those produced on women. The importance of the gonadotropic substance present in normal pregnancy is absolutely unclear if the woman and the immature monkey are equally sensitive, and the factors involved in ovulation and luteinisation are in that case unknown. Courrier and Gros [1934 a, b] say that gonado-

11 The Reaction of Female Monkeys to Gonadotropic Extracts 213 tropic hormones of chorionic origin are active in Macaca ecaudatus, a species closely related to those used by us, and active in causing not only follicular enlargement and cestrus, but also ovulation and luteinisation. Whether this is a species difference, or whether the animals used by the French investigators were older than ours, cannot be stated with certainty. SUMMARY. 1. Gonadotropic substances of pituitary origin, extracted from hog's pituitary, human pituitary, and human menopausal urine, produce growth of the ovarian follicles, but no ovulation or luteinisation, in immature Macaca irus or Macaca mulatta. 2. Gonadotropic substances of chorionic origin, extracted from human placenta and human pregnancy urine, are inactive in all these respects. 3. No luteinisation occurs in follicles when, after previous stimulation by a pituitary preparation, a preparation of human pregnancy urine is administered. 4. It is impossible to augment the activity of a pituitary preparation in immature female monkeys by the synergistic or the Maxwell technique. 5. The pituitary glands of the experimental monkeys induced follicle ripening and luteinisation when implanted into the immature female rat. 6. The serum of untreated immature animals contains no antiactivity against gonadotropic hormones of chorionic origin. 7. It is possible to inhibit the activity of the gonadotropic hormones present in the human pituitary, by the simultaneous administration of serum of a goat treated for several months with gonadotropic preparations of human pregnancy urine. This serum does not inhibit the activity of hog pituitary extract. [TABLE.

12 214 de Fremery C)C CS 6. En rn c, X, C.*v=:~ C." mc -~.~, 0 C C... 4 O C.frCC 0>0 ~ 00 -.,I. 1-i _Y Y1 -: ' -: -~ -~ ' I-, 1-1 FH- <- I-- F- I'l 1; 1- r, e1 F1 F1 -, -, ri <1 F - 7r - I- I-.= = Z- :-, z ti -,) trz --,> x v: - - = r- "Z ll-. lll.l -1. L'Z oo.; ::c.fz x m fz L t- -,.,H O~ ti oc --,.4 --Iq -to.,o C-- ez --t, cll. -Cl M. m --q cli C.11 CI. --I ls. ct,- I.r I--, 1- x xv: x x or, 00 + z 1-4 X, cr, D X_ Y- Z -f Y- -4 Z_ + + -OC-' v z r 1. :: t. ti -1- -,Z.4 ; ;.: ; -- = -,-.) =. :.. -- f'.) t, -4, -4 :. :,.:, CCC + ) 0 C. r O ++ t - Z E 5 C; 0 C.c - - O L o C If. C. -f).-.- 7! t.t :1. -t-. t, t-- :,. :t. Z'z z Z3 Z.) :t t. -:: ;:,-.- ;:_ ;a... = = Z: Z: = C. 71 t - z ;= =.: ;4 - = ::.: E; =.. ;..1;1.-Il " = x N , m C..z =,. -.1_, = t " cl I ---. Ti ".11

13 The Reaction of Female Monkeys to Gonadotropic Extracts 215 REFERENCES. ANSELMINO, K. J., and HOFFMAN, F. (1935). Zbl. Gynrdk. 59, BACHMAN, C., COLLIP, J. B., and SELYE, H. (1934). Proc. Soc. exp. Biol. N.Y. 32, 544. CASIDA, L. E., MEYER, R. K., and MCSHAN, W. H. (1937). Anat. Rec. 70, 47. COURRIER, R., and GROS, G. (1934 a). C. R. Soc. Biol. 116, COURRIER, R., and GROS, G. (1934 b). Ibid. 116, ENGLE, E. T. (1932). Proc. Soc. exp. Biol. N.Y. 29, ENGLE, E. T. (1932). Endocrinology, 16, 513. ENGLE, E. T. (1933). Amer. J. Physiol. 106, 145. ENGLE, E. T. (1934). Ibid. 108, 528. EVANS, H. M., MEYER, R. K., and SIMPSON, M. E. (1931). Proc. Soc. exp. Biol. N. Y. 28, 845. FEVOLD, H. L. (1936). Endocrinology, 20, 40. FREMERY, P. DE, and SCHEYGROND, B. (1937). Acta brev. Neerl. 7, 8. GUSTUS, E. L., MEYER, R. K., and DINGLE, J. H. (1935). Proc. Soc. exp. Biol. N. Y. 33, 257. HAMBLEN, E. C., and Ross, R. A. (1936). Amer. J. Obstet. Gyncec. 31, 14. HAMBURGER, C. (1936). Endokrinologie, 17, 8. HAMLETT, G. W. D. (1937). Amer. J. Physiol. 118, 664. HISAW, F. L., FEVOLD, H. L., and LEONARD, S. L. (1931). Proc. Soc. exp. Biol. N.Y. 29, 204. HOUSSAY, B. A. (1921). C. R. Soc. Biol. 85, 33. JOHNSON, C. E. (1935). Amer. J. Obstet. Gyncec. 29, 120. LAROCHE, G., and SIMONNET, H. (1936). C. R. Soc. Biol. 121, 449. MAXWELL, L. C. (1934). Amer. J. Physiol. 110, 458. ROCK, J. (1935). Endocrinology, 19, 269. ROSS, R. A. (1937). Amer. J. Obstet. Gyncec. 34, 780. ROWLANDS, I. W., and PARKES, A. S. (1937). Lancet, 232, 924. SAIKI, S. (1932). Amer. J. Physiol. 100, 8. ST6CKL, E. (1934). Zbl. Gynak. 58, WESTMAN, A. (1935). Ibid. 59, WOLFE, H. R., and MEYER, R. K. (1937). Anat. Rec. 70, 46. ZUCKERMAN, S. (1935). Amer. J. Physiol. 110, 597. DESCRIPTION OF FIGURES. Ovaries of monkeys receiving various types of gonadotropic extract.- All x 141. VOL. XXIX., NO