Talk:Paper - Variation in the activity of the rabbit hypophysis during the reproductive cycle (1934)
SPECIES VARIATION IN THE GONADOTROPIC ACTIVITY OF THE HYPOPHYSIS.
By R. TOWNER HILL (National Research Council Fellow). (From the National Institute for Medical Research, London.)
(Received July 28, 1934.)
Ir is recognized that the hypophyses of different species differ greatly in their power to stimulate the gonads of test animals. Thus Smith [quoted by Bugbee, Simond and Grimes, 1931] noted that the pituitaries of both pigs and sheep were more potent than those of cattle, and according to Wallen-Lawrence and van Dyke  the difference may be as much as ten times. Recently Hellbaum  stated that horse pituitaries are about four times more active than ox pituitaries. Age and sex differences in the activity of the anterior lobe, as well as changes due to castration and injection of sex hormones, have been described by many writers [references in Allen, 1932]. Most, if not all, of this work, however, has been done with immature rats or mice, and these test animals, as pointed out by Lipschiitz, Reyes and Vinals  and Hill, Parkes and White  give entirely different results from those observed on cestrous rabbits. The rabbit ovulation test has now been put on a quantitative basis [Hill, Parkes and White, 1934] and it seemed desirable to re-examine these apparent species and sex differences. The power to induce ovulation in the cestrous rabbit is a well-defined criterion of activity, and is much more satisfactory than the multiple reaction of the immature rat or mouse.
Leonard , who tested several preparations on both rabbits and immature rats, suggests a possible separate ovulation substance, unconnected with any previously described anterior lobe hormone. However, in the light of the experiments of Wolfe , Wolfe and Cleveland , Hill , Hill , Evans and Simpson [1929 a, 5] and of many others, it seems probable that ovulation-producing activity is directly connected with the principles known to stimulate follicular growth and luteinization. 138 R. T. HILL.
I. MATERIAL AND METHODS.
Preparation of animals and material. The preparation of test rabbits is similar to that described by Hill e¢ al.  for routine assay. Ten cestrous animals were used for the assay of each preparation except where otherwise specified (Table I). The rats used were 23-25 days old when injections were started. They were given two daily subcutaneous injections for 5 days, and killed on the sixth day. Increase in ovarian weight (over normal controls) was taken as a sign of stimulation.
Taste I. Activity of the hypophyses of various species assayed on cestrous rabbits.
Av. wt. acetone No. of desiccated Wt. of Units pituitaries pituitary unit per Species Sex obtained mg. mg. g. Notes
Cat 3 5 6-6 0-32 312 — Baboon 3 2 27-0 0-40 2500 _ Leopard Q 1 23-0 0-50 2000 _ Sheep 3? _ —_ 0-54 1865 A.L. only Pig 3? — ~ 0-64 1560 A.L. only Rabbit 9 23 78 0-64 1560 _— Rat 3 102 1-5 0-75 1330 — Dog Q 8 11-0 0-81 1230 _ Horse Q —_ — 0-81 1230 _ Rabbit 3 15 4:8 0-81 1230 _— Rabbit* 2 9 3-4 0-81 1230 _ Rat Q. 76 1-6 0-94 1060 — Cat Q 10 6-3 1-2 800 — Human 3 1 71-0 1-6 620 64 years old Dog 3 10 10-1 1-9 510 _ Deer Q 1 74-0 3-0 330 _ Guinea-pigt Q 24 1-9 3-4 290 - _ Ox 3? —_— — 6-1 160 A.L. only Guinea-pigt 3 51 2-1 10-9 90 - _—
- 3-4 months of age.
+ Assay on seven cestrous rabbits. } Assay on eight cestrous rabbits.
The pituitaries of a large number of animals were collected (see Tables I and II for list) immediately after death, except for a very few cases in which there was a lapse of a few hours. The glands were dropped into a large excess of acetone for 24 hours, and then dried in warm air, ground, sifted through an 80-mesh sieve, and stored at 0° C. until use. The whole pituitary was taken except in the case of the glands from ox, sheep and pigs, of which only the anterior lobe was obtained. In Tables II and III the figures for horse glands are calculated for anterior lobe only, based on the observed fact that the posterior lobe amounts to about 20 p.c. of the whole. The glands from cattle, pigs and sheep were collected at random, ACTIVITY OF THE HYPOPHYSIS. 139
and may have included pregnant females and castrated males. The horse material was from non-pregnant mares and castrated males. Some of the female rats may have been pregnant. Otherwise the sex and condition were known of all animals from which material was collected.
Taswz II. Activity of horse, ox, sheep and pig anterior hypophyses assayed on immature rats.
Average No. of ovarian Rabbit units immature Source of | Amount given increase per amount Tats used pituitary mg. mg. given* 3 Horse 2-0 3 3 3 ” 4-0 16 6 3 ” 8-0 66 12 3 ” 12-0 174 19 3 ” 16-0 171 25 3 ” 24-0 175 37 3 Pig 10-0 10 16 3 ” 20-0 18 31 3 ” 40-0 41 63 3 ” 80-0 74 125 3 Sheep 15-0 58 27 3 % 30-0 127 © 54 3 Ox 20-0 5 3 3 ” 40-0 2 7 3 ” 100-0 6 16
- To nearest whole number.
Tastz III. Relative potency of the anterior hypophyses of horse, ox, sheep and pig.
Amount required to produce 60 mg. increase in im- Rabbit units Relative po- Relative
Source of mature rat per amount tency in potency in pituitary ovary (mg.) given rats rabbits Horse 6-7 12 143-200 9 Swine 60 90 17-25 9 Sheep 16 28 62-100 1l Ox 1000-1500 165-200 1 1
All injections were made in the form of suspensions of the desiccated powder, to avoid extraction, which would have introduced a variable factor and which would in any case have been difficult where the amount of tissue was small. Two sets of control experiments were carried out with reference to this technique.
Efficiency of acetone desiccation. The question of loss of activity during acetone desiccation was investigated as follows: 100 g. of fresh ox anterior lobes were dissected out and minced. 50g. were put through routine acetone desiccation, and 50g. were immediately extracted with N /20 NaOH for 18 hours at 0° C., after which the preparation was neutralized 140 R. T. HILL.
and centrifuged. The acetone dried material was subjected to the same extraction procedure. Assay of the two extracts gave similar results by the rabbit ovulation test and showed that no loss had occurred during desiccation.
Stability of desiccated preparation. Coincident with the assay of a large number of samples, it was decided to test the effect of heat on our dry powders. For this purpose about 0-3 g. of powdered horse pituitaries was put in a glass tube which was then allowed to stand in a desiccator over P.O; for 1 week at a pressure reduced to 15 mm. Hg. At the end of this time the tube was sealed off, and immersed in boiling water for 5 hours. Assay proved that this heated material retained full potency in the ovulation-producing principle, and confirmed the results of Askew and Parkes  on urine of pregnancy preparations. Sterile preparations have been made by suspending the heated dry powder in aqueous or other media. Emulsions made in this manner have remained sterile after 10 days’ incubation at 37° C.
Expression of results. All results are expressed as units [see Hill, Parkes and White, 1934] per gram of dry gland tissue, and where useful the weight of the unit of different preparations and the number of units per pituitary are also given.
II. Assay ON GSTROUS RABBITS.
Throughout the experiments there is a marked lack of similarity between the potency of the glands of the two sexes of a species, or between the glands of closely related species. Assay of male cat pituitaries gave an extremely high result (Table I) at 3120 units per g., while female cat glands only showed 800 units. A female leopard pituitary gave 2000 units per g. In dogs, the gland of the female was the stronger, with 1230 units per g. as against 510 in the male.
Rodents were found to be very variable: female cestrous rabbit, 1560; male rabbit, 1230; male rat, 1330; female rat, 1060; female guinea-pig, 290; and male guinea-pig, 90 units per g. This work, although carried out with acetone desiccated glands instead of fresh material, supports the finding of Wolfe and Cleveland  that the pituitaries of immature rabbits (3-4 months old) are as potent as those from full cestrous adults. Our results show that immature rabbit pituitaries contain 1230 units per g., and those of adult females 1560 units per g.
Ruminants likewise varied greatly, with sheep at the high figure of 1860 units per g., and ox and deer at the opposite end of the scale with ACTIVITY OF THE HYPOPHYSIS. 141
160 and 330 units per g., respectively. No correlation was found in the two primates tested, man and the Hamadryas baboon, with 620 and 2500 units respectively.
III. Assay ON IMMATURE RATS.
In only four species was the material sufficient to assay on immature rats, namely: horses, sheep, pigs and cattle. The results are summarized in Table IT. The greatest efficiency of this type of assay is when the dose given causes about a 60 mg. increase in ovarian weight of the test animals (immature rats). With the ox anterior lobes, however, it was not found possible to produce so large an increase. In Table III are shown the theoretical amounts of horse, pig, sheep and ox anterior lobes to produce a similar ovarian weight increase in immature rats. The last two columns of Table III also give the relative potencies of the anterior lobes as assayed on rats and rabbits. The ox glands, which are comparatively inactive, are considered as 1, and comparable figures are given for horse, sheep and pig anterior lobes. Horse and swine anterior lobes are equally potent by the rabbit ovulation test, being nine times more potent than ox. Sheep are slightly better at about eleven times greater potency. Assayed on immature rats, horse glands are much the strongest, followed by those of sheep, swine and ox respectively (Table III).
IV. EFFECT OF TREATMENT ON OVULATION-PRODUCING ACTIVITY OF THE HYPOPHYSIS.
Male and female rabbits were gonadectomized to determine the effect on the ability of the pituitary to induce ovulation in the cestrous rabbit. The pituitaries from male rabbits which had been castrated 1 month showed
Taste IV. Effect of gonadectomy on the activity of the rabbit hypophyses.
Av. wt. of acetone Loss in N °. of dried U Wt. of Units units pituitaries pituita: nits per unit r r Species Sex Treatment obtained ® mg. v pituitary mg. y y Rabbit é Normal 15 4:8 5-9 0-81 123 —
- 3 1 month 6 5-3 5-5 0-96 1040 -190
castrate 9 3 ” 7 6-3 4-2 1-50 670 — 560 ” 2 Normal 23 78 12-2 0-64 1560 _—
- 2 1 month 12 77 9-5 0-81 1230 ~—330
castrate ” g 2 4 ll 9-5 9-9 0-96 1040 - 520 Cat 3 Normal 5 6-6 20-6 0-32 3120 _ » 9 Longtime 4 11-0 13-6 0-81 1230 -1890
castrate 142 R. T. HILL.
a decline of 190 units per g., and after two months a still greater decrease of 560 units from their normal controls (Table IV). Ovariectomy in female rabbits had a similar effect. The values at 1 and 2 months after castration showed a decline of 330 and 520 units per g. respectively from the normal estrous animals.
In the course of collecting cat pituitaries several castrated male cats came to hand, probably one or more years after the operation. The drop in potency of the pituitaries is very significant, 1890 units per g. less being obtained than for normal male cat glands.
160014004 “uy, Leo 12004 — e M, _ 10005 Kes aD fot & 8007 ‘a p 600+ 4007 2007 0 1 2 Months after gonadectomy
Fig. 1. Effect of gonadectomy on the potency of the rabbit hypophysis.
General acceptance has led to the use of the term “ gonadotropic” activity. With the rapid advancement of research, the word “gonadotropic” must certainly be used with discretion; a substance which may prove gonadotropic in one test animal may not prove so with another species and a different criterion of activity. The inadequacy of the term is shown by the effects of gonadectomy on hypophyseal activity. Evans ACTIVITY OF THE HYPOPHYSIS. 143
and Simpson [1929 a, 6], Domm , Hill  and others have all mentioned the increased “gonadotropic” potency of the pituitaries from castrated donors, using immature rats or fowl as test animals. The present work, where the cstrous rabbit is the test animal, gives an opposite result (Table IV, Fig. 1). The greater potency of the anterior lobes of castrated donors, when tested on immature rats and mice, must be due primarily to their greater power of eliciting follicular growth as compared with the glands of normal animals. It seems likely that the decreased ability of the pituitary from a castrated donor to cause ovulation in the cestrous rabbit is due to a decrease in the substance causing luteinization. On the other hand, since a positive test in rats consists of follicular growth and subsequent luteinization, and in rabbits of the ovulation of a naturally mature follicle, it is possible that different substances enter into the reactions. It seems most probable, however, that ovulation in the rabbit depends on principally the luteinizing substance, or possibly on a delicate balance between the luteinizing and follicle-growing principles.
Evans and Simpson [1929 a, 6] and Nelson  found male rat pituitaries more potent than those of females, as tested by implants into immature female rats. Domm  reported the same result for fowl. On the rabbit ovulation test the hypophyses of female rabbits and dogs are more potent than those of the males, while in cats and rats the reverse is found.
Further evidence of the difference in the results obtained from assay on immature rats and cestrous rabbits is provided by the fact that whereas horse and pig glands are about equally potent on the latter, horse glands are nearly ten times more potent on immature rats. It is of interest that ox glands, by far the most common source of material, are shown to be extremely weak by either system of assay.
1. The comparative gonadotropic potency of the pituitaries of a large number of species has been determined by the rabbit ovulation test. No correlation is found between related species or between the two sexes of single species. In cats and rats the pituitaries of males are stronger than those of female, while in rabbits and dogs the reverse is true.
2. Gonadectomy of the animals tested (male and female rabbits and male cats) causes a significant decrease in the power of the pituitary to cause ovulation in the rabbit. The relationship between assay on immature rats and on cestrous rabbits is discussed. 144 R. T. HILL.
The author wishes to thank Colonel A. E. Hamerton, Pathologist to the Zoological Society; Prof. G. H. Wooldridge, Royal Veterinary College of London; Dr D. 8. Russell of the London Hospital; Chapple Brothers of Rockford, Illinois, U.S.A., and Parke, Davis and Co. of Detroit, Michigan, U.S.A., for their willing cooperation in the collection and supply of pituitaries.
His best thanks are due to Dr A. S. Parkes for valuable suggestions and criticisms during the course of the work.
Allen, E. (1932). Sex and Internal Secretions. Williams and Wilkins Co., Baltimore. Askew, F. A. and Parkes, A. S. (1933). Biochem. J. 27, 1495.
Bugbee, E. P., Simond, A. E. and Grimes, H. M. (1931). Endocrinology, 15, 41. Domn, L. V. (1931). Proc. Soc. exp. Biol., N.Y., 29, 308, 310.
Evans, H. M. and Simpson, M. E. (1929 a). Amer. J. Physiol. 89, 371.
Evans, H. M. and Simpson, M. E. (1929b). Ibid. 89, 375.
Hellbaun, A. A. (1933). Proc. Soc. exp. Biol., N.Y., 30, 641.
Hill, R. T. (1933). Endocrinology, 17, 414.
Hill, R. T. (1934). J. Physiol. 83, 129.
Hill, R. T., Parkes, A. S. and White, W. E. (1934). Ibid. 81, 335.
Leonard, S. L. (1931). Amer. J. Physiol. 98, 406.
Lipschiitz, A., Reyes, G. and Vinals, E. (1932). Soc. Biol. 111, 852.
Nelson, W. O. (1933). Anat. Rec. 56, 241.
Wallen-Lawrence, Z. and van Dyke, H. B. (1931). J. Pharmcol., Baltimore, 43, 93. Wolfe, J. M. (1931). Amer. J. Anat. 48, 391.
Wolfe, J. M. and Cleveland, R. (1931). Anat. Rec. 51, 213.