MITOTIC ACTIVITY I N THE ANTERIOR HTPOPHYSIS O F FEMALE R,ATS THOMAS E. HUST IkpUit?&?nt of Anatomy, university of Alabamu INTRODUCTIOX Mitotic activity in the pars glandularis of the hypophysis of sexually mature rats is usually considered to occur infrequently. Wolfe ('35a), for instance, reports no more than a n average of three mitoses per section in the rat hypophysis and others have reported even fewer or none in normal animals. Only in experimental animals have considerable numbers been found. Wolfe ( '35 b) and Halpern and D'Amour ('36) described numerous mitotic figures in the pituitary of rats after administration of oestrin and TYolfe ('35c) reported an increase after injections of pregnancy urine. Ponierat ( '41) has found considerable mitotic activity in immature and young mature, unoperated and castrated male rats after injections of colchicine. It is surprising that cell divisions have not been found also in the normal animal since the hypophysis of rats (Hatai, '13) continues to increase considerably in size and weight aftcr sexual maturity is reached. Furthermore, according to Rirkman ('37), cells are lost in the guinea pig hypophysis through degeneration or through the holocrine type of secretion. A priori, one can say that mitoses should take place t o compeiisate for the cells lost as well as to account for growth of the gland; therefore failure to observe mitoses may mean that they were not looked for at the right time rather than that they do riot occur at all. It is obvious that there is not Aided by a grant from the TJniversity of Alabarnrt Research Fiind. 263 T H E ANA'COMIC'AC HRCOltn VOL. 8 2 . XI. M A R C H . 1942 264 T H O M A S E. H U N T a sigiiificunt, continuous mitotic activity but tlie possibility remains that cells may divide during a certain period of the estrous cycle. Tlie fact that estrogens may stimulate mitosis suggests that cell divisions occur at a time when estrogens are normally at a high level. Wolfe ('35 a), in fact, does state that there are more mitoses in the rat hypophysis during estrus than at other times but the nuniber that he reports is not great. There have been other attempts t o correlate mitotic activity in various organs with the estrous cycle. Chouke, Friedman and Loeb ( '35) reported that the maximum number of mitoses in the thyroid of the guinea pig was found in the lutein phase or approximately tlie fifth day of the cycle. Chouke, however, more recently ('41) has stated that upon reexamining the material he found that there is iiot a statistically significant difference in mitotic activity at different periods of the cycle. In the adrenal cortex of the rat I have found ('40) that niitoses occur more frequently at diestrus than at other phases. Thc work reported here shows that mitoses do occur in significant numbers in the hypophysis and that there is a cyclic mit,otic activity that map be correlated with the estrous cycle. ~\TATRRIATIANT, METHODS Tlic rats used were from the colony of Dr. C. 31. Goss w-hich was started through the generosity of Dr. P. E. Smith and included black, gray, hooded and albino types. The color did not seem to have any significant bearing on tlie number of mitoses found. The animals were fed on a dict of Purinachow supplemented with lettuce and milk. Food was available constantly except f o r one group in which food was withheld for 24 hours. The animals were free from Bartonella niuris infection, which may cause an increased mitotic activity in the reticuloendothelial system. This mas shown by the fact that splenectomy had no effect on the survival of a series of six j-oung rats selected at random from differcnt cages of the colony. MITOTIC ACTIVITY IX T H E H Y P O P H Y S I S 365 The stage of the estrous cycle in the mature animals was ascertained by making vaginal smears daily for 2 or 3 weeks. The leIigtli and character of the individual cycles was thus determined approximately. Any animal not showing regular cycles was discarded. I n the latter part of the series several successive smears were made a t intervals of 2 or 3 hours before killing an animal in order to determine more exactly the stage of the cycle. The majority of animals (exceptions noted in tables 2 and 3) were killed between 11 and 1 2 A.M. This was done since many reports have stressed the variability of mitotic activity at different periods of the day. Animals were killed by severing the cervical spinal cord with bone forceps. The brain was quickly removed from the base of the’skull and the latter with the hypophysis in situ was dropped immediately into fixing fluid. After fixation for 1 2 to 24 hours the gland was dissected away from the skull. The fixative used was Allen’s B-15 fluid which was found to be much superior to Bouin’s and other fixatives for studying mitosis. The glands were cut serially in the coronal plane at 3 micra and mounted at representative intervals. I n the older glmcls every twenty-fifth section was taken, in the younger ones every twelfth section. Every section was mounted from glands less than 39 days old. The sections were stained for the most part with iron hematoxylin followed by acid fuchsin and anilin blue (Masson’s method). The number of mitoses per square millimeter of section was determined since the expression of mitotic activity by tlic number found per section does not allow for very accurate comparisons. Sections taken at different levels of a gland may vary from 2 to 6 sq.mm. and unless the different glands are cut in exactly the same plane another variable is encountered. The area of a section was calculated after measuring with a planimeter the area of the section projected at a magnification of 50 diameters. I n making the counts a 4 mni. ~ were used. The latter had an ocular objective and a 7 . 5 ocular disc marked with twenty-five 5 mm. squares that covered 266 THOMAS E. H U N T I/lo sqmni. of the section. By running the section from one square t o the liest the entire section was examined without duplicating fields. The stage of mitosis and, insofar as possible, the type of cell was noted in each case. RESULTS The mitotic activity was determined in two main groups of rats, the immature and the mature. Mitotic activity irt the hypophysis of immature female rats In this series of nineteen animals (table 1) ranging from 1 to 50 days of age there is a decline from eighty-five to ninety-nine mitoses per square millimeter of section on the first day to one to six per square millimeter on.the fiftieth. There is some variability between individuals that are litter mates as in the case of 182a, b and c but the difference is not great. There is likewise some difference in the count from glands from different litters of the same age as between 110. 104b (eighty-five mitoses per square millimeter) and no. 186 (ninety-nine mitoses per square millimeter). These differences, however, are minor ones and the principal point is that there is a gradual decline in the mitotic activity as the animals become older which continues until sexual maturity is reached. A similar decline in mitotic activity was noted by Pomerat ('41) in young male rats which had been injected with colchicine. Mitotic activity in the hypophysis of mature female rats I n those animals which are mature there is a clear cut difference in the mitotic activity at different stages of the estrous cycle. Animals in proestrus and diestrus show relatively few mitotic figures, but during the latter half of estrus (table 2, LE), there are surprisingly large numbers of dividing cells. In one specimen as many as seventy-two mitoses per square millimeter of section were counted (no. 76, table 2) - a total of 357 in R T,-nim. iirea, 3 micra in thickness. 267 MITOTIC ACTIVITY 1X THE HYPOPHYSIS EXPLANATION O F TABLES ’ Under the coluinn designated, ‘ ‘ Mitoses per square millimeter’ the quotient is given of the total iinniber of mitoses counted h oiic gland divided by the total area of sectioiis examined. The “Mitoses per sertion” is used t o allow comparison with results presented i n t h a t ii-vay by other workers. In making such comparisons, how-eyer, it should be remembered t h a t others may have cut sections a t a different thickness and in different planes. The abbreviations f o r color of the animal are as follows: G - gray, B -black, W -white, GH - gray-hooded, BH - black-hooded. Under “Hour killed,’’ thc time given is hours a f t e r 1 2 midnight. Thus, hour 15 is used t o designatc 3 P.N. All immature animals werc killed between 11 mid 1 2 A.X. The abbreviations f o r the stages of the cycle a r e as follows: EE -early estrus, LE - late estrus, E - estrus (exact stage unknown b u t definitely estrns), M - metestrus, D - diestrus, P - proestrus. TABLE 1 Jlitotic activity in immature f e m a l e rats. I NO. OF RAT AGE 1N EXAMINED DAYS MITOSES COUNTED JIITOSES PER SQUARE MILLIYETER SECTIONS EXAMINED YILLIIIETERS I 186 104:l 104b 178a l78b 182a 182b 18% 174% 1741) l7Oa 1701) 17OC 165% I65b 77 12.5 90 89 19 4.50 2.43 1.69 6.93 3.23 3.81 4.81 5.33 6.00 5.09 3.68 3.31 7.35 33.59 12.46 6.72 10.89 13.59 6.01 1 1 1 7 7 10 10 10 14 14 16 16 16 20 20 24 39 50 51 447 212 144 550 242 225 242 269 232 169 163 92 199 244 186 30 I50 91 8 99.3 87.2 85.2 79.4 74.9 59.1 50.5 50.3 38.7 33.2 28.7 27.8 27.1 18.0 15.0 4.5 11.6 6.7 1.3 I I 123 3.895 1 11 13 10 10 MITOSES PER SECTION 41 16 14 55 27 25 2T 30 17 01 20 10 25 31 31 5 30 I1 9 9 9 9 14 8 8 8 8 8 6 6 5 8 7 1 - 268 THOMAS E. HUNT TABLE 2 Mitotic QCtiVity in mature female rats. - ~ - 8:: * K M f. 0 E - ~ 141 83 84 191 1.93 194 86 69 74 190 203 198 199 202 66 119 121 68 s 0 $4 i 17.03 21.7@ 9.01 11.79 6.85 16.71 5.99 21.74 32.74 13.85 12.23 17.10 18.39 9.83 30.77 10.91 12.77 34.50 53 62 69 73 76 87 90 92 97 101 103 105 105 106 126 135 135 185 171 262 103 78 16 93 45 197 463 37 72 307 91 57 175 87 152 59 10.0 12.1 11.4 6.6 2.3 5.6 7.5 9.1 14.1 2.7 5.9 18.0 5.0 5.8 5.7 8.0 11.9 1.7 4 8 2 4 4 4 2 11 8 4 4 4 4 3 - 4 5 8 43 33 51 20 4 23 28 18 52 9 18 77 33 19 25 22 30 7 G G w W G G B w W w B c* :H w w G G c: - 142 Ii5 175 160 158 180 180 165 li5 160 214 195 195 1ii 180 153 159 224 13 12 12 6 20 20 11 12 11 5 12 18 19 11 12 11 11 12 10 12 4 4 12 16 9 9 16 EE EE EE EE EX EE EE EE El3 EE EE EE EE EE EE EE BE El7 ~ 18 126 139 142 140 116 81 105 213 76 214 75 196 107 $04 2,465 8.34 15.90 17.84 12.28 14.73 19.90 23.66 19.06 24.76 15.59 20.05 13.51 30.23 431 864 837 835 967 782 1,068 565 1,777 524 687 149 81 7 45 52 52 53 62 63 80 86 87 92 101 104 111 90 J I .7 54.3 46.9 68.0 65.7 39.3 45.1 29.6 71.8 33.6 34.3 11.0 27.(J 6 4 4 4 6 4 5 4 6 4 7 3 8 72 !16 !09 !09 61 96 114 .41 196 31 98 50 02 cr 126 138 149 148 135 165 15.5 198 160 G 170 W G 180 189 LV 1i5 G w G G w G R G 12 12 12 12 12 11 11 15 12 1.5 12 9 12 ~ ~ 13 ~ 2.16 10.303 65 - - 18 24 24 30 30 LE LE LE JAE LE LE LE IX TIE LF: LE 22 LE TJF, TABLE 3 * Mitotic activity in mature female rats. __ ~ z$ I C $ ii $s “;Bwa 3aEx O P B O ii” ~ 55 40 54 61 56 64 62 63 60 57 109 131 194 213 215 241 331 339 342 560 32.41 30.89 42.00 39.82 44.66 11.61 22.08 35.27 32.54 31.12 138 304 112 127 169 29 37 52 46 21 - 8c: 2,. c :as, 3;a B $3, 0 __ 4.3 9.8 2.7 3.2 3.8 2.5 1.7 6 7 6 8 6 3 3 6 8 6 1.5 1.4 .7 P5 r’ X - 185 182 235 255 220 225 245 220 270 208 11 12 11 12 11 12 18 12 11 11 187. 166 212 180 180 12 12 9 12 17 130 160 190 205 170 21 9 180 190 215 204 212 215 225 218 12 12 19 11 12 11 11 12 12 11 12 11 12 12 D D D D D D D R 1Go R 155 178 216 17-1. 170 210 12 12 12 12 12 12 12 P P P P P P P G B W G BH G W 23 43 18 16 28 10 12 9 6 3 E w B B E E E I3 E E E E E E ~ 322 10 195 192 212 45 48 94 94 114 151 156 ,035 59 12.25 9.21 16.96 24.90 19.11 46 3.8 144 15.6 3.0 50 111 4.5 129 6.8 82 480 3 4 4 6 5 G 15 w 36 12 18 26 W G G 22 24 40 M M M M Ikf ~ 5 82 87 88 99 85 215 39 41 46 44 47 50 53 43 66 68 68 93 96 114 131 131 202 206 206 223 222 223 13 16 28 2 19 10 4 2 0 0 0 2 3 0 204 14 163 80 !62 161 65 67 52 17.87 10.21 14.13 15.45 18.75 22.14 10.64 10.62 8.22 15.04 12.86 18.40 17.37 12.17 61 63 81 94 126 126 219 31 172 24 13 7 14 19 4 4 .1 4 1.0 .5 .4 .2 .2 5 4 2 2 2 4 2 4 6 0 4 0 0 0 .1 1.8 7.2 1.1 .6 .4 .8 .3 273 163 ~ 1.6 4.0 7.0 .5 4.0 2.5 2.0 1.0 0 0 0 .5 B R R B B B w G G W w .5 R 0 0 cr n n D n D D D 55 ~ c 8 .7 1.6 2.0 99 17.49 33.96 21.10 22.21 17.94 18.70 41.51 ~ 22 7 6 6 5 5 5 7 41 - 4.4 30.0 4.0 2.6 1.4 2.8 3.7 cr G w W G 269 - 2'70 THOMAS E. HUNT Although this was the highest count there are enough other instances approaching it to indicate that it is not exceptional. During proestrus and diestrus (table 3) the number of cell divisions is uniformly low. The only exception is rat no. 80 which has seven and two-tenths mitoses per square millimeter. This animal had become sexually mature a few days previously and it is possible that the larger number of mitoses is an expression of the factors responsible for mitotic activity before maturity. I n all other animals the number of mitoses counted is not greater than two per square millimeter and is usually less. After it was ascertained that considerable mitotic activit;v occurred during the latter half of estrus, the attempt was made to determine more accurately when it began and when it ceased. I n the cases listed (table 2, LE) it may be assumed that some of them have reached or a t least approached the maximum activity. I n about half the cases the beginning of estrus was known to liave occurred 18 to 30 hours previously. In the others the time when cornified cells first appeared in the smears is not known exactly but it is certain that the animals were killed late in estrus. The smears in such cases are composed of typical cheesy masses of cornified cells and the uterus is 110 longer swollen. The exact time in hours after the onset of estrus when mitotic activity begins cannot be stated due to the individual variations in the length of the cycles. This is illustrated by animals nos. 198 and 199 (table 2, EE) which have eighteen and five mitoses per square millimeter respectively. Both of these animals were killed approximately 9 hours after cornified cells were first found in the vaginal smear. They were litter mates and had the same weight but no. 198 had a cycle of 4 days and 110. 199 had a cycle of 5 days. Otlicr similar cases lead me to believe that rnitotic activity may appear sooner in animals with short cycles. I n animal no. 194 which was killed 4 hours after cornified cells were first found in the smear there arc five and six-tenths mitoses per square millimeter. This is distinctly above the average for proestrus MITOTIC ACTIVITY I N TIIE HYPOPIIPSIS 271 and diestrus and it may be assumed that mitotic activitj- niay appear as early as this in some animals that run short cycles. Mitotic activity diminishes and practically ceases during metestrus (table 3). This is shown by the considerably decreased activity during this phase as compared with the latter part of estrus and the essential lack of mitoses during diestrus. Metestrus, which is characterized by the appearance of leucocytes among the cornified cells in smears, was found to occur from 22 hours (no. 195) to 40 hours (no. 212) after the beginning of estrus. Long and Evans ('22) found the range of time t o be 12 to 81 hours with the average length 27 hours. While it is not possible to say with certainty, it seems likely that mitotic activity continues over a period of 18 to 30 hours, the difference depending on the length of estrus. It also seems probable that in cases where there is a long period of estrus the maximum mitotic rate is not as great as in those cases where there is a short period. If this is true, the total number of cell divisions occurring in a single cycle might be the same in two cases that had markedly different mitotic rates. The age of the animal is another cause of variation in the mitotic activity. I n general it has been found that the older the animal the less frequent are mitoses. A sufficient number of glands have been examined t o show that this is true. I t is probable, however, that the maximum activity has not been found in animals more than 111 days old. The older animals were for the most part the first to be examined and not as great care was used in determining the exact stage of the cycle as later in the series. It is only known that they were in some stage of estrus (table 3, E). The greatest mitotic activity found in these older animals is in general less than the least activity found in the group under 111 days of age. The number of hours after ingestion of food map be a factor in determining the mitotic rate in the hypophysis as Rlumenthal ('40) found in the thyroid and adrenal gland of the guinea pig. It is certainly not the primary factor, however. Rats nos. 119 and 121 (table 2, EE) were killed 24 hours 2i2 THOMAS E. H U N T after food had been removed from the cage and the mitotic activity is essentially the same as in comparable animals that had food available a few hours before killing. Rats nos. 4<3 and 48 were killed 6 and 24 hours respectively after feeding. The one without food for 24 hours has a somewhat greater number of mitoses, to be sure, but the discrepancy can be attributed more readily to other factors. Furthermore, it is contrary to Blumenthal’s results which indicate that the greatest activity is between the fourth and the twelfth hour after feeding. The time of day at which the animal is killed is likewise not the primary factor that determines mitotic activity since practically all glands were obtained between 11 and 12 A.M. Animals killed at other times have about the same number of mitoses provided the stage of estrus and age are the same. The cell in which mitosis occurs in the mature animals is usually the chromophobe. I n glands where mitochondria and granules can be identified satisfactorily it is found that only 5 to 10% of the mitoses occur in the granular acidophiles. Of all the mitoses counted in the series none was identifled with certainty in a basophile. The number of dividing acidophiles is usually greater in the younger mature animals than in the older ones and in the ininiature animals it is sometimes considerably greater. I n one case (no. 90,50 days of age when sacrificed) there are as many as 50% of the dividing cells that are acidophiles. I n some cases only a few fuchsinophilic granules are present in the dividing cells. Whether such cells represent a transformation from chromophobe to chromopliile or vice versa could not be ascertained. DISCUSSION It is difficult to explain why mitoses in large numbers have not been reported previously in a n organ studied as intensively as the rat hypopliysis. There are several possible explanations. I n the first place, few studies have been made that have been correlated with the estrous cycle. An esception MITOTIC ACTIVITY I S THE HYPOPHYSIS 273 is the work of Wolfe ('35 a) who looked for mitoses in a total of ninety-seven glands from mature rats. Of these, twenty glands were from animals in estrus but it is not stated how many of these were taken late in estrus nor is the age of the animal given. While the average number of mitoses that he observed at this phase is only three mitoses per section, this number is higher than that found at other times. Other investigators have studied the hypophysis from a cytological point of view with or without regard to the phase of the cycle. Usually the fixatives used in such cases were not adapted to the study of mitotic activity. It is not surprising that mitoses have not been generally observed in animals that have a longer sex cycle than the rat. The period during which mitotic activity occurs in these longer cycles may be so protracted that only a few mitotic figures would be found at any one time. The question arises as to the primary factor responsible for stiiiiulating mitoses in the hypophysis. Light, time of feeding and time of day, which are usually mentioned in connection with mitotic activity, can not be considered to be of primary importance although they may be secondary factors. The close correlation of mitotic activity in the hypophysis and the changes in the ovary points to a direct or indirect relationship between the two. Further, since mitotic activity is greatest when the follicles are large or when ovulation has recently occurred, it seems probable that an estrogen may be involved. The fact that administration of an estrogen (TTTolfe, '35b; Halpern and D'Amour, '36) results in an increased mitotic activity in the chromophobes of the hypophysis substantiates such a view but the dosage given to produce this effect is greatly in excess of the physiological amounts during estrus. Whatever the stimulus may be that causes mitotic activity in the hypophysis, it does not cause such activity simultaneously in any other organ so far as known. I have found that the number of mitoses in the intestine does not vary signi- 274 THOMAS R. HUNT ficantly during different phases of the cycle. I n the adrenal cortex the greatest mitotic activity occurs during diestrus (Hunt, '40). I n the reproductive tract mitotic activity in the uterus and vagina occurs earlier in the cycle (Long and Evans, '22) than it does in the Bypophysis. The stimulus thus appears to act specifically on the hypophysis. It is probable that the high mitotic activity in the hypophpsis provicles new cells to replace those that have degenerated. The activity observed in diestrus and proestrus, since it is coniparable t o that found in glands shortly before maturity, is sufficient to account for the normal growth of the gland. This difference in the observed mitotic activity in late estrus and that required for growth of the gland necessitates the assumption that newly forming cells will need additional space that can be acquired only by a loss of a comparable volume of degenerated cells. Degeneration of cells has been obserred by Kirkman ('37) and otliers but it seems likely that the process is even more extensive than that so far described. There is also the possibility that the greatly increased mitotic activity observed in the latter part of estrus may be correlated with the shift in cell proportions that occurs during the estrous cycle. During estrus the percentage of granular basophiles drops considerably in the rat, dog and sow (Wolfe and co-workers) and in the guinea pig (Kirkman). The reappearance of these cells has been explained by their reversion to chromophobes which again become basophiles. Severinghaus ( '37) has shown by cytological studies that such a transition may take place and if mitoses were as rare in the lij-popliysis as is generally believed such an explanation would be almost necessary. With the observation of large numbers of mitoses, however, a second explanation is equally tenable, namely, that the basophiles instead of reverting to chromophobes disappear by degeneration and are replaced by dividing chromophobes which in turn differentiate into ti new set of ba sophiles. MITOTIC ACTIVITY IN THE HYPOPHYSIS 275 SUMMARY AND CONCLUSIOXS 1. Mitotic activity in the hypophysis of immature female rats declines from eighty-five to ninety-nine mitoses per square millimeter of section on the first day to one to six mitoses on the fiftieth. 2. I n mature female rats mitotic activity varies during the estrous cycle as follows: ( a ) Mitotic activity begins and increases during tlie first 12- to 16-hour period of estrus; (b) The greatest number of mitotic figures is found 18 to 30 hours after the onset of estrus (as many as seventy-two mitoses per square millimctcr in a section 3 micra in thickness). (c) Activity declines and practically ceases during metestrus. (d) During diestrus and proestrus mitotic activity is at a low level (usually less than two mitoses per square millimeter of section). 3. There are indications that mitotic activity in sexually mature rats declines as the animals become older. 4. The majority of mitoses occur in the chromophobes. Only 5 to 10% occur in thc acidophiles. None were found in basophiles. 5. It is concluded that tlie mitotic activity in sexually mature rats is greater than that necessary to account for the growth of the gland and that it serves to compensate for cells lost by degeneration. LITERATURE CITED RTXMENTHAL,HERMANT. 1940 The influence of time of feeding on the periodicity in activity in thyroid and adrenal gland of normal male guinea pigs. Endoerin., vol. 27, pp. 481-485. C'IIOUKE, I<. 8. 1941 Relationship of the proliferative activity of the thyroid gland to the oestrons eyelo in the gninea pig. Anat. Ree., vol. 79 (suppl.), p. 14. C'IIOUKE, JTALPERN, s., H I L D A F R I E D M a N A N D LEO LOER 1935 Proliferative activity of tho thyroid gland of the female guinea pig during tlie sexual cycle. Anat. Rcr., rol. 63, pp. 131-137. I<. 5. R., AHD F. E. D'AMOUB 1936 Studies on the gonad-hypophyseal complex in cstrin-injected rats. Am. a. Phpsiol., rol. 115, pp. 229-238. 276 T H O M A S E. H U X T HATAI,SITINKISHI 1913 On the weights of the abdominal and the thoracic viscera, the sex glands, ductless glands and the eyeballs of the albino rat (Mus norregicus albinus) according t o body weight. Am. J. Anat., TO]. 15, pp. 87-119. HEST, THOMASE. 1940 Mitotic activity of the adrenal gland of rats in different phases of the sexual cycle. Anat. Rcc., vol. 78 (suppl.), p. 152. KIRKIT .ZN, HADLEY1937 A cytological study of the anterior hypophysis of the guinea pig and a statistical an:~Iysis of i t s cell typrs. Am. .T. Anat., rol. 61, pp. 233-287. LONG,JOSEPHA., BND HERBERT If. EVANS 1922 The oestrous cycle in the rat and its sssociatcd phenomena. Memoirs Unir. of Cal., vol. 6, pp. 1-148. PoMcRAI,, R. 1941 Mitotic activity in the pituitary of the white rat following castration. L4m.J. Anat., vol. 69, pp. 89-121. GERARD SmmtIxGFr4cs, A. E. 1937 Ccllular chaiiges in the anterior hypophpsis with special reference t o its secretory artirities. Physiol. Rev., vol. 17, pp. 556-588. WOLFE,J. M. 1935 a The normal level of the various cell types in the anterior pituitaries of mature and immature rats and further observations on cyclic histologic variations. Anat. Rrc., vol. 61, pp. 321-330. 1935 b Reaction of anterior pituitaries of mature female rats to injections of large amounts of oestrin. Proc. Soe. Exp. Bid. and Med., vol. 30, pp. 1192-1195. 1935 c Morphologic reaction of the anterior pituitaries of mature female rats to prolonged injectinus of pregnancy urine extracts. Anat. Rec., ml. 63, pp. 1-11.