FIVE FIGURES IPITTRODITCTION The cliaiiger Tvliich talic placc~in the o v a r i f l h during the rcproductire. c-j-clc have bccii studied cxte.imiwly in mammals, such as the. rat and mouse, which liaw i*clatirrlysliort oestrous cycles. Evans and S ~ v e z y ( '31) have. rccordcd that during each cycle a coiisidemhle gronp of follicles hegins growth. Some oS tliese complcte their clift'ercii tiation sncl rnpt1ii.e ; the majority undergo retrogressiou. h the m o u e it secms c+ar that this periodic iiiitiatioii of activity ill tlir germ wlls inrolves thc germirial cpitlicliiim ( L1llen, '23 ; Allen aiitl Cwaclick, ' 3 7 ) . hi the bats of thu temperate zone t h c w is only one rcyroductive cycle each year. T ~ species P M yol is lucifugus lucifngus, which is il cave residont in Missouri (luring thc wiiitct(Gnthrici, '33 a ) , €allows the general p a t t t ~ nf o r such rriariimals. OvuIatioii is spoiitancwis a n d o c ~ u r somctirne s in April, w h e n ~ v e iilici ~ feniales l e a w ttic (*ares. A siiigle tei-tiarv, or \~esicida~ follicle ~, cwiitaiiiing a small antrum is Pwsciiit i r i October at t h e onsrt of the (?aw-duelJing period a n d grows but littlcl, if at all, until the prrovulation pe.rioc1 i i t ilir spring. Primary folljcles, consisting of a sirigle layer of squamous cells, arid both niiilaminar. a r i d multilaminar srcoiidary 01' growing follicles are conspjcnoiis t Iiroughout thc so-ca1lcc.l * 'rhe multilaminar srcondary follii-les corrcrpond to folliclc typrs 2 to 5 (I!. qcribetl b.r I'iiicus : ~ n dE1i7rriaiin ( ' 3 7 ) for the r:~bkiit. 'r1it.y do iiot rceognize the uni1artiin:rr serondarp folhcle, in nhicli t h e s q i i a m o ~ i scells of the primarp follicle lrarr 'x)rcoinr cuboidal, a s ii t j p ~ . 47'7 hibernating mouths. Insemiriation occur-s in tho autumn, but ferriales appareiitly again hecomr rcwplive to males iu ~ ) e r i o d s of activity as the u-iiiter advaxiccls. Sperm arc phagoc~-tizcti iii the uterus and eliminated tlirougli the vagiiia (luring the winter (Gutlii*ic, '33 1 1 ) . The prwcnccl of the vesicular follicle aiid the evidence of ~ ~ ~ u r r ciiisemiiiationr iit before its rupture made it seem lilic~lythat these aiiimals were in coiitiiiuons oestixs from Octoher t o April (compare ('afier, '34; M K e 1 aucl Kolbow, '34). Granliiig this possibility, xvhat woiilcl be the effect up011 tlie gimvth of mx)iiilar-y follicles ? TTould a ln?o~i~leclge of the total follicle popillation he of valur in clarifying our untlc*rstan(liiig of tlie i*ept-oductivccycle ? I n order to aiis\ser tliese questions a c'eiisu5 of all normal gro\viiig follicles a r i d follicles in carlv stages of retnogrcssion was made in lioth ovaries of a series of females in which a c y t o l o g i d study of the ovaries had bceii made (Guthi-ie arid *Jeffers, '38 a ) . ('omparable oll?sers.ations have bee11 made 011 ovaries of Al . jii*iwsc~ns collected at intervals thro1igliout the Tear arid will be prescnlecl elsewhcrc. G R O W T H O F FOLLICLES IN T H E BAT 479 a solution of 3% potassium bichromate f o r 1 day each. Dehydration and embedding in parafin wcrc carried out in the usual way, using xylol as a clearing agent, and serial sections were cut at 3 or 4 p . Sections were stained bv Kull's acid fuchsin-thionin-aurantia method (Lee, '37, p. 306). The smallest follicles included in the counts consisted of a single layer of cuboidal cells around an oocyte, that is, they mere unilaminar secondary or growing follicles. Primary follicles were not considered. Two diametcrs of cach follicle were measured and the average taken as the diameter of the structure ; the theca was not included. Measurements were made at a magnification of x 600 with an ocular micrometer, each unit of which was equal to 4 p at this magnification. OBSERVATIONS Normal follicles The number of normal growing follicles in both ovaries of this bat varies in different individuals from 53 to 305, exclusive of the singlc vesicular follicle (table 1). The average number of follicles is 157. I n 22.2% of the bats studied, less than 100 follicles are present; in 46.7%, between 100 and 200 follicles occur; and in 31.1%, over 200 follicles arc found. I n only one individual (2.2%) are thcre more tlian 300 follicles. To find the relationship between the age of bats and the number of follicles in their ovaries, the forty-five animals were divided into groups on the basis of the amount of wear on their teeth. I n the group of the youngest bats, with no wear on the teeth (twenty individuals), the number of follicles varies from 53 t o 305 (average 182.3) ; in bats showing some wear on the teeth (elcven individuals), the variation is from 87 t o 284 (average 168.7) ; and in the fourteen bats in which the teeth are conspicuously worn, from 65 to 173 follicles occur (average 110.1). In order to detcrrnine wheiher there is a preponderance of follicles in a single ovary of the bat, the percentages of the total normal follicles occurring in the right ovary and in the THE ANATOMICAL RECORD, VOL. 71. N O 4 TABLE 1 Counts of follicles in individual bats 1 I SERIAL DATE O B S O . OF cnI,mc- BAT TION XVXBER OF FOLLICLES . ..._.. + DIAMETER .-ca E 2 4 !4 ~~ 164 to 1 8 3 /r 598 614 1128 640 13 31 639 1129 1085 668 672 687 689 710 734 736 1157 738 1159 1165 1170 756 1090 1089 1092 776 778 1183 1176 1175 830 1095 1100 1192 1199 1191 1193 1209 1214 1210 956 954 1224 1225 947 1024 10-19-31 10-28-31 10-31-34 11- 8-31 11- 9-34 11- 8-31 11- 9-34 11-10-33 11-21-31 11-21-31 12- 3-31 12- 3-31 12-12-31 12-26-31 12-26-31 12-27-32 12-2&31 12-27-34 1-14-35 1-14-35 1-16-32 1-22-34 1-22-34 1-22-34 2- 6-33 9- 6-32 2-21-35 2-21-35 2-21-35 2-20-32 3-10-34 3-10-34 3-14-35 3-14-35 3-14-35 3-14-35 3-2 9-3 5 3-29-35 3-29-35 4-16-32 4-16-32 4-22-35 4-22-35 4- 2-32 4- 6-33 68 85 80 28 103 96 130 178 31 54 54 43 67 43 121 86 167 183 116 133 55 194 86 154 114 82 78 88 103 53 169 145 97 114 56 43 76 112 71 113 157 197 74 128 52 31 57 47 21 83 61 95 I) 4 2 1 2 94 16 45 19 20 50 29 54 41 49 45 27 58 28 84 26 64 66 57 44 34 63 18 91 57 32 39 21 16 48 47 29 25 72 83 49 61 21 - 5 1 2 14 4 28 16 17 15 10 9 11 2 11 in 1 1 1 1 5 16 2 8 6 4 1 5 5 2 8 8 2 ~ 1 28 7 16 10 1 1G 8 27 11 14 8 23 4 7 14 1 8 8 28 3 9 5 2 1 15 12 4 5 15 14 - a 26 8 1 4 30 16 33 22 5 22 9 ti 20 5 11 7 12 8 13 G 14 14 6 12 4 17 7 10 8 16 22 21 6 11 14 9 10 17 9 5 8 56 31 10 3 - 2 2 1 2 2 1 ~ 108 144 141 . 3i 214 173 242 287 57 108 84 63 128 82 203 134 232 238 144 207 92 305 123 222 188 162 126 128 170 72 268 210 129 181 81 68 129 161 101 173 241 284 128 204 87 2 30 9 6 31 16 35 22 5 27 9 9 20 5 13 7 13 9 13 6 1(i 14 6 15 6 18 12 10 8 31 24 29 12 15 16 14 15 19 9 5 16 64 33 10 4 - 345 358 305 3,54 418 359 348 371 376 2 80 308 279 322 373 388 350 368 394 273 359 318 400 382 3 74 342 382 328 422 388 402 384 324 337 417 x 3.53 345 388 318 484 326 375 361 c.1. c.1. EXPLANATION: Animals are arranged chronologically in the order in which they are plotted iu figure 3. A space separates the groups collected at comparable dates. c.l., corpus luteum; *, vesicular follicle damaged. 480 G R O W T H O F FOLLlCLES I N THE EAT 481 ovary coiitaiiiiiig the vesiculai. follicle were calculated for each individual. I n 49% of tlie bats tlie vesicular follicle is in the right ovary.2 The data on the number of follicles in a single ovary have been plotted (fig. l), and it is seen from the graph that the follicles are not constantly more numerous in one ovary than in the other. The curves s h o ~ vthat in a single animal as little as 39.6% or as much as 60.4% of the total follicular population may he present in oiie ovary. HoweT-er, if Fig. 1 Graph showing the percentage of normal growing follielcs present iu the right w a r y and in the ovary containing the single vesicular follicle in individual bats. the figures for the forty-five animals are combined, it is found that 49.5% of tlie total number of follicles in these animals is in the ovary with the vesicular follicle, while 50.8% is in the right ovary. In neither case is the deviation from a 1:1ratio of statistical sigiiificaiice. Tlie bats were grouped together on tlie basis of the dates on which they were collected, and the average number of total 2While the vesicular follicle occurs a s oftcn in the left ovary as in the right, implantation in hundreds of hfyotis observed is always in the riglit horn of the uterus. After the feniales reach seuual inaturitv the right horn is larger than the left throughout the year. 482 M. J. GUTIll1IE AKD K. R. JEFFEEIS normal follicles calculated for each date. Figure 2 shows that the average number varies throughout the minter and spring months. The curvc reaches peaks in early November, in late December and January, and again in late March and April. This curve is in many respects similar t o that of the distribution of the smallest follicles measured (compare fig. 4). Fig. 2 Graph showing average numbers of all normal growing follicles and of follicles in early stages of retrogression in bats grouped in accordance with the dates of collection. The normal secondary follicles in the ovaries of M. lucifugus lucifugus range in size from 24 to 1 5 7 p in diameter. These follicles were classified in four groups on the basis of size. The percentage of the total follicular population found in each group mas calculated for each individual and the results plotted (fig. 3 ) . The marked individual variation in the distribution of follicles of different sizes is shown by the graph. The combined data for individuals killed at comparable dates are shown graphically in figure 4. The curve for fol- 483 G R O W T H O F FOLLICLES I N T H E BAT size groups in individual bats arranged in accordance with the dates Of collection. % . 10 i-Lu 3- 5 4 - 3 2 I- m m 5% s 3 :5 J, e - 0 3-$srJ g.2 0 U- 3 Fig.4 Graph showing percentage of normal growing follicles in the different size ranges in bats grouped in accordance with the dates of collection. THE ANATOXICAL RECORD, VCI,. 71, NO. 4 484 M. J. GUTHRIE AND K . R. JEFPEXS licles 24 to 43 in diameter shows that there are periodic variations in the relative numbers of these follicles. They are very numerous toward the end of October, during late December and throughout January, and, again, during the last of February and most of hlarch and Spril. I n the two animals in which ovulation had occurrcd, thcre is a dccrease in the number of these small follicles. The curve for follicles 44 to 83 11 in diameter also shows periodic variations. It has a conspicuous peak in early December, which we interpret as being conditioned by the peak in the curve of follicles 24 to 43 p in diameter seen in late October. There is another rise in the curve in Fehruary, following the December and January increase in the smaller follicles. The remainder of the curve is without conspicuous variation. The curve f o r folliclcs 84 to 123 p in diameter reveals two marked rises in the relative numbers of these follicles; the first occurs toward the end of November, the second is a t its height early in February and follows the December peak in the curve for follicles 44 to 83 in diameter. The curve f o r the largest normal secondary follicles, 124 to 163 p in diameter, indicates no marked variation in the relative numbers of these follicles, although the slight rise in Deeembcr, following the November increase in follicles 84 t o 124 p in diame may be significant. The diameter of the vesicular follicle varies during the winter months from 273 to 422 p (table 1). The largest follicle observcd, from a bat killed April 16th (no. 956), had a diameter of 484 p and contained an oocyte with a meiotic spindle. There is no indication that any marked growth of the tertiary follicle occurs until the period just before ovulation. The average diameter of seren vesicular follicles in November is 358 p and of cight similar follicles in llhrch is 358.5 p. Retrogre.ssi.lzg follicles Follicles a t all stages of degeneration occur in the ovaries of M. lucifugus lucifugus during the months when collections were made (sec Guthrie and Jeffers, '38a, for criteria of retrogression with stages and types). Only those in early GR OWTH O F FOLLICLES I N T H E BAT 485 stages of retrogression of type I were measured and counted ; their diametcrs have riot yet decreased as a result of disintegration of the granulosa cells, and it is possible to determine how large they were when retrogression began. From 2 t o 64 of these follicles are found in different individuals (table 1); L a 40 - 30 - a n 6 z Fig. 5 Graph showing average numbers of all follicles in early stages of retrogression and of such follicles in different size ranges in bats grouped in accordance with the dstcs of collection. the average number for the forty-five bats studied is 15.7. In 40% of the animals, 10 o r less occur; in 37.8%, 11t.o 20 are found; in 11.1%,there are 2 1 to 30; in 8.9%, 31 to 40 are present; and in only one bat (2.2%), do more than 40 such follicles occur. 486 111. J. GLTTHRTE AXD K. R. JEFFEBS The counts of follicles in earl7 stages of retrogressioii for bats collected on comparable dates were combined, tlie a r e r age computed, and the data plotted (fig. 2). From the graph it is seen that, aside from the two individuals killed April 22nd in each of which atypical persistence of the vesicular follicle iws noted, there is no pronounced variation in the number of degenerating follicles during the months in which collections coulcl be made. However, slight increases in retrogression of follicles occur early in November and about the middle of March. The size at which follicles begin to degenerate varies markedly. The smallest follicles in which degenerative changes a r e found i n the granulosa a r e 67 p in diameter, the largest 182 p. Three-tenths per cent of the retrogressing follicles measured a r e 64 to 83 p in diameter; 55.5% a r e 84 to 123 p ; 42.77., 1 2 1 to 163 11; and 1.5%, 164 to 183 p in diameter. If the average number of degenerating follicles of different sizes is calculated for bats collected at comparable dates (fig. 5) i t becomes evident that from October until the middle of February approximately two-thirds of the follicles beginning to degenerate are 124 to 163 p i n diameter; about onethird are 84 to 123 p in diameter. A few retrogressiiig follicles 164 to 183 p in diameter ( a total of ten) a r e found during this period. After February Gth, there is a marked increase in the number of follicles 84 to 123 p in diameter that begin to degenerate and a decrease in the number of retrogressing follicles 124 to 163 p in diameter. nrscussIoR The number of normal follicles found in tlie ovaries of the bat is considerahly smaller than that reported f o r the rat (Arai, '20; Swezy, '33 a ) , the mouse and ape (Blotvogel, '32), and the rabbit (Desaiw, ' 3 5 ) . H O W ~ V for C tlie ~ , niialysis presented in this paper, only the secondary growing follicles were counted ; the primary follicles and the tertiary follicle were not included. F o r this reason the counts gireii here a r e not comparable with tliose of most other investigatoi*s. Arai ('20) reported that the ratio of the number of oocytes in the right ovary to that in the left was 1.03: 1.00; for the cntire series of rats, 50.676 of the oocytes occurred in the right ovary. F o r the forty-five 31. lucifngus lucifugus studied, it was found that 50.8% of the normal growing follicles were in the right ovary. I n the rat, Arai ( ?20) determined by counting all the oocytea in both ovaries of thirty-nine individuals 1 to 947 days old that the number of oocytes decreased rapidly u p to 20 days of age, after which the decrease was inore gradual. The counts i n 31. lucifugus lucifugus suggest that a similar gradual decrease i n the number of growing follicles occurs with age, but t.he number of indkiduals in each age group is small and the varjation in tlie number of follicles is great. Since only the growing follicles i n the ovaries of the bat wcre counted, the data presented can throw light only on the growth of follicles, not on the seasonal variations in the proliferatioii of new oocptes, although differentiation of cells of the germinal epithelium is belicved t o occur throughout adult life (Guthric aiid Jeffers, '38 a ) . Allen ( '23) counted the mitoses in tlie cells of the germinal epithelium of the ovaries of mice at different stages of the oestrous cycle. A cj-die proliferation of the cells of thc germinal epithelium, which had its peak at ocstrus, was found. H e regarded this as the first stage of ovogenesis. These results have becn confirmed by Allen and Creadick ( ' 3 7 ) . Evans and Swezy ( '31) reported that the proliferation of oocptes and the growth and degeneration of follicles were cyclic phenomena which c o n e spondccl in tlie rat, guinea pig and dog with the clianges occurring during the oestrous cycle (compare Swezy, '33 b). Whilc new sex cells were formed at all periods of the oestrous cycle, the proliferation of new oocytes was most marked during metoestrus, the number of growing follicles \\-as at a maximum during anoestrus and at a minimum during oestrus. Myers. Young and Dempsey ('36) have reported that, in the guinea pig, follicles began to grow a t all stages of the oestrous c p l e . However, only those follicles heginning growth during a lim- 488 M. J. BUTHRIE AND Ei. R. JEFFERS ited interval reached maturity and ruptured a t the following oestrous period; all others degenerated. I n contrast t o these findings, the connts of normal growing follicles in the ovaries of &I. lucifugus lucifugus indicate that, instead of a single waiTe of growth or a continuous iiiitiation of growth in primary follicles, there are at lcast four peaks of follicular growth in this bat during the oestrous cycle. The curve for follicles 24 to 43 p in diameter reveals thrce waves of growth in them during the 6 months studied (fig. 4). That these three are probably preceded by a t lcast one other growth wave is suggested by the pronounccd rise in the curve f o r Polliclcs 84 to 123 p in diameter late in November. The peaks of the three growth waves shown by the curve for follicles 24 t o 43 p in diameter occur at intervals of 60 to 75 days, and the peaks in the curves for follicles 44 to 83 and 84 to 123 p in diameter appear at intervals of about 75 days. It is possible, by means of the data plotted in figure 4, to estimate the rate a t which folliclcs p o w in 31. lucifugus lucifugus during the period of so-called hibernation. The peak in the curve of follicles 24 to 43 p in diameter for October 28th is followed in 45 days by a peak in the curve for follicles 44 to 83 p in diameter, Similarly, about 43 days after the December and January increase in the number of follicles 24 to 43 p in diameter there is an increase in the number of follicles 44 to 83 p in diameter. A comparison of the curve f o r follicles 44 to 83 p in diameter with that for follicles 84 to 123 p in diameter shows that there is an interval of a little over 60 days between the corresponding peaks in the two curves. It requires, therefore, approximately 105 days for follicles 24 to 43 p in diameter to become 84 t o 123 p in diameter, which is near the limit of size attained by follicles before they begin to degenerate. No conspicuous wave-like variations in the numbers of degenerating follicles have been found to occur in the bat between Octoher and the middle of April. However, it is interesting to note that there is a marked change beginning in February in the size attained by the secondary follicles before GROWTH O F FOLLICLES IN TIIE BAT 489 they begin to degenerate. During the months from October to the middle of February, follicles may become relatively quite large before they degenerate ; two-thirds of the retrogressing follicles are over 124 p in diameter. S f t e r the middle of February, the majority of the degenerating follicles are smaller than 124 [I in diameter. It appears that something is limiting the growth of follicles beyond a certain size but not increasing the number of retrogressing follicles. The reproductive cycle of the bat is known to have many peculiarities. First to be recognized were the facts of autumnal insemination and vernal ovulation. It has generally been held that the eggs are fertilized by sperm retained in the tract throughout hibernation (compare Hartman, '33). Observations on tho tracts of maiiy recently killed females (Guthrie, '33 b), the behavior of artificially ovulated eggs (Guthrie and JeEers, '38 b), and the presence of abundant sperm in males of the species throughout the winter (Miller, '36, '37) have convinced us that, under the climatic conditions of central Missouri, spring copulations are the rule in these bats. Where the winters arc more severe and temperatures more constant it seems possible that different reactions may prevail in the bats. With the information accumulated over a period of years we may now formulate a n explanation of the correlation of certain activities related to reproduction in the female bat. Since the cells of the pars distalis of the hypophysis of this bat have been shown to be understandable as stages in the differentiation of a single type of secretory cell (Guthrie, Jeffers and Sawyer, '36; Sawyer, '36) we shall assume that one secretion is produced by these cells. It is obvious that the preparation of however many hppophyseal extracts producing varied responses in several regions of the body does not establish that the active principles of such extracts exist as such in the intact gland (cornpare van Dyke, '36, pp. 174-175). Nor does the observation that a particular region, which is undergoing differentiation, seems to respond at one time to one fraction and at another time to a different fraction necessarily 490 M. J. GCTHBIF, ANT) K . R. J E F P E R S suggest more tliaii that cells are capable of diflereiit reaction* and have different thresholds of response when in successive stages of differentiation. The effector cells are not static : the reaction is conditioned not only by the stimulus but by the effector (compare Maxwell, '34 ; Engle, ' 3 2 , pp. 7%-'796 1. Lillie and his associates 1iaT.e made use of the concept of growth rate and hormone threshold in their brilliant analysis of feather patterns (compare Domm, Gustavson and Juhn, '32, pp. 636-637). Zuckerman ( ' 3 7 ) has suggested that a variation in threshold to ocstrogcnic hormone is exhibited by the uterine endometrium of the monkey. When females of M. lucifugus lucifugns enter the eaves in the autumn there is a single follicle mitli a small antrum in the ovaries of each individual, the vaginal epithelium is cornif i ~ d and , ~ the uterus contains sperm. These individuals are in a state of oestrns as judged b ~ the - criteria of vagiiial reaction and receptivity to males. However, the uteriis is not fully differentiated nor does ovulation immediately ensue. Let us assume that this state has been coriditioned in the following may. During the summer, when these bats arc actirc and feeding each night, follicles grow under the influence of the gonadotropic hormone of the hypophysis. At least oiie of these follicles reaches the stage of differentiation at whicli an oestrogen is prodneed in a quantity sufficient to condition raginal cornificatioii a i d the response to males, but not to initiate differeritintion in tlie uterus o r tubes. These animals appear to correspond to Pfeiffer's ( ' 3 3 ) 'single constant oestrous' rats, except that the mating reaction has been induced. There is nothing to suggest that progestin has been responsible f o r tlie onset of receptivity, as Young ( ' 3 i ) has postulated f o r the guinea pig, since no luteiiiization of follicles is observed. We shall use the phrase 'submaximal oestrus' Statements concernirig tlie condition of thc reproductile tiact are based on unpublished obserratioiis by Elizabeth 31. Recdcr, P1i.D. thesis, Unir. of Mo., 1935. 4 W e have no evidence that more than onc tertiary follicle is produced in 11. lucifugus lucifugus since v-e h a ~ eno specimens taken a t the season n hen aevcrd follicles with antra are f o u n d in the ovarics of M. grisesceiis (compare Gutlirrc and Jeffers, '38 a ) . G R O W T H U F FOLLICLES IA- T H E BAT 499 (Witschi ailti Pfriffer, '35) to designate the coiiditioii of female bats which have entered the cams. At the lower metabolic level of the early liibernating periocl (November) the gonadotropic hormonal output of the liypophysis is reduced and the average number of growing follicles is lowered while retrogression is conspicuous in the larger secondary follicles. The single tertiary follicle undergoes no detectable change, and retrogression of the secondary follicles is not accompanied 1))- luteinization of the granulosa. It is interesting to note that Foster, Foster and Hisaw ( ' 3 T ) report a comparable manner of retrogression of follicles in hypoplipsectomized adult rabbits and find that small follicle^ with antra may persist and be capable of stimulation to g r o ~ v f o r about 30 days after the removal of the gland. By December the average number of growing follicles in these bats is greatly reduced, and the wave of retrogression is subsiding. One may assume that follicles growing at a certain rate require and take available hormone in a certaiii qimntitp. TThen the hormone has fallen below that level and many follicles have undergoiie retrogression the net result is a relative increase in the hormone available. The response to this is a gradual increase in growing follicles of the smallest size raiige. Such follicles have been stated by a nnmloer of workers to be independent of any hypophyseal iiiffuence as a result of studies on immature, growing animals or hypophysectoniized adults. This matter is discussed in another place where the results of injection of hypophyseal extract upon these smallest growing follicles are presented ((luthrie and Jeff ers, '38 In). Foster, Foster and Hisaw ( '37) reported that a few primordial follicles persisted in the rabbit ovary for as long as 90 days after bypophysectomy. This observation, together with the long survival of follicles with small antra under the same circumstances, suggests that the total secretion of the hypophysis may not act directly on the gonad but through an intermediary. After hypophysectomy a low level of gonad stimulating substance from such a soiirce maintains a limited number of follicles at certain stapes of differentia- 492 M. J. GUTEIRIE A&-D B. H. JEPFEXS tiori f o r a considerable time. The smallest 91-owi13g follicles are capable of respoiisc to very small quantities of this substance. The number of smallest growing follicles in tlie bat undergoes a relative increase until about the middle of January when there is a rise in the percentage of larger secondary follicles ~ h i c his conspicuous early in February. This is believed to be induced by an incrcasc in the amount of gonadotropic hormoiie available, wliicli is correlated with changing temperature conditions and a n elevation of metabolic level in the bats as indicated by evidence of shifting populations at this time (compare Gnthrie, '33 a ) . Along with this increase in number of larger secondary follicles the output of the oestrogenic substance increases and the females are again receptive to males in periods of activity. It is during this time that spontaneous ovulation under laboratory conditions begins (Guthrie and Jeffers, '38 b). By the end of February, however, the number of growing follicles has become too large for tlie amount of gonadotropic hormone arailable and retro;0 ression is again on the increase, this time among smaller follicles. T17ith the release of this drain on tlie supply of gonadotropic hormone the smallest follicles show a relative increase. Toward the end of Ifarch, as the time of emergence from the caves nears, there is an increase in the average number of growing follicles again, but the tertiary follicle shows no definite trend toward increase in size so long as the females remain in the caves. We have by chance obtained one individual (no. 956) in which preovulatory growth has occurred. TTTe believe that with an increase in metabolic activity the amount of gonadotropic hormone finally rises until the threshold of response of the tertiary follicle is reached. When this happens the tertiary follicle undergoes very rapid growth, and ovulation and smelling of uterus and tubes occur within 24 hours in many individuals removed from the caves at this time of year. This hypothesis that preovulatory growth and ovulation require a very high level of available gonadotropic hormone is consistent with the observation that the large G R O W T H O F FOLLICLES I N TI-IE BAT 493 graafian follicles are the first to respond t o diminution of the hormone in the hypophysectomized rabbit (Foster, Foster and Hisaw-, ' 3 7 ) , and that a large final intravenous injection of the follicle stimulating or of the luteinizing fraction of hypophyseal extract appears necessary in order to induce ovulation in the anoestrous cat (Foster and Hisaw, '35). Witschi and Yfeiffer ('35) used a large dose of the luleinizing fraction of the gonadotropic hormone in order to produce ovulation in their rats which were in submaximal oestrus. In the bat there seems no reason t o postulate the presence of more than one gonadotropic hormone if the possibility of differing thresholds of response in follicles at the several stages of differentiation be granted. There is no indication of any lnteinixation except in the granulosa of the follicle of oviilation. There is nothing wliich suggests to us that a swing from F.S.H. secretion to L.H. secretion occurs under the influence of an ocstrogenic substance (compare Witschi and Pfeiffer, ' 3 5 ; Fevold, Hisaw and Breep, '36). A single gonadotropic hormone which is produced or becomes available in a limited quantity and for which follicles compete at all stages of growth but at different thresholds is adequate t o explain the peculiarities and duration of the oestrous period in the bat. Evans and Swezy ('31) found for several mammals that the period when most follicles underwent retrogression was when a few completed the preovulatory growth. They found, also, that gestation did not inhibit the c j d i c growth and degeneration of follicles, even though ovulation did not occur. Follicles growing during gestation sometimes luteinizcd in the r a t but not in the guinea pig. Dempsey ('37) could find no effect of oestrogenic substance (progynon B, Schcring) on rhythmicity of ovarian response in the guinea pig. The hibernating female bat is in submaximal oestrus as a consequence of an inadequate quantity of available gonadotropic hormone. x.J. GUTHEIE a m 494 I<. I:. JEYFERS SUMMARY 1. The nuinber of normal growing follicles in the ovaries of 11.lucifugus lucifugus ranges from 53 to 305 ; the average is 157 follicles, sdiich are cqually clistributed in the two ovaries. The average iiiimber of normal growing follicles decreases somewhat with age. 2. The periodic variations in the relative iiumbers of follicles of clifferent sizes which occur in the ovary during the months from October through April are interpreted as indicating follicular growth waves. Three peaks of growth are evident in follicles 24 to 43 p in diameter, and these are followed by peaks of growth in the larger follicles. A fourth growth waw just prior to hibernation is suggested by the data. 3. Follicles in which retrogression is beginning vary in number from 2 to 64 in different individuals ; the average is 15.7. From October until the middle of February, approximately two-thirds of the degenerating follicles are 124 to 183 p in diameter; from February 21st on, the majority of the retrogrcssing follicles are 64 to 123 p in diametei-. 4. The four growth waves of the secondary Iollicles occur during a. single ocstrous cycle which terminates at the time of rupture of the one vesicular follicle present throughout the so-called hibernating season. 5. Tlie hibernating female bat is in a state of submaximal oestrns conditioned by an inadequate quantity of a single gonadotropic substance. LITERATT'BE CITED 1923 O~ogeiicsis during sexual inaturitv. Am. J. Ailat., vol. 31, pp. 4 3 9 4 8 2 . ALLEN, E., AND R. S.CREADICK 1937 Ovogeiieuis during sexual maturity. The first stage, mitosis in the germinal epithelium as shown by tlit colrliicine techuiync. Anat. Rce., rol. 69, pi'. 191-196. ARAI, H. 1920 0 1 1 the postnatal dcvelopment of the ovary (albino r a t ) , ~ i t h cspccial reference to the number of ova. Am. J. Anat., vol. 27, pp. ALLEN, E. 40.5462. 1932 Die Follikelatresie be1 hlaus und Affe. Gd. 7.5, S. 122-128. BIJorroom, W. Anat. AIIL Erg., GROWTH OF FOTJJCT,ES Ihi T H E RAT 493 C'AFFIER, P. 1934 Horinonwle S c h \ ~ a l i g e r s c h a f t s e z ~ ~ u ~ uliwi l l g dcr winterschlafendrn Fledermans. Zentralbl. f. Qyniik., Jalirg. 58, S. 2354-2363. CAPFIEX. P.,A N ] ) €1. 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E. 1936 The reproductive cycle in the males of some cave bats. Anat. Rec., vol. 67 (suppl. l), p. 62. 1937 Further observations on the reproductive system in male bats. Anat. Bee., vol. 70 (suppl. l), p. 84. LIIYERS, 1%.I., W. C. Youiw ASD 3:. W. I)XMPSE~Y 1936 Graafian follicle development throughout the reproductive cycle in the guinea pig, with especial reference t o changes during oestrus (sexual receptivity). Anat. Rcc., vol. 65, pp. 381402. PBEIFFER, C. A. 1935 Origin of functional differences between male and female hypophyses. Proc. Soe. Exp. Biol. and Med., vol. 32, pp. 603-605. PINOUS, G., AKD E. V. EYZXANN1937 The growth, mnturation and atresia of ovarian eggs i n the rabbit. J. Morph., vol. 61, pp. 351-384. SAWYER, ELIZABETH L. 1936 The cytology of the hypophysis cercbri of the bat. J. Morph., vol. GO, pp. 127-157. S w E Z Y , OLIVE 1933 a Ovogenesis and its relation t o the hypophysis. Science Prcss, Lancastcr, Pennsylvania. 1933 b The changing eonccpt of ovarian rhythms. Quart. Rev. Biol., vol. 8, pp. 4 2 3 4 3 3 . VAN DYKE,H. 13. 1936 The physiology and pharmacology of the pituitary body. Univ. of Chicago Press, Chicago. WITSCHI,E., AND C. A. PF'EIFFER 1935 Tlic hormonal control of oestrus, ovulstion a n d mating in the fenlalc rat. Anst. Rec., vol. 64, pp. S.5-105. YOUNG, W. C. 1937 The vaginal smear picture, sexual receptivity and the tinic of ovulation in the guinea, pig. anat. Rec., vol. 67, pp. 30.5-323. ZUCKERMAN, 8. 1937 Cyclical fluctuations in oestrin threshold. Nature, vol. 139, p. 628.