AUTHOR'S ABSTRACT OF THIS P.4PER ISSUED DY T H E BIBLIOGRAPHIC SERVICE. OCTORER 22

RACIAL AND FAMILIAL CYCLIC INHERITANCE AND OTHER EVIDENCE FROM THE MOUSE CONCERN-

ING THE CAUSE OF OESTROUS PHENOMENA

EDGAR ALLEN Department of Anatomy, Washington University, St. Louis

SIX CHARTS

It is a general observation that certain species and races of mammals have varying degrees of fecundity. Since several strains of mice were used for this study of the oestrous cycle (Allen, '22), it has seemed worth while to examine available data for possible variations in cycle length in races and families. More than ninety animals were studied, some for extended periods of time, to furnish data as to cycle length. Uniform environ- mental conditions and a plentiful supply of food and water were maintained throughout the work but still there was great varia- tion in the length of the cycles recorded.

The oestrous cycle in the mouse usually requires from four to six days (graph 1). The curve represents diagrammatically variation in the blood supply, and alternate periods of acceler- ated growth and degeneration in the genital tract. During the dioestrous interval (D) the generative organs are relatively quiescent and anemic. The prooestrum (P) is a period of hyper- emia, growth, and hypersecretion which culminates in oestrus (0), the period of sexual desire. This is closely followed by ovulation if that is to be spontaneous. Then follows the met- oestrum ( M ) or period of degeneration and leucocytosis of the uterine and vaginal epithelium which destroys much of this growth of the preceding periods and returns these organs to a dioestrous condition of relative quiescence.

There are two principle types of variation of this usual 4 to 6- day cycle. 1) Instead of the rapid recurrence of a second cycle,

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THE AMLRICAN .JOURNAL OF ANATOMY, VOL. 32, NO 3

294 EDGAR ALLEN

a long dioestrous interval may intervene, resulting in an appar- ently normal cycle of eight or more days' duration (D', cycle represented by dotted curve). At times in such a cycle there may be slight indications of oestrus in the smears at the midintend suggesting possibly that the failure of the expected heat period is only partial. 2) There may be a continuation of oestrus, usually evident for one day or .less, for two, three, or more days (O", cycle represented by dot-and-dash curve). Due chiefly to these two types, there is great variation in the oestrous cycle.

Graph 2 represents a total of 563 cycles. The length of the cycle in days is plotted against the number of cycles of a certain duration. When data from different strains are lumped together any racial or familial differences are obscured in a fairly uniform, unimodal curve. The mode falls at four and a half days.

Oestrous cycle i n the louse.

D , dioestrous interval P, prooestrum 0, oestrus

KEY TO LET'PERTh'G O F GRAPHS

Ou?, sporadic Pu, puberty, attainment of P, follicles, growing; contained ova

ill, inetoestrum approaching maturation (D ' ) , extended dioestruni, long cycle FA, follicles, large, atretic

(0"); extended oestrus, long cycle type

00, ovulation

type 1

I1 tion

CLO, corpora lutea of oestrus RO, retention of ova a t failure of ovula-

CONCERNING THE CAUSE O F OESTRUS 295

In graph 3, composed of data from sixty-four animals, the cycle length is plotted for different coat colors. The mode of the curve for browns is between five and six days. This strain has the greatest number of long cycles of type 11, i.e., with an extended heat period. They are a very prolific strain. The curve of the albinos has a distinct mode at four days. Al- binos in our stock are browns minus the color factor. Has the loss

Curve of cycle length

Length of cycles in days

Graph 2

of the color factor for brown shortened the cycle? The mode for blacks also falls at 4 days. Clear-cut modes of the curves for yellows and grays fall at five days. Grays are recessive deriva- tives of the yellow strain. In dealing with such a variable unit as the oestrous cycle such evidence cannot be considered con- clusive, but it at least suggests that a genetic factor, possibly linked with color factors, may be accountable for some of the variation in cycle length among different strains of mice.

296 EDGAR ALLEN

If such a genetic factor is operating to partly determine cycle length, one would expect less variation in litter sisters of a pure strain. Table 1 consists of data from four litter sisters (albino mice) which were examined for 146 days. This provided 24 to 30 cycles in each from which to determine an ayerage

Variations in cycle length in different strains

80

70

60

3 50 A u c 0 r40 3 E

K30

20

10

--- brown black -- - -

\ albino - -- yellow

\ gray -.._- -

2 3 4 6 6 7 8 9 1 0 Length of cycles in days

Graph 3

The first line shows that the average cycle length varies some- what. 53W had no unusually long cycles: NL had 4; NR, 1; SE, 2. These cycles, however, are approximately twice the aver- age length for 53W. They apparently result from the failure of an expected oestrus. The average of these seven long cycles is 9.8 days, or exactly twice as long as the total average if the long cycles be omitted, 'in which case there is a cycle length varying

CONCERNING THE CAUSE O F OESTRUS 297

Average cycle length.. . . . . Number of unusually long

cycles.. . . . . . . . . . . . . . . . . . .

Average minus long cycles. .

only one-tenth of a day in these litter sisters. This family average of 4.8 days is more striking sincz these four sisters are albinos and the mode for albinos falls at four days, while this is nearer five.

It may be concluded that when different strains of mice are kept side by side under uniform enrironmental conditions they do not show racial differences and in some instances familial similarities in the length of the oestrous cycle. This evidence suggests the operation of a genetic factor in at least partially determining cycle length even within a single species. It directs attention to the ova themselves as a possible cause of oestrus.

TABLE 1

Oestrous record o f f o u r litter sisters (albz'no mice) (146 days: $4 to 30 cycles)

53 w

4.8

0

4.8

5.7 1 5.1 1 5.2

4.8 4.9 1 4.9

4 (9, 11, 11, 10 1 (8 days) 2 (10, 10 days) I days)

ANIMAL@

53 WNL ~ 53 WNR I 531E 1 *A',","-

5.2

9.8

4.85

FURTHER EVtDENCE FROM THE MGUSE CONCERNING THE CA4USE OF OESTRUS

In 1900 Heape believed oestrus to be caused by a hypo- thetical substance, gonadin, derived from certain foods and circulating in the blood stream. At that time it was believed that oestrous phenomena might occur independently of ovarian influences. Now, however, convincing evidence has established the conclusion that oestrous changes are no longer evident after double ovariotomy. Therefore the cause of oestrus must be sought for in the ovaries.

Many attempts have been made to localize ovarian function in specific structural units. Marshall ('14) concluded from experi- mental evidence that ovarian interstitial tissue is responsible for oestrus. To the corpora lutea of oestrus have also been attributed causative functions. There is some evidence from the mouse bearing on this attempt to localize the ovarian factor influencing the uterus and vagina.

298 EDGAR ALLEN

1. Interstitial tissue. In mouse ovaries interstitial tissue is inconstant. No variation in it with different phases of the cycle has been established. Consequently it is difficult to consider it seriously as a causative factor in oestrous phenomena.

2. Corpora lutea of oestrus. There are three points of evidence from the mouse bearing on the possible participation of the corpora lutea of oestrus as causative factors in these cyclic changes.

a. In animals that ovulate spontaneously at oestrus it is the exception to find ovulation before the attainment of puberty (defined as the first oestrus at which the opening of the vagina

Oestrous curve at the a:tainnzent of pubertg

Graph 4

occurs). A long prooestrum or growth phase precedes this (graph 4; key to lettering, p. 294), but since there has been no ovulation there are no corpora lutea in the ovary. How can they then cause the growth phase in the genital tract?

b. I n some mature animals after recording several normal cycles, histological examination of the ovaries shows them to be free from corpora of oestrus, proving that ovulation has not occurred at the recorded heat periods (graph 5 ; key to lettering, p. 294). Often after recording three or four oestrous periods only one or two sets of corpora are to be found in the ovaries. There- fore ovulation may occur at one period but at the next the folli- cles may fail to rupture. Yet in the absence of corpora the ana- bolic and catabolic phases of the cycle as manifested in the genital tract may succeed each other as in spontaneously ovu- lating animals.

CONCERNING THE CAUSE O F OESTRUS 299

c. In mice where ovulation is spontaneous at each oestrus, because of the short cycle and the persistence of the corpora of oestrus through several cycles, lives of corpora from several successive ovulations overlap (graph 6; key to lettering, p. 294). There may be several sets of corpora in the ovaries at one time and still cycles succeed each other regularly.

Oestrous curve when oaulation i s not spontanecus at oeslrus or only sporadically SO

Graph 5

Oestrous curve when ovulation i s spontaneous at oestrus

P 0 hf D Graph 6

Therefore, in the absence of corpora lutea of oestrus,l or in the presence of several overlapping sets, anabolic and catabolic changes in the genital tract may occur in fairly regular succession. Consequently it is rather difficult to see how the corpora of oestrus in the mouse can be very important causative factors in oestrous phenomena .

1 The distinction is clearly drawn between the corpora lutea of oestrus and those of pregnancy: no reference is made to the latter in this paper.

300 EDGAR ALLEN

3. The follicles during the anabolic phase of the cycle there are always follicles approaching rupture size in the ovaries. An ex- ‘onded oestrous period seems to he correlated in some cases with a ictention of mature ova (o”, dot-and-dash curve, graphs 1 and 5). La1 ge follicles are absent through ovulation, or atretic, during the metoestrum. In the absence of definite evidence for the partici- pation of the corpora of oestrus and interstitial tissue as causative factors, there seems to be nothing left but the follicles. Therefore the follicles must be considered most important. Ovulation, or atresia if this fails to occur, is the dividing line between the anabolic and catabolic phases of the cycle. This observation and the suggestion from racial and familial cyclic inheritance point to the ova themselves as the ultimate cause of oestrous phenomena.

A further analysis of the ovarian follicle may offer a suggestion as to function. The blood vessels, of course, do not penetrate the follicular epithelium. Consequently all nutriment from the blood stream must reach the ovum through the mediation of the follicle cells. Therefore they have been called nurse cells to the ovum. When the maturation mitoses of the ovum begin the corona radiata forms in the follicle cells immediately sur- rounding it (Robinson, ’18). The nuclei of these cells migrate away from the egg membrane, greatly elongating the cells in a direction perpendicular to a tangent a t its surface. Robinson has inteipreted this maturation phenomenon as a severance of this sacretory function of the follicle cells to the ovum preparatory to ovulation. He observes a similar phenomenon at the same time in the follicle cells bordering the theca interna. If these cells have been secreting a hormone, his interpretation that a corona-like formation indicates a discontinuance of the nor- monal function of the follicles seems logical (plates V I I to X in Robinson’s paper).

The evidence from the mouse and Robinson’s observations and interpretation make possible the statement of the following working hypothesis. Under the influence of the ovum the follicle cells secrete a hormone which causes the hyperemia, growth, and hypersecretion in the genital tract during the anabolic phase

CONCERNING THE CAUSE OF OESTRUS 301

of the oestrous cycle. When the influence of the ovum is removed at ovulation (or atresia) there is a cessation of the elaboration of this hormone or at least it is no longer secreted into the blood st,ream. Its withdrawal allows the degeneration of the excessive growth of the preceding stage and the metoestrum (probably analogous to menstruation) sets in and removes this necrotic tissue.

In an attempt to follow up these conclusions experimentally Dr. E. A. Doisy, of the Department of Biochemistry, and I have been able to isolate an active substance from the follicles of hog and cattle ovaries which, injected into previously sptiyed animals, causes typical oestrous changes.2 At this time as in the normal animal an acceleration of growth occurs in the genital tract. In the vagina this greatly thickens the walls and leads to the formation of a cornified layer similar to that in the epidermis. As the superficial cells are slowly sloughed into the lumen they provide a definite succession of specific cell types which con- stitutes a reliable test of the oestrous condition of the living animal (Stockard and Papanicolaou, '17; Long and Evans, '22; Allen, '22). During this artificially induced oestrus animals experience mating instincts, the female at times taking the initiative in the courtship, which culminates in copulation and the formatim of a typical vaginal plug. Oestrous changes as de- scribed in the papers cited above also occur in the uterus. When the effect of the injections wears off a heavy leucocytic infiltra- tion sets in and removes much of the tissue which resulted from the injections of this follicular hormone.3

This demonstrates the conclusions previously arrived a t concerning the cause of oestrous phenomena in the genital tract

2 R. T. Frank has reported inducing oestrous conditions in two rabbits by injections of liquor folliculi. These animals were not spayed, however; con- sequently it is difficult to be certain that his results were obtained from t h e injections or from direct ovarian influences. Also no such reliable criterion of oestrus as applied in our work was used to determine his results. Frank gives an extensive review of the present status of ovarian therapy. Ifis conclusions concerning the value of commercial ovarian extracts are substantiated by their failure to produce results in our test animals.

3 This evidence will be published in detail a t an early date.

302 EDGAR ALLEN

in the absence of pregnancy and shows that but one factor is necessary. The alternate presence and absence of a hormone from successive groups of maturing follicles is sufficient to explain this mechanism. Although the corpora lutea of oest>rus may exert a secondary regulatory influence it is doubtful if they have any primary, causative (hormonal) function in oestrous phenom- ena in the genital tract of non-pregnant animals.

It seems preferable to interpret the anabolic changes in the uterus as merely the well being of the ducts of the gonads at the time when the sex cells are approaching maturation and the cata- bolic phase as a degeneration of an excessive growth of tissue no longer supported by a necessary hormone. This interpretation tends to the application of the same fundamental principles to mammals as to the lower vertebrates (i.e., seasonally breeding fishes and birds, etc.).

The question as to why certain groups of ova mature at rather definite intervals is still unanswered. The ovary is considered a a reservoir in which ova are stored away before birth or a least before the attainment of puberty until some unknown cause brings about their maturation during sexual maturity. In the mouse, however, there is evidence that new generations of ova are added by proliferation of the germinal epithelium during sexual maturity (illlen, '23). These ovogenetic periods begin by mitotic division of germinal epithelial cells during the protiestrum, rise to a maximum during oestrus, and continue into the metoestrum. During the dioestrous interval ovogenesis wanes or ceases alto- gether. There is some evidence that the same condition prevails in the ferret (Robinson), the rat (Xrai, '21), (Allen, unpublished), and the guinea-pig (Sun, '23) , and also in other mammals al- though as yet the latter has not been definitely correlated with the oestrous cycle. This evidence furnishes a distinct age differ- ence in sets of maturing ova, thus providing a rational explanation for the maturation of successive groups of follicles which alone is sufficient to account for oestrous phenomena in a placental inamnial as high as the mouse.

CONCERNING THE CAUSE O F OESTRUS 303

SUMMARY AND CONCLUSIONS

1. Cyclic changes in the genital tract in the mouse can be resolved into alternate anabolic and catabolic phases. The ana- bolic phase parallels the growth of the follicles and the matura- tion of their contained ova. The catabolic phase returns the genital tract to the relatively quiescent condition of the dioestrous interval.

2. There are two common variations in the oestrous cycle. These occur when uniform environmental conditions and a plentiful food supply are maintained. There is some evidence for racial differences and familial similarity in cycle length, This suggests that a genetic factor may be operating to partially determine cycle length.

3. There is no evidence that the corpora lutea of oestrus bear any primary causative relation to oestrous phenomena in the genital tract in the mouse. Three points against such partici- pation by the corpora of oestrus are cited. 4. Interstitial tissue is too inconstant in mouse ovaries to

receive serious consideration. 5 . The follicles are the most important source of the ovarian

influence on the genital tract in the absence of pregnancy. As ovulation is the dividing line between the anabolic and catabolic phases of the oestrous cycle, the presence of maturing ova in large follicles and their absence after ovulation or atresia is sufficient to explain the mechanism of these phenomena.

6. A functional analysis of the follicle is attempted. 7 . Following these conclusions we have isolated a hormone

from the ovarian follicles of hogs and cattle which, injected into previously spayed animals, causes typical oestrous changes in the genital tract. During this induced oestrus typical mating instincts are experienced and copulation occurs. After the effect of these injections wears off a typical metoestrum sets in and returns the genital tract to a dioestrous condition.

8. It is probable that this hormone is produced by the matur- ing ova or by the follicle cells under the influence of the ova. It is not species specific, but probably common to maturing ova

304 EDGAR ALLEN

of all female animals. Its presence and absence, due to the periodic development of successive sets of follicles, is sufficient to explain the mechanism of oestrous phenomena.

9. The continuation of ovogenesis during sexual maturity is stressed as establishing a distinct age difference in successive generations of ova. This is a logical reason for their maturation at definite intervals.

LITERATURE CITED

ALLEX, E D G ~ R 1922 The oestrous cycle in the mouse. Am. Jour. Anat., vol. 30, no. 3. 1923 Ovogenesis during sexual maturity. Am. Jour. Anat., vol. 31, no. 5.

ARAI, Hyam On the postnatal development of the ovary (albino rat) with especial reference to the number of ova. Am. Jour. Anat., vol. 27.

FRANK, I<. T. 1922 J.A.M.A., vol. 78, p. 181. LONG, J. A,, AND EVANS, 1-1. M. ‘rile oestrous cycle in thc ra t apd nssoci-

ated phenomena. I~OBINSON, ARTHUR 1918 The formation, rupture, and closure of ovarian

follicles in ferrets and ferret-polecat hybrids and some associated phenomena.

STO~~KARD, C. R., AND PZPAXICOJAOU, G. N. 1917 Existence of a typical oes- trous cycle in t>he guinea-pig with a study of its histological and phvsio- logical changes. Am. Jour. Anat., vol. 22.

SUN, Y. C. 1923 Post-pubertd ovogenesis in the guinea-pig. i lnat . Rec.. vol. 25, no. 3, p. 114.

1921

1922 Univ. of Calif. Pub.

Trans. Roy. SOG. Edinburgh, vol. 5’2, pt . 2, no. 13.