THE OESTROUS CYCLE I N T H E M A R E AND SOME ASSOCIA TED PHENOMENA EDWIN SEABORN London, Canada CONTENTS .......................... Introduction . . . . Description of the cases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Survey of the oestrous cycle , . . ....................... A. Uterus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Ovaries . . . . . . . . . . . . . . . . . ................ Fundamental identity of the oestrou mammals . . .......................... Fertilieation . . . . . . Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Suprarenals . ....................................... Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bibliography ...................................... 277 279 282 282 283 283 284 285 283 285 287 INTRODUCTION A very favorable opportunity of studying the oestrous cycle in the mare, together with the gross and microscopic findings in the ovaries and uteri of these animals at definitely known periods throughout the cycle, was afforded the writer while in Paris, France, in the summer of 1923. Inasmuch as such material is difficult to obtain under ordinary circumstances, especially material from mares where the exact day in the oestrous cycle is known, it is felt that this study may be of value to workers in this field, particularly since interest in the phenomenon of oestrus in the mammals is now undergoing a marked reawakening. The mares were kept in the stable under good conditions and were under observation for some time, so that their sexual behavior could be carefully noted and the exact time of the oestrous period could be accurately determined f o r each 277 278 EDWIN SEARORN animal. Willingness to accept the male was taken as an indication of the oestrous period, and the animals were presented to the male daily, without allowing copulation to occur except in two cases-one killed immediately and the other on the second day following. The accurate determination of the exact stage of the cycle is of the utmost importance, for it enables us to correlate with the greatest exactness the external objective manifestations of the various stages with the gross and microscopic anatomical changes of the sexual and other organs taking place in these stages. The oestrous cycle in the mare lasts twenty-four days. It may be divided into a period of rest, lasting eight days; a prooestrous period of three days, during which the animal exhibits external evidences of heat, but does not accept the male ; an oestrous period of three days, characterized by decrease in amount of mucous discharge from the vulva and willingness to mate, and a metoestrous period of ten days. Animals whose time of oestrus had been accurately determined were killed at various times throughout the cycle, so that material from all stages was available. The uteri and ovaries were removed, their gross appearance noted, and microscopic preparations made from them. I n the gross examination of the uterus, it was first opened and the condition of the mucosa carefully noted and recorded. Particular attention was addressed to its looseness and flabbiness, color, vascularity, and oedematous condition. The ovaries of every animal were weighed separately, both untouched and after the follicles had been evacuated. The following report is based upon the findings in this accurately timed and wellpreserved material. The Fallopian tubes, suprarenals, thyroids, hypophysis, and a portion of the vagina were also removed and examined. I n all, nineteen mares were so studied. While all of these could be utilized to show the progression of changes in the cycle, for the sake of brevity only six will be described in some detail, since these are all typical cases, illustrating all the important stages of the oestrous cycle. The stage of the 279 OESTHOUS CYCLE I N THE MARE cycle a t which the mare was killed and the number of each specimen are given in the following table : Fourteenth day following oestrus (rest), First day of prooestrus, First day of oestrus, Third day of oestrus, Second day following oestrus, Eighth day following oestrus (metoestrus), mare mare mare mare mare mare no. no. no. no. no. no. 404 398 4\59 51 468 416 DESCRIPTION O F THE CASES Mare 404-kiJled on the f o u r t e e f l t h d a y following oestrus The opened uterus shows a mucosa of a faint bluish color, containing a minimum of moisturc. The folds of the mucosa are not voluminous and do not hang down of their own weight when the uterus is held reversed. When the mucosa is cut, the tissue does not spread away from the cut edge and extend itself on the table-a process which we have called ‘flowing.’ The blood vessels of the cut mucosa are not visible with the naked eye. Under the microscope, sections show the following characteristics: the superficial cells are cuboidal, with a small content of cytoplasm; the proportion of stroma is small and there is an almost total absence of lymphocytes. The left ovary weighs 55 grams and contains four small Graafian follicle cysts, together with a corpus luteum very f a r advanced in degeneration. After evacuation of the cysts the ovary weighs 47 grams. The right ovary weighs 38 grams and contains six small Graafian follicle cysts. After emptying the latter the ovary weighs 31 grams. The uterus and ovaries are considered to be in that state of low activity characterized as ‘rest.’ Mare 398-killed 041,the firsl d n y of prooestrus The uterine mucosa is of faint bluish color and is somewhat firm in texture. I t is not pendulous and on section does not ‘flow.’ XJnder the microscope, we find great mitotic activity in the epithelium and beginning degeneration of the superficial stroma. Lymphocytes are frequently seen. 280 EDWIN SEABORN The left ovary weighs 33.50 grams and, after emptying the cysts, 29.50 grams. The right ovary weighs 28.50 grams with, and 25.50 grams without, the fluid of the follicles. Neither ovary shows any enlarging Graafian follicle, nor any follicle which is approaching the hilum of the ovary, as occurs in all cases in which a follicle is about to rupture (Seaborn and Champy, ’23). Mare 459-killed o n thc first d a y of oestrus The uterine mucosa is covered by a small amount of nearly colorless fluid, which under the microscope shows no blood cells o r other formed elements. When the uterus is reversed, the folds do not hang down of their ow7n weight, but they are more voluminous than in mares 404 and 398. The mucosa is rosy in color. The main characteristics, under the microscope, a r e : degeneration in the cells of the superficial epithelium, collagenous degeneration of the stroma, and increase in the number of lymphocytes. The left ovary contains four small Graafian follicle cysts and a very large follicle which is on the point of rupturing. An exceedingly thin membrane marks the point of imminent rupture. The ovary weighed 126 grams and, after evacuation of the cysts, 80 grams. The fluid of the mature Graafian follicle weighed 39 grams. On microscopic examination the walls of the large follicle showed lutein transformation of the cells of the theca interna and of the membrana granulosa. The right ovary weighed 70 grams before, and 65 grams after sectioning. It contained five small cysts and a degenerated corpus luteum. ,Ware 51-killrd o n the third day of oestrus The uterine folds are most vo1umiiious, are semigelatinous, and show collections of a yellow fluid under the surface. There is a small amount of fluid free i n the uterus, but this fluid does not contain blood cells or other formed elements. On section the mucosa ‘flows,’ and the blood vessels and OESTROUS CYCLE I N THE MARE 281 ducts of the glands are visible with the naked eye. On reversing the uterus, the folds hang down of their own weight. The main microscopic characteristics are : the irregularity of the epithelial cells, increase in degeneration of the stroma, and great increase in the number of lymphocytes. The left ovary shows a large Graafian follicle which has just ruptured and from which fluid can be expressed in a small stream. The point of rupture is partially occluded by a minute blood clot. The ovary weighed 132 grams, and decreased to 75 grams upon evacuation of the cysts. The right ovary weighed 124 grams and, after evacuation of the cysts, 72 grams. It contained several large cysts and a corpus luteum in regression. illare 468-killed on the second day following oestrus The uterus shows less moisture and is not as gelatinous; when reversed, the folds hang down much less than in no. 53, of the third day of oestrus. In the cut edges the blood vessels and ducts of the glands can be indistinctly made out. The main microscopical characteristics are : lessening of the proportion of stroma to gland tissue, decrease in degeneration of the stroma as shown by diminution of the collagenous tissue, and reduction in the number of lymphocytes. The left ovary weighed 120 grams, contained eight cysts each the size of a cherry, and a corpus luteum 2 em. by 3 em., which consisted of an outer zone of a mottled dark plum color, with blood vessels coursing through it, and an inner zone of a purplish-red color and a consistency of jelly. The right ovary contained several moderate-sized cysts. It weighed 101 grams and, after sectioning, 75 grams. Mare 416, kiLled 0% the eighth day followirzg oest'rus The uterus does not show the yellow color of no. 51, of the third day of oestrus, but is bluish, resembling no. 404, showing the condition of rest, of the fourteenth day following oestrus. It contains no free fluid. When the uterus is re- 282 EDWIN SEABORN versed, the folds hang down of their own weight for a short distance only. The main microscopic characteristics are : continued lessening of the proportion of stroma to gland tissue; the more regular form of the cells of the surface epithelium and of the glands, and reduction in the lymphocytes. The left ovary weighed 62 grams and, after sectioning, 55 grams. It contained a few small cysts. The right ovary weighed 112 grams and contained several cysts and a corpus luteum showing an outer zone of a dark plum color, and an inner zone of a light yellow color and the consistency of jelly. After evacuation of the cysts, the ovary weighed 80 grams. S U R V E Y O F THE OESTROUS CYCLE B. Uterus Surveying the uterine mucosa of the entire oestrous cycle of the mare, we find in the rest period a relatively thin bluish membrane, which upon the onset of prooestrus gradually changes to rosy color, becomes moister, and shows thickening and increase in size of the folds. As the three-day period of oestrus wears on, the much-thickened mucosa becomes yellowish in color, with heavy, wet, and flabby folds. The third day of oestrus having been left behind, the mucosa returns by degrees t o normal, there being evident a thinning and dehydration of the mucosa, a shrinking of the folds, a reduction in size of the blood vessels and gland ducts, with a change in the general appearance from pink to bluish. The uterine mucosa shows microscopically, in the prooestrus stage, an awakening growth activity in the epithelial cells manifesting itself in numerous mitotic figures. This proliferation is followed by degeneration of the epithelial cells in the oestrous period, with gradual return to normal following this. As prooestrus progresses, there is considerable collagenous degeneration in the stroma, especially superficially, which is even more pronounced in oestrus, and which OESTROUS CYCLE I N THE MARE 283 gradually gives way to the normal condition in the metoest ruum. Lymphocytes, scanty in rest, become progressively more numerous until the end of oestrus, when their number is gradually reduced following this stage. B. Ovaries Briefly considering the changes in the ovaries which are correlated with the above-described uterine phenomena, we find during oestrus a considerable increase in the weight of these organs, this being due to the augmentation of fluid in the ripening follicle and hypertrophy of ovarian tissue. Rupture of the single ripened follicle occurs during the second or third day of oestrus; since this is the case, it is evident that the corpus luteum arising from this follicle cannot have initiated the prooestrual and oestrnal uterine changes immediately preceding the rupture, nor can it be held accountable for the sexual behavior of the animal during these stages. All of these phenomena would seem more probably to be related to a hormone of the liquor folliculi or the follicular epithelium, as found by Allen et al. to be the case in spayed mice and rats injected with extract from the liquor folliculi of the pig. Champy and I ('23) brought about the occurrence of rut in unspayed rabbits by injecting into them liquor folliculi from ripe follicles of the mare. I n this connection it was noted that in the mare's ripe follicles lutein transformation of the cells of the theca interna and of the membrana granulosa was found as early as the first day of oestrus. Lutein cells disappear from the corpus luteum when the uterus returns to rest. FUNDAMENTAL IDENTITY O F THE OESTROUS CYCLE I N T H E VARIOUS MAMMALS It would appear, from a review of some of the most outstanding contributions to the subject, that the cycle of oestrus in the mare is essentially the same as that in the other mammals. Stockard and Papanicolaou for the Guinea-pig, Mar- 284 EDWIN SEABORN shall and Robinson for the ferret, Givkovitch for the sow, Hill and O’Donoghue for Dasyurus viverrinus, Van der Stricht, Van Beneden, Courrier, Benecke, and Eimen for the bat, Heape for the monkey, and Schroeder for man, together with many others, have recorded their observations, and although they use different names for the various stages of the cycle, and although the cycle shows wide individual variations, as in such animals as the bat and the Guinea-pig, yet the fundamental ground-plan is the same throughout the mammals, as shown by a careful perusal of their gross and microscopic findings, coupled with the consideration of the sexual behavior of the animals studied. Most interesting of all, perhaps, is the cycle in the human female, which has been a subject f o r speculation for so many ages and about which so many erroneous ideas have gained credence. Although there are some features of it which are obscure when interpreted with reference t o the cycle in other mammals, yet it seems evident that the stages which have been described in the mare are all represented in the woman and in the same order. Thus the oestrous cycle of the lower animals is equivalent to the menstrual cycle in women. This view is in line with the statement of Corner in his recent excellent review of oestrus, ovulation, and menstruation (p. 479) : “it may fairly be stated that the trend of recent work is to suggest the basic similarity of the primate reproductive cycle t o that of the other mammalia.” It seems probable that oestrus of the mare includes more than three periods of the cycle in of woman, according to Schroeder ’s terminology-‘middle interval, ’ ‘end of interval, ’ ‘beginning of pre-menstruum,’ and part of ‘end of pre-menstruum.’ This would push menstruation proper into the metoestruum of the mare. FERTILIZATION It has been noted that the signs of ‘heat’ last some six days, but that during the first three days, the prooestrous stage, the mare does not accept the male willingly. Ordinarily, if a male be available, the mare is covered as soon as oestrus OESTROUS CYCLE I N T H E MARE 285 proper begins. Rupture of the Graafian follicle, as noted, occurs during the second o r third day of oestrus properthus it is a day or so after the usual time of copulation. The distance from the external 0s uteri to the ampulla of the Fallopian tube is some 48 em., and it seems probable that the spermatozoa would take one o r two days to travel this distance. Thus, in the ordinary course of events, the spermatozoa and ovum would arrive in the ampulla at about the same time. PREGNANCY If the ovum lives, i.e., if it is impregnated, the mucoitl degeneration and swelling of the stroma continue and the proportion of stroma to gland tissue becomes very much greater. This increase of degeneration was shown in mare no. 32, killed on the fourth day following her oestrus. She had been covered twice during the oestrus. If the ovum dies, however, the degenerated stroma is gradually absorbed, the lymphocytes disappear, and the uterus in time returns to a condition of comparative rest. 8 IJPRAKENALS As to the changes in other organs of the mare during the oestrous cycle, nothing will be said here except to remark that the suprarenal glands in some cases increase in weight during the oestrous period by a s much as 100 per cent. Macrnscopically, the glands show an increase of the yellow material which forms the peculiar rays of the cortex. Microscopically, the principal changes consist in an increase of the spongiocytic tissue of the cortex. This tissue is probably formed by the dissolving of the labile fats by the reagents employed in the preparation of the specimen. CONCLUSION8 1. The oestrous cycle in the mare lasts twenty-four days and can be divided into four periods: rest-about eight days; days, and metoesprooestrus-three days ; oestrus-three trus-about ten days. 286 EDWIX SEABORN 2. ‘Heat’ lasts six days, and in its first half, the prooestrous stage, although the signs are well marked, there is an unwillingness to accept the male; in the second half, or oestrus proper, the mucous discharge from the vulva has diminished and the mare is willing t o accept the male. 3. I n heat there are well-marked uterine changes. In the prooestruum the mucosa becomes hypertrophied, there being mucoid degeneration of the stroma, multiplication of the epithelial cells, and augmentation of the lymphocyte content. In the oestruum these changes are very outspoken, and on the second and third days, synchronous with the rupture of the Graafian follicle, the mucosa is very thick, wet, and yellowish, with heavy, flabby, and loose folds. Blood vessels and gland ducts are seen with the naked eye and there is much degeneration of the epithelial structure and of the stroma. These changes appear to be in the nature of a preparation of the uterus for the reception of the fertilized ovum. 4. Correlated with these uterine phenomena we find an increase in weight of the ovaries in heat, the ripening of a follicle, and, at the middle o r end of oestrus, ovulation. The suprarenals may double their weight in some cases. 5. Since the corpus luteum is not found until after oestrus is well advanced and degenerates before the next oestrus begins, it is probably not concerned with the initiation of oestrus. The uterine changes and sexual behavior assocciated with heat would seem more probably to be an effect of the influence of the maturing Graafian follicle. 6. Since ovulation doesnot occur until the middle or end of oestrus proper, the probability of fecundation is increased, doubtless, if the mare is covered during the first day that she is willing to accept the male, for this allows time for the sperm to make their way up the uterus and tube. 7. The oestrous cycle in the mare is fundamentally identical with that in other mammals and also with the menstrual cycle of woman. I n conclusion, I wish to extend thanks to Institut Pasteur, University of Paris, St. George’s Hospital of London, and rniversity of Western Ontario. OESTROUS CYCLE I N T H E MARE 287 BIBLIOGH APHY BYRONF. FRANCIS, LEROYL. ROBERTSON, CLEON E. COLGATE, ALLEN, EDGAR, CHARLESG . JOHNSTON, EDWARD A. DOISY, WILLIAMB. KOUNTZ,AND HARRYV. GIBSON 1924 The hormone of the ovarian follicle; its localization and action in test animals and additional points bearing upon the internal secretion of the ovary. Am. Jour. Anat., vol. 34, no. 1, pp. 133-181. 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