Physiology of reproduction in the rat. (1954)

the laboratory rat problem another than any other mammal in this film is internal and external changes which occur during a single reproductive cycle numerous observations have shown that the changes which occur during the reproductive araya t of methods and at close intervals without disturbing their normality mature female rats separated from males repeat the estrous cycle throughout the year at intervals of approximately four days the period of heat lasts on the average for 13 hours and it is only during this interval that the female will mate the interval between the two periods of heat is usually called daya strim it is during the die astrum that the ovarian follicles grow and secrete hormones so as to prepare the animal for the following period of heat and ovulation the most rapid growth of the follicles occurs during the period of heat approximately 10 hours after the onset of heat the follicles rupture releasing the eggs and ovulation has then been accomplished let us now examine some of the methods which may be used for timing the various changes in the reproductive cycle if a female rat is confined to an activity cage observations of her running activity may give a clue as to the time of the period of heat if we record the number of revolutions of the drum for each day of the reproductive cycle and plot these data in the form of a graph we will observe that the animal runs in the activity caves for a considerable distance each day but that she will run for a particularly long distance approximately every fourth day it has been established that this great increase in running activity coincides with the period of heat since the female rap may begin running several hours before she will mate this method of observation is only of relative value in determining either the onset of heat or the time of ovulation the actual onset of the may determined easily and accurately by placing a female with a male as seen here a female rat which is not in heat will resist all mating attempts of the male she will likewise be resistant to manual stoking of the pudendal area and will invariably attempt to escape however at the onset of heat the behavior of the female changes greatly she will no longer attempt to escape but will respond to the mountings of the mail by remaining stationary by arching her back and by raising the pudendal area and tail an identical response may be elicited by manually stroking the pudendal area of the female in heat as may be observed in the next few scenes the response obtained by manually stroking the pudendal area of a female in heat is identical to that displayed when the male mounts the female the onset of heat may be accurately determined by this method and it is therefore a reliable basis for determining the time of ovulation and various other reproductive phenomena another change which may be observed in the external behavior of the female in heat is the ear quiver shortly before the onset and during heat the ears may quiver whenever the female is stimulated in the pudendal area the ear quiver gradually subsides toward the end of heat and is not present during the daya strim still another change which may be observed externally as the period of heat approaches is the tumescence and coloration of the folds of the external vaginal orifice several hours before the onset of heat and during heat the folds become swollen and engorged with blood giving them a bluish color when compared to an animal not in heat the tumescence of the vaginal folds is a useful method for predicting which of the females in the colony will come into heat during the succeeding hours the rack is a nocturnal animal and its greatest activity including the period of heat and ovulation occurs between the hours of six pm and six a.m. here is a graph indicating the onset of heat in 609 females eighty percent of them came into heat between 7 p.m. and 11pm another method for timing the events in a reproductive cycle is by examining the vaginal smear let us now observe the alterations which occur in the vaginal mucosa during the cycle and which account for the changes in the cell types which may be observed in the stained smear preparations for orientation let us examine a section through the wall of the vagina removed on the first day of the cycle the epithelium is composed of several layers of cells the number of cell layers increases rapidly by mitotic division at this time of the cycle leukocytes are making their way through the epithelium and into the lumen of the vaginal canal cells which are slough from the surface in making the smear are round and nucleated an actual smear made at this time shows both nucleated epithelial cells and leukocytes a section of the vagina examined on day three of the four day cycle will show an increase in the number of layers of epithelial cells leukocytes are no longer passing through the epithelium the superficial epithelial cells are still nucleated in turn actual smear made at this time only nucleated cells are present as the period of heat approaches the superficial epithelial layers gradually become stratified and cornified the nuclei of the superficial cells become thick nautic and disappear a smear made of these superficial cells will show only flattened non nucleated plaques wood stain quite differently from the cells of the deeper layers near the end of heat and for a period thereafter leukocytes invade the superficial epithelium and large numbers of them migrate into the lumen thus the smear will again show the cornified cells but in addition large numbers of leukocytes will be present after the period of heat the superficial cornified layers slough off and are lost from the vagina the changes in the vaginal smear during the cycle are only of relative value in accurately determining the time of ovulation and various other reproductive phenomena before examining the more obvious changes in the reproductive organs of the wrath especially during the period of heat it is desirable to understand the normal anatomy of the genital tract the ovaries are enclosed in a thin transparent membrane the perio varial sac the highly coiled oviducts connect the period of aerial spaces with the uterine cornea the right and left cornu enter separately into the cranial vagina during the period of heat the corn you have become distended with a clear fluid the cervix undergoes sustained contraction to retain the uterine fluid here may be seen the left and right cornea of a living animal which was not in heat peristaltic and added peristaltic activity is minimal very little fluid may be aspirated from the lumen of the cornu of a female rat not in heat in contrast the cornea are considerably distended with fluid in a female examined during heat muscular activity of the uterine horns has increased greatly peristaltic and airy peristaltic contractions waves are readily visible the corner of the female in heat contains two to three tenths of a cubic milliliter of clear fluid during heat several of the loops of the oviduct as seen on the right become dilated with fluid and it is in this region that the ovulated ova our first stored and fertilized we will now observe the method whereby the oval ated eggs are transported into the oviduct this drawing is for orientation of the next few scenes if the perio varial sac surrounding the ovary is opened we may observe the fimbriated end of the oviduct extending into the perio varial space on the left is the ovary the coils of the oviduct actually lie outside of the perio varial sac membrane the ovum with its accompanying granulosa cells has been super vitally stained with methylene blue we will observe the egg being moved by ciliary activity toward the ostium where a combination of ciliary and muscular activity carries it into the oviduct in this specimen the granulosa cell mass with its enclosed egg is moved rapidly over the surface of the fimbriae t'attend of the oviduct and enters the ostium it is then transported into the oviduct by means of both ciliary and muscular movements if we remove a small segment of the mucosa of the fimbriated end of the oviduct and examine it with phase contrast microscopy we may observe the appearance and activity of the cilia in this sequence to ova arrive at the ostium which is temporarily hidden from view by a large corpus luteum contraction of the oviduct will bring the ostium into focus the two ova appearing as translucent vesicles are now easily visible in the center of each granulosa cell mass the end of the oviduct appears to dilate as the eggs pass into the ostium as has been mentioned earlier mating in the rat will occur only during the period of heat at the moment of ejaculation approximately 50 million spermatozoa are catapulted directly into the uterine lumen where they are transported almost immediately by the anti peristaltic activity of the cornea to the uterine hand of the oviduct ordinarily less than 50 spermatozoa arrived at the site of fertilization and it requires approximately one and a half hours for them to travel to the upper dilated loops of the oviduct the site of fertilization when sperm reached the ova they immediately begin to penetrate the granulosa cell mass how this is accomplished is as yet unknown it is assumed that the spermatozoa release an enzyme hyaluronidase which d polymerizes the hyaluronic acid intercellular cement substance here are several spermatozoa penetrating the granulosa cell mass their rate of forward movement is [ __ ] due to the barrier of the cells not the great activity of the tail as the sperm pushes into the granulosa cell mass sperm may be observed within the pair of a tell'n space of an egg without a noticeable change in the granulosa cell mass several spermatozoa may penetrate into the pair of at elland space but only one enters the OL plasm to affect fertilization in this specimen the fertilizing sperm has become quiet but the accessory spermatozoan is actively moving about in the pair of a tell'n space after a sperm penetrates into the Auto plasm it soon becomes quiescent and its head which contains the nuclear material is transformed into the male pronucleus by means of time-lapse photography we will observe the transformation of the sperm head into the male pronucleus the high contrast of the head is gradually lost beginning first in the area nearest the flagellum proceeding in a cephalic direction soon the pro-nuclear cytoplasm becomes visible and the development of the nucleoli by fusion of smaller nuclear leur units may be observed the male and female pronuclei grow rapidly in size and migrate toward each other the male pronucleus is always the larger of the two at approximately 24 hours after sperm penetration fusion of the pronuclei occurs when this has been accomplished the fertilization prostate and segmentation division begins the telophase stage of the first segmentation division may be seen in the center of this cell the egg then quickly divides into two cells and is on its way to develop into a new animal as has been shown here the reproductive process of the rat is a very efficient biological phenomenon the cyclic nature of which can be readily determined by observing the various external and internal changes which occur during a single reproductive cycle