Paper - The breeding habits, maturation of eggs and ovulation of the albino rat (1913)
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Kirkham WB. and Burr HS. The breeding habits, maturation of eggs and ovulation of the albino rat. (1913) Amer. J Anat. 15(2): 291-319.
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The Breeding Habits, Maturation of Eggs and Ovulation of the Albino Rat
W. B. Kirkham And H. S. Burr
Sheffield Biological Laboratory, Yale University
The present work was started by the senior author under the supervision of Professor W. R. Coe in the spring 1907, and in 1908 Professor Coe published in Science a brief statement of what had been found. In the summer of 1911 the junior member, Mr. Burr, took up the work. In the interim the literature of the subject had been enriched by three papers, and since then two additional ones have appeared. Lantz in 1910 contributed to a United States government report, on the economic importance of the rat, a short paper on the natural history of the animal. The author describes the different species of rats, their distribution, and general habits, but pays little attention to the details of their reproduction.
Sobotta and Burckhard ('10) made a careful study of the maturation and fertilization of the egg of the albino rat, and they describe and figure the ovarian egg in the stages of the first polar spindle, and first polar body with the second polar spindle, and the tube egg in the stages of the second polar spindle, fertilization, second polar body, and the pronuclei. Ovulation is stated to occur independent of pairing within thirty-six hours after the birth of a litter, and the eggs fertilized nine to twelve hours after copulation. Sobotta and Burckhard found the mature rat egg, in the ovary, to measure in preserved material 0.06 to 0.065 mm. in diameter; practically the same as the mouse egg. These investigators never saw a definite first polar body associated with an egg in the tube.
Newton Miller's paper ('11) on reproduction in the brown rat is based solely upon observations of the living animals. He found that both sexes become sexually mature at least by the end of the fourth month," that the litters contain from six to nineteen young apiece, and that these animals breed the year, round.
Mark and Long ('12) devote most of their contribution to an extended description of the elaborate warm chamber they have devised for the study of living mammalian eggs. When it comes to the results obtained with their apparatus they have, at present, little to say. Living eggs of rats and mice obtained in a manner similar to that described by one of us (Kirkham '07) were placed on the stage of the microscope in the warm chamber and spermatozoa added, the mouse eggs underwent no change, but the rat eggs within five minutes to two hours began the formation of the second polar cell. Cleavage has never been observed, and after twelve hours the eggs begin to degenerate.
The latest contribution to the literature on the subject of rat breeding is by Helen Dean King ('13) who records for the albino rat somewhat the same phenomena previously observed by Daniels ('10) in mice. The normal period of gestation for the albino rat, according to Miss King, is twenty-one to twentythree days. If six or more young are being carried while a previous litter of five or less are still suckling the period of gestation may be prolonged, while if more than six young are suckling the period is always prolonged, regardless of the number being carried. Unlike the mouse, the albino rat appears not to exhibit any exact relation between the number of young either suckling or borne and the extent of prolongation of the gestation period. This paper also contains evidence that the eggs of a given oestrus cycle in the albino rat may be discharged from the ovaries in two sets, with an interval of two to three days, and also that in very rare instances this interval may be extended to two weeks. Miss King would like to interpret the latter cases as instances of a distinct oestrus cycle occurring during pregnancy.
The present paper has as its object the filling in, as far as possible, of such stages as have not previously been described, and the presentation of evidence regarding the time relations in the development of individual eggs. The authors' thanks are due to Prof. W. R. Coe for the use of the notes and drawings of the eggs of the brown rat, and to Dr. T. B. Osborn of the Connecticut Agricultural Experiment Station for the designs of the cages used and for the animals with which the work was started.
About 150 albino rats were under observation at different times during the investigation. One' large cage was used for all rats not at the time under special care. For individuals two types of cages were employed, one, a cylindrical cage of wire netting of sufficient size to accommodate two rats at a time, and the other a much larger, rectangular cage of galvanized iron, with wire netting only on the front and bottom. This second type of cage was designed primarily as a breeding cage and was large enough to house a mother rat and a litter of the largest size until the latter were sexually mature.
The food of the animals consisted of oats, corn, wheat, sunflower seeds, and dog-biscuit, together with bits of lettuce, string beans, bread, and various kinds of cooked meat and fish.
All cages were kept as clean as possible, but except when absolutely necessary litters less than two weeks old were never disturbed. At the times when we were inspecting them the rats were encouraged to come out of their cages and run about the room, and to this faixdliarity with us as well as to the additional exercise thus secured we attribute much of our success in rearing large litters without their being maimed or eaten by the parents.
Usually females were isolated in breeding cages as soon as they were seen to be pregnant, but in the few instances when males were left with such females until several days after the birth of the litter no mortalitv occurred. This fact leads us to agree with Miller ('11) and King ('13) that mother rats, unless they are in an unhealthy condition, or have been frightened in some way, rarely if ever kill or maim their young.
Albino rats give birth to young in all seasons of the year, but it is only from April to October that ovulation as a rule occurs within 48 hours after parturition; during the remaining months they are apt to skip oestrus cycles, ovulation not occurring until some three weeks after parturition.
The senior author showed in 1910 that the albino rat o\tilates regardless of whether pairing has previouslv taken place, and when males are continuously present copulation may occur before the ripest eggs in the ovaries have formed the first polar spindles. On several different occasions we have seen the actual pairing. It differs markedly from the condition described by Sobotta ('95) for the mouse, since the male albino rat is not prostrated by the sexual act, but walks slowly away. When a previously isolated female who is in heat is placed in a cage wdth several males they will all pair with her in rapid succession.
The period of gestation in the albino rat is twenty-two days when the female is not nursing a previous litter, in which event the period may be lengthened as found by King ('13). The litters varied in number from four to twelve and the birth usually took place in the late afternoon or the early evening, although probably it may occur at any hour of the day, since we have observed it at noon. The process of parturition is briefly as follows : The female in order to aid in the expulsion of the foetus flattens herself against the bottom of the cage while a series of wave-like muscular movements pass posteriorly along the body starting just behind the shoulder. As soon as the young rat is free from her body, the female rises up on her haunches, seizes in her forepaws the button-like placenta? which is still attached to the offspring by the umbilical cord, and devours first it and then the cord, cutting off the latter as close to the body of the young animal as she can get with her teeth. The female then again flattens herself out against the bottom of the cage preparatory to the appearance of the next young rat. The process is repeated until all have been brought forth. Then, and not before, does the mother assemble the young, cleaning them up with her tongue, after which they lie close together under her to keep warm. From this time on until the young are able to crawl around by themselves the mother never leaves the nest until she has carefully covered her litter. On returning she always looks around for any that may have rolled or crawled out in her absence, and such offenders are quickly seized in her jaws and hauled back into the nest.
The albino rat becomes sexually mature, at least in some cases, as early as fifty-five days after birth, since in one instance a litter was born to rats that were only seventy-seven days old.
Maturation and Ovulation
The paper by Sobotta and Burckhard ('10) on the maturation and fertilization of the albino rat is by far the most complete account of the subject that has so far been published. However this report left a number of things to be cleared up.
Working with material from 81 rats we have attempted to investigate and make clear the following points: (1) the early development of the egg previous to the formation of the first polar spindle, (2) the formation of the first polar body, (3) the condition of the egg at ovulation, (4) the process of fertilization and second polar body formation.
At first the rats were watched and killed at short intervals up to forty-eight hours after pairing. This gave no data that could be depended upon for determining the stage which either the ovarian or the tube eggs had reached. However, by relating the time of killing the female to the time of parturition it was found that tiie approximate development of the egg could be predicted without much difficulty. We say approximate because even though the exact hour of parturition be known it is impossible to say that at a given interval of time the eggs are in a given stage of development.
The parturition of a female caged with a male having been observed, she was killed twenty-four hours later. This female yielded unfertilized tube eggs, indicating that ovulation had recently occurred. Cases such as this show that ovulation usually occurs about twenty-four hours after parturition. The individual variation is so great that any complicated apparatus for determining the exact date of parturition is valueless. We have obtained the best results by killing the females at half hour intervals, beginning in the later afternoon and continuing through the early evening. By doing this practically all the stages of maturation can be obtained.
In a number of instances the senior author dissected out the Fallopian tubes, and after placing them in warm salt solution, by slitting the tubes he was enabled to obtain two eggs fertilized but unsegmented, three eggs in the two cell stage and three eggs so obscured by follicle cells as to prevent any exact information as to their condition. The technique of this operation is so simple, requiring only a binocular microscope, two needles, some warm physiological salt solution, and a female rat that has given birth to a litter at least twenty-four hours before and not more than five days previously, that we recommend the rat as highly as the mouse for obtaining live mammalian eggs for class demonstration.
In all other instances the ovaries (and also the tubes, wherever ovulation was thought to have occurred) were fixed in either Zenker's 'fluid or in a strong solution of Flemming, imbedded in paraffin, cut serially into sections 0.010 mm, thick and stained in Delafield's haematoxylin. Such sections as were found on subsequent examination to be worthy of detailed study were later decolorized with acid alcohol and restained with Heidenhain's iron-haematoxylin.
A study of the ovaries of the above rats showed that there is a progressive development of the egg until it is ready to leave the ovary at ovulation. The developing eggs of any adult ovary can be readily divided into six groups. The first of these (fig. 1) includes all those eggs that are in the resting condition. These vary considerably in size, as do also their follicles. The earlier stages show a small egg with a follicle consisting of from one to three layers of radially arranged follicle cells with scattered cells lying between the layers, the later stages lie in larger follicles with many more layers of cells. The egg nucleus presents a constant appearance, a clearly defined nuclear membrane, scattered chromatin and a deeply staining nucleolus.
The second group includes those eggs which differ from those of Group I only in their size and in the fact that they lie in much larger follicles, the latter consisting of a large number of cells closely packed but showing no radial arrangement except in the layer immediately surrounding the egg. Such an egg is shown in figure 7.
The third group includes a much smaller number of eggs which lie in follicles similar to the preceding, except that the cells lying in or near the center of the follicle show a marked tendency to separate, leaving a clear space. This condition may, however, be found in follicles belonging to eggs of Group II, for the factors governing the growth of the follicle are not, according to our observation, constant, since growth may set in when the egg has reached the stage of development included in either Groups II, III or IV (figs. 2, 3 and 4). The nuclei of the eggs of this third group show a marked change. The nuclear membrane is still distinct, but the chromatin is less scattered and the nucleolus has become partially vacuolated, since it shows much less affinity for the stain. Figure 8 shows an egg of this group.
Fig. 1 Normal resting follicle. X 630
The fourth group shows further modifications. It is at this point that the maximum growth in the size of the follicle takes place. While growth may have started in either of the two preceding groups, the greatest growth occurs with the egg in this stage of development. Eggs of this group have been observed with follicles similar to those of the two preceding groups, and also with follicles of nearly the maximum size. The nuclei of these eggs show a diminution in the amount of chromatin present and a complete vacuolization of the nucleoli, the latter showing no affinity whatever for the stain. Such an egg is shown in figure 9.
Fig. 2 Earliest observed maturation phenomena — increase in size of follicle. X 90.
Fig. 3 Follicle of egg with first polar spindle shown in figure 10 of Plate II. X90.
Fig. 4 Follicle of egg with first polar body and second polar spindle shown in figure 11 of Plate III. X 90.
The fifth group consists of the eggs with first polar spindles. The follicles here are typical, showing a slight tendency to be thinner in the region where the follicle is nearest to the surface of the ovary. The nucleus of the egg has disappeared, and in its place lies the first polar spindle (fig. 10).
The sixth group shows no change in the size of the follicle. The first polar body has been extruded and a second polar spindle formed (fig. 11).
In all the above divisions, with the exception of the sixth group, wherever a distinct zona radiata can be seen, very fine protoplasmic bridges can readily be distinguished crossing froni the follicle cells to the egg. The presence of these very distinct filamentous processes of the follicle cells seems to have been entirely overlooked by previous investigators.
One striking thing is to be noted with regard to the above divisions — never were all six found together in one ovary at a given time. As was to be expected. Group I, since it included all the resting eggs, was present in all ovaries. Group II, on the other hand, was seen to drop out on the appearance of Group IV and to reappear on the disappearance of the latter. Group V also appeared on the disappearance of Group IV. When Group V dropped out, Group VI appeared. Group III was found in all ovaries.
From the fact that perfectly normal eggs of Groups II and III were found in the ovary just at, and also just subsequent to ovulation, it was evident that more than one oestrus cycle was necessary for the development of the egg from the resting stage to the stage of the first polar body and second polar spindle, at which stage the egg leaves the ovary, for, if the above changes occurred in one oestrus cycle, ail normal eggs in the above condition would go out of the ovary at ovulation, leaving only Group I eggs in the ovary. This condition was not seen. Hence we were forced to find some other explanation of the facts.
Figure 5 shows in the form of a table the facts described above. The vertical readings show the groups of eggs. The horizontal readings show the periods into which the oestrus cycle is divided. Period a is the division of the oestrus cycle extending from ovulation to the twenty-first subsequent day and covers a period of time in which there is little change in the personnel of the ovary. Period h covers the succeeding six hours; period c, the next six, and period d, the last six hours remaining before ovulation. The above figures are only approximate, as the individual variation is too great to permit of any exact data.
By studying the figure it will be seen that Group IV disappears at period c. At the same time the ovary contains Groups I, II, III and V, IV and VI being absent. During the interval between periods c and d Groups I, II and III remain unchanged, but Group V disappears and Group VI appears.
After ovulation we find in period a, Groups I, II and III only. But in period h, II disappears and IV appears.
From the above data we were led to believe that the development of an egg follows the arrows in the diagram. That is, that Group II comes from I in period c, remains unchanged through d and a, becomes transformed into III during period h, remains unchanged through c, d and a, grows to IV in 6, to V in c and to VI in d, and so out at ovulation.
The above explanation of the facts rests on the assumption that the normal rate of development is approximately the same for all eggs. This assumption we think is warranted, for if II developed into IV during period h instead of remaining unchanged until the next oestrus cycle, the number of Group III eggs found should be very small, since the change would be a rapid one. On the other hand, if the development involved a longer period of time — that is, if Group III became a second resting stage— one would expect to find a comparatively large number from resting stage stages in developindicate probable of such eggs in an ovary at any given time. This, however, v^^as not the case, the number of Group III eggs found being relatively close to the number of Group I eggs.
Fig. 5 Diagram showing probable development of an egg through ovulation. Roman numerals I-VI indicate successive ment of eggs; a-d indicate periods in oestrus cycle. Arrows course of development of individual eggs.
Showing the relative number of eggs in the various stages of development at different periods of the oestrus cycle, as found in individual ovaries
168 145 / 171 \ 167 j 152.2 \ 152
Table I is compiled from a count of all the follicles in six ovaries, representing each of the four periods. It shows the relative number of eggs in each group present at the same time in a given ovary. The count can only be an approximation, owing to the occasional loss of a critical section and the frequent difficulty in determining with accuracy whether or not an egg was normal, but is sufficiently exact for this purpose.
We were unable to obtain any stages that intervene between the eggs of Group IV and those with the first polar spindle, so we cannot say whether the nuclear membrance disappears before or after the first appearance of the first polar spindle. With regard to this spindle, however, there are a number of details worthy of attention. It is short and broad, with well defined fibers which do not come to a sharp focus (fig. 10). The possibility of centrioles being present was mentioned by Coe ('08), but these are apparently lacking in polar spindles of the albino rat. The chromosomes are numerous, crowded, and never found in a definite equatorial plate. Moat of the first polar spindles seen are parallel to the surface of the egg, and this appears to be the position in which the spindle waits for the stimulus that leads to the formation of the first polar body (fig. 6 a) . When this stimulus comes the spindle rotates on its long axis, coming to lie more or less radially (fig. 6 b and 6 c).
The next stage we were able to obtain is shown in figure 11. This is an ovarian egg with the first polar body and the second polar spindle. As in the case of the mouse, the nuclear material is never gathered into a resting stage between the time of exti-usion of the first polar body and the formation of the second polar spindle.
Fig. 6. Reconstructions of three spindles showing gradual rotation from the paratangential position (a), through (6), to the radial position (c).
The first polar body is rarely seen in eggs outside of the ovary, but there is absolutely no reason to doubt that it is always formed, since it is almost invariably present beside normal ovarian eggs, possessing a se'cond polar spindle. Even in the ovary, however, its protoplasm displays its characteristic tendency to uiidergo rapid disintegration. In such fully matured eggs as have failed to escape from the ovary and are just starting to degenerate, as well as in those about to be discharged, the second polar spindle may be sharply defined, yet a careful search fails to reveal a trace of the first polar body. The chromatin in the first polar body is always scattered, and when first formed this polar body is, in all probability, always larger than the second, though disintegration may set in immediately upon its formation. The second polar spindle as seen in the ovary is much longer and narrower than the first, but resembles the first polar spindle in having open ends and no centrioles. The chromosomes in the second polar spindle are almost always spherical.
The living unsegmented egg of the albino rat measures about 0.079 mm. in diameter (the exact size varies a few thousandths of a millimeter in different specimens), and is surrounded by a zona of transparent jelly about 0.022 mm. in thickness. The two unsegmented rat eggs that were obtained sufficiently free from follicle cells to be available for detailed study, both possessed two polar bodies, measuring in one specimen 0.019 and 0.0132 mm. in diameter respectively, and in the other specimen 0.008 and 0.0065 mm. These eggs while translucent were filled with highly refracting globules scattered through the protoplasm. In one egg there was a clear area near the center, where we thought we could distinguish the two pronuclei lying side by side.
The rare occurrence of the first polar body associated with the egg in the tube is to be attributed to its rapid disintegration, which, as already stated, begins almost as soon as it is formed, and may lead to its complete disappearance before ovulation occurs. A stained and sectioned tube egg, accompanied by the first polar body, is shown in figure 12. This polar body is very small, contains only a little stainable chromatin scattered through it, and its protoplasm is much denser than that of the egg.
Until after fertilization, and if this fails to take place until it degenerates, the chromatin of the second -polar spindle remains in a clearly defined equatorial plate, but in the egg in the Fallopian tubes, this spindle always appears much longer and thinner than in the ovarian eggs.
The rat spermatozoon has an exceedingly long tail (fig. 16 a), and like that of the mouse carries more or less of its tail with it when it enters the egg, a fact mentioned by Coe, and by Sobotta and Burckhard. As soon as the sperm head begins to penetrate the cytoplasm of the egg the formation of the second polar body is started.
In the albino rat the second polar body is characterized by having the chromatin content massed, while the chromatin of the first polar body is always scattered through the cytoplasm. This distinction, however, does not hold for the Norwegian rat, of which two eggs are shown in figures 17 and 18. The chromatin left in the egg after the formation of the second polar body rounds itself up and becomes surrounded by a membrane, thus forming the female pronucleus. The sperm head on its entrance swells up and likewise assumes a rounded form with a nuclear membrane, as is shown in figure 16.
- Male albino rats rarely, if ever, are responsible for the killing or maiming of their young. Diseased condition or fright are probably the chief causes of the destruction or injury of their offspring by the females.
- Albino rats give birth to young the year round, but only from April to October do the females regularly ovulate twenty to forty-eight hours after parturition.
- Albino rats of both sexes are sexually mature when less than two months old.
- Living rat eggs are easily obtainable during the four days following ovulation by dissection of the Fallopian tubes.
- The maturing eggs in the ovary are joined to the surrounding follicle cells by very definite cell bridges.
- The development of eggs can be traced in the ovary through two oestrus cycles preceding their discharge.
- The first polar spindle is short and broad, and is usually formed less than twenty-four hours after parturition.
- The first polar body is always formed in rat eggs, but its protoplasm is very unstable, and disintegrative processes often bring about its complete disappearance about the time the egg reaches the Fallopian tube.
- The second polar spindle is long and narrow. Its appearance marks the end of maturation phenomena in the ovary, and the termination of all development of the egg unless fertilization occurs.
- In albino rats the chromatin of the first polar body is scattered, that of the second polar body is massed.
- The very long middle piece of the sperm tail follows the head into the cytoplasm of the egg.
CoE, W. R. 1908 The maturation of the egg of the rat. Science, N. S., vol. 27, no. 690.
Daniel, J. F. 1910 Observations on the period of gestation in white mice. Jour. Exper. Zool., vol. 9.
Donaldson, H. H. 1912 The history and zoological position of the Albino rat. Jour. Acad. Nat. Sci., Philadelphia, vol. 15, 2d series.
King, H. D. 1913 Some anomalies in the gestation of the albino rat (Mus norvegicus albinus). Biol. Bull., vol. 24.
Lantz, D. E. 1910 Natural history of the rat. Treas. Dept., Pub. Health and Marine Hosp. Service U. S., Washington.
Mark, E. L.,and Long, J. A. 1912 Studies on early stages of development in rats and mice. No. 3. Univ. California Pub. in Zool., vol.9, no. 3.
Miller, N. 1911 Reproduction in the brown rat (Mus norvegicus). Amer. Naturalist, vol. 45.
SoBOTTA, J. 1895 Die Befruchtung und Furchung des Eies der Maus. Arch. f. Mikr. Anat., Bd. 45.
SoBOTTA, J., u. BuRCKHARD, G. 1910 Reifuug und Befruchtung des Eies der weissen Ratte. Anat. Hefte, Bd. 42.
Figures 1 to 4 and 7 to 18 were drawn with the camera lucida. All figures, except 17 and 18 were drawn with Zeiss no. 4 oc. and xV oil immersion obj. giving a magnification of 1000 diameters. Figures 17 and 18 were drawn with a no. 6 oc. and tV oil imm. obj., giving a magnification of 1760 diameters. These figures are reduced one-third, giving a magnification in the finished plate of 1174. All other figures are reproduced at the size drawn.
EXPLANATION OF FIGURES
7 Shows an ovarian egg in the resting stage. The egg (Group II) has attained approximately its greatest diameter. Nucleolus solid and deeply staining. Protoplasmic bridges well marked. A follicle cell is shown dividing at^the left.
8 An ovarian egg (Group III) at a slightly later stage showing the vacuolization of the nucleolus well started.
EXPLANATION OF FIGURES
9 A later stage than fig. 8, showing the complete vacuolization of the nucleolus (Group IV).
10 A radial first polar spindle (Group V) showing the blunt ends.
EXPLANATION OF FIGURES
11 An ovarian egg (Group VI) showing the first polar body with a spindle, and the early type of short, thick second polar spindle within the egg. The protoplasmic bridges have at this stage disappeared.
12 A tube egg, showing the first polar body at the right, and the long slender type of second polar spindle at the lower left-hand margin of the egg.
EXPLANATION OF FIGURES
13 A tube egg, showing the second polar body in the process of formation. Th^ enveloping follicle cells still retain their continuity.
14 A tube egg, showing the first polar body at the top, the second polar body in the process of formation at the lower left-hand margin of the egg, and the sperm head at the right, with a portion of the tail.
EXPLANATION OF FIGURES
15 A tube egg, showing the second polar body at the left and the sperm head at the right.
16 A series of drawings showing the changes in the sperm head: a, a spermatozoon in the tube. The head was drawn from a stained section — the tail was added from data of a living spermatozoon, b to g, various stages in the transformation of the sperm head within the egg.
EXPLANATION OF FIGURES
17 A tube egg of a brown rat, showing the second polar body at the top, below the deeply staining female pronucleus, the entrance cone to the right, the male pronucleus with the sperm aster in the lower left-hand margin and the sperm tail extending diagonally through the egg.
18 A somewhat later stage of the- egg of the brown rat, showing in addition to the above-mentioned points the nucleolus vacuolated in the female pronucleus.
Figures 17 and 18 were drawn by Dr. W. R. Coe, and are here published with his kind permission.
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