Book - The Development of the Albino Rat 2
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Huber GC. The Development of the Albino Rat (Mus norvegicus albinus). (1915) J. Morphology 26(2).
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Ovulation, Maturation and Fertilization
When this study was projected, it was the purpose to begin it with the stages of maturation and fertilization. During the time of material collection, there appeared the contribution of Sobotta and Burckhard: "Reifung und Befruchtung des Eies der Weissen Rate," covering these stages fairly completely. Duplication of their work did not seem necessary, so that my own studies begin with the pronuclear stage, to which stage the above mentioned investigators had carried their observations. Therefore, as concerns the process of ovulation, maturation, and fertilization as observed in the albino rat, I am confined for my data to the literature; from which a brief resume is here made.
The normal gestation period for non-lactating albino rats may be roughly estimated as from 21 to 23 days. As has been shown by King, the period of gestation of lactating albino rats varies from a minimum of 24 days to a maximum of 34 days. The average number in a litter is six. In lactating females suckling five or less young and carrying five or less young, the period of gestation usually does not exceed 23 days and may thus be considered as normal. In lactating females suckling five or less young, while they are carrying more than five young, the period of gestation may be prolonged from one to six days. In lactating females suckling more than five young, the period of gestation is always prolonged, and may be prolonged to a maximum of 34 days. Daniel's studies on the white mouse lead him to formulate the following law: "The period of gestation in lactating mothers varies directly with the young suckled." Such exact relation between the number of young suckled and the extent of the prolongation of the gestation period was not observed by King for the albino rat.
In the albino rat, ovulation occurs spontaneously and is not dependent on copulation, which act, however, may precede or follow ovulation. Kirkham and Burr state that ovulation usually occurs about 24 hours after parturition and that the developing ova can be traced in the ovary through the two oestrus cycles preceding their discharge. Long, in his study No. 3, by jNlark and Long, finds that ovulation must occur in the albino rat on an average not less than 18 hours after parturition. Sobotta and Burckhard state that ovulation always occurs within 36 hours post partem, though at very variable periods, often only a few hours after the completion of parturition; again, much later. A second ovulation period apparently occurs some 30 days post partem, as would appear from the successful pairings conducted by Dr. Stotsenburg. This agrees with the observations of Melissinos, who found that pairings were more numerous when attempted 29 days after parturition, than when attempted 20 to 21 days after parturition, as practiced by Sobotta. Semination probably takes place in the ampullar portion of the oviduct. Relatively few spermatozoa enter the oviducts and Sobotta and Burckhard estimate that the life of the spermatozoa in the genital tracts of the albino rat is only about 10 hours.
The phenomena of maturation and feiliUzation in the albino rat have been carefully studied by Sobotta and Burckhanl, from whose account the following brief summary is taken : The behavior of the ovum of the albino rat with i-espect to the formation of polar bodies is very similar to that of most other mammals studied. The first polar body is given off within the ovarian follicle, the second in the oviduct and only after semination. The first maturation spindle, developed from the nucleus of the oocyte of the first order, forms usually innnediatcly after parturition. Kirkham and Burr state "it is usiuilly [oiiiumI less than 24 hours after parturition." It is short and broad, with the chromatin scattered. The first maturation spindle lies near the center of the ovum, then passes toward the surface assuming a tangential position, and only with the beginning of metakinesis, takes a radial position. The chromosomes of the first maturation spindle, estimated as numbering 16, appear in the form of modified rings, which are divided transversely across to form short rounded rods with a longitudinal direction in the diaster stage. The first polar body is formed in the ovarian follicle and appears to be relatively large. It is evident only in the ovarian ovum, and appears to be lost soon after its formation. Its fate is doubtful. The first polar body is nearly always missing in tubal ova. Kirkham and Burr state that the rare occurrence of the first polar body associated with the egg in the tube is to be attributed to its rapid disintegration, which begins as soon as it is formed, and may lead to complete disappearance before ovulation occurs." The second maturation division begins immediately after the completion of the first, without an intervening resting phase. The spindle formed is narrower and longer than the first, with the chromatin massed. In its monaster stage, it lies in a tangential position, with the chromatin in diads, and with the lines of division at right angles to the axis of the spindle. The appearance of the second maturation spindle in the monaster stage marks the end of the maturation phenomena in the ovary. The monaster stage of the second oocyte division was not observed in the ovary by Sobotta and Burckhard, but was seen by Kirkham and Burr. The first division Sobotta and Burckhard regard as a reduction division, a heterotypic longitudinal division; the second as an equatorial division, a homeotypic longitudinal division. Ovulation probably occurs during the monaster stage of the second maturation division.
The tubal ova are surrounded by a relatively thin oolemma to which are adherent a variable number of discus cells. They are smaller than the ovarian ova; the latter measuring 60 /x to 65 IX, the tubal ova 55 n to 60^. The recently discharged tubal ova are to be found in the distended ampullar portion of the oviduct, where they are found clumped together surrounded by discus cells. Semination takes place in this region. The spermatozoa usually enter while the tubal ova are in the monaster stage of the second maturation division, after which metakinesis begins. The secondmaturation spindle assumes a radial position in the metakinotic phase. The second polar body is smaller than the first, and usually lies compressed between the oolemma and the ooplasm, and is evident during fertilization and segmentation. The spermatozoan head penetrates the thin oolemma and the ooplasma; the long middle piece and tail following the head into the ooplasma, as has been shown by C'oe, and Kirkham and Burr. The long middle piece, soon after penetrating the ooplasma, presents an increase in stainability, and its spiral thread becomes evident. The spiral thread, as Duesberg has shown, has its origin in the mitachondria of the spermatid. It may be, therefore, that the male sexual cell introduces mitachondria to the egg cell at the time of fertilization. Some little time after the penetration of the sperm head, this enlarges and becomes vacuolated, and diplosomes with polar rays become evident. As the sperm head begins to metamorphose, tending to the formation of the male pronucleus, the chromosome group of the dispireme of the second maturation spindle, undergoes metamorphosis to form the female pronucleus. This enlarges rapidly to form a vesicular nucleus which lies free in the ooplasm, while the metamorphosing male pronucleus, usually smaller, is accompanied by a deeply staining thread-like structure, derived from the middle piece. The centrosomes of the first segmentation spindle are by inference derived from the sperm centrosome. The data here given, as concerns the maturation and fertilization phenomena pertaining to the albino rat, unless otherwise credited, have been drawn from the account of Sobotta and Burckhard, whose account is accompanied by excellent figures.
Long has studied in living ova of mice and rats the phenomena of maturation and fertilization. Tubal ova were placed in Ringer's solution on an especially constructed slide and spermatozoa introduced. It was possible to seminate the ova of rats with rat spermatozoa and to observe the formation of the second polar body. The formation of the second polar body, usually near the first polar cell, may begin within five minutes to two or more hours after the spermatozoa are introduced. The constriction may be finished three-fourths of an hour later. The first appearance is an elevation clearer than the rest of the cell. The swelling becomes higher, and at one side of the elevation there appears a depression which is the beginning of the constriction which presently encircles the whole swelling and cuts it off from the egg." Nothing could be said as to the changes which the chromatin undergoes after the spermatozoa have penetrated the egg. The eggs remained alive and apparently normal for about twelve hours, after which they began to degenerate.
As has been stated, my own observations on the development of the albino rat (Mus norvegicus albinus) begin with the pronuclear stage. The material at hand for this stage is listed in table 1, page 258.
Thus there are present in the series 34 ova showing a pronuclear stage and 9 ova showing the second maturation spindle in the monaster phase. The latter may be dismissed with the brief statement that they represent unfertilized ova. In rat No. 108, with 7 ova in the stage of the second maturation spindle, killed 24 hours after the observed copulation, there was found no trace of spermatozoa in the oviduct. Two reasons may be offered for the non-appearance of fertilization in this case:
|Hours after Beginning
|Stage of Development|
|Pronuclear|| Second maturation|
|109||24 hours, 15 min.||9||8||1|
|110||24 hours, 15 min.||8||8|
Ovulation may have occurred so late that the spermatozoa may have died before the ova reached the ampullar portion of the oviduct. This explanation, it would seem, is invalidated by the fact that the position of the ova in the oviduct, as shown by graphic reconstruction, is essentially the same as in the other four rats studied, and in which fertilized ova were found, so that ovulation must have preceded the killing of the animal by some hours. The other reason, more plausible, attributes non-fertilization to a pathologic condition of the genital tract. In this rat, one ovary was distinctly pathologic, with periovarian capsule greatly distended with a sanguineus liquid, while the upper end of the uterine horn with adjacent oviduct on the other side, as seen in sections, presented evidence of inflammation and epithelial desquamation, in part occluding the lumen. It seemed evident, therefore, that the spermatozoa introduced in the genital tract were unable to penetrate to the oviduct and consummate fertilization. The other two unfertilized ova, found with ova in the pronuclear stage, were in oviducts in which no spermatozoa were found. Both in the mouse and the rat, relatively few spermatozoa reach the upper end of the oviduct ; too few, it would seem, to consummate fertilization of all the ova in certain cases. In all of the ova which contained the second maturation spindle, this was in the monaster phase and in tangential position. In size, shape, and chromatin configuration, all presented the characteristics described and figured by Sobotta and Burckhard and Kirkham and Burr, therefore, need not be considered further.
The stage of pronuclei was observed in over 100 ova of the white rat by Sobotta and Burckhard. According to these observers, the two pronuclei show in the earlier stages of their development, large chromatin-like nucleoli, the number of which varies. Some little time later, one or several such chromatoid nucleolar bodies with irregularly formed chromatin masses arranged on the linin network are to be observed. At a still later time, the chromatin becomes distributed over the linin network, throughout the nuclear space, giving the appearance of a fine chronxatin network. One of the pronuclei is, as a rule, somewhat smaller than the other. This is regarded as the male pronucleus, since near it the 'sperm centrum' was now and then observed. The pronuclei lie in about the center of the ovum. The pronuclear stages of my own material, observed in 34 ova, obtained 24 hours after the beginning of insemination — thus at the end of the first day of development — all present essentially the same stage of metamorphosis. As may be seen in .\ of figure 1, the nuclei are distinctly membraned, and are of relatively large size. The ovum here sketched measures in the stained preparation 70 /x by 62 ix, and is, therefore, of slightly oval form. Sobotta and Burckhard give 55 m to 60 m as the size of the tubal ova, and 60 ^ to 65 m as the size of the ovarian ova in the white rat. Kirkham and Burr give the diameter of the Hving unsegmented egg of the rat as of 0.079 mm. As may be seen from A and B, of figure 2, the tubal ova, even when free in the oviduct, are not of necessity spherical in shape, but often slightly compressed, as may be clearly seen in four models of tubal ova in the pronuclear stage, reconstructed at a magnification of 1000 diameters, in my possession. Depending on the plane of section, the diameter of a tubal ovum may thus vary to the extent of 5 ^ to 8 /x. The two nuclei in the preparation shown in A of figure 1, measure, the larger one, regarded as the female pronucleus, 23 ix by 16 m, the smaller 17 ^ by 15 ^l. Essentially all of the chromatin is distributed over the linin network in fine granules, the larger nucleus presenting one large, faint staining chromatoid nucl(M)lus. The ooplasm is finely granular, distributed so as to give the section a slightly mottled appearance. When compared with figures given by Sobotta and Burckhard (figs. 21 to 24, plates 9-10) showing pronuclear stages of the ova of the rat, my own seem to fall in about the middle of this series, thus some little time after their formation, but not immediately preceding the stage of segmentation spindle formation. In the albino rat, and perhaps in other mammals, the pronuclear stage, in its various phases of nuclear metamorphosis, must constitute a stage covering a relatively long period. If it is assumed that semination occurs about 10 to 12 hours after the beginning of insemination, such assumption being justified by the observations of Sobotta and Burckhard, according to whom the life of the spermatozoa in the genital tract of the white rat is only about 10 hours, and if it is recalled that in living rat ova J.oiig found that the constriction of the second polar body may be completed three-fourths of an hour after its inception, then it must be evident that the pronuclear stage extends through a period which exceeds 10 to 12 hours, since in none of my pronuclear stages obtained 24 hours after insemination was evidence of first segmentation spindle observed.
In order to determine accurately the relative position of the ova within the oviduct during the pronuclear stage and the stages of segmentation, oviducts containing ova were reconstructed after the Born wax plate method. In form, relations, and general structure, the oviduct of the albino rat is essentially the same as that of the mouse as described by Sobotta. The oviduct of the rat measures from fimbriated end to termination in the uterine horn from 2.5 cm. to about 3.0 cm. It presents eight to ten fairly constant major folds, the middle group of which is closely applied to the ovarian capsule. The upper or distal folds pierce the capsule, ending in the fimbriated end found within the capsule, while the lower or proximal folds, proximal with reference to the uterine horn, effect connection with the uterine horn. These relations are essentially the same as those described by Sobotta for the oviduct of the mouse. This observer recognizes four segments in the oviduct of the mouse, characterized by epithelial lining, nature and extent of folding of the mucosa, and thickness of the musculature. The first segment, which falls to the infundilxilum, presents a thin musculature and high mucosal folds with epithelial lining consisting of relatively short cylindrical cells with distinct cuticular border and long cilia. As characteristic of this portion of the tube there are further described accessory nuclei compressed between the epithehal cells. Only this portion of the oviduct is ciliated. In the second segment, the lumen is large and the folds of the mucosa prominent. They are covered by a non-ciliated epithelium, without distinct cuticular border. The musculature is relatively thin. In the third segment the musculature is well developed with circularly and longitudinally disposed cells. The lumen is narrow and the folds are nearly absent, while the epithelium is of a simple columnar variety. The fourth segment, not so well characterized, consists of the loops which make connection with the uterine horns, with folds and epithelium much as in the third segment, and a prominent musculature. In all essentials, this description applies to the oviduct of the albino rat, except that in the first segment the accessory nuclei described by Sobotta as found between the epithelial cells were not evident in the rat. In figure 3, is reproduced a model of a wax reconstruction of the right oviduct of rat No. 106, killed 24 hours after the beginning of insemination, and containing eight ova in the pronuclear stage. This oviduct measured from fimbria to termination in the uterine horn 3.2 cm. It presents 10 major folds, which folds may be recognized with more
or less clearness in all the models made and here reproduced. The slight difference in the relative position of these folds as seen in the several figures may be accounted for by the varying degrees of tension to which the tissues were subjected prior to fixation. In rat No. 106, the ovaries with oviduct and upper end of the uterine horn, were excised and placed in the fixing fluid without applying any tension. Of these 10 major folds, the four distal ones, those beginning with the fimbriated end, fall to segments one and two of Sobotta's designation, having a wide lumen and folded mucosa. In the figure, the position of the ova is indicated by small black circles. By reason of the relation of the folds, only five of the.,eight ova can be brought to view in the aspect of the model skclclicd. 'l\\v position of the first and the hist of the series is correctly gi\(Mi. 'IMic ova are situated in a loop of the oviduct which is about S mm. from the fimbriated end. By the end of the first day after the beginning of insemination, the ova have thus travelled about onefourth the length of the oviduct. In figure 4 is reproduced a model of a detailed reconstruction of that portion of the oviduct containing the ova, representing a loop of the tube with one side cut away, this to show the extent and character of the mucosal folds, the width of the huiien and the relative position of the several ova.
The figure presents these facts so clearly that lengthy description is deemed unnecessary. The several ova are distributed through a tube segment measuring about 2.5 mm. in length. They lie free in the lumen, apparently bathed in a fluid from which there is only a small amount of precipitation at the time of fixation. Their position in the oviduct at this stage, free in the lumen, is well shown in figure 5, which is from a longitudinal section of a loop from the left oviduct of rat No. 109, showing three ova, with but few remaining discus cells and a thread of coagulum linking the ova together, an appearance quite characteristic at this stage. The figure was drawn by aid of camera lucida from a single section. All of the ova, of which there are seven, distributed through this loop, contain two pronuclei; in none of the ova figured do the two pronuclei fall in the same section, My series contains seven oviducts with pronuclear stages, with accompanying ovary, cut serially. Only one of the oviducts, rat No. lOG, was reconstructed in wax. In the other six, graphic reconstructions were made. This permits analysing the loops, determines their sequence, but does not readily admit of measuring their length. In the six oviducts graphically reconstructed, the position of the ova, the number of which varies from one to seven in the several tubes, is essentially as in the wax reconstruction figured. It would appear, therefore, that in the albino rat, 24 hours after the beginning of insemination, the ova are to be found in the pronuclear stage, with the ova distributed in the end of the third to the beginning of the fourth major loop of the oviduct, a portion of the oviduct having a relatively wide lumen and lined by a much folded mucosa and possessing a relatively thin muscular wall, having thus migrated about one-fourth of the length of the oviduct.
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Cite this page: Hill, M.A. (2018, November 19) Embryology Book - The Development of the Albino Rat 2. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Book_-_The_Development_of_the_Albino_Rat_2
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