AN UNFERTILIZED TURAL OVUM FROM MACACUS RHESUS

EDGAR ALLEN Department of Anatomy, Vniuersity of ,Wissouril

ONE FIGURE

There are still relatively few observations upon unfertil- ized tubal ova of primates. None has yet been recovered from woman. Corner (’23) recovered the first from the monkey, Macacus rhesus, and also obtained a degenerating ovum from the uterine cavity. Two additional tubal ova, also from Macacus rhesus, were recovered by Allen ( ’27) . Recently, while removing the ovaries preparatory to experi- ments with an ovarian hormone, the finding of a fresh rupture point of a recently ovulated follicle on the surface of one of the ovaries led to the recovery of a fourth unfertilized primate ovum.

Several interesting observations were made upon the fresh ovum in Ringer’s solution and measurements taken while fresh to add further data as to size.

Of the first three ova, two were recovered on the fourteenth and the third on the tenth day of the menstrual cycle. Un- fortunately, the recovery of the fourth ovum cannot be related to any particular time in the menstrual cycle. This animal had had two periods of menstruation, each three days in duration, but the onset of the last period was more than five and one half months (169 days) previous to the opera- tion. During this interval the ‘sexual skin’ surrounding the ex-

‘This work was supported by a grant from the Committee for Research in Problems of Sex of the National Research Council. The assistance of G. C. Arvin and H. E. Allen, research assistants under this grant, is acknowledged.

351 THB A N 4 T O X I C A L RCCORD, VOL. 37, NO. 4

FEBRUARY. 1928

352 EDGAR ALLEN

ternal genital organs had been red and rough, the intensity of these phenomena undergoing five or six rather definite fluctu- ations, first fading and then coloring more deeply. Since these phenomena have been induced in ovariectomized ani- mals by injected ovarian hormone (Allen, ’27) , the deepening of the ‘ sexual skin’ color indicated increased ovarian secre- tion, probably dependent upon the growth of one or several follicles. At the time of tlie operation, the reddening mas quite intense, although not at its maximum, and the skin in some regions was roughened and swollen and the hair stand- ing on end. It is probable that the development of tlie follicle from which this ovum was extruded was concerned with the latest phases of coloring and swelling of the ‘sexual skin.’

The ovary and the tube were removed and rinsed in Ringer’s solution. The deep red fringe of the fimbria of the tube sui-rounded one half of the ovary. On this surface, quite near the line of entrance of the vessels into the ovary, was the red rupture point, and from it protruded the usual ‘plug’ of follicular tissue. The plug was quite firm.

A hypodermic needle was inserted through tlie ostium, ligatured in place, and slow injection begun. The first six drops of fluid from the uterine end of the tube were collected in a first watch-glass, and as injection %‘as continued a series of watch-glasses were used, not more than ten drops to each, until a total of 1.5 cc. had been injected through the tube. A search of less than two minutes through the contents of the first watch-glass under the low power of the microscope located the ovum. It had been flushed out by the first six drops injected.

The ovum u7as completely free from follicle cells-a con- trast to the three other tuba1 ova previously recovered. This is interpreted as indicating a longer interval elapsed since ovulation. It is planned to check this point in a histologic study of the degree of transformation of the walls of the ruptured follicle (newly formed corpus luteum) from which this ovum was extruded. The ovum was surrounded by a thick, clear, highly refractive, and sharply defined zona

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pellucida, the inner third of which appeared to be tinted a pale iridescent blue (fig. 1). Within the zona pellucida was a second clear zoiiular area of slightly greater width sur- rounding the ovum proper. The most outstanding char- acteristic of the ovum was the yellowish tint of the cytoplasm. Under the low power of the microscope, this appeared vesicu- lar rather than granular. Under high magnification, it ap- peared grayish and granular. A clear polar body (as con-

Fig. 1 Camera-lucida drawing of unfertilized t u h l oviim in Ringer’s solutioii. x 900. z.P., zona pellucida; c.m., yellow cytoplasmic mass; PA., polar body; fs., fluid-filled space. Compare with Corner ( ’23, pl. VI) and Allen ( ’27, pl. 17).

trasted with the yellow cytoplasm of the ovum) lay against the outer surface in the clear space beneath the zona pel- lucida. None of the details of nuclear structure of the polar body was visible. There was no polar spindle apparent in the ovum, as seen in the fresh condition.

The high-power objective was carefully focused down on the ovum, measurements taken at a magnification of 650 X, and a camera-lucida slietch made. After a time, some addi- tional Ringer’s solution was added which carried the ovum

354 EDGAR ALLEN

toward one side of the field. As this happened, the ovum proper (yellow cytoplasmic sphere) moved toward one sur- face of the zona pellucida. By drawing one point of a small pair of forceps through the surrounding fluid, which was slightly viscous, the ovum in its zona could be moved around and rolled over without risking actual contact with the for- ceps. This motion showed quite clearly that the second zone, or space between the zona pellucida and surface of the yellow cytoplasm, was fluid. The lagging back of the ovum proper made it appear as if it were afloat in the transparent zona pellucida.

Further movement of the forceps point started the ovum proper rolling slowly within the zona pellucida. This re- vealed a cap of lighter clear protoplasm making up about one-third of the egg. A s soon as the motion was stopped, this inner cytoplasmic sphere righted itself with the clear cap on top, like a floating barrel with one side weighted. In this position the clear cap was no longer visible. This rolling of the cytoplasmic mass within the zona pellucida with the appearance of the clear cap and the righting into equilibrium when motion ceased was observed repeatedly. The polar body was attached to the ovum and rolled with it inside the zona pellucida. It seems quite probable from these observa- tions, that a definite polarity existed in this ovum.

The zona pellucida was nearly spherical, the outside diame- ters were 177.8 to 179.2 p, average 178.5. It was 15 p thick. The ovum proper was not exactly spherical, the diameters varied from 102.2 to 106.4p, average 104. These measure- ments are listed in the table for purposes of comparison with the primate ova previously recovered.

It should be noted in comparing the sizes of these ova that the zona pellucida did not fit ovum IV tightly as it did tuba1 ova I, 11, and 111. The thickness of the zona of ovum IV (15 p ) is quite comparable to that of ova I1 and 111, all of which are slightly thicker than that of I. The size of ovum IV (yellow cytoplasmic mass, exclusive of the zona) is exactly the same as ovum 11, while the other ova are much

UNFERTILIZED TUBAL OVUM FROM MACACUS RHESUS 355

smaller (83 to 87 p). May this possibly mean that there are primate ova of two sizes, indicating perhaps differences in yolk-storage potentiality similar to the condition described by Riddle for pigeon eggs? Or are the size differences merely due to degenerative changes?

After the foregoing observations, which required nearly an hour, an attempt was made to make a whole-mount prepa- tion of the egg. Some of the Ringer's solution was drained off and Bouin's fluid diluted with Ringer slowly added, the egg being observed meanwhile under the low power of the microscope. As the yellow wave of fixing solution engulfed

TABLE 1

Measurements of unfertilized ova of Macacua r h e a s made just after recovery and before fization

0YL.M

Corner '5, tubal I Corner '5, degenerating uterine Ovum, tubal I1 Ovum, tubal I11 Ovum, tubal IV

-4VERAGE6, IN MICRONS

Outer diameter, including zona

pellucida

109 137 138 115 178.5

Diameter of ovum, excluding zona

86 83 104 87 104

Thickness of zona

11.5 Not uniform

17 14 15

the ovum, the zona pellucida collapsed to nearly one-half its former thickness, and finally ruptured, emitting a small amount of clear fluid. After the initial relief of pressure, the remaining material in which the ovum proper floated in the zona pellucida seemed to set firmly and the yellow cytoplasm became opaque, the yellowish color changing to brown. After fixation the zona was only 8.4 p thick and its greatest diame- ter was 148 p. The ovum proper had shrunken to an average diameter of 73 p.

The ovum was then stained in dilute haematoxylin. During subsequent gradual dehydration the ovum broke up into sev- eral fragments. All of these parts could not be located and

356 EDGAR ALLEN

identified? but a half of the zoiia pellucida was retained in the field. In the fixed condition this appeared to be pitted; it was impossible, however, to determine whether the pits were really perforations.

The question arises as to whether this ovum was normal or had already begun to degenerate. The fact that there were no follicle cells adherent to the zona pellucida suggests a con- siderable interval after ovulation. The observation that the zona did not fit tightly about the ovum as in tuba1 ova I, 11, and 111 might be considered a degenerative change. A corpus luteum at a slightly later stage of development than the others previously described is t o be expected.

LITERATURE CITED

ALLEN, EDGAR 1927 The menstrual cTcle of tlie monkey, Macacus rliesus: Observations on norinal animals, the effects of removal of the ovaries and tlie effects of injectioiis of ovarian and placental extracts into tlie spayed animals. Contributions to E m l q ology, vol. 19, Cariiegie Inst. Wash. Pub. no. 380, pp. 1-44.

CORNER, G. W. 1923 Ovnlatioii and mc7iistruatioii in Macacus rhesus. Contri- butions to Emhryologj, 101. 1.5, Cariiegie Inst. Wash. Pub. 110. 332, pp. 79-101.