A LMOST ANY MAMMAL, at any age, has been found to have some abnormal

Transcription

1 Ovulation and Fertilization of Abnormal Ova of the Golden Hamster HARRY A. KENT, JR., Ph.D." A LMOST ANY MAMMAL, at any age, has been found to have some abnormal ovarian structures. Those structures are of two types, polyovular follicles and multinucleate ova. As might be expected,4 the abnormalities are more prominent in the immature ovary. The incidence of abnormalities has been determined in the ovaries of a number of immature laboratory animals including the Swiss mouse,6 the Sprague-Dawley rat,7 the golden hamster,8 and the guinea pig.3 Each study consisted of an analysis of the prevalence of abnormal structures in the ovary, at each week of age, from the third through the tenth week. It was found that all four species demonstrated fluctuations in abnormality levels with statistically significant high values somewhere near the fifth to sixth weeks of age. The work on the hamster has been confirmed by Bodemer and Warnick, who theorized that the product of abnormal follicles might be ovulated at a rate in direct proportion to the rate of ovulation of the contents of normal follicles. The hamster is an ideal animal for studies on ovulation abnormalities because of the relatively high incidence of abnormalities at specific ages. It is to be expected, in such an animal, that a sizeable number of the products of ovarian abnormalities might be found in the extraovarian portions of the reproductive tract if they are, indeed, released from the ovary. A study of the ability to be ovulated and fertilizability of polyovular follicles and multinucleate ova of the hamster is reported here. METHODS Animals ranging in age from 29 to 48 days were bred on the evening before collection of the experimental materials. This age range was chosen as being representative of high abnormality levels (Fig. 1 and 2). Breeding The investigation reported in this article was conducted at the Worcester Foundation for Experimental Biology, Shrewsbury, Mass., and was supported by Grant GM from the U. S. Public Health Service. *Present address: Zoology Department, University of Georgia, Athens, Ga. 591

2 592 KENT FERTILITY & STERILITY consisted of allowing the animals to mate for 20 min. between 9:00 and 11 :00 P.M. The onset of heat for each animal was determined on the basis of time of initiation of the lordosis response. In accordance with the findings of Harvey et al. it was assumed that ovulation occurred 8 hr. after the onset of heat. No consideration need be given either sperm age or sperm ascent time since only normal conditions of fertilization were utilized. The animals were ovariectomized-salpingectomized between 9:00 and 11:00 A.M. The time of operation for each animal was determined on the basis of the onset of heat, so that the resultant zygotes were at an age of approximately 8 hr. In some cases collection was delayed for up to 55 hr. post-fertilization, a time interval in accord with the finding of Venable that fertilized ova remain in the oviduct for at least 2 days and undergo one or two divisions during that time. The ovary and oviduct were imbedded in paraffin, sectioned serially, and stained with hematoxylin and eosin. The numbers of fertilized ova in the oviduct were counted, as were the numbers of corpora hemorrhagica in the accompanying ovary. In cases involving later post-fertilization structures, corpora lutea were counted. Special attention was given to the number of pronuclei per fertilized ovum. RESULTS AND DISCUSSION The number of ova released in relation to the number of corpora hemorrhagica present in the corresponding ovary suggests that from the fourth to sixth weeks of age a sizeable number of polyovular follicles have ruptured and provided fertilizable ova (Table 1 and Fig. 1). Variations in the determination of numbers of ruptured polyovular follicles are inescapable since there is no possibility of knowing whether the excessive numbers of fertilized ova in an oviduct represent the product of one or more than one follicle. As an example, there might be seven ova produced from an ovary containing five corpora hemorrhagica; in this case the two extra ova could have derived from two biovate follicles or from one triovate follicle. Consequently, the maximum and minimum values for the number of ruptured polyovular follicles were calculated and the most likely value between the extreme values was assumed to be a valid indication of the true value. Since biovate follicles represent 75% of all polyovular abnormalities in the ovary, 8 the median value obtained from the present work is weighted to the maximum figure for number of ruptured polyovular follicles by a ratio of 3:1 (Table 1). A small number of multinucleate ova were produced and fertilized, as evidenced by the presence of tripronucleate ova (Table 2, Fig. 2). Chang stated

3 VOL. 15, No.6, 1964 ABNORMAL HAMSTER OVA 593 TABLE 1. Occurrence of Excess Ova, Based on Ovarian Post-rupture Structures, and of Tripronucleate Fertilized Ova Age at Total No. 0/0 triproinsemination No. of of corpora Weighted nucleate (days) animals hemorrhagica 0/0 polyovular follicles mean ova , 13.0, , 16.7, , 17.8, , , 17.7, , 11.8, 14.7, 17.7, , S !',:(~~t\ a., '\~+~ T AGE IN DAYS Fig. 1. Comparison of percentages of ruptured polyovular follicles with those of ovarian polyovular follicles. Solid line indicates polyovular follicles in ovary of the golden hamster, expressed as percentages of total numbers of follicles; circles, fertilized ova from polyovular follicles, expressed as percentages of total numbers of follicles ruptured.

4 594 KENT FERTILITY & STERILITY TABLE 2. Comparison of Numbers of Corpora Lutea with Numbers of Zygotes in Advanced Stages of Development Age at Collection time No. of Zygotes per oviduct insemination (hours corpora lutea No. of ova (days) post-ovulation) per ovary 1-cell 2-cell 4-cell produced t f t *Division of the product of polyovular follicles. tfusion of the pronuclei of zygotes derived from polyovular follicles. that polyspermy in the hamster is extremely rare. On that basis, the relatively high percentages of tripronucleate ova on the thirtieth to thirty-second days may be considered the result of ovulation and fertilization of binucleate ova. A logical consideration pertains to the ability of fertilized products of abnormal follicles to continue development. Table 2 provides data in support of the thesis that subsequent divisions did indeed take place. In general, 2-cell and 4-cell stages persisted from 33 to 42 hr. after ovulation. In two instances, at 33 and 42 hr. post-ovulation the fertilized product from abnormal follicles had undergone cell division. An indication of potential divisional capability may be the fusion of pronuclei, which had occurred in all fertilized ova, even those from polyovular follicles, by 31 hr. postovulation. The observed timing of fusion of pronuclei and cell division agreed perfectly with the excellent and complete study conducted by Venable on the hamster. Venable's work was further corroborated since by 55 hr. postovulation (corresponding to his 2.5 days post-fertilization) the zygotes were no longer in the oviduct. We may make a few assumptions on the basis of the present work. The rupture of polyovular follicles may be assumed to proceed at a rate similar to that observed for normal follicles. Multinucleate ova are not produced by

5 VOL. 15, No.6, 1964 ABNORMAL HAMSTER OVA 595 the ovary during or immediately after the period of their greatest incidence in the ovary. Instead, they are released from the ovary only during a period when they are present in low amounts, and quite early in the reproductive life of the animal (Fig. 2). It must be assumed that atresia of the multi ~ 4 w o It: 3 ~ AGE IN DAYS Fig. 2. Comparison of percentages of fertilized multinucleate ova with those of ovarian multinucleate ova of the golden hamster. Solid line indicates golden hamster ovarian multinucleate ova, expressed as percentages of total numbers of follicles; circles, fertilized binucleate ova, expressed as percentages of total numbers of ruptured follicles. nucleate forms is the common occurrence since the percentage values observed in the ovary are of sufficient magnitude to permit expectation of ovulation of reasonable numbers of multinucleate ova (Fig. 2). It is permissible to ignore the possibility that multinucleate ova may be converted into polyovular follicles in the ovary, since the hamster appears to be unique in its development of polyovate structures prior to multinucleate ova. It is of interest that the products of abnormal follicles and the multinucleate ova are capable not only of release from the ovary but of subsequent fertilization. SUMMARY A study of the ability to ovulate and the fertilizability of multinucleate ova and the products of polyovular follicles in the golden hamster is presented. On the day following insemination the ovary and its attendant oviduct were sectioned and analyzed together. Statements correlating num-

6 596 KENT FERTILITY & STERILITY bers of post-rupture ovarian structures with numbers of fertilized ova were obtained. Close attention was paid to the number of pronuclei per fertilized ovum. Analysis of the data indicated that both the excess ova from a follicle and binucleate ova are capable of being fertilized. In addition, the product of polyovular follicles successfully divided, while all fertilized abnormal ova demonstrated at least pronuclear fusion. Zoology Dept. University of Georgia Athens, Ga. REFERENCES 1. BODEMER, C. W., and WARNICK, S. Polyovular follicles in the immature hamster ovary. Fertil. & Steril. 12:159, CHANG, M. C., and FERNANDEZ-CANO, L. Effects of delayed fertilization on the development of pronucleus and segmentation of hamster ova. Anat. Rec. 132:307, COLLINS, D. C., and KENT, H. A., JR. Polyovular follicles and multinucleate ova in the ovaries of young guinea pigs. Anat. Rec. 148:115, ENGLE, E. T. Polyovular follicles and polynuclear ova in the mouse. Anat. Rec. 35: 341, HARVEY, E. B., YANAGIMACHI, R, and CHANG, M. C. Onset of estrus and ovulation in the golden hamster. J. Exper. Zool. 146:231, KENT, H. A., JR. Polyovular follicles and multinucleate ova in the ovaries of young mice. Anat. Rec. 137:521, KENT, H. A., JR. Polyovular follicles and multinucleate ova in the ovaries of young rats. Anat. Rec. 142:25, KENT, H. A., JR. Polyovular follicles and multinucleate ova in the ovaries of young hamsters. Anat. Rec. 143:345, VENABLE, J. H. Pre-implantation stages in the golden hamster. Anat. Rec. 94:105, 1946.