IN THE course of our studv of the hormonal factors involved in miosis and

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1 Arrival of Fertilizing Sperm at the Follicular Cell of the Secondary Oocyte A Study of the Rat R. Moricard and J. Bossu IN THE course of our studv of the hormonal factors involved in miosis and "' fertilization in mammals, we observed sperm in the zona radiata of perioocytary cells. We were able to count the number of spermatozoa present at the exact time of fertilization. The evolution of the second miotic division and formation of the pronuclei made it possible to establish the chronology of phenomena accompanying fertilization. In the case of 14 rats it was shown that the swarming of sperm found near the fertilized ova follows fertilization, and that at the time of fertilization the number of sperm in the ampulla may be less than the number of oocytes. - HISTORICAL BACKGROUND In 1911, Sobotta and Burkhart in their research on fecundation in rats, found the ampulla to be the site of fertilization. They did not, however, succeed in observing the actual arrival of fertilizing sperm. Later Hartman and Ball described the rapid ascension of sperm in the uterus, and Pincus observed the action of sperm on dispersion of follicular cells in vitro. Leonard and Kurzrok were able to observe the influence of hyaluronidase on this dispersion, whereas, in 1947, these same authors with Perlman determined that in vivo hyaluronidase has no influence on the dispersion of perioocytary follicular cells in the tube. In 1948, with Dalemans From the Laboratoire d'hormonologie de l'ecole des Hautes Etudes a la Clinique Gynecologique, Hopital Broca, Paris. 260 t t

2 Vol. 2, No.3, 1951] ARRIVAL OF FERTILIZING SPERM 261. ~ l Ampulla of the tube with three oocytes. No dispersion of follicular cells. 2. Three oocytes and only three spermatozoa. Each is marked with ink. One is a lateral view and the two others are sectioned. No other spermatozoa are seen near these oocytes. FIGURE 3. Sperm, marked with ink, near the oocyte without dissociation of the follicular cells. FIGURE FIGURE we noted that in vitro hyaluronidase has no influence on the perioocytary follicular cells of primary oocyte (mouse). In 1948, together with Bossu, we were able to see the arrival of a single

3 262 MORICARD & BOSSU [Fertility & Sterility spermatozoon near the follicular cells and observed it passing through the zona radiata of the undissociated follicular cells of a secondary oocvte in vivo. MATERIAL AND TECHNIC In order to count the spermatozoa at the time of fertilization, the ovaries and tubes of 14 rats were serially sectioned and stained according to Feulgen's method (Fig. 1). In most cases the exact time between mating and the removal of the ovaries and tubes was known. By the injection of about 5 I. U. of gonadotropin, ovulation was induced in 5 of the rats at the very beginning of diestrus, as well as in 3 others with persisting diestrus. ARRIVAL OF FERTILIZING SPERMATOZOON IN SPONTANEOUS OVULATION In the case of rat No , 11 secondary oocytes were found in the tubes ten hours after mating. Both tubes looked the same. Five oocytes were fertilized, and 6 were not. Only 6 spermatozoa were found near the oocytes in the ampullae. Thirteen hours after mating rat No , fertilizing sperm were observed in the cluster of undissociated perioocytary follicular cells. The secondary oocyte was in the metaphase of the second maturation division. Five oocytes were fertilized, and 8 sperm were seen in the ampullae. Fourteen hours after mating, 7 oocytes were found in a single tube, 2 were unfertilized, and 4 more fertilized. One spermatozoon was seen near another of the oocytes. There were 7 sperm in the ampulla and 7 oocytes. When the other ovary was removed fifteen minutes after mating, some sperm were found in the juxtauterine portion of the tube, but none in the medial portion nor in the ampulla. The tubes of animals No , No , No , and No which were removed twenty-four hours after mating, contained a definite increase in the number of sperm in the ampullae. The more numerous the sperm, the more complete was the dissociation of the perioocytary cells. When the formation of the pronuclei was in progress, this dissociation was usually complete. It is Significant that the sperm were not free in the secretion of the tubes, but were in contact with those follicular cells that were more or less dissociated from the fertilized ova. The author wishes to record his thanks to Dr. John Rock for editorial assistance in revising this manuscript.

4 .t.. 11 ~ 1. ~ t """" J y ---- TABLE 1. Spontaneous Ovulation and Fecundation in the Rat (Periodic diestrus) Hours Follicular Cells Sperm 1st 2nd Animal After Ova Mat. Fert. Begin. End of Total In Polar Meta- No. Coitus' No. Crown" Crown'! Dissoc. 4 Dissoc." Free Ova Body phase Generally there was but a single coitus. In the 24-hour cases, it may have occurred many times. 2 Maturation crown: Zona radiata within the cumulus as seen just before ovulation. 3 Fertilization crown: only 3 to 4 strata of granulosa cells around the ovum. 4 Beginning of dissociation: a portion of the viteline membrane is free of cells. G End of dissociation: there are no more follicular cells in contact with ovum. 2nd Anaphase Nuclei 2nd Polar Body Fem. Male Prontlc. Prontlc

5 264 MORICARD & BOSSU [Fertility & Sterility ARRIVAL OF FERTILIZING SPERMATAZOON AFTER OVULATION INDUCED BY GONADOTROPIN In rat No oocytes were counted in the tube, and a spermatozoon was observed in the cluster of follicular cells surrounding the oocyte. When the tubes were removed sixteen hours after mating rat No , there were 10 eggs and a single fertilizing sperm was among the follicular cells. In 5 oocytes the second polar body was forming while in the other oocytes the formation of pronuclei was already in progress. Three animals were examined twenty-four hours after mating. In one of them, No , 7 fertilized ova were found with only 3 sperm nearby. The follicular cells were not dissociated although the pronuclei had already. formed. In another animal, rat No ,6 ova were found with 60 sperm nearby. The formation of pronuclei was in progress, and the dissociation of the perioocytary cells was complete. INTERPRETATION At the time of fertilization-ten to fifteen hours after mating-the number of sperm in the ampulla may be less than or equal to the number of oocytes (Fig. 2). Twenty-four hours after mating, the number of sperm present increases considerably, and 100 or more sperm may be found among the dissociated follicular cells. When the sperm are not very numerous, the dissociation of follicular cells is not complete, even twenty-four hours after mating. The elective localization of the sperm near the follicular cells suggests that the follicular cells surrounding the oocyte exert chemotaxis on the sperm. The progressive increase in the number of sperm seems to indicate a complex mechanism drawing the sperm from the uterus toward the ampulla. It is possible that in the human female, phenomena of this kind would condition the opening and closing of the sphincter of the tubal ostium. SUMMARY We have observed the approach of a single sperm toward the undissociated follicular cells surrounding the secondary oocyte in the ampulla immediately before fertilization (Fig. 3). The number of sperm at this time may be less than or equal to the number of oocytes. Fertilization takes place

6 ~ : '"., ~ <!-! " '",. -Ir.. l -+ " TABLE 2. Induced Ovulation and Fecundation in the Rat Nuclei Follicular Cells Sperm Hours 1st 2nd 2nd 2nd Animal After Ova Mat. Fert. Begin. End of Total In Polar Meta- Ana- Polar Fem. Male No. Coitus 1 No. Crown 2 Crown3 Dissoc. 4 Dissoc. 5 Free Ova Body phase phase Body Pronuc. Pronuc. AT START OF -DIESTRUS DURING PERSISTENT DIESTRUS All abbreviations as defined in Table l.

7 266 MORICARD & BOSSU [Fertility & Sterility between ten and sixteen hours after mating. A hundred or more sperm may be present twenty-four hours after mating near the follicular cells dissociated from the ova migrating in the tube. REFERENCES 1. Hartman, C, G., and Ball, Josephine: Proc. Soc. Expel'. BioI. & Med. 28:312, Leonard, S. L., and Kurzrok, R: Endocrinology 37:171, Leonard, S. L., Perlman, P. L., and Kurzrok, R: Proc. Soc. Exper. BioI. & Med. 66:517, Moricard, R, and Dalemans, Louise: Gynec. et obst. 47:186, Moricard, R, and Bossu, J.: Compt. rend. Soc. de bioi. 142:303, Pincus, G.: Anat. Record 54:275, Sobotta, J., and Burkart, G.: Anat. Hefte 42:435, t