Detailed Time Course of Ovum Transport in the Rhesus Monkey (Macaca mulatta)

Transcription

1 BIOLOGY OF REPRODUCTION 13, Detailed Time Course of Ovum Transport in the Rhesus Monkey (Macaca mulatta) CARLTON A. EDDY, RAUL G. GARCIA, DUANE C. KRAEMER2 and CARL J. PAUERSTEIN3 Departments of Obstetrics and Gynecology and Physiology Center for Research and Training in Reproductive Biology4 The University of Texas Health Science Center at San Antonio 7703 Floyd Curl Drive, San Antonio, Texas ABSTRACT The time course of ovum transport was studied in a colony of untreated spontaneously cycling female rhesus monkeys. Plasma estrogen values were determined daily by radioimmunoassay starting on Day 8 of the menstrual cycle to provide predictive indication of impending ovulation. After detection of a well-defined preovulatory increase in estrogen production (levels in excess of pg/mi) serial laparoscopies were initiated and performed every 12 to 24 h to monitor ovarian follicular development and to visually confirm the occurrence of ovulation. Laparotomies were performed approximately 24, 48, or 72 h post-ovulation and the reproductive tracts were flushed in Situ to recover ova. The ampulla, isthmus and uterus were flushed separately, allowing the position of the ova within the tract to be determined. Ova were consistently recovered from the tubal ampulla at 24 and 48 h postovulation. By 72 h ova were recovered from both the tube and the uterus. Our findings, based upon accurately timed ovulation, indicate that normal tubal ovum transport time for the untreated, spontaneously cycling rhesus monkey is approximately 72 h. INTRODUCTION Knowledge of the normal time course of tubal ovum transport is basic to an understanding of tubal function and is a prerequisite for the development of future contraceptive techniques operative at the tubal level. Because of its anatomic, physiologic, and endocrinologic similarity to man, the nonhuman primate constitutes a potentially valuable model in which to investigate tubal transport phenomena. Although the detailed time course of ovum transport has been determined for various laboratory species, most notably the rabbit, similar information for man and nonhuman primates is lacking or incomplete. This lack of knowledge reflects the inherent complexities associated with the use of the primate in ovum transport studies, chief among which is the difficulty in accurately determining the time of Accepted July 16, Rockefeller Foundation Postdoctoral Fellow in Reproductive Biology. 2 Present Address: College of Veterinary Medicine, Department of Veterinary Physiology and Pharmacology, Texas A & M University, College Station, Texas Recipient of NICHHD Research Career Development Award K04 HD Supported by the Rockefeller Foundation. ovulation. A variety of indirect criteria has been evolved to determine the time of ovulation in the nonhuman primate. These include rectal palpation of the ovaries (Uartman, 1932;Mahoney, 1970), sequential changes in vaginal cytology (Mahoney, 1970; Mauro et al., 1970; Uutchinson, 1970) and in cervical mucus composition (Mahoney, 1970; Ovadia et al., 1971), and sex skin changes (Uagino and Goldzieher, 1970). However, extensive variability between and within animals combined with equivocal changes in the above parameters renders temporal correlation of such events with ovulation difficult. Periovulatory changes in gross ovarian follicular morphology observed at laparotomy have been described in the rhesus monkey (Betteridge et al., 1970) and have obviated much of the difficulty in diagnosing ovulation in this species. When such observations are made serially, it becomes possible to both diagnose and time the occurrence of ovulation with accuracy. Serial laparoscopy has been used to time ovulation in the primate for use in ovum transport studies (Jainudeen and Hafez, 1973). These investigators encountered difficulty in recovering natural ova, and speculated 363

2 364 EDDY ET AL. that the low recovery reflected interference with the mechanism of ovum pickup due to frequent and repeated laparoscopic manipulation of the reproductive tract. Thus, attempts to recover natural ova were decreased and emphasis were shifted to the recovery of surrogate ova (Sephadex beads and rabbit ova). Thus the time course of tubal ovum transport of natural primate ova remains largely undefined. Because the primate ovulates spontaneously, an excessive number of serial laparoscopic observations may be required if laparoscopy is initiated prematurely. In the rhesus monkey the preovulatory surge of LH is preceded by a gradual increase in circulating estrogen of approximately three days duration (Uotchkiss et al., 1971). Estrogen levels then rise sharply, peak 9-15 h prior to the LU surge, then decline prior to ovulation. Ovulation follows the estrogen peak by approximately 37 h (Weick et al., 1973). Measurement of a characteristic preovulatory peak in circulating estrogen may be used to predict impending ovulation and thereby decrease the number of laparoscopies required to verify ovulation. The present study was undertaken to define the detailed time course of tubal ovum transport in the spontaneously ovulating rhesus monkey using serial laparoscopy, initiated following detection of a preovulatory estrogen peak, to time ovulation. in place of methanol for precipitation of plasma proteins. The estrogen antibody was prepared and made available by Dr. Burton V. Caldwell. Laparoscopy Following the detection of a preovulatory peak in estrogen (levels in excess of pg/mi), serial Iaparoscopies were initiated in order to monitor sequential changes in follicular morphology. Under Sernylan anesthesia (3-5 mg/kg body weight) the MATERIALS AND METHODS Sexually mature female rhesus monkeys (Macaca mulatta) weighing kg were individually caged under controlled conditions of 75#{176} F and 50 percent relative humidity and exposed to a 14 h fluorescent light photo-period. Commercially prepared pelleted monkey chow was supplemented twice weekly with fresh fruit and vegetables. Tap water was available ad lib. Vaginal swabs were taken each morning and examined for menses. The first day of menstrual bleeding was designated Day 1 of the cycle. Each animal was allowed to establish regular, spontaneous, cyclic activity prior to use. Daily blood samples (2-3 ml) were drawn each morning beginning on Day 8 by femoral puncture with a heparinized syringe following tranquilization with phencyclidine hydrochloride (Sernylan, Parke-Davis, mg/kg body weight, i.m.). The heparinized blood was centrifuged and the plasma assayed for estrogen. Samples were drawn through Day 16 or until a preovulatory estrogen peak was detected. Estrogen Assay Total plasma estrogen levels were determined by radioimmunoassay as described by Wu et al. (1973) with minor modification. Absolute ethanol was used FIG. 1. In situ flushing technique for the recovery of ova from various portions of the Macaca mulatta reproductive tract. (a) Ampullary flush; (b) Isthmic flush; (c) Uterine flush. (Insert: details of fenestrations applied to tip of intracatheter cannula.) )

3 OVUM TRANSPORT IN THE RHESUS MONKEY 365 C a, 0 0 U E 100 a- (2) (6) : ) 6 Days (8) (8) From Ovulation FIG. 2. Mean plasma estrogen pattern of nine ovulatory menstrual cycles in which ova were recovered in Macaca mulatta. Values are normalized to the day of ovulation. Vertical lines about each point represent the standard error of the mean. Numbers in parentheses represent the number of observations. Hatched area defines the average interval of time during which ovulation occurred (range ± 6-15 h). possible. The isthmus, excluding the intramural position and a 3-5 mm proximal segment, was flushed with 3-5 ml of saline, and the effluent collected through the ampulla. To flush the uterus, an 18 gauge polyethylene intracatheter fenestrated at the tip was introduced through the uterine fundus into the uterine cavity. A second intracatheter was inserted through the ventral aspect of the uterus into the uterine cavity at a point several millimeters above the internal cervical Os. Syringes were attached to each intracatheter and successive 6 ml quantities of saline were flushed through the uterine cavity from alternating syringes. At each flushing the effluent was collected in the corresponding empty syringe. The use of a cervical clamp prevented transcervical leakage of fluid during uterine flushing. In the 72 h post ovulation group, the uterus was flushed first, with the TUJ occluded, to avoid artifactual dislocation of ova located in the proximal isthmus into the uterus during tubal flushing. Tubal and uterine effluents were examined for the presence of ova with a binocular dissecting microscope. RESULTS entire surface of both ovaries was visualized using a pediatric laparoscope. The time of ovulation was arbitrarily designated as the midpoint between two successive lararoscopic observations, the former preovulatory and the latter postovulatory. Using this rationale, the time of ovulation was accurately defined within a range of ± 6-15 h. Ovum Recovery Midventral laparotom ies were performed under Sernylan anesthesia.3-5 mg/kg) 24, 48 or 72 h post ovulation and the reproductive tracts flushed in situ to recover the unfertilized ova (Fig. 1). In the majority of cases a polyethylene cannula (P.E. 60, id in., o.d in.) was inserted into the ampulla to a point just beyond the infundibulum and ligated in place. In the remaining cases, the oviduct was flushed directly into a receptacle (Mastroianni and Rosseau, 1965). The ampullary isthmic junction (AIJ) was located and a short, 30 gauge needle mounted on a syringe filled with normal saline was inserted into the lumen of the ampulla at the level of the AlJ or several millimeters to the ovarian side of it. The tubo-uterine junction (TUJ) was digitally occluded and 3-10 ml of saline was flushed through the ampulla. The needle was then introduced into the isthmus as close to the TUJ as Timing of Ovulation Figure 2 shows the periovulatory peripheral plasma estrogen levels of nine ovulatory menstrual cycles in which ova were recovered following in situ flushings of the reproductive tract. In each cycle a well defined preovulatory peak of estrogen in excess of 200 pg/mi (range pg/mi) was observed h prior to ovulation. Serial laparoscopy was initiated h after detection of each peak and was repeated every h until post ovulatory changes were observed. In eight cycles two laparoscopies were sufficient to confirm and time ovulation. In the remaining cycle, two additional laparoscopies were required. During the initial observation the ovary containing the Graafian follicle was easily distinguished from the contralateral nonovulatory ovary. The former was much larger, the entire ovary assuming a fluid-filled, bluish-pink opaque appearance which could occasionally be localized to a specific area which subsequently TABLE 1. Ovum recovery rates following in situ flushing of the reproductive tract of Macaca mulatta. Time ovulation after (hrs) Number flushings of Number of ova recovered Percent recovery Total

4 366 EDDY ET AL. TABLE 2. Results of in situ flushing of the reproductive tract following timed ovulation in Macaca rnulatta. Animal number Time post ovulation Tubal flush Uterine flush Location ovum of ± Ampulla ± Ampulla ± Ampulla ± Ampulla ± Ampulla ± Ampulla All 72 ± Uterus A13 72 ± Tube A17 72 ± Uterus proved to be the site of ovulation. In agreement with Betteridge et al. (1970) we uniformly failed to observe persistent, well defined, protuberant Graafian follicles with preovulatory stigmata (Johnson et al., 1968). Instead, final follicular maturation and ovulation, visually observed as the transition from poorly defined Graafian follicle to protuberant, hemorrhagic, raw, post ovulatory corpus luteum (CL), generally encompassed a period of only hours. Although variety in periovulatory follicular morphology was encountered, the diagnosis of ovulation was always unequivocal, due primarily to the opportunity to view progressive changes, before and after ovulation. Ovum Transport A total of 24 in situ flushings were performed following laparoscopic timing of ovulation and resulted in recovery of nine ova (Table 1) yielding an overall recovery rate of 37.5 percent. In contrast, recovery rates for individual time groups ranged from 75 percent to 25 percent. The location of ova recovered through in situ flushing of the reproductive tract at various times post ovulation is summarized in Table 2. Ova were consistently recovered from the ampulla at both 24 and 48 h after ovulation, in all cases still in cumulus (Figs. 3, 4). By 72 h post ovulation, ova were entering the uterus. Two ova were recovered from this site whereas a third was flushed from the tube. In the latter recovery, the precise location of the ovum within the tube is not known with certainty. Uterine ova were still surrounded by cumulus cells which persisted in much smaller numbers than the cumulus mass seen at 24 and 48 h (Fig. 5). This same sparse cellular investment characterized the single ovum recovered from the tube at 72 h (Fig. 6). On the basis of cumulus investment, it would appear that this FIG. 3. Macaca mulatta ovum recovered 24 h after ovulation. This ovum was surrounded by a cumulus mass when flushed from the ampulla. FIG. 4. Macaca mulatta ovum recovered 48 h after ovulation. This ovum was located in the ampulla and still retained a dense cellular investment of cumulus cells.

5 OVUM TRANSPORT IN THE RHESUS MONKEY 367 FIG. 5. Macaca mulatta ovum recovered from the uterus 72 h after ovulation. Unlike the tubal ova recovered from the anipulla 24 and 48 h after ovulation, this uterine ovum was surrounded by comparatively few cumulus cells. ovum had traveled beyond the AIJ and was located in the isthmus, if denudation occurs during passage through the isthmus. DISCUSSION Discussion of Limitations of Methods As amplified below this report represents, to the best of our knowledge, the first description of the detailed time-course of tubal transport of ova in the rhesus monkey. Before discussing our conclusions, it is pertinent to review possible limitations imposed upon the interpretation of our data. First it may be argued that the administration of transquilizing doses of Sernylan to FIG. 6. Macaca mulatta ovum recovered from the tube 72 h after ovulation. Note the same sparse cellular investment as that seen surrounding the ovum in Fig. 5. facilitate daily blood collection may have modified the process of ovum transport. However, use of Sernylan in Macaca mulatta in conjunction with serial blood drawing has been reported to restore normal fertility in females previously experiencing lowered fertility due to blood collection (Hamner, 1974). The use of Ketamine in conjunction with serial blood drawing has also been shown to have no effect on normal follicular maturation and ovulation in Macaca mulatta (Channing, 1975). Thus, we do not think that the blood sampling interfered with ovulation and ovum transport. Second, one must confront the possibility that the serial laparoscopic examinations, so essential to the accurate determination of ovulation, might have interfered with ovum transport. Jainudeen and Hafez (1973) speculated that excessive manipulation of the Macaca fascicularis genital tract during laparoscopic examination might have interfered with ovum pickup. Although the number of laparoscopies required to time ovulation was not specified in their study, the arbitrary initiation of serial laparoscopy on Day 11 and its continuation until ovulation was confirmed may be expected to have at least sometimes necessitated an excessive number of laparoscopies, which might have contributed to the recovery of only three natural ova. In the present study daily measurement of plasma estrogen levels furnished accurate prospective indication of impending ovulation. As a result, the number of laparoscopics performed was minimized. This fact notwithstanding, the overall recovery rate was only 37.5 percent. Obviously some of the failures to recover ova may reflect limitations of the recovery technique. However, during one recovery attempt an ovum was aspirated from the cul-de-sac 72 ± 12 h post ovulation, prior to any attempt to flush the tract itself. Since only two laparoscopies, 24 h apart, were required to time ovulation in this case, this finding may reflect normal variations in the efficiency of the ovum pick up mechanism. This possibility is strengthened by the successful recovery of a uterine ovum at 72 h post ovulation despite four antecedent laparoscopies. Third, the recovery rates varied with the time of examination. Ova were recovered in 3 of 4 attempts at 24 h (all in the ampulla), in 3 of 8 attempts at 48 h (all in the ampulla) and in 3 of 12 attempts at 72 h (2 from the uterus and one from the tube). Failure to recover ova in 5

6 368 EDDY ET AL. of 8 attempts to flush the isthmus may reflect technical inefficiency in flushing the isthmus. Thus we might have failed to recover those ova which passed into the isthmus prior to 48 h. By the same reasoning, failure to recover more ova from the uterus at 72 h postovulation (9 out of 12) may reflect their continued presence in the isthmus. Recovery rates of uterine ova significantly lower than those for tubal ova have also been repeatedly remarked upon in reports of investigations of ovum transport in the rabbit. Finally, the described time-course is synthesized by noting the location of recovered ova in different animals at each time interval, and thus is intrinsically inferior to tracking the complete transport of individual ova through the same animal. Discussion and Results In the majority of mammalian species studied, tubal transport of the ovum into the uterus requires 3 to 4 days. The few reports of tubal ovum transport in the spontaneously ovulating primate have generally dealt with the overall time of transport from ovary to uterus, and were not based on accurate knowledge of the time of ovulation. Hartman (1944) used rectal palpation to time ovulation and determined tubal ovum transport in the rhesus to require somewhat over three days based on recovery of ova from the uterus. Mastroianni et al. (1967) using ferning of cervical mucus to time ovulation noted the location of ova in the reproductive tract of spontaneously ovulating rhesus monkeys 24 h after presumed ovulation. All ova recovered were in the tube. Marston et al. (1969) using morphologic dating of the CL at the time of flushing of the entire tube noted the persistence of ova within the rhesus oviduct 4 to 7 days postovulation. Ova were also recovered from the uterus during this interval. In none of these studies was the detailed time course of ovum transport investigated. Jainudeen and Hafez (1973) attempted to provide such a detailed description in Macaca fascicularis by excising and segmentally flushing oviducts at 24 h intervals following lararoscopically timed ovulation. Unfortunately early in the experiment they abandoned the attempt to recover natural ova and substituted surrogate ova. Their results therefore are largely predicted upon the movement of artificial ova which were not proved to accurately mimic the transport of natural ova. They noted transport through the distal two-thirds of the tube within 48 h, retention at the AIJ for an additional 48 h, followed by transport through the proximal third of the tube in an additional 48 h. These results are in contrast to the time-course of ovum transport in the rabbit, where newly ovulated ova arrive at the ampullary-isthmic junction within minutes and are delayed at that location for some h, before entering the isthmus. The journey through the isthmus to the uterus takes about 36 h, so that entry into the uterus usually takes place 66 to 72 h after injection of human chorionic gonadotropin. Within the limits discussed above we may safely conclude from our data that at least some rhesus ova are still in the ampulla 48 ± 15 h after ovulation and that some ova arrive in the uterus within 72 ± 12 h after ovulation. Thus, although the overall time-course of ovum transport in Macaca mulatta is apparently not greatly different from that in the rabbit, the details of transit are. Our data suggest that rhesus ova remain in the ampulla much longer and traverse the isthmus much more quickly than do rabbit ova. In addition the cumulus cells persist much longer around unfertilized Macaca mulatta ova than around unfertilized rabbit ova. Our data are in good agreement with those obtained in our own laboratories and by Croxatto et al. (1972) concerning the timecourse of egg transport in women, and support the contention that the rhesus is a good model for studies of human ovum transport. ACKNOWLEDGMENT The authors gratefully acknowledge the excellent technical assistance of Thomas C. Turner and Elizabeth Menchaca, Department of Obstetrics and Gynecology. This work is supported by the Agency for International Development, Subcontract PARFR-56 from the University of Minnesota. REFERENCES Betteridge, K. J., Kelly, W. A., and Marston, J. H. (1970). Morphology of the rhesus monkey ovary near the time of ovulation. J. Reprod. Fertil. 22, Channing, C. P. (1975). Personal communication. Croxatto, H. B., Fuentealba, B., Diaz, S., Pastene, L., and Tatum, H. J. (1972). A simple non-surgical technique to obtain unimplanted eggs from human uteri. Am. J. Obstet. Gynecol. 112, Hagino, N. and Goldzieher, J. W. (1970). Regulation of gonadotrophin release by the corpus luteum in the baboon. Endocrinology 87, Hamner, C. (1974). Personal communication. Hartman, C. G. (1932). Pelvic (rectal) palpation of the female monkey, with special reference to the ascertainment of ovulation time. Am. J. Obstet.

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