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a Unidad de Reproducción y Desarrollo, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because of ethical and logistic limitations in investigating sperm migration beyond the cervix in women, the influence of hormonal fluctuations on sperm migration in the upper segments of the genital tract is unknown. In order to explore whether sperm migration through the oviduct is influenced by the cyclic variations of the endocrine milieu, we used the rat. This species presents a short estrous cycle of 4 days, with wide fluctuations in the plasma levels of sex steroids [4]. Furthermore, the spermatozoa of the rat migrate from the uterus into the oviduct in approximately 15 min and traverse the oviduct approximately 3 h after mating [5].
We conducted a quantitative study of sperm recovery from rat oviductal segments at 3 and 8 h after artificial intrauterine insemination, performed at various stages of the estrous cycle. The effects of treatment with a GnRH antagonist to suppress the estrous cycle and with exogenous estradiol-17ß (E2) and progesterone (P4) on sperm migration were also investigated.
Some of the results presented here were previously reported in abstract form by Orihuela et al. [6].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sprague-Dawley rats were used. The animals were kept under controlled temperature (21–24°C), and lights were on from 0700 to 2100 h. Water and pelleted food were supplied ad libitum. Males were 4–5 mo old, weighing 400–450 g, and were of proven fertility. Females weighing 200–220 g were selected from among those that had had at least two regular cycles of 4 days. The regularity of the cycles was verified by daily vaginal smears. The day of estrus was considered Day 1 of cycle. The care and manipulation of the animals were in accordance with the ethical guidelines of our institution.
Treatment
E2 (5 µg) or P4 (5 mg) were injected s.c. as a single dose dissolved in 0.1 ml propylene glycol or olive oil, respectively. The production rate and plasma levels of these hormones in the rat differ by approximately 1000-fold [4], and these doses are sufficient to produce estrogenic and progestogenic responses in the uterus of immature or ovariectomized rats [7, 8]. Although these are pharmacologic doses for a normal rat, they are not excessive, particularly in ovarian-suppressed animals. The GnRH antagonist Nal-Glu was injected s.c. at a daily dose of 300 µg dissolved in 2.5 µl 0.05 M acetic acid and 97.5 µl distilled water. Nal-Glu was synthesized at the Salk Institute (under contract NO1-HD-0–2906 with NIH (Bethesda, MD) and made available through the Contraceptive Development Branch, Center for Population Research, NICHD.
Preparation of Spermatozoa for Insemination
Each morning, two males were killed by decapitation in order to obtain their spermatozoa. The four caudae epididymides were removed and cut into several pieces, which were placed in culture dishes containing 1 ml sterile saline, overlaid with mineral oil at 37°C, to let spermatozoa diffuse into the medium. The concentration of spermatozoa in the suspension was determined in a Neubauer chamber (Cambridge Instruments, Buffalo, NY), and to preserve motility, further dilution was avoided. All samples used contained between 10 and 20 million spermatozoa per 0.1 ml. Each sperm suspension was used to inseminate four females that represented different stages of the estrous cycle or different treatment groups.
Insemination
Females were anesthetized with ether at 0900 h. The uteri were exposed through flank incisions, and 0.1 ml of sperm suspension, containing 10–20 million spermatozoa, was injected into the upper third of each uterine horn. The uteri were returned to the peritoneal cavity, and muscles and skin were sutured. The number of spermatozoa inseminated is 3-fold lower than the number observed in the rat uterus after mating [9]; but as shown below, it was sufficient for the purpose of this study. In order to mimic the normal amount, a large number of males would have to be killed because of the low yield of epididymal sperm recovery. Preliminary experiments showed that inseminating with 10–20 million spermatozoa in each uterine horn allowed recovery of spermatozoa from the oviduct in numbers only one third lower than the number found after natural insemination (mean ± SE 1548 ± 293, n = 8; unpublished results). Furthermore, we found that 54% of ova recovered from the genital tract 72 h after insemination were morulae, as previously reported by Ortiz et al. [10]. The proportion of inseminated spermatozoa that migrated into the oviduct fluctuated between 0.001% and 0.003%, and the number of spermatozoa recovered from individual oviducts of estrous females was independent of the number inseminated within the range 10–20 million (r2 = 0.03, n = 5).
Recovery of Spermatozoa
Females were killed with an overdose of ether, and the oviducts were removed free of fat tissue. In separating oviduct from uterus, care was taken to leave the intramural segment attached to the oviduct. Spermatozoa were recovered from the oviduct as reported by Smith and Yanagimachi [11]. Their original technique consists of flushing the oviduct three times: the first two with 20 µl saline to recover spermatozoa lying free in the lumen and loosely attached to the mucosal surface, and the third with 20 µl saline + 0.5% Triton X-100 to dislodge spermatozoa adhering to the epithelium in the mucosal crypts. Smith and Yanagimachi determined the origin of the sperm cells in the flushings by direct observation through the oviductal wall. In our preliminary experiments, after the first flushing with 20 µl or 50 µl saline, no spermatozoa were recovered when the second flushing was performed with saline alone; in the third flushing, performed with saline + 0.5% Triton X-100, sperm cells were recovered. Therefore, the second flushing was deleted and we flushed the oviduct only twice, the first time with 50 µl saline and the second time with 50 µl of saline + Triton X-100. Oviducts were divided into ampulla and isthmus (containing the intramural segment), and each segment was flushed twice, as described above, on a glass slide. On the basis of the experiments of Smith and Yanagimachi [11], we assumed that the first flushing removed spermatozoa lying free in the lumen and that the second one removed sperm cells adhering to the oviductal epithelium. In the original technique, the flushings were diluted with water to immobilize spermatozoa and avoid counting them twice. In our work, this was not necessary: the majority of spermatozoa recovered from the oviduct had very low motility so that the probability of counting them twice was minimal. The counting was done using brightfield microscopy at a magnification of x250. No attempt was made to assess motility or viability.
Experiment 1
The first experiment was designed to establish the effect of various stages of the estrous cycle on sperm migration into and through the oviduct. Rats in proestrus, estrus, metestrus, or diestrus were artificially inseminated, and 3 or 8 h later the ampullary and isthmic segments were flushed to determine the number of spermatozoa lying free in the lumen or adhering to the epithelium. Because spermatozoa traverse the oviduct nearly 3 h after mating [5], this was the first point in time selected, and a second point at 8 h was considered appropriate for detection of a change in the number or distribution of spermatozoa.
Experiment 2
The second experiment was designed to establish the effect of estrous cycle suppression on sperm migration into and through the oviduct. For this, we used Nal-Glu since it has been shown to antagonize the effects of GnRH in rats [12, 13]. Rats were injected s.c. with 300 µg/day of Nal-Glu for 7 days starting in diestrus, and each day a vaginal smear was taken to confirm suppression of the estrous cycle. In the morning of Day 7 of treatment, rats were inseminated, and 3 h later the number of spermatozoa in the oviducts was determined as in experiment 1.
Experiment 3
The third experiment was designed to determine the effect of E2 and P4 on sperm migration into and through the oviduct. Rats were treated with Nal-Glu as in experiment 2, and in the morning of Day 7 of treatment they received an s.c. injection of vehicle, E2 (5 µg), P4 (5 mg), or E2+P4 (Time 0). In order to determine the effects of E2 or P4 alone, rats were inseminated at 0, 6, or 12 h after steroid injection; to determine the effect of concomitant administration of E2 and P4, rats were inseminated only at 6 h. Three hours after insemination, rats were killed, and the number of oviductal spermatozoa was determined as described above.
Statistical Analysis
The results are presented as mean ± SEM. Nonparametric statistics were used because the data exhibited non-normal distribution. Overall analysis was done by Kruskal-Wallis test, followed by Mann-Whitney test for pair-wise comparisons when overall significance was detected.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At 3 and 8 h postinsemination, more sperm cells were recovered in proestrus and estrus than in the other two stages, with the highest number recovered in estrus. Furthermore, more spermatozoa were recovered at 3 h than at 8 h postinsemination from proestrus to metestrus (Fig. 1). At both times, the highest number of spermatozoa was recovered from the isthmus in estrus (Fig. 1). At 3 h postinsemination, the number of spermatozoa in the ampulla was similar across the stages (range for individual animals: 6–71); an exception was in diestrus—at this stage, no sperm were recovered from this segment (Fig. 1). At 8 h postinsemination, the number of spermatozoa recovered from the ampulla had decreased in proestrus and metestrus and remained stable in estrus (Fig. 1). A separate analysis of the second flushings showed that sperm cells were recovered only in those obtained from the isthmus in proestrus and estrus (Fig. 2). Furthermore, the highest number of adhering spermatozoa was seen in proestrus, and the number decreased remarkably from 3 h to 8 h after insemination.
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Experiment 2
During the 7 days of treatment with Nal-Glu, rats remained in diestrus. The total number of spermatozoa recovered from the oviducts of rats treated with Nal-Glu (6.1 ± 3.0) was not statistically different from the number recovered from diestrous rats in experiment 1 (3.8 ± 1.7).
Experiment 3
In rats inseminated at 6 or 12 h after E2 administration, the total number of spermatozoa recovered from the oviducts was higher than in their corresponding control groups (Fig. 3). The highest number of sperm was recovered when insemination was done at 6 h posttreatment; this number was similar to the number found in proestrus in experiment 1. Furthermore, after E2 treatment the largest number of spermatozoa was recovered from the isthmus (data not shown), but no adhering spermatozoa were recovered at any time studied. No significant differences were found in the recovery of total sperm in rats treated with Nal-Glu and P4 (range for all three time points 10.3 ± 2.3–9.8 ± 1.3) in comparison with the control groups (10.3 ± 2.0–11 ± 0.3), and no adhering spermatozoa were found in either group (data not shown). Treatment with E2+P4 increased significantly the recovery of total sperm in comparison with Nal-Glu alone, but this increase was smaller than that caused by E2 without concomitant P4. The majority of spermatozoa were recovered from the isthmus, and adhering spermatozoa were found in this segment in numbers similar to the those obtained in estrus in experiment 1 (Fig. 4). The proportion of total spermatozoa that were adhering to the isthmic segment (16%) was similar to the proportion found in proestrus in experiment 1 (15%).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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More spermatozoa entered the oviduct during estrus; there was then a gradual reduction in their number in metestrus and diestrus until it dramatically increased again in proestrus. Thus, the various biological environments generated in the female genital tract along the estrous cycle affect sperm migration into the oviduct differently. Most likely this is accomplished in part through functional changes in the uterotubal junction, since the uterotubal junction controls the number of spermatozoa passing from the uterus into the oviduct [15]. It is also possible that changes in myometrial contractions during the estrous cycle [16] affect the migration of spermatozoa into the oviduct or that sperm motility or viability in the uterus changes throughout the cycle. The recovery of a higher number of oviductal spermatozoa in estrus as compared to other stages is consistent with the concept that during estrus the female genital tract presents a biological environment that is most favorable for gamete transport and fertilization.
In order to identify differences in sperm migration within the oviduct with respect to the estrous cycle, we analyzed the distribution of spermatozoa between ampullary and isthmic segments. At all stages, more sperm cells were found in the isthmus, suggesting that this segment acts as a sperm reservoir independently of the stage of the cycle. In a variety of other species, including the cow [17], sheep [18], pig [19], hamster [20], guinea pig [21], and mouse [22], spermatozoa accumulate in the isthmic segment during the preovulatory period. However, our data show that this feature, at least in the rat, is not limited to the preovulatory period but is present at all stages.
In the majority of the rats, the number of sperm cells entering the ampulla was in the range of 10–20 and was similar throughout the cycle except during diestrus, when no sperm were found at the 3-h time point. While during proestrus and metestrus, the number of ampullary spermatozoa decreased from 3 to 8 h, this number remained stable during estrus. At this stage, the number of spermatozoa that were recovered from the ampulla was of the same order of magnitude as the normal number of oocytes available for fertilization, corroborating previous findings in the rat [5] and in other species (reviewed in [23]). The retention of sperm cells in the ampulla during estrus was clearly associated with the presence of newly ovulated oocytes, since these spermatozoa were found attached to the zona pellucida.
Sperm adherence to the epithelium was found to be limited to the isthmic segment and to proestrus and estrus, indicating that this is a stage- and segment-specific phenomenon. Possibly the oviductal secretions that vary during the estrous cycle in the rat [24] influence the binding of spermatozoa to the epithelium. On the other hand, the population of bound spermatozoa did not exceed 15% of total recovered from the oviduct, which suggests either that there are a small number of binding sites in the isthmus or that only a small subpopulation of spermatozoa have binding ability. Another possibility is that spermatozoa that were bound prior to flushing had been released at the time of recovery. Since we did not examine the oviductal epithelium by histology after the second flushing, we cannot exclude the possibility that some adhering spermatozoa remained attached and were not counted.
The abrupt and transient increase in the number of adhering spermatozoa from diestrus to proestrus suggests that adhesion molecules for spermatozoa are expressed in the epithelium of the isthmus only for a very short time preceding ovulation. The reduction of sperm adherence from 3 to 8 h after insemination in proestrus may be explained by changes in the sperm membrane induced by the contact with the epithelial cells, which leads to detachment and capacitation. The capacitation status of the sperm is known to play an important role in the ability of sperm to bind to the oviductal epithelium [25, 26].
Lefebvre et al. [27] found in cattle that the number of spermatozoa bound to oviductal explants was not affected by the stage of the estrous cycle or by the anatomic origin of the explants; Baillie et al. [28] found only a small difference in the number of spermatozoa that attached to human tubal epithelium in vitro when ampulla and isthmus were compared and no differences when menstrual cycle stages were compared. These results contrast with our study and are probably due to the fact that the other authors' in vitro experimental model does not mimic the in vivo conditions. Another possibility is that the timing and site of expression of the putative adhesion molecules or the binding ability of sperm differ among the species.
The functional significance of sperm binding to oviductal epithelium is that binding preserves the motility and fertilizing ability of sperm until the time of ovulation [29–31], and there is evidence that the population of unbound spermatozoa does not supply the fertilizing sperm [9, 32]. Recent investigators have attempted to characterize the molecular bases of sperm attachment to the epithelium. In hamsters, sperm binding to the isthmic epithelium is through a specific ligand/receptor interaction that includes sialic acid [33]. The binding of stallion sperm to oviductal explants is inhibited in vitro by galactose [34], and bovine sperm binding to oviductal epithelium in vitro involves fucose recognition [35] and is mediated by a Ca2+-dependent lectin present on the surface of bull sperm [36]. On the other hand, it has been postulated that the binding of sperm to the oviductal epithelium may be the primary factor for considering the isthmic segment a sperm reservoir in the mammalian oviduct [22, 27]. However, while the isthmic segment accumulated spermatozoa at all stages of the cycle, we observed sperm adherence only during proestrus and estrus and only in relation to a small proportion of the sperm cells. Thus, sperm adherence does not account for the accumulation of spermatozoa in the isthmus. The present findings and data from others [17–22] indicate that the isthmic segment stops the majority of spermatozoa that reach this segment from passing beyond.
Sperm migration from the uterus into the oviduct appears to be regulated by sex steroids during the estrous cycle. When the normal oscillations of circulating levels of E2 and P4 were suppressed in Nal-Glu-treated rats (acyclic), sperm migration into the oviduct was as observed during diestrus. Furthermore, in acyclic rats treated with E2, sperm migration into the oviduct was as observed during proestrus, suggesting that E2 regulates the uterotubal junction. This effect of E2 was evident at 6 h and had partially waned by 12 h. On the other hand, P4 administration did not directly affect sperm migration into the oviduct but partially counteracted the effect of E2 in acyclic rats. When acyclic rats were treated with E2 or P4, no adhering spermatozoa were recovered; but when E2 and P4 were administered concomitantly, recovery of adhering sperm was restored. One plausible explanation is that E2 may induce the expression of adhesion molecules for attachment of spermatozoa in the oviductal epithelium and that P4 acts directly on the sperm membrane [37, 38], influencing the ability of sperm cells to bind to the epithelium.
In summary, this study shows for the first time that the pattern of sperm migration into and through the oviduct varies with the day of the cycle in which insemination is performed, and also that the attachment of spermatozoa to oviductal epithelium is stage- and segment-specific and sex steroid-inducible. Furthermore, our results indicate that E2 acutely facilitates sperm migration from the uterus to the oviduct, that P4 modulates this effect of E2, and that the interaction of E2 and P4 stimulates adhesion of spermatozoa to the oviductal epithelium. These observations demonstrate that sperm migration into and through the oviduct is subjected to hormonal regulation, lending support to the hypothesis that the pattern of sperm migration may differ through the primate oviduct depending on the timing of coitus.
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FOOTNOTES |
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2 Correspondence: H.B. Croxatto, Unidad de Reproducción y Desarrollo, Facultad de Ciencias Biológicas, Pontifica Universidad Católica de Chile, Casilla 114-D, Santiago, Chile. FAX: 562 222 5515; hbcroxat{at}genes.bio.puc.cl
Accepted: November 17, 1998.
Received: July 30, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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