Sperm migration into and through the oviduct following artificial insemination at different stages of the estrous cycle in the rat

In order to examine whether sperm migration into and through the oviduct follows an invariable pattern or is subject to regulation, rats in proestrus, estrus, metestrus, or diestrus were inseminated in the upper third of each uterine horn with 10-20 million epididymal spermatozoa. Three or eight hours later, the numbers of spermatozoa free and adhering to the epithelium in the ampullary and isthmic segments were determined. A significantly higher number of spermatozoa were recovered in estrus than in other stages, at 3 h than at 8 h, and at all stages from the isthmus than from the ampulla. Spermatozoa adhering to the epithelium were observed only in proestrus and estrus and in the isthmus. The effect of exogenous estradiol-17beta (E2) and progesterone (P4) on sperm migration was investigated in rats in which the estrous cycle was inhibited pharmacologically. E2 facilitated sperm migration into the oviduct and P4 antagonized this effect, whereas P4 alone had no effect. Concomitant treatment with E2+P4 induced adhesion of spermatozoa to the oviductal epithelium. In conclusion, the pattern of sperm migration into and through the rat oviduct varies with the stage of the cycle, being dependent on E2 and P4. The adhesion of spermatozoa to the rat oviductal epithelium is stage- and segment-specific and requires the combined action of both hormones.     

Distribution and number of spermatozoa in the oviduct of the golden hamster after natural mating and artificial insemination

A group of female hamsters was mated with males of proven fertility either several hours before or during ovulation. Another group of females was artificially inseminated several hours before ovulation. Females were killed at various times after the onset of mating or artificial insemination, oviducts were fixed and sectioned serially, and spermatozoa were counted individually as to their location in the oviduct. Regardless of the type or time of insemination, the vast majority of spermatozoa that entered the oviduct remained in the lower segments of the isthmus (the intramural and caudal isthmus) without ascending to the ampulla. The lower segments of the oviduct, particularly the caudal isthmus, appeared to be acting as a "sieve" and/or "sperm reservoir." In females mated or artificially inseminated prior to ovulation, virtually no spermatozoa reached the cephalic isthmus or ampulla until the commencement of ovulation. Although a few spermatozoa reached the ampulla by 1 h after the onset of mating, they were the exception rather than the rule. When females were mated during ovulation, spermatozoa spent a minimum of about 3 h in the caudal isthmus before ascending to the ampulla. The number of spermatozoa that entered the oviduct after artificial insemination was considerably lower than in naturally mated animals, but this low number was apparently large enough to ensure complete fertilization.     

Impact of ACTH during oestrus on the ultrastructure of the spermatozoa and their environment in the tubal reservoir of the postovulatory sow

This study investigated whether injections of synthetic ACTH (simulating short-term stress) in sows during standing oestrus have a negative effect on spermatozoa and the local intraluminal environment in the utero-tubal junction (UTJ) and isthmus. Seven of the 14 sows were given ACTH through a jugular catheter every 2 h from the onset of standing oestrus until the sow ovulated (ACTH-group), while the other seven sows were given NaCl solution (C-group). All sows were artificially inseminated before ovulation. Six hours after ovulation (detected with transrectal ultrasonography) the sows were anaesthetised, the right oviduct was fixed in toto by vascular perfusion with glutaraldehyde, and the UTJ and specimens from the isthmus were prepared for scanning electron microscopy (SEM). SEM revealed that a seemingly viable population of spermatozoa remained in the UTJ 6 h after ovulation. A majority of sows in the ACTH-group had moderately to exaggerated amounts of mucus in the intraluminal environment of the sperm reservoir. In conclusion, stress simulated by exogenous ACTH in sows may alter the intraluminal environment of the sperm reservoir.     

The cause of infertility of male c-ros tyrosine kinase receptor knockout mice

Male homozygous transgenic c-ros knockout mice are sterile by natural mating, lack a part of their epididymis, and the epididymal sperm exhibit tail angulation in vivo and in vitro. To ascertain if this abnormal tail form caused the infertility, the number and nature of sperm in the tract of females mated to knockout and wild-type mice were determined. Percentage motility and numbers of sperm in the uterus 1 h after mating were similar between genotypes. The majority of the uterine sperm from the wild-type males had straight flagella, whereas 46-86% of knockout sperm were bent at the cytoplasmic droplet even when motile. Motile knockout sperm showed a 54 and 37% reduction in the straightline and curvilinear velocities compared with straight wild-type sperm. Sequential flushings of the oviduct 4 h after mating with the wild-type males contained sperm: 591 +/- 119 free, 371 +/- 70 loosely, and 122 +/- 47 tightly bound to the epithelium, but no knockout sperm were recovered from the oviduct or observed within the uterotubal junction in tissue sections. The infertility of c-ros knockout male mice can be explained by the sperm's inability to enter the oviduct, as a result of their bent tails forming the entangled sperm mass and their compromised flagellar vigor within the uterus.     

Distribution of spermatozoa in the female reproductive tract of the domestic cat in relation to ovulation induced by natural mating

The purposes of this study were to demonstrate the localization of spermatozoa in the reproductive tract of female domestic cats before (30 min and 3 h after mating) and after ovulation (48 and 96 h after mating), and to evaluate the efficiency of two techniques for studying sperm distribution. Estrus was induced in twenty-four female cats using 100 IU eCG and the females were divided into four groups with six females per group. The same male cat was used for mating with all the females. One group of six females was mated once; the others were mated four times in 1 h. Ovariohysterectomy was performed at 30 min, 3 h, 48 h, and 96 h after mating and the excised reproductive tracts were divided into seven segments on each side: infundibulum, ampulla, isthmus, uterotubal junction (UTJ), cranial and caudal uterine horn, and uterine body. The vagina and the lumina of the segments from one side were flushed with 0.5 ml PBS. The flushed and the non-flushed segments from the contralateral side were then fixed in 3% neutral buffered formalin and processed for routine histology. The numbers of spermatozoa in the flushings and in 40 histological sections from each segment were counted. Before ovulation, the majority of spermatozoa was detected in the vagina and the uterine segments, whereas after ovulation, significantly higher numbers of spermatozoa were present in the uterine tubal segments. The decreasing gradient in sperm numbers at 30 min and 3 h after mating between the vagina, the uterine segments, including the UTJ, and the uterine tubal segments indicated that the cervix and the UTJ served as barriers for sperm transport in the cat. The UTJ and the uterine crypts acted as sperm reservoirs before ovulation whereas the isthmus was a sperm reservoir around the time of ovulation. There was no difference in sperm numbers in the tissue sections between flushed and non-flushed segments, implying that the flushing technique only recovered some intraluminal spermatozoa while most of the spermatozoa remained in the epithelial crypts. This was further supported by the finding that significantly higher numbers of spermatozoa were recovered in the flushings at 30 min and 3 h after mating, when more spermatozoa were free in the lumina, than at 48 and 96 h after mating, when the majority of the spermatozoa were entrapped in the uterine epithelial crypts.     

Sperm transport in the female reproductive tract of the brushtail possum, Trichosurus vulpecula, following superovulation and artificial insemination

This study investigated sperm transport following superovulation and artificial insemination (AI) in the common brushtail possum, Trichosurus vulpecula. Females were superovulated by treatment with 15 IU pregnant mare serum gonadotrophin (PMSG) then 4 mg luteinizing hormone (LH) 78 h later. Inseminations were performed 27 h after LH (4 million motile spermatozoa/uterus). At 1.5, 3, 6, 9 and 12 h after AI (n=5 per group), females were euthanised and reproductive tracts removed for examination and flushed for sperm. No ovulations had occurred by 1.5 h, but 20% of animals had ovulated by 3 or 6 h, and 80% by 9 or 12 h. The mean numbers of spermatozoa recovered ranged from 249 to 275x10(3) in the uterus; 16-51x10(3) in the isthmus; 8-11x10(3) in the middle segment; and 6-16x10(3) in the ampulla at 1.5, 3 and 6 h after AI. Sperm numbers in all regions decreased at later times (P<0.05) except the isthmus, where 100x10(3) sperm were recovered by 12 h. Highly motile thumbtack sperm (a putative indicator of capacitation in marsupials), were recovered from the isthmus (20%), middle segment (50%) and ampulla (90%) at all sampling times, but not from the uterus. The epithelium of the oviduct segments contained mucus-secreting and ciliated cells and peak secretory activity was observed in the ampulla at 6 h. At 3, 6 and 12 h, many spermatozoa were found in epithelial folds within the isthmus. The present study has provided basic information on sperm transport and storage events within the female reproductive tract of T. vulpecula following superovulation and AI. It is concluded that this model may be useful to better understand pre-fertilization sperm maturation events in the possum, which could facilitate the development of IVF technology.     

Initiation of hyperactivated flagellar bending in mouse sperm within the female reproductive tract

To determine where and when hyperactivation is initiated in vivo, the flagellar curvature ratios (fcr) of mouse sperm within the female reproductive tract were measured from videotape recordings and compared with those of epididymal sperm incubated under capacitating conditions in vitro. The fcrs and linearities of trajectory were significantly lowered after 90 min of incubation in vitro, indicating that hyperactivation had been initiated by that time. The flagellar curvature ratios of sperm at the colliculus tubarius, within the uterotubal junction, and in the isthmus, measured at 1-2 h postcoitus and approximately 1 h before and 1 h after ovulation, were found to have fcrs that were not different from those of sperm incubated for 90 min in vitro. It was concluded that the tract sperm had initiated hyperactivated flagellar bending before the time of ovulation and before entering the oviduct. Only sperm in the lower isthmus 1 h before ovulation had fcrs that were significantly different from sperm incubated for 90 min in vitro, but not from sperm measured at the beginning of incubation in vitro. This could be the result of motility suppression in the lower isthmus.     

Distribution of embryos and 500-microM microspheres in the rabbit oviduct: controls for acute motion analysis during transport

Distributions of embryos and 500-micron diameter microspheres were measured in the cleared oviducts of 32 rabbits at 8 intervals post coitus (pc) and normalized to percentage of isthmic length. By 18 h pc, 46% of the embryos had entered the isthmus and were denuded of the cumulus, while 55% of the microspheres had entered the isthmus. By 24 h pc, all embryos and microspheres were in the isthmus. At 72, 78, and 84 h pc, 6.5, 37, and 93% of embryos and 21, 73, and 95% of microspheres were in the cornu, respectively. The mean positions of embryos and microspheres progressed at approximately 1% of isthmic length per hour between 24 and 72 h pc. Throughout isthmic transport, embryos and microspheres in individual oviducts were tightly grouped and had similar statistical distributions. Although microspheres began to transfer into the uterine horns earlier than embryos, the data suggest that 500-micron microspheres can be used in studies to quantify discrete movements within the oviduct prior to 72 h pc. The data further suggest that opposing forces may be generated by contractile events that keep embryos grouped and possibly control their rate of progress through the oviduct.     

Sperm Localization in the Oviducts of Artificially Inseminated Dairy Cattle

Eight animals, 3 heifers and 5 primiparous cows, were artificially inseminated by intrauterine deposition of frozen-thawed semen. The insemination dose comprised 20×106 or 200 × 106 spermatozoa, frozen in French mini straws. Four animals were inseminated at fixed time interval (72 or 84 h) after cloprostenol injection. The remaining 4 animals were inseminated in spontaneous oestrus. Slaughter took place 2 or 12 h after insemination. After fixation the oviducts were cut into segments, which were serial-sectioned and stained. Six sections per segment were examined under the microscope for sperm recovery. The number of spermatozoa recovered from the oviducts varied considerably among animals. Recovery was poor (less than 50 spermatozoa) in 4 animals. Recovery was low when insemination took place in induced oestrus and with the lower sperm number (20×106). In animals in which more than 50 spermatozoa were found the distribution varied both between animals and between oviducts within the same animal. Overall, more spermatozoa were found in the lower (UTJ, isthmus and AIJ) than in the upper (ampulla) parts of the oviducts. In 3 out of 4 animals more spermatozoa were recovered from the left than from the right oviduct. Only in 1 animal were the majority of spermatozoa found in the oviduct ipsilateral to the follicle-bearing ovary.     

Conference lecture: influence of stress on estrus, gametes and early embryo development in the sow

Systems with loose-housed sows have become common. Regrouping, which is commonly done after weaning and may coincide with many important reproductive events, causes stressful situations with elevated blood cortisol concentrations. Depending on group size, approximately 2-7 d are required for a new group of sows to become relatively stable. In a series of studies, the social stress after regrouping was simulated with repeated adrenocorticotrophic hormone (ACTH) treatments for approximately 48h. Sows were allocated into control and experimental groups, fitted with jugular catheters, and blood samples were collected every 2 or 4h. Follicular development and ovulation were monitored by transrectal ultrasonography every 4h. Simulated stress during pro-estrus prolonged estrus and disturbed the follicular growth and ovulation. Giving ACTH during estrus elevated concentrations of cortisol and progesterone, and changed the intraluminal environment, including exaggerated amounts of mucus in the UTJ and isthmus. Although ACTH had no effect on the time of ovulation (relative to onset of standing estrus), or on embryo development, fewer oocytes/embryos were retrieved from the ACTH group than from the control group (51% vs. 81%, P<0.05), and there was a tendency towards faster embryo transportation to the uterus. Short-term fasting after ovulation had an unfavourable effect on sperm numbers in UTJ/isthmus, cleavage rate of fertilized ova, as well as ova transport through the isthmic part of the oviduct. Treatment with ACTH after ovulation reduced numbers of spermatozoa at the zona pellucida and retarded cleavage rate of fertilized ova. Therefore, the timing of stress seemed to be an important factor regarding effects on reproductive events.     

Fertilization efficiency of in vitro matured oocytes transferred to oviducts of inseminated goats: a model to assess in vivo fertilization performance of goat spermatozoa

An alternative to conventional in vivo validation of sperm assays might be to assess the fertilization rate of multiple oocytes transferred to the oviducts of inseminated females. Increasing the number of oocytes increases the egg-sperm ratio in the oviduct under an unaltered endocrine milieu, setting the basis for picking up statistical differences between treatments in small populations. The study evaluated the model by transferring oocytes to females inseminated under conditions that are known to modify the fertilization rate in the field. The study then evaluated the use of cattle oocytes to replace goat oocytes for assessing sperm function under this model. In Experiment 1, 12 females were inseminated at estrus with either 100 or 300 million spermatozoa 20 h before transferring homologous oocytes into the oviduct ipsilateral to the ovulation point. In Experiment 2, 10 females were inseminated either once or twice; 10-20 h later, homologous oocytes were transferred into the oviduct ipsilateral to the ovulation point. In Experiment 3, 13 bilateral-ovulated females were inseminated and 20 h later goat and cattle oocytes were transferred to contralateral oviducts. Then, 16-20 h later, oocytes were flushed from the oviduct, cleaned of spermatozoa and stained to assess the fertilization rate. The fertilization rate was improved by increasing sperm numbers at insemination (P < 0.04) and by increasing the number of inseminations (P < 0.02). The results in Experiment 3 showed that fertilization rates were similar for goat and cattle oocyte (P > 0.05) and that fertilization values were highly correlated (r = 0.811, P < 0.001). Results suggest that the model can be used for in vivo validation of in vitro sperm assays by facilitating the expression of statistical differences in small number of animals. In addition, cattle oocytes can be used to replace goat oocytes to study in vivo sperm function in goats.     

Differences in sperm migration through cervical mucus in vitro relates to sperm colonization of the oviduct and fertilizing ability in goats

Sperm migration in estrous cervical mucus can be used to measure the ability of spermatozoa to migrate through the genital tract. The relationship of this test with the sperm colonization of the isthmus, and its impact on fertility has not been evaluated in goats. Our objectives were to determine the differences among spermatozoa of different bucks in their ability to penetrate homologous cervical mucus in vitro and to determine the relationship between sperm displacement through cervical mucus and the ability of spermatozoa to colonize the oviduct and penetrate IVM oocytes, in vivo. Sperm migration in cervical mucus was assessed in flat capillary tubes with a phase contrast microscope. In the first experiment, fresh semen was used to establish differences between males in the ability of their spermatozoa to migrate in cervical mucus. In the second experiment, goats in estrus were inseminated with fresh spermatozoa from males with significant differences in mucus migration ability, and sperm numbers were evaluated at the UTJ. In the third experiment, the fertilization efficiency of IVM oocytes transferred to the oviduct of estrous females inseminated with semen from the same males as earlier, was used to assess the relationship between the mucus migration test and the in vivo fertilization performance of their spermatozoa. Spermatozoa from different males varies significantly in sperm migration efficiency in cervical mucus (15.5a +/- 1.2; 14.9a +/- 1.4; 17.5ab +/- 1.2; 17.0ab +/- 1.5; 19.7b +/- 1.2; 20.1b +/- 1.4 mm; media +/- S.E.M. for males A-F, respectively, P < 0.05). Spermatozoa from males with different mucus migration efficiency values produced different sperm populations at the oviduct reservoir of inseminated females (1,233 +/- 92.3 versus 28.8 +/- 17.0 spermatozoa of males with high and low relative migration efficiency, respectively, P < 0.02). Spermatozoa from males with different mucus migration efficiency values have different fertilization rates of IVM oocytes transferred to oviduct (47/96 (49.0%) versus 25/91 (27.5%) for males with high and low relative migration efficiency, respectively, P < 0.05). Cumulative results suggest that sperm migration in cervical mucus is related to the ability of spermatozoa to colonize the oviduct and to fertilize matured oocytes in vivo.     

Unexpected flagellar movement patterns and epithelial binding behavior of mouse sperm in the oviduct

In order to better understand how sperm movement is regulated in the oviduct, we mated wild-type female mice with Acr-EGFP males that produce sperm with fluorescent acrosomes. The fluorescence improved our ability to detect sperm within the oviduct. Oviducts were removed shortly before or after ovulation and placed in chambers on a warm microscope stage for video recording. Hyperactivated sperm in the isthmic reservoir detached frequently from the epithelium and then reattached. Unexpectedly, most sperm found in the ampulla remained bound to epithelium throughout the observation period of several minutes. In both regions, most sperm produced deep flagellar bends in the direction opposite the hook of the sperm head. This was unexpected, because mouse sperm incubated under capacitating conditions in vitro primarily hyperactivate by producing deep flagellar bends in the same direction as the hook of the head. In vitro, sperm that are treated with thimerosal to release Ca(2+) from internal stores produce deep anti-hook bends; however, physical factors such as viscous oviduct fluid could also have influenced bending in oviductal sperm. Some sperm detached from epithelium in both the ampulla and isthmus during strong contractions of the oviduct. Blockage of oviduct contractions with nicardipine, however, did not stop sperm from forming a storage reservoir in the isthmus or prevent sperm from reaching the ampulla. These observations indicate that sperm continue to bind to oviductal epithelium after they leave the isthmic reservoir and that sperm motility is crucial in the transport of sperm to the fertilization site.     

Expression of TLR2/4 in the sperm‐storing oviduct of the Chinese soft‐shelled turtle Pelodiscus sinensis during hibernation season

The initiation of innate immunology system could play an important role in the aspect of protection for sperms long‐term storage when the sperms got into oviduct of turtles and come into contact with epithelium. The exploration of TLR2/4 distribution and expression in oviduct during hibernation could help make the storage mechanism understandable. The objective of this study was to examine the gene and protein expression profiles in Chinese soft‐shelled turtle during hibernation from November to April in the next year. The protein distribution of TLR2/4 was investigated in the magnum, isthmus, uterus, and vagina of the turtle oviduct using immunohistochemistry, and the gene expression of TLR2/4 was analyzed using quantitative real‐time PCR (qRT‐PCR). The results showed positive TLR2 protein expression primarily in the epithelium of the oviduct. TLR4 immunoreactivity was widely observed in almost every part of the oviduct, particularly in the epithelium and secretory gland membrane. Analysis of protein, mRNA expression revealed the decreased expression of TLR2/4 in the magnum compared with the isthmus, uterus, and vagina during hibernation. The protein and mRNA expression of TLR2 in the magnum, isthmus, uterus, and vagina was decreased in April compared with that in November. TLR4 protein and mRNA expression in the magnum, isthmus, uterus and vagina was decreased in November compared with that in April. These results indicated that TLR2/4 expression might protect the sperm from microbial infections. In contrast to the function of TLR2, which protects sperm during the early stages of hibernation, TLR4 might play a role in later stages of storage. The present study is the first to report the functions of TLR2/4 in reptiles.     

Sulphated glycosaminoglycans (S-GAGs) and syndecans in the bovine oviduct

In vivo, bull sperm capacitation seems to occur mainly in the oviduct. Capacitation of bull spermatozoa can be triggered in vitro by exposure to heparin, a heavily sulphated glycosaminoglycan (S-GAG). We determined the concentration of S-GAGs in oviductal fluid from dairy heifers, collected over the course of several oestrous cycles via surgically implanted intraluminal catheters. We also investigated the presence of syndecans, i.e. heparan sulphate proteoglycans, in the bovine oviductal epithelium of Swedish dairy cattle during standing oestrus and the luteal phase of the oestrous cycle, using immunohistochemistry for three different polyclonal antibodies raised against human syndecan-2 and rat syndecan-1 and syndecan-2, respectively. The concentration of S-GAGs in oviductal fluid obtained from the ampullar segment of the oviduct was significantly higher (P=0.0026) than it was in fluid from the isthmic segment during the functional period, i.e. from prooestrus to metaoestrus (73.5+/-10.49 mg/L in ampullar ODF, compared to 43.2+/-10.74 mg/L in isthmic ODF); least square mean (L.S.M.)+/-standard error of the mean (S.E.M.). There was also a significantly higher concentration of S-GAGs in the fluid from the oviduct ipsilateral to the ovulation side 73.5+/-10.54 mg/L on the ovulation side, compared to 43.1+/-10.71 mg/L in the oviduct on the contralateral side (L.S.M.+/-S.E.M., P=0.0026) during this period. Both syndecan-1 and syndecan-2 were present in the epithelial cells lining all studied segments of the bovine oviduct, i.e. the UTJ, isthmus and ampulla, during both standing oestrus and dioestrus. The syndecans and S-GAGs found may influence the gametes, while they reside in the oviduct; the amounts of S-GAGs found in the bovine oviduct seem sufficient to act as capacitating factors in vivo.     

Hyperactivated sperm progress in the mouse oviduct.

Sperm from naturally mated mice were observed and videotaped moving within mouse oviducts. The typical pattern of sperm progress involved intermittently breaking free and swimming a short distance, then reattaching to the epithelium. The proportion of sperm that swam freely (were not attached to the epithelium) was calculated and analyzed for effects of oviductal region, ovulation status, and sperm location relative to the lumen. A significantly higher proportion of sperm were free in the ampulla than in the isthmus (26.3% +/- 0.8% vs. 11.8% +/- 1.0%; p less than 0.0001) and in post-ovulatory than pre-ovulatory (16.2% +/- 2.0% vs. 10.6% +/- 1.6%; p less than 0.05) oviducts. Flagellar curvature ratio values showed that free sperm (0.716 +/- 0.024) had more sharply curved tails than stuck sperm (0.782 +/- 0.013). While this difference is significant (p = 0.01), the effect of attachment status interacted significantly (p less than 0.05) with the oviductal region such that there was a greater difference in the isthmus than in the ampulla. Only sperm using the more curved tail beats of hyperactivation were seen to break free from the epithelium and to progress along the oviduct. These results indicate that hyperactivation plays a role in moving sperm out of the isthmic reservoir and to the site of fertilization.     

Quantitative comparison of the passage of homologous and heterologous spermatozoa through the uterotubal junction of the golden hamster

A quantitative method was used to determine whether the spermatozoa of foreign species could pass through the uterotubal junction (UTJ) of the hamster as efficiently as homologous (hamster) spermatozoa. Estrous female hamsters were artificially inseminated with epididymal spermatozoa of homologous and heterologous (foreign) species. The number and distribution of spermatozoa in the oviduct were determined several hours after insemination (shortly before ovulation). The passage of immotile (dead) hamster spermatozoa through the UTJ was also examined. It was found that the spermatozoa of all foreign species tested (rat, mouse, guinea pig, and rabbit), as well as immotile hamster spermatozoa, could pass through the UTJ but did so in much smaller numbers compared to live hamster spermatozoa. This was not specifically due to poor survival of foreign spermatozoa in the hamster uterus, as the viability of all inseminated spermatozoa (including hamster spermatozoa) was considerably reduced by 1 h after insemination. While a large number of live hamster spermatozoa were distributed throughout the caudal isthmus at the time of examination, none or only a very few foreign spermatozoa had advanced this far. The few foreign and immotile spermatozoa that reached the caudal isthmus were confined to the first ascending loop of this segment. Some possible causes for the small number and retarded advance of foreign spermatozoa in the hamster oviduct were discussed.     

Detection of Fas ligand in the bovine oviduct

Presence of a Fas-Fas ligand (FasL) system defines the immune-privileged status of certain tissues such as placenta. This study examined the fluids and tissue(s) of the bovine oviduct, where both spermatozoa and early embryos escape elimination by the female immune system, for the presence and the distribution of Fas and FasL, which might provide an explanation for the immune-privileged site of this organ. In the present study, the immunolocalisation of FasL and Fas, as well as the gene expression of FasL, were determined in the uterotubal junction (UTJ), isthmic (I) and ampullar (A) segments of the oviduct during oestrus and the luteal phase of the oestrous cycle. The degree of apoptosis of oviductal epithelium was examined by the TUNEL method. Oviductal fluid (ODF), collected chronically via indwelling catheters from the I or A segments during both non-luteal and luteal phases of the cycle, was analysed for the presence of FasL. The Fas immunostaining was scattered along the epithelium of all regions of the oviduct and cycle stages investigated, whereas FasL immunolabelling was more conspicuous in oestrous samples. This staining disappeared during the luteal phase, which was particularly evident in the sperm reservoir (UTJ and I). There were fewer TUNEL-positive cells than Fas- or FasL-positive cells in the oviductal epithelium, suggesting that tubal Fas and FasL are not directly involved in epithelial apoptosis. Western blot analyses detected FasL in ODF collected from both I and A, most conspicuously as a 24-27kDa band but also at a 40-45kDa band level. FasL mRNA was expressed in the epithelial cells from the sperm reservoir and A during both non-luteal and luteal phases. However, the level of expression differed significantly between segments during the luteal phase. The results provide novel evidence that the Fas-FasL system is present in the bovine oviduct and could be involved in mediating survival of spermatozoa and early embryos.     

Capacitation status of hamster spermatozoa in the oviduct at various times after mating

Female hamsters were mated shortly after the onset of oestrus or immediately after ovulation. At various times after mating, spermatozoa were flushed from the isthmus of the oviduct using a modified Tyrode's medium supplemented with 20% hamster serum. Cumulus oophorus-free eggs were introduced into the suspensions of isthmic spermatozoa. Some eggs were removed every 30 min and examined for evidence of fertilization. For females mated shortly after the onset of oestrus, spermatozoa recovered from the oviducts 8 h after mating (about 1.5 h after ovulation) could penetrate eggs within 30 min and were considered fully capacitated. When spermatozoa were recovered at earlier times (1, 2, 4 and 6 h after mating) they required additional time (2, 1.5, 1 and 1 h respectively) in vitro before penetrating eggs. Therefore, when mating occurs shortly after the onset of oestrus, spermatozoa in the oviduct do not appear to become fully capacitated until about the time of ovulation. For females mated immediately after ovulation, spermatozoa recovered from the oviducts at 4 h after mating could penetrate eggs within 30 min. Spermatozoa recovered at 1 and 3 h after mating required 2 and 1 h respectively in vitro before penetrating eggs. These results suggest that sperm capacitation proceeds at a faster rate when mating occurs after ovulation.     

Effect of ovulation on sperm transport in the hamster oviduct

When hamsters mate shortly after the onset of oestrus (4.5-6 h before the onset of ovulation), spermatozoa are stored in the caudal isthmus of the oviduct until near the time of ovulation. At this time, a few spermatozoa ascend to the ampulla to fertilize the eggs. Superovulation resulted in a significant increase in the number of spermatozoa in the caudal isthmus at 6 h post coitus (p.c.) and in the ampulla and bursal cavity at 12 h p.c. Precocious ovulation resulted in a highly significant reduction in the total number of spermatozoa in the oviduct at 3 and 6 h p.c. This effect was completely overcome by intrauterine artificial insemination, suggesting lack of cervical patency as the block to sperm transport in precociously ovulated animals. Ligation of the ampulla-infundibulum junction in naturally ovulating hamsters resulted in significantly fewer spermatozoa in the caudal isthmus and ampulla at 12 h p.c. Preclusion of ovulation also resulted in fewer spermatozoa in the caudal isthmus and ampulla at 12 h p.c., suggesting that the products of ovulation stimulate sperm transport in the oviduct.