Attachment and release of spermatozoa from the caudal isthmus of the hamster oviduct

Female hamsters were mated shortly after the onset of oestrus. At 3 or 6 h after mating, the right oviduct was flushed in situ with 30, 90 or 180 microliters medium to remove spermatozoa from the lumen, leaving only those firmly attached to the isthmic mucosa of the oviduct. When eggs were recovered from oviducts at 20 h after flushing the majority were fertilized, indicating that the spermatozoa that were firmly attached to the mucosa were capable of detaching and ascending to the ampulla to fertilize eggs. Neither the time of flushing nor the volume of flushing medium had a significant effect on the percentage of spermatozoa that remained in the isthmus after flushing. These results suggest that there is no change in the surface of the oviduct mucosa that causes the release of spermatozoa from the caudal isthmus near the time of ovulation. When incapacitated spermatozoa were introduced into the oviduct, many of them attached to oviductal mucosa, while capacitated spermatozoa did not. This indicates that it is a change in the sperm surface, rather than the mucosal surface, that causes the release of spermatozoa, i.e. spermatozoa remain attached to the isthmic mucosa until they become capacitated and then detach and migrate to the ampulla to fertilize the eggs.     

Acrosome-reacted guinea pig spermatozoa become fusion competent in the presence of extracellular potassium ions

Guinea pig spermatozoa are able to undergo capacitation and the acrosome reaction in a K+-free (-deficient) medium. However, they are unable to fuse with eggs unless they are exposed to a millimolar concentration of extracellular K+ during or after the acrosome reaction. Apparently, the plasma membrane over the equatorial segment gains the ability to fuse with eggs in the presence of K+ during and/or after the acrosome reaction. Once it becomes fusible, the membrane retains its fusibility even in a K+-deficient medium. Rb+ is almost as effective as K+ in rendering the sperm membrane fusible. Li+ and Cs+ are less effective. The molecular mechanism by which K+ renders acrosome-reacted spermatozoa fusion competent is unknown, but it may involve K+-mediated efflux of H+ from the spermatozoa.     

The viability of hamster spermatozoa stored in the isthmus of the oviduct: the importance of sperm-epithelium contact for sperm survival

When hamsters mate shortly after the onset of estrus, spermatozoa are stored in the lower oviduct (isthmus) during the preovulatory period. The present study was performed to determine what proportion of the spermatozoa in the isthmus survive until fertilization. Females were mated 5 to 6.5 h before ovulation. When spermatozoa in the isthmus were observed through the wall of oviducts excised 2 h after the onset of mating, spermatozoa were seen free in the lumen, attached to the mucosal surface of the wall, and in crypts. The vast majority of spermatozoa in the lumen were immotile, whereas most of those attached to the mucosal surface of the wall and almost all of the those in the crypts exhibited flagellar movement. This suggested that attachment to the mucosa and/or storage in the crypts is beneficial to the survival of spermatozoa. Sequential flushing of an oviduct at various times (2-8 h) after mating was used to remove spermatozoa from the lumen (first flush), from the mucosal surface (second flush), and from the crypts (third flush). The highest number of spermatozoa was always contained in the first flush, the next highest in the second flush, and the smallest in the third flush. When Trypan blue was included in the flushing medium to differentiate live and dead spermatozoa, the first flush recovered the smallest percentage of liver spermatozoa (2-22%), the second flush slightly more (16-37%), and the third flush the highest (51-69%), regardless of the time after mating. These data indicate that the majority of spermatozoa stored in the hamster isthmus die before ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of IGF-I and porcine oviductal secretory protein (pOSP) secretion into the pig oviduct in the peri-ovulatory period,and effects of previous nutrition

The mechanisms regulating oviduct function were investigated. In Experiment 1, porcine oviductal secretory protein (pOSP) mRNA, and pOSP and insulin-like growth factor (IGF-I) in oviductal flushings, decreased through the peri-ovulatory period. In Experiment 2, higher plasma steroids in oviductal veins, ipsilateral (INT), rather than contralateral (OVX), to the remaining ovary in unilaterally ovariectomized gilts, were associated with higher pOSP in INT oviductal flushings. In Experiment 3, oviduct function was assessed as part of a collaborative study in cyclic gilts. Feed restriction in the late, compared to the early, luteal phase reduced estradiol concentrations in oviductal plasma, pOSP mRNA in oviductal tissue, and IGF-I concentrations and pOSP abundance in oviduct flushings. Previous insulin treatment differentially affected oviduct function. These data provide the first direct evidence for effects of previous feed restriction and insulin treatment on the oviduct environment in the peri-ovulatory period, which may contribute to nutritional effects on embryonic survival.     

Acid glycosidases in the isthmus of the hen oviduct and egg shell membranes

Specific activities of seven acid glycosidases: beta-hexosaminidase, alpha- and beta-galactosidase, alpha- and beta-mannosidase, alpha-glucosidase and alpha-fucosidase were determined in various parts of the domestic hen oviduct (infundibulum, isthmus, shell gland and vagina). The activity of most enzymes was the highest in the isthmus. Multiple forms of all acid glycosidases from the isthmus were separated by strong anion exchange chromatography at pH 6.0. The isoelectric points of the isthmus forms of beta-hexosaminidase, beta-galactosidase and alpha- and beta-mannosidase were determined by chromatofocusing. For the first time the high beta-galactosidase activity was found in hen egg shell membranes.     

The presence of the mu-opioid receptor in the isthmus of mare oviduct

The presence of the mu-opioid receptor and the type of glycosylation in the third extra-cellular loop of this receptor was investigated in the isthmus of mare oviduct during oestrus by means of immunoblotting and immunohistochemistry combined with enzymatic (N-glycosidase F and O-glycosidase) and chemical (beta-elimination) treatments. Immunoblotting analysis showed that the mu-opioid receptor consists of two peptides with molecular weights of around 65 and 50 kDa. After N-deglycosylation with N-glycosidase F an additional immunoreactive peptide was observed at around 30 KDa. The cleavage of O-glycans by O-glycosidase failed in immunoblotting as well as in immunohistochemistry investigations, revealing that the third extra-cellular loop of the mu-opioid receptor expressed in mare isthmus oviduct contains some modifications of the Galbeta(1-3)GalNAc core binding to serine or threonine. Immunohistochemistry revealed the mu-opioid receptor in the mucosal epithelium, some stromal cells, muscle cells and blood vessels. In ciliated cells the mu-opioid receptor showed N-linked glycans, since the immunoreactivity was abolished after N-glycosidase F treatment, whereas it was preserved in the apical region after beta-elimination. Most non-ciliated cells expressed the mu-opioid receptor with both N- and O-linked oligosaccharides, as revealed by the abolition of immunostaining after N-glycosidase F and beta-elimination. Stromal cells, endothelial and muscle cells of blood vessels expressed the mu-opioid receptor containing both N- and O-linked oligosaccharides. Myosalpinx myocytes expressed the mu-opioid receptor with O-linked oligosaccharides. The immunopositive myocytes formed a circular coat in the intrinsic musculature, whereas they were arranged in some isolated, oblique bundles in the extrinsic musculature. In conclusion, the mu-opioid receptor could have a role in the production and the movement of isthmus lumen content that contributes to ensuring the effective condition of the sperm in the mare oviduct.     

Capacitation of bovine spermatozoa by oviduct fluid

Oviduct fluid collected from chronically cannulated oviducts of heifers was evaluated for its effect on capacitation of bovine sperm in vitro. Capacitation was determined by the ability of sperm to fertilize bovine oocytes in vitro and to undergo an acrosome reaction (AR) upon exposure to lysophosphatidylcholine (LC). After incubation of sperm with 0-25% (v/v) estrual oviduct fluid (collected +/- 1 day from estrus) for 4 h, addition of LC (100 micrograms/ml) for an additional 0.25 h resulted in an increasing percentage of acrosome-reacted sperm as the concentration of oviduct fluid increased. Sperm incubated 4 h with 25% estrual oviduct fluid fertilized more oocytes than sperm incubated in medium alone (p less than 0.05) but was not different from sperm incubated with 10 micrograms/ml heparin (p greater than 0.05). Glucose inhibited the ability of LC to induce ARs in sperm incubated 4 h with heparin or estrual oviduct fluid. Incubation of sperm with 25% oviduct fluid collected at various days over the estrous cycle demonstrated that peak capacitating activity was found at estrus but was also present +/- 1 day from estrus. The active capacitating factor in oviduct fluid was found to be heat stable. In addition, when extraction procedures were applied in sequential order, oviduct fluid capacitating activity was resistant to protease digestion, precipitable by ethanol, size-excluded by Sephadex G-25, and destroyed by nitrous acid. These results suggest that a heparin-like glycosaminoglycan from the oviduct is a potential in vivo capacitating agent in the bovine.     

Spontaneous motility of the oviduct in the anaesthetized pig

Intraluminal pressure microtransducers were placed at the uterotubal junction, the proximal isthmus, the ampullary-isthmic junction and the mid-ampulla. Spontaneous motility occurred throughout the oestrous cycle in all segments. During oestrus there were regular, high amplitude peristaltic waves in all segments, superimposed on basal activity. On Day 1 of the cycle the pattern was mostly antiperistaltic, presumably related to sperm transport. During the periovulatory period the number of peristaltic and antiperistaltic waves became equal, perhaps in relation to the transport of gametes to the fertilization site. During Day 3 there was no peristaltic activity; the motility patterns of the isthmus and ampullary-isthmic junction were similar (regular phasic contractions of high frequency and amplitude) while the ampullary motility was low. On Day 4, when the eggs enter the uterine lumen, the ampullary-isthmic junction and particularly the isthmus showed strong contraction waves (mostly peristaltic) superimposed on the basal phasic activity. This suggests an active role of the smooth muscle of the lower oviducal segments in ovum descent. During the mid- and late-luteal phases, the isthmus remained motile, with an irregular base line, but lost the pattern of basal contractions that dominated the activity during the first 4 days of the cycle. The ampulla showed low levels of spontaneous motility throughout the rest of the cycle.     

Oestradiol and progesterone receptors in the pig oviduct during the oestrous cycle

The concentrations of the tissue receptors for oestradiol (E) and progesterone (P) in the porcine oviduct at different stages on the oestrous cycle have been investigated by in vitro binding and exchange methods. Both hormones bound to specific cytoplasmic (Rc) and nuclear (Rn) receptor proteins with high affinity. The concentrations of ERc and ERn were two-fold higher in the ampulla as compared to the isthmus. The amount of ERc in the isthmic portion of the oviduct did not vary throughout the oestrous cycle. However, the ampullar ERc concentrations increased during prooestrus, showed a maximum at standing oestrus, thereafter decreasing. Significant variations in the amount of oviductal ERn were observed. Despite the differences in ERn amounts between segments, the concentration of ERn increased significantly during late prooestrus, attaining a three-fold elevation and remaining elevated during the period of standing oestrous and early luteal phase (days 3-4), thereafter returning to basal levels. No significant variations in the amount of isthmic PRc were found throughout the period studied. The ampulla, however, showed a significant increase in PRc concentrations during standing oestrus, thereafter decreasing. The concentrations of PRn in isthmus and ampulla were of about the same magnitude and varied significantly during the oestrous cycle, increasing in concentration from standing oestrous onwards. The temporal relationships between the variations in levels of oestradiol and progesterone receptors in oviductal tissues and those of the circulating plasma levels were established. The data obtained in this study suggest a relationship between the changes in the levels of oestradiol and progesterone oviductal binding during the first days of the oestrous cycle, and the gamete and embryo transport throughout the oviduct in the porcine species.     

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.     

Migration of spermatozoa in the oviduct of hens following intravaginal, intramagnal and intraabdominal insemination

When hens were inseminated intravaginally with 200 million spermatozoa, the largest number of sperm cells reaching the infundibulum was observed 2 d after insemination (41.64 x 10(3) per female), even though a few sperm (0.23 x 10(3) per female) had already been detected within the first hour following insemination. After 2 d, the number of spermatozoa rapidly decreased and only 4.09 x 10(3) sperm per female were present in the infundibulum on Days 14 and 21, respectively. However, when the same dose of spermatozoa was inseminated intramagnally, a large number of spermatozoa (8.01 x 10(3) per female) was found in the infundibulum within 4 h after insemination. A parallel study showed that extensive migration of spermatozoa towards the abodminal cavity had already occurred.     

Effect of cocaine and other drugs on sensitivity of the estrogen-dominated isthmus of rabbit oviduct to noradrenaline.

The influence of the adrenergic innervation on the magnitude of responses of the isthmus of rabbit oviduct to (-)-noradrenaline was examined. Tissues were obtained from estrogen-dominated animals, and isometric contractions of longitudinal and circular muscle layers separately recorded. Longitudinal muscle was significantly more sensitive to (-)-noradrenaline. Cocaine potentiated responses of both corcular and longitudinal muscle to (-)-noradrenaline, circular muscle being more potentiated. Similar results were obtained with desipramine. Tissues obtained from 6-hydroxydopamine pretreated animals did not respond to tyramine, and longitudinal and circular muscles were equisensitive to (-)-noradrenaline. Cocaine did not potentiate responses to (-)-noradrenaline in such tissues. Responses to (-)-noradrenaline were not altered by propranolol, hydrocortisone, or oxytetracycline. It was concluded that responses resulted from a predominant effect on alpha-receptors. The magnitude of responses to (-)-noradrenaline was mainly influenced by neuronal uptake of amine. Indirect evidence was obtained for a greater degree of adrenergic innervation to the circular muscle layer of the isthmus, in keeping with histological studies.     

Sperm transport and reservoirs in the pig oviduct in relation to the time of ovulation

The rate of establishment of a population of viable spermatozoa in the oviducts was studied using a technique of post-coital transection in conjunction with subsequent examination of the proportion of eggs fertilized. Gilts were mated early in oestrus (before ovulation) or on the 2nd day of oestrus (after ovulation), and 30, 45 or 60 min later the reproductive tract was sectioned just above or below the utero-tubal junction in a total of 48 animals; these were slaughtered 1 or 2 days after the operation. Some fertilized eggs were recovered from 40 animals, and 72.3% of the 679 eggs examined were fertilized. Mean percentage fertilization increased overall (a) with the time elapsing from mating to transection, (b) with transection below the utero-tubal junction compared with in the caudal isthmus, and (c) with a post-ovulatory versus pre-ovulatory mating. In a further 6 gilts, the results of transection in the lower third of the oviduct 3 h after mating at the onset of oestrus indicated that spermatozoa were initially sequestered in the caudal portion of the isthmus. It is concluded that a population of spermatozoa sufficient to give maximum fertilization is established in the oviducts within 1--2 h of mating, thereby affording protection from the uterine invasion of polymorphonuclear leucocytes.