Isthmic Fluid Research Articles

Role of the oviduct in sperm capacitation

Following insemination of spermatozoa pre-ovulation, the mammalian oviduct ensures, by the formation of a functional sperm reservoir (SR), that suitable (low) numbers of viable and potentially fertile spermatozoa are available for fertilization at the ampullary isthmic junction (AIJ). As ovulation approaches, a proportion of the SR-stored spermatozoa is continuously distributed towards the AIJ and individually activated leading to step-wise capacitation and the attainment of hyperactivated motility. This paper reviews in vivo changes in the intra-luminal milieu of the oviduct of pigs and cows, in particular the SR and the AIJ which relate to the modulation of sperm capacitation around spontaneous ovulation. In vivo, most viable spermatozoa in the pre-ovulatory SR are uncapacitated. Capacitation rates significantly increase after ovulation, apparently not massively but concurrent with the individual, continuous sperm dislocation from the SR. Bicarbonate, whose levels differ between the SR and the AIJ, appears as the common primary effector of the membrane destabilizing changes that encompasses the first stages of capacitation. Sperm activation can be delayed or even reversed by co-incubation with membrane proteins of the tubal lining, isthmic fluid or specific tubal glycosaminoglycans, such as hyaluronan. Although the pattern of response to i n vitro induction of sperm activation – capacitation in particular – is similar for all spermatozoa, the capacity and speed of the response is very individual. Such diversity in responsiveness among spermatozoa insures full sperm viability before ovulation and the presence of spermatozoa at different stages of capacitation at the AIJ, thus maximizing the chances of normal fertilization.

Expression and presence of osteopontin and integrins in the bovine oviduct during the oestrous cycle

Osteopontin and integrin alpha(v)beta(3) are known to mediate cell-cell attachment and cell migration. Western blot analysis was used to demonstrate the presence of osteopontin in oviductal fluid collected from ampullar and isthmic regions. Three different osteopontin isoforms of 55 kDa, 48 kDa and 25 kDa were detected in the oviductal fluid. Each isoform was observed during the luteal and non-luteal phases and in both ampullar and isthmic fluids. The 25 kDa osteopontin was the most prevalent isoform in oviductal fluid except in isthmic fluid during the non-luteal phase of the oestrous cycle. RT-PCR was performed with RNA from oviductal cells collected from cows in the post-ovulatory, early to mid-luteal, late luteal or pre-ovulatory stages of the oestrous cycle to reveal the oviduct as a site of osteopontin and integrin synthesis. Only one osteopontin mRNA transcript was detected, and amounts did not vary throughout the oestrous cycle. In contrast, the relative expression of the integrin subtypes alpha(v) and beta(1) during the late luteal phase was lower compared with the other oestrous cycle phases. Integrin beta(3) mRNA content increased significantly from the lowest level during the late luteal phase to the highest level before ovulation. In conclusion, differential presence of osteopontin isoforms and integrins in the bovine oviduct throughout the oestrous cycle indicate that osteopontin-integrin interactions have functional roles in normal oviduct physiology which may potentially influence interactions between the gametes, the embryo, and the epithelium.

Expression and presence of osteopontin and integrins in the bovine oviduct during the oestrous cycle

Osteopontin and integrin alpha(v)beta(3) are known to mediate cell-cell attachment and cell migration. Western blot analysis was used to demonstrate the presence of osteopontin in oviductal fluid collected from ampullar and isthmic regions. Three different osteopontin isoforms of 55 kDa, 48 kDa and 25 kDa were detected in the oviductal fluid. Each isoform was observed during the luteal and non-luteal phases and in both ampullar and isthmic fluids. The 25 kDa osteopontin was the most prevalent isoform in oviductal fluid except in isthmic fluid during the non-luteal phase of the oestrous cycle. RT-PCR was performed with RNA from oviductal cells collected from cows in the post-ovulatory, early to mid-luteal, late luteal or pre-ovulatory stages of the oestrous cycle to reveal the oviduct as a site of osteopontin and integrin synthesis. Only one osteopontin mRNA transcript was detected, and amounts did not vary throughout the oestrous cycle. In contrast, the relative expression of the integrin subtypes alpha(v) and beta(1) during the late luteal phase was lower compared with the other oestrous cycle phases. Integrin beta(3) mRNA content increased significantly from the lowest level during the late luteal phase to the highest level before ovulation. In conclusion, differential presence of osteopontin isoforms and integrins in the bovine oviduct throughout the oestrous cycle indicate that osteopontin-integrin interactions have functional roles in normal oviduct physiology which may potentially influence interactions between the gametes, the embryo, and the epithelium.

Identification of ampullary and isthmic oviductal fluid proteins that associate with the bovine sperm membrane.

The objective of this study was to define the complement of ampullary and isthmic oviductal fluid proteins that associate with the sperm membrane during the non-luteal and luteal stages of the oestrous cycle. Oviductal fluid was obtained from three dairy cows via indwelling cannulae in the ampulla and isthmus of the same oviduct. Daily samples of oviductal fluid were combined by region and stage of the cycle to create pools consisting of non-luteal isthmic, luteal isthmic, non-luteal ampullary, and luteal ampullary oviductal fluid. An aliquot from each oviductal fluid pool was biotinylated by incubation in biotin reagent. Spermatozoa from three fertile bulls were pooled, washed and incubated for 4 h in each of the four pools of biotinylated oviductal fluid. After incubation, sperm membrane proteins were solubilized, and proteins subjected to one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The biotinylated oviductal fluid proteins that associated with the sperm membranes were detected by avidin-horseradish peroxidase and diaminobenzidine substrate. Eight oviductal fluid proteins with apparent molecular weights of 97, 75, 66, 55, 48, 34, 28 and 24 kDa were consistently detected to associate with the sperm membrane. Differences in ampullar and isthmic fluid proteins that associated with sperm during the luteal and non-luteal stage of the oestrous cycle were not detected.

Monoclonal antibodies against boar sperm zona pellucida-binding protein AWN-1. Characterization of a continuous antigenic determinant and immunolocalization of AWN epitopes in inseminated sows.

Boar spermadhesin AWN-1 is a sperm surface-associated 14.7-kDa lectin and a major protein of porcine seminal plasma. AWN-1 binds to beta-galactosides and to porcine zona pellucida glycoproteins, suggesting that this protein might play a role in the primary binding of spermatozoa to the egg's external glycoprotein matrix. We have produced a collection of murine monoclonal antibodies against purified AWN-1. Five monoclonal antibodies recognized sequential antigenic determinants. All these epitopes were located at the C-terminal region of AWN-1 (residues 109-123) by competitive ELISA using overlapping synthetic peptides that cover the complete 133 amino acid sequence of the lectin. In a structural model of spermadhesin AWN-1, the polypeptide stretch 109-123 is fully solvent-exposed, providing a reasonable explanation for its high immunogenicity. In addition to epitope mapping, we have employed anti-AWN monoclonal antibodies for immunolocalization of the protein in the genital tract of inseminated sows. Clusters of AWN epitopes were occasionally found attached to the epithelium of the uterotubal junction and the adjacent lower isthmus. However, neither AWN-1 nor other seminal plasma proteins were found in the isthmic fluid collected 10-26 h after insemination. These results suggest that the whole amount of seminal plasma proteins are absorbed by the epithelium of the female genital tract, supporting the claim that removal of seminal plasma components from spermatozoa might be a major event in both in vitro and in vivo sperm capacitation.

Cholesterol, phospholipid and phospholipase activity of ampullary and isthmic fluid from the bovine oviduct.

Cholesterol and phospholipid concentrations and phospholipase activity were measured in fluid from cannulae collected from the bovine oviductal isthmus and ampulla at different stages of the oestrous cycle. The cholesterol concentration and cholesterol normalized by protein were significantly (P = 0.03) greater in isthmic oviductal fluid (224.3 +/- 42.7 micrograms ml-1 over all stages) than in ampullary oviductal fluid (164.5 +/- 11.3 micrograms ml-1), and maximal concentrations (284.5 +/- 25.5 micrograms ml-1) were found during the luteal stage (serum progesterone concentration > or = 1.5 ng ml-1). The concentrations of the phospholipids sphingomyelin and lysophosphatidylcholine increased at different stages of the cycle and in different regions. In the ampulla, the concentration of sphingomyelin was significantly (P < 0.05) greater in oviductal fluid collected during the luteal phase (12.1 +/- 2.7% of total phospholipids) than in fluid collected near oestrus and ovulation (7.5 +/- 1.5% and 6.9 +/- 1%, respectively). The concentration of lysophosphatidylcholine was greater (P < 0.01) in ampullary (19.2 +/- 1.6% of total phospholipids) than in isthmic oviductal fluid (9.9 +/- 1.1%) collected near ovulation. The ratio of cholesterol to total phospholipid was highest in oviductal fluid collected from the isthmus during all stages (2.3 micrograms ml-1:% total phospholipid), while the minimal ratio was found in ampullary fluid collected near ovulation (1.5). Phospholipase activity was higher (P = 0.03) in isthmic oviductal fluid (20.4 +/- 3.2% product formed) than in ampullary oviductal fluid (14.6 +/- 1.4%); the lowest activity (12.6 +/- 1.7% product formed) was in fluid collected during the phase of the oestrous cycle immediately before ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)

A possible role for potassium and pyruvate in the modulation of sperm motility in the rabbit oviducal isthmus.

Spermatozoa were recovered from the isthmus of the rabbit oviduct at 4 and 11 h post coitum using several defined flushing media. The motility of spermatozoa in the isthmic flushings was subsequently analysed from video recordings. There was little sperm movement in the native isthmic fluid, but vigorous flagellar activity and hyperactivated movement were induced by flushing the isthmus with 0.25 M-sucrose, apparently an effect of dilution. Flushing with a complex culture medium resulted in similar stimulation of sperm movement. When pyruvate was present in the medium, hyperactivated flagellar bending was stimulated, whereas these movements were virtually absent when glucose alone was present. The stimulating effect of dilution was less pronounced when the flushing medium contained 50 mM-potassium. When the isthmus was flushed with media containing 50 mM-K+ and the K+ concentration was lowered to 5 mM during a washing procedure, large-amplitude flagellar movements were sequentially suppressed and restored. The restoration of large-amplitude movements was enhanced when pyruvate was present in the 5 mM-K+ washing medium. These results suggest that alterations in the concentration of both K+ and pyruvate may have a role in regulating the motility of rabbit spermatozoa in the oviducal isthmus, K+ being inhibitory and pyruvate stimulatory.

The movement characteristics of rabbit spermatozoa before and after activation

AbstractSpermatozoa were flushed with mineral oil from the lower isthmus of the rabbit oviduct at four hours postcoitus (pc) and 11 hours pc. Videotapes were made of sperm behavior in the native isthmic fluid and after dilution of the fluid with culture medium. The tapes showed that, initially, spermatozoa in the native isthmic fluid were virtually immotile, but immediately resumed movement on contact with the culture medium. Isthmic sperm motility then evolved over a five‐ to 10‐minute interval into the characteristic biphasic pattern of activated movement. Cine films of isthmic spermatozoa taken with a high‐speed camera were analyzed to determine flagellar beat frequency, maximum flagellar curvature, and swimming velocity. Progressiveness ratios and hydrodynamic power outputs were then calculated for individual spermatozoa. Two phases of activated sperm movement, a whiplash phase and a progressive phase, were identified and characterized. The power output of activated spermatozoa increased twentyfold in comparison with the preactivated state. The power output of activated spermatozoa did not differ between the two phases of activated movement.