Determination of glycosidase activity in porcine oviductal fluid at the different phases of the estrous cycle

Abstract

Sperm-oocyte binding and gamete-oviductal epithelium interactions are carbohydrate-mediated events occurring in the oviductal fluid (OF). Thus, knowledge about the activities of glycosidases (enzymes catalyzing hydrolytic cleavage of terminal sugar residues) in this milieu would help us understand the molecular mechanisms involved in these events. This work was carried out to investigate the glycosidase activity, protein content, and volume of OF collected from gilts and sows. Oviducts were classified into four phases of the estrous cycle (early follicular, late follicular, early luteal, and late luteal) based on the appearance of the ovaries. OF was aspirated, centrifuged, measured for volume, and frozen until assay. Substrates conjugated to 4-methylumbelliferyl were used to screen the activities of seven different glycosidases at physiological pH (7.2). a-L-Fucosidase and b-N-acetyl-glucosaminidase activities increased at the late follicular phase to decrease after ovulation. b-D-Galactosidase, a-D-mannosidase, and b-N-acetyl-galactosaminidase showed higher activities at the early follicular phase, which decreased after ovulation. N-Acetyl-neuraminidase and a-D-galactosidase did not show activity at any phase of estrous cycle neither in sows nor in gilts at pH 7.2, although it did at acidic pH (4.4) in the follicular and luteal phase samples. Total protein also changed during the cycle showing the maximum secretion at the late follicular phase (2118.6G200.7 mg/oviduct). The highest volumes of OF were collected from the oviducts at the late follicular phase (50.7G1.3 ml/oviduct). These results indicate that OF from sows and gilts shows glycosidase activity varying throughout the estrous cycle suggesting a role of these enzymes in carbohydrate-mediated events. Reproduction (2008) 136 833-842

Establishment of conditions for ovum pick up and IVM of jennies oocytes toward the setting up of efficient IVF and in vitro embryos culture procedures in donkey (Equus asinus)

“…The oviducts were dissected free from surrounding tissues. The oviductal content from the ampulla was expelled by gentle squeezing using a sterile microscope slide and collected by introducing the tip of a pipette into the ampulla and aspirating while making a manual ascendant pressure from the isthmus to the ampulla [37]. After centrifugation at 10,000 Â g for 15 minutes, the supernatant containing secreted and intracellular components was immediately stored at À20 C until use as "oviductal fluid".…”

Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species

“…The cyclic stage of females was assessed on the basis of ovarian morphology on both ovaries from the same female. Oviducts were classified as early follicular, mid follicular, late follicular, or luteal phase, according to the diameter of follicles and the presence of corpora lutea, as described previously (Carrasco et al 2008). Porcine oviducts at the preovulatory or post-ovulatory stage were collected from slaughtered Meishan gilts from our experimental pigsty (Unité Expérimentale de Physiologie Animale de l'Orfrasière, UEPAO, Nouzilly, France).…”

“…The oviductal fluid from the ampulla was expelled by gentle squeezing using a sterile microscope slide. It was collected by aspiration with an automatic pipette by introducing the tip into the ampulla and making a manual ascendant pressure from the isthmus to the ampulla (Carrasco et al 2008). After centrifugation at 10 000 g for 15 min, the supernatant containing secreted and intracellular components was immediately stored at K20 8C until use as 'oviductal fluid'.…”

Effects of oviductal fluid on the development, quality, and gene expression of porcine blastocysts produced in vitro

“…Oviducts were washed twice, transferred to Petri dishes on an ice bath and dissected. bOF was collected by aspiration with an automatic pipette using a tip for a maximum 200 ml volume and centrifuged at 7000 g for 10 min at 4 8C to remove cellular debris (Carrasco et al 2008). The supernatant was stored at K80 8C until use.…”