Induction of Dog Sperm Capacitation by Glycosaminoglycans and Glycosaminoglycan Amounts of Oviductal and Uterine Fluids in Bitches.

Abstract

ABSTRACT. Ejaculated sperm collected from 12 beagle dogs were incubated in canine capacitation medium (CCM), supplemented with 5 µg/ml chondroitin sulfate A (CS), 5 µg/ml hyaluronic acid (HA), or 5 µg/ml heparin (HP) for 7 hr at 38°C in a 5% CO2 in air atmosphere to investigate the effects of glycosaminoglycans (GAGs) on dog sperm capacitation. The percentages of motile sperm, hyperactivated sperm (%HY), and acrosome-reacted sperm (%AR) in all media were examined after 4 hr and 7 hr of incubation. The oviducts and uteri of 9 anestrous and 18 estrous beagle bitches were removed under halothane inhalation anesthesia to measure the total GAG amounts in oviductal and uterine fluids. The lumens of the ampulla of the oviducts, isthmus of the oviducts, and the uterine horns were each flushed with 1 ml HEPES-EDTA fluid. Total GAG amounts in the flush fluids obtained were measured with a spectrophotometer. Sperm motility (51-59%), %HY (79-86%), and %AR (31-36%) in CCM supplemented with CS, HA, or HP were significantly higher after 7 hr of incubation than when incubated in CCM without GAGs (P<0.01 or 0.05). The mean total GAG amounts in the fluids from the ampulla and isthmus of the oviducts and the uterine horns in the estrous bitches were higher than in the anestrous bitches. These results indicate that GAGs in the oviductal and uterine fluids in estrous bitches are associated with in vivo sperm capacitation. -KEY WORDS: canine, capacitation, glycosaminoglycan, sperm.J. Vet. Med. Sci. 62(1): 65-68, 2000 investigate the relationship between in vivo capacitation of dog sperm and the concentrations of GAG. MATERIALS AND METHODS Animals:Twelve male beagle dogs, aged 2-6 years with normal semen quality, cared for in our university, were used to collect the semen for this experiment. The dogs were housed in pens with ample runs under natural lighting. Commercial dry dog food was provided twice a day and access was given to water ad libitum. Nine anestrous and eighteen estrous beagle bitches, aged 4-8 years, cared for in our university and at a pet food company, were subjected to ovariohysterectomy under halothane inhalation anesthesia.Semen collection and evaluation: The sperm-rich second fraction of ejaculated semen [10] was collected by digital manipulation and immediately transported to our laboratory. The concentration of sperm in the semen was determined by hematocytometer counts. Sperm motility, i.e., the percentage of actively motile sperm, was evaluated by using a sperm motility examination plate and a warm-plate. Semen samples with excellent sperm motility (>90%) were used.Sperm incubation: The spermatozoa in the semen were washed twice by centrifugation at 400 g for 5 min in 5 ml of canine capacitation medium (CCM) [20] warmed to 38°C. The final sperm pellet was diluted in CCM supplemented with 5 µg/ml CS, 5 µg/ml HA, or 5 µg/ml HP to a concentration of 5 × 10 6 sperm/ml. It has been reported that 10 µg/ml HA for hamster sperm [22] and 10 µg/ml HP for bovine sperm [25] incubation were used. Therefore, the Sperm c...

Oocyte biology and challenges in developing in vitro maturation systems in the domestic dog

“…However, most of the epithelial cells were derived from diestrous bitches, which may well lack the proteins essential for promoting meiotic maturation because oviductal secretory cells are mostly active during estrus (not diestrus). Intraoviductal concentrations of glycosaminoglycans are higher in estrual compared to anestrous bitches (Kawakami et al, 2000). In fact, culturing dog oocytes with oviductal cells recovered from an estrual bitch improved the proportions of oocytes resuming meiosis and developing to the MII stage (Bogliolo et al, 2002;Luvoni et al, 2003).…”

Composition and significance of glycosaminoglycans in the uterus and placenta of mammals

“…The high concentration of GAGs during estrum foregrounds the uterus´s macroscopic increase, since these molecules trigger tissue hydration and, at the same time, tissue hyperplasia (Cubas et al 2010). Studies on bitches during the anestrus phases or estrus and the comparison of GAGs rates in the fluids of the ampulla and isthmus of the uterine tubes and the lumen of the uterine horns showed that GAGs concentrations in these organs are greater in the bitches in the estrus when compared to those in the anestrus phase (Kawakami et al 2000). This is due to estrogen on the genital organs during the estrus cycle.…”

Microenvironnement tubaire: rôle dans la maturation des ovocytes canins in vivo et in vitro

“…Une seule publication a trait à la composition du liquide tubaire canin en glycosaminoglycanes et à leur rôle dans la capacitation spermatique (Kawakami et al 2000). Les analyses biochimiques réa-lisées chez la vache , la truie (Buhi et al 1990), montrent que les sécrétions tubaires sont constituées d'un mélange de composés transsudés d'origine sérique et de composés spécifiques sécrétés par l'épithélium tubaire, dont la composition est sensiblement identique dans ces espèces…”