Xavier Druart

Contents: Sperm transit in the female tract is a critical event for the success of fertilization. From their deposition in the vagina to final migration in the oviduct, sperm pass through the different compartments of the genital tract in which they encounter different environments. The cervix and the uterotubal junction (UTJ) are two barriers with different relative importance according to the species. The protein composition, the degree of glycosylation and the hydration of the cervical mucus change during the oestrous cycle. Several sperm surface proteins are associated with their migration through the cervical mucus and the UTJ. Data regarding the interaction of sperm with secretions of the epithelial tissue lining the different compartments of the female genital tract during the sperm transit are reviewed, with a particular emphasis on the migration of sperm through the cervix.

The fertility of ram semen after cervical insemination is substantially reduced by 24 h of storage in liquid form. The effects of liquid storage on the transit of ram spermatozoa in the ewe genital tract was investigated using a new procedure allowing direct observation of the spermatozoa in the genital tract. Ejaculated ram spermatozoa were double labeled with R18 and MitoTracker Green FM, and used to inseminate ewes in estrus either cervically through the vagina or laparoscopically into the base of the uterine horns. Four hours after insemination, the spermatozoa were directly observed in situ using fibered confocal fluorescence microscopy in the base, middle and tip of the uterine horns, the utero-tubal junction (UTJ) and the oviduct. The high resolution video images obtained with this technique allowed determination of the distribution of spermatozoa and individual motility in the lumen of the ewe's genital tract. The results showed a gradient of increasing concentration of spermatozoa from the base of the uterus to the UTJ 4 h after intra-uterine insemination into the base of the horns. The UTJ was shown to be a storage region for spermatozoa before their transfer to the oviduct. The in vitro storage of spermatozoa in liquid form decreased their migration through the cervix and reduced the proportion of motile spermatozoa and their straight line velocity at the UTJ and their transit into the oviduct.

Hypotonic resistance of boar spermatozoa was investigated by measuring the ratio of live/dead spermatozoa (SYBR-14/propidium iodide) by flow cytometry after hypotonic stress. The survival rate of ejaculated spermatozoa incubated in hypotonic solutions ranging from 3 to 330 mmol/kg followed a sigmoid curve that fitted a simple logistic model. The critical osmolality value (Osmcrit) at which 50% of spermatozoa died was determined with this model. Hypotonic resistance of spermatozoa increased with temperature between 15 and 39 °C and decreased after hydrogen superoxide treatment, but was not modified during 8 days of preservation in Beltsville thawing solution. Hypotonic resistance markedly decreased during epididymal maturation and after ejaculation as Osmcrit at 15 °C was 54.7±3.2, 68.5±10.6, 116.7±2.1 and 194.3±3.7 mmol/kg for the caput, corpus, cauda and ejaculated spermatozoa respectively. Hypo-osmotic stress of 100 mmol/kg revealed a sperm subpopulation exhibiting increased hypotonic resistance compared with the whole ejaculate (Osmcrit=67.8±2.1 mmol/kg). Consistent differences were observed between lean and standard breeds (Pietrain versus Large White) and between boars within the same breed. According to data collected by artificial insemination centers during a large-scale field trial, hypotonic resistance of ejaculates was found to be positively correlated with in vivo fertility.