Seminal Plasma Characteristics and Expression of ATP-binding Cassette Transporter A1 (ABCA1) in Canine Spermatozoa from Ejaculates with Good and Bad Freezability.

The composition of seminal plasma and the localization of the ATP-binding cassette transporter A1 (ABCA1) in spermatozoa from good and bad freezers were compared to frozen-thawed spermatozoa from the same dog. Ejaculates were obtained from 31 stud dogs, and the sperm-rich fraction (SRF) was kept for analysis. One aliquot was used for the analysis of concentration, progressive motility (P; CASA), viability (V; CASA) and leucocyte count, and the analysis was performed by flow cytometry (FITC-PNA/PI), SCSA and HOST. In seminal plasma, concentration of albumin, cholesterol, calcium, inorganic phosphate, sodium, potassium, zinc and copper was measured. Semen smears were prepared and evaluated for the expression of ABCA1. The remainder of each ejaculate was frozen. After thawing, the quality assessment was repeated and further smears were prepared. According to post-thaw semen quality, dogs were assigned to good freezers (n = 20) or bad freezers (n = 11), the latter were defined as < 50% progressive motility and/or > 40% morphologically abnormal sperm and/or < 50% viability. Bad freezers were older than good freezers (5.3 vs 3.4 years, p < 0.05). In bad freezers, the percentage of sperm with ABCA1 signal in the acrosome was lower (26.3% vs 35.7%, p < 0.01) and the percentage of sperm with complete loss of ABCA1 signal higher (46.7% vs 30%, p < 0.01); the percentage of dead spermatozoa was higher (36.1% vs 25.5%, p < 0.05), and the concentration of cholesterol and sodium in seminal plasma was lower than in good freezers (p < 0.05). We conclude that in thawed bad freezer sperm, an increase in acrosome damages coincided with an increased loss of cholesterol transporters and cell death, and a lower cholesterol concentration in seminal plasma. Follow-up studies revealed whether a relation exists between these findings.

Immunolocalization of the cholesterol transporters ABCA1 and ABCG1 in canine reproductive tract tissues and spermatozoa.

The mammalian sperm membrane undergoes cholesterol efflux during maturation and fertilization. Although ATP-binding cassette (ABC) transporters are known to transport cholesterol through cell membranes in other organs, their presence in canine testis, epididymis and sperm has not been proven to date. Hence, the aim of the present study was to localize the ABC transporters ABCA1 and ABCG1 in canine testicular and epididymidal tissue as well as in spermatozoa membranes. To this end, semen samples from 12 dogs as well as testicles and epididymides of four young and healthy dogs were prepared for immunohistochemistry, respectively. Capacitation and acrosome reaction (AR) were induced in aliquots of the semen samples before immunostaining to assess changes in the expression of ABCA1 and ABCG1. Evaluation by confocal microscopy revealed the presence of both ABCA1 and ABCG1 in canine testicles and of ABCA1 in the epididymides. In spermatozoa, only ABCA1 immunoreactivity was detected, mainly in the region of the acrosome and midpiece. After induction of capacitation, ABCA1 signal persisted in the acrosome but disappeared after AR, indicating a loss of ABCA1 with the loss of the acrosome. We conclude that ABCA1 and ABCG1 are expressed in canine testis, whereas only ABCA1 is expressed in epididymis and spermatozoa membrane, both transporters probably contributing to the regulation of membrane cholesterol content.