Abstract
BackgroundThe ATP binding cassette (ABC) transporters participate in the cholesterol and phospholipid transport within and through cell membranes of many cells including spermatozoa. Cholesterol efflux is important for capacitation of spermatozoa. ABCA1 expression has been assessed in canine spermatozoa previously but its role in capacitation still has to be determined. The aim of the study was to test whether inhibition of ABCA1 (1) decreases capacitation in ejaculated and epididymal canine sperm samples and (2) decreases cholesterol efflux in the same samples. Twenty-one ejaculates and sperm from 22 epididymal tails were collected from healthy dogs. Motility was measured by CASA and viability assessed after staining with SYBR-14/PI. Samples from ejaculated sperm and sperm from epididymal tails were aliquoted. One part was incubated with the ABCA1 inhibitor probucol, the other served as a negative control. In all samples, capacitation was evaluated by chlortetracyclin (CTC) assay and cholesterol was measured by cholesterol efflux assay and colorimetric enzymatic assay.ResultsIn ejaculated sperm, blockade of ABCA1 with 100 µM of probucol/mL of sample resulted in a significantly higher percentage of uncapacitated and acrosome reacted spermatozoa (P < 0.001 and P = 0.031), capacitation was significantly decreased (35% in probucol samples vs 54.2% in controls, P < 0.001). In probucol inhibited sperm samples from epididymal tails, the percentage of capacitated spermatozoa did not differ between groups but the percentage of acrosome reacted spermatozoa increased significantly (P = 0.014). The cholesterol measurement revealed significantly lower cholesterol concentration in the probucol group when compared to the controls (P = 0.035), however only in ejaculated sperm samples.ConclusionsCTC assay and cholesterol measurement revealed significant differences between groups; we conclude that inhibition of ABCA1 significantly decreased capacitation and cholesterol efflux in ejaculated canine spermatozoa. The inhibition was not complete but ABCA1 is supposed to contribute to capacitation in canine ejaculated spermatozoa. ABCA1 is probably not important for capacitation of epididymal spermatozoa but might exert other functions during spermatozoa ripening.
Highlights
The ATP binding cassette (ABC) transporters participate in the cholesterol and phospholipid trans‐ port within and through cell membranes of many cells including spermatozoa
These molecules can transport cholesterol to sites of need [9]. This is a model and might not be applicable for all ABC transporters. Single members of this transporter family have to be examined or to be functionally grouped; which ABC transporters participate in this cholesterol redistribution seems to be species-specific and to our knowledge so far no data were available for the situation in dog spermatozoa
A significant higher number of spermatozoa were uncapacitated and a lower number of sperm were capacitated (Fig. 2b) than in the controls (35% vs 54.2%, P < 0.001, Fig. 3)
Summary
The ATP binding cassette (ABC) transporters participate in the cholesterol and phospholipid trans‐ port within and through cell membranes of many cells including spermatozoa. ABCA1 expression has been assessed in canine spermatozoa previously but its role in capacitation still has to be determined. The aim of the study was to test whether inhibition of ABCA1 (1) decreases capacitation in ejaculated and epididymal canine sperm samples and (2) decreases cholesterol efflux in the same samples. The ABC transporters are believed to transport cholesterol to certain micro-domains (rafts) in the outer spermatozoa membrane, mainly located in the apical region of the head, where the cholesterol molecules are accumulating and imported into HDL molecules. These molecules can transport cholesterol to sites of need [9]. Single members of this transporter family have to be examined or to be functionally grouped; which ABC transporters participate in this cholesterol redistribution seems to be species-specific and to our knowledge so far no data were available for the situation in dog spermatozoa
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