BODIPY-cholesterol can be reliably used to monitor cholesterol efflux from capacitating mammalian spermatozoa.

Capacitation is the final maturation step spermatozoa undergo prior to fertilisation. The efflux of cholesterol from the sperm membrane to the extracellular environment is a crucial step during capacitation but current methods to quantify this process are suboptimal. In this study, we validate the use of a BODIPY-cholesterol assay to quantify cholesterol efflux from spermatozoa during in vitro capacitation, using the boar as a model species. The novel flow cytometric BODIPY-cholesterol assay was validated with endogenous cholesterol loss as measured by mass spectrometry and compared to filipin labelling. Following exposure to a range of conditions, the BODIPY-cholesterol assay was able to detect and quantify cholesterol efflux akin to that measured with mass spectrometry. The ability to counterstain for viability is a unique feature of this assay that allowed us to highlight the importance of isolating viable cells only for a reliable measure of cholesterol efflux. Finally, the BODIPY-cholesterol assay proved to be the superior method to quantify cholesterol efflux relative to filipin labelling, though filipin remains useful for assessing cholesterol redistribution. Taken together, the BODIPY-cholesterol assay is a simple, inexpensive and reliable flow cytometric method for the measurement of cholesterol efflux from spermatozoa during in vitro capacitation.

Ram seminal plasma and its functional proteomic assessment.

Ejaculation results in the confluence of epididymal spermatozoa with secretions of the accessory sex glands. This interaction is not a prerequisite for fertilisation success, but seminal factors do play a crucial role in prolonging the survival of spermatozoa both in vitro and in vivo by affording protection from handling induced stress and some selective mechanisms of the female reproductive tract. Reproductive biologists have long sought to identify specific factors in seminal plasma that influence sperm function and fertility in these contexts. Many seminal plasma proteins have been identified as diagnostic predictors of sperm function and have been isolated and applied in vitro to prevent sperm damage associated with the application of artificial reproductive technologies. Proteomic assessment of the spermatozoon, and its surroundings, has provided considerable advances towards these goals and allowed for greater understanding of their physiological function. In this review, the importance of seminal plasma will be examined through a proteomic lens to provide comprehensive analysis of the ram seminal proteome and detail the use of proteomic studies that correlate seminal plasma proteins with ram sperm function and preservation ability.