Seminal plasma proteins interacting with sperm surface revert capacitation indicators in frozen-thawed ram sperm

Abstract

This study was conducted to evaluate the effects of interacting seminal plasma proteins (iSPP) obtained by AV or EE on frozen-thawed ram sperm in order to test the hypothesis whether this fraction could be sufficient to emulate the effect of complete seminal plasma (SP). Additionally, we evaluated whether these proteins have a differential effect between spermatozoa from high and low fertility rams and between breeding and non-breeding seasons. We assessed sperm motility, quality parameters (intracellular reactive oxygen species, membrane fluidity, plasma membrane permeability and mitochondrial activity) and capacitation status. The main findings from this work were: i) iSPP had no effect on sperm motility, whereas SP (AV or EE) addition produced the highest values of total motility (74.13±2.99 and 72.27±2.99 for AV and EE, respectively) and progressive motility (64.97±2.64 and 63.73±2.64 for AV and EE, respectively); ii) iSPP had no effect on sperm quality parameters (p>0.05), but whole SP improved all parameters evaluated. Moreover, SP collected by AV yielded significantly higher viability (44.60±2.87) and sperm with stable plasma membrane (44.56±2.49) comparing with the addition of SP collected by EE (35.80±2.47 and 36.67±1.71, respectively); iii) iSPP and SP collected by EE, but not by AV, reverted molecular signals of capacitation as protein tyrosine phosphorylation caused by freezing temperatures; iv) there were no effects of fertility or season in sperm quality parameters evaluated. This study demonstrated that, although the iSPP have a clear decapacitating effect, including the ability to revert cryo-capacitation indicators, they are not sufficient to emulate the effects of complete SP regarding sperm functional parameters.

Markers for Sperm Freezability and Relevance of Transcriptome Studies in Semen Cryopreservation: A Review

“…Even though post-thaw sperm motility is a good indicator of viability, it is not always an accurate fertility predictor of an AI-semen dose [9]. Evaluations of sperm motility characteristics have been improved by the incorporation of the computer-assisted semen analysis (CASA) system, which measures several motility and motion parameters of spermatozoa that are closely related to fertility compared with subjective motility measurements [21][22][23][24]. Besides motility analysis, studies have conirmed that the velocity parameters, such as velocity straight line (VSL), velocity curvilinear (VCL), and velocity average path (VAP), are associated with the fertilizing capacity of frozen-thawed spermatozoa [21,24].…”

“…In recent years, several luorescent probes have shown that the cryopreservation process compromises the sperm plasma membrane integrity (PMI), resulting in reduced fertilizing capacity of postthaw semen [3][4][5][23][24][25][26][27][28][29]. Post-thaw sperm PMI has been assessed with diferent luorescent membrane probes, such as the dual SYBR-14 and propidium iodide (PI) assay [25,28] or membrane-permeable substrate carboxyluorescein diacetate (CFDA), a nonspeciic esterase substrate [24].…”

“…Post-thaw sperm PMI has been assessed with diferent luorescent membrane probes, such as the dual SYBR-14 and propidium iodide (PI) assay [25,28] or membrane-permeable substrate carboxyluorescein diacetate (CFDA), a nonspeciic esterase substrate [24]. The chlortetracycline (CTC) luorescence assay has been used to detect capacitation-like changes in frozen-thawed spermatozoa, which may compromise their fertilizing ability [6,10,[22][23][24]29]. Cryo-induced changes in the acrosome membrane integrity (AMI) have been monitored by speciic Giemsa-staining technique [25] or with luorescent dyes, such as luorescein isothiocyanate (FITC)-conjugated PNA (peanut agglutinin) or conjugated PSA (Pisum sativum agglutinin), known as plant lectins, which bind to glycoproteins in the outer acrosomal membrane [4,5,26,27].…”

“…In a recent study, it has been demonstrated that FITC-PSA/PI-staining protocol can detect marked deterioration in both the PMI and AMI in frozen-thawed bull spermatozoa [4]. Earlier changes in the membrane permeability of frozen-thawed spermatozoa have been monitored with the calcium-dependent binding of Annexin-V/FITC/PI [21,24,26] or YO-PRO-1 assay [23,25,28], which is an impermeable membrane probe. Moreover, triple staining with YO-PRO-1, ethidium homodimer (Eth), and SNARF-1 (YO-PRO-1/Eth/SNARF-1) has been shown to give similar results with respect to PMI assessment of frozen-thawed spermatozoa compared with the Annexin-V/ FITC/PI assay [27,28].…”

“…Besides the R123/PI and JC-1/PI assays, there are a plethora of luorescent dyes that can be used to microscopically or cytometrically assess the sperm mitochondrial membrane function (MMF). Some luorescent MitoTracker probes, such as MitoTracker Deep Red, MitoTracker Red, MitoTracker Orange, and MitoTracker Green, have been used efectively to assess the MMF on frozen-thawed spermatozoa [23,26]. Boars with good and poor semen freezability ejaculates were identiied using several sperm function parameters, including total and progressive motility (TMOT and PMOT, respectively), and rapid movement (RAP) analyzed by the computer-assisted semen analysis system, mitochondrial membrane function, MMF (JC-1/PI assay) and PMI (SYBR-14/PI assay) (Figure 1).…”