Identification of capacitation inducers customized to sperm retrieved from inbred mouse epididymis
Introduction
In the process of producing fertilized eggs in vivo or in vitro, sperm experience several biochemical and physiological alterations to ensure successful fertilization processes including implantation [1], [2], pregnancy [3], embryogenesis [4], [5], transfer of the haploid genome [6], [7], induction of oocyte activation [8], [9], and offer of the centriole [10]. These biochemical and physiological alterations, called sperm capacitation, are part of a complex process that induce changes in sperm in the female genital tract to acquire fertilization capacity [11], [12]. During capacitation, sperm experience phospholipid alterations [13], [14], cholesterol efflux [13], [15], [16], ion channel activation [17], [18], upregulation of intracellular calcium and cAMP levels [19], [15], [20], and alterations in flagellar activity and protein phosphorylation [21], [22], resulting in the generation of mature sperm with hyperactivated motility that have undergone acrosome reaction against specific stimuli [23], [24]. Therefore, this process is a critical event for successful fertilization, both in vivo and in vitro.
To date, diverse candidate substances, such as calcium [25], [26], [21], progesterone [27], [28], bovine serum albumin (BSA) [21], [29], heparin [30], [31], and lysophosphatidylcholine (Lyso-PC) [32], have been considered as sperm capacitation inducers. However, despite the fact that the type of capacitation inducer and the concentration are strongly dependent on species, there are no reports on the customization of sperm capacitation inducers to specific species by comparing the efficiency of sperm capacitation among several inducers. Therefore, using inbred mice, we attempted to determine the type and concentration of an effective capacitation inducer through comparative analysis among a variety of substances. Motile sperm collected from the epididymis of male mice were capacitated by several inducers at various concentrations, and the proportion and fertilization competence of sperm experiencing an acrosome reaction by capacitation were evaluated and compared.
Section snippets
Animals
Eight-to nine-week-old male ICR mice, which were used as sperm donors, were purchased from DBL (Eumseong, Korea). All animal housing, handling, and experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (IACUC approval no. KW-140904-1) and carried out in accordance with the Animal Care and Use Guidelines of Kangwon National University.
Preparation of sperm
The cauda epididymis was excised from mice sacrificed by cervical dislocation and the
Identification of an effective capacitation inducer of sperm retrieved from inbred mouse epididymis
We identified an inducer that maximizes the capacitation induction of sperm derived from inbred mouse epididymis by comparative analysis of candidates at optimized concentrations after identification of the concentration that induced the maximal acrosome reaction for each inducer. As shown in Fig. 1, 2.7 mM calcium had the highest ratio (1.70 ± 0.02 fold of control) of sperm experiencing the acrosome reaction post-capacitation, compared to other calcium concentrations (1.22 ± 0.11 to
Discussion
In this study, we exposed sperm retrieved from the epididymis of inbred mice to known capacitation inducers by culturing in various concentrations of each substance. There was a significant increase in the ratio of sperm experiencing the acrosome reaction post-induction of capacitation in 2.7 mM calcium and in the percentage of zygotes produced through in vitro fertilization with sperm experiencing the acrosome reaction post-induction of capacitation in 2.7 mM calcium or 0.3% (w/v) BSA.
Acknowledgements
This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning (NRF-2014R1A1A3052964). Also, this study was supported by 2013 Research Grant from Kangwon National University (No. 120131999).
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