Cyclodextrin Removes Cholesterol from Mouse Sperm and Induces Capacitation in a Protein-Free Medium1

Abstract

Cyclodextrin, which stimulates cholesterol efflux from cells, was examined for its ability to induce capacitation of mouse spermatozoa. A chemically defined, protein-free medium was used for in vitro fertilization of cumulus-free mouse eggs. Fertilization did not occur in modified Krebs-Ringer bicarbonate medium (TYH) supplemented with 1 mg/ml polyvinylalcohol instead of BSA. However, fertilization was observed when spermatozoa were preincubated with methyl-beta-cyclodextrin (MBCD); fertilization rates increased dose-dependently from 0.25 to 0.75 mM MBCD. The fertilization rate decreased when 0.75 mM MBCD was added to both preincubation and fertilization media versus only the preincubation medium (21% vs. 53%); in sharp contrast, fertilization increased when 4 mg/ml BSA was present in both of the media versus the preincubation medium only (66% vs. 25%). At 0.75 mM, 2-hydroxy-beta-cyclodextrin had a lower ability to capacitate spermatozoa in vitro than MBCD (14% vs. 41%). Eggs fertilized by spermatozoa treated with MBCD (0.75 mM) developed to blastocysts (45%, 36 of 80) when cultured in KSOM. When 160 fertilized eggs were transferred to ICR recipients, 62 live offspring were born. After incubation of mouse spermatozoa for 90 min in 0.75 mM MBCD in TYH medium, the cholesterol content of the spermatozoa was significantly (p < 0.01) lower than that of the control (2.27 +/- 0.09 vs. 4.13 +/- 0.09 nmol unesterified cholesterol/10(7) sperm; mean +/- SEM, n = 5). The proportion of capacitated (B pattern) spermatozoa determined by chlortetracycline fluorescence was higher with MBCD treatment for 90 min than for the control (45% vs. 15%; p < 0.01). The proportion of acrosome-reacted (AR pattern) spermatozoa was not different between MBCD treatment and the control. Therefore, MBCD increased capacitation rather than the acrosome reaction of spermatozoa.

SPINK3 modulates mouse sperm physiology through the reduction of nitric oxide level independently of its trypsin inhibitory activity

“…Cholesterol efflux induced in vitro by BSA or methyl-b-cyclodextrine (Choi & Toyoda 1998) and in vivo by progesterone (Costello et al 2009) and female tract neighborhood promotes the remodeling of the plasma membrane and triggers signaling pathways leading to different sperm processes that account for sperm fertilizing ability. Extracellular Ca 2C influx might induce Ca 2C /CaM-dependent NOS activity, increasing NO production.…”

“…Unless indicated, methyl-b-cyclodextrin was used as a replacement of 3% BSA for capacitation, since the latter may affect the fluorescence in the DAF-FM DA assay. The use of methyl-b-cyclodextrine has been already reported (Choi & Toyoda 1998). Sperm concentration was adjusted to the concentration indicated for each assay.…”

Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm

“…Non-physiological cholesterol acceptors such as cyclodextrins (cyclic oligosaccharides consisting of 7a (1-4)-glucopyranose units) have been shown to also alter the cholesterol content of sperm cell membranes (Ohtani et al 1989, Choi & Toyoda 1998, Flesch et al 2001 and induce an increase in PY (Osheroff et al 1999, Visconti et al 1999c. Interestingly, the cholesterol complexing agent, methyl-b-cyclodextrin (MbCD), does not require cholesterol oxidation in order to effectively remove cholesterol from the sperm plasma membrane (Boerke et al 2013).…”

Chromosome analysis of mouse zygotes produced by intracytoplasmic injection of spermatozoa exposed to acrosome reaction inducing agents methyl-β-cyclodextrin and calcium ionophore A23187

“…It has been demonstrated that methyl-β-cyclodextrin (MβCD) allows mouse spermatozoa to capacitate by promotion of cholesterol efflux from the plasma membrane [13][14][15]. Calcium ionophore A23187 is commonly used to induce the acrosome reaction in mammalian spermatozoa.…”