In vitrofertilisation of Chinese hamster oocytes by spermatozoa that have undergone ionophore A23187-induced acrosome reaction, and their subsequent development into blastocysts

Abstract

SummaryTo enhance potential use of the Chinese hamster, Cricetulus griseus, in developmental and cytogenetic studies of mammalian gametes and embryos, techniques for in vitro fertilisation and embryo culture were developed in the species. Spermatozoa were recovered from the vasa deferentia of mature males, and incubated in modified TYH medium for 1 h at 37°C under 5% CO2 in air. They were then treated with ionophore A23187 (20¼M) for 10min to induce the acrosome reaction. Following ionophore treatment, superovulated oocytes were collected from hormonally stimulated females and incubated with the acrosome-reacted spermatozoa for 2 h at 37°C under 5% CO2 in air. In this study, 245 oocytes ova (98.0%) were determined to be monospermic. The monospermic ova were then cultured in TYH supplemented with 1mM hypotaurine under the same gas phase. Within 30h of fertilisation, 182 ova (93.8%) cleaved to the 2-cell stage, and subsequently 163 ova (84.0%) developed beyond the 2-cell stage. Thus, obstinate developmental arrest at the 2-cell stage(‘2-cell block’) was not observed in this species. Ultimately, 65.5% of monospermic ova reached morula to blastocyst stages.

Sperm acrosome status before and during fertilization in the Chinese hamster (Cricetulus griseus), and observation of oviductal vesicles and globules

“…Thus, most of the acrosomal contents were localized in the lateral bulges. Immediately after treatment with the Ca‐ionophore, spermatozoa became motionless, and the acrosomal caps of some thinned (collapsed) (Figure 5b), as shown previously (Tateno & Kamiguchi, 1996). The thin acrosomes positively reacted with PNA‐FITC, regardless of fixation and permeabilization (Figure 5b′ and 5b″), and the acrosomal contents appeared to have decreased.…”

“…Table 1 shows the results of IVF experiments using spermatozoa collected from the lower oviductal isthmus and uterus. The results of IVF using Ca‐ionophore‐treated spermatozoa (Tateno & Kamiguchi, 1996) are also provided. Because a small number of spermatozoa were collected from the lower isthmus, the sperm concentration during insemination was relatively low.…”

“…The pH values of these media were 7.3–7.4 when equilibrated with 5% CO 2 in the air. When spermatozoa were treated with calcium‐ionophore A23187 (Ca‐ionophore) (Calbiochem‐Merck), a modified TYH (m‐TYH) medium was used, in which the amount of CaCl 2 ·2H 2 O was doubled, and sperm motility factors, i.e., 20 µM d ‐penicillamine (Sigma‐Aldrich), 100 µM hypotaurine (Sigma‐Aldrich), and 1 µM epinephrine (Sigma‐Aldrich) were supplemented (Tateno & Kamiguchi, 1996). In addition, HEPES‐buffered TYH (H‐TYH) medium was used to recover spermatozoa from the oviduct.…”

“…Ca‐ionophores are known to induce the acrosome reaction in Chinese hamster spermatozoa, rendering them capable of fertilizing cumulus‐enclosed and cumulus‐free oocytes (Tateno & Kamiguchi, 1996). Therefore, we treated spermatozoa with a Ca‐ionophore to determine the relationship between the morphological alteration of the acrosomal cap and the progress of the acrosome reaction.…”

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

“…When spermatozoa were treated with ionophore, the concentration of calcium in TYH was doubled to effectively induce the acrosome reaction [17]. This modified TYH with two-fold of Ca 2+ was designated as mTYH.…”