Activation of pig oocytes by intracytoplasmic injection of strontium and barium

Abstract

Ovulated mouse oocytes are activated by exposure to culture medium containing Sr2+ or Ba2+ or by intracytoplasmic injection of the divalent cations. It is known that in vitro matured pig oocytes are activated by the intracytoplasmic injection of Ca2+. In this study, we examined the effect of exposure and of intracytoplasmic injection of Sr2+ or Ba2+ on in vitro matured pig oocytes (MII-oocytes). When MII-oocytes were exposed to the medium containing divalent cations, no oocytes were activated. However, in the case of oocytes that were injected with Sr2+, Ba2+ and Ca2+, at 6 h after injection, 64%, 71% and 86% of the oocytes had been released from MII-arrest, and 51%, 67% and 84% formed female pronuclei, respectively. The initial transient in intracellular Ca2+ concentration ([Ca2+]i) was measured by the Ca2+ indicator dye fluo-4 dextran. Microinjection of Sr2+, Ba2+ or Ca2+ induced a rapid elevation of [Ca2+]i. The exocytosis of cortical granules was examined by staining with fluorescein isothiocyanate (FITC)-labelled peanut agglutinin. After an injection of divalent cations, a release of cortical granules was observed in the oocytes. Maturation promoting factor (MPF) activity declined to a low level after 6 h in all the oocytes injected with divalent cations. To test their developmental ability, injected oocytes were treated with cytochalasin B and then cultured for 168 h in NCSU23 medium. After 168 h, 29% (Sr2+), 29% (Ba2+) and 51% (Ca2+) of the oocytes had developed to the blastocyst stage. These results indicate that intracytoplasmic injection of Sr2+ and Ba2+, like that of Ca2+, induces in vitro matured pig oocytes to be released from MII-arrest and leads them into a series of events related to oocyte activation.

Activation and Development of Pig Oocytes after Microinjection of Crude Sperm Extract

“…In the present study, cortical granules were released within 6-8 h after electro-stimulation or microinjection of sperm extract, and the result was consistent with the previous report [11]. The exocytotic patterns were similar to those of the oocytes injected with divalent cations such as Ca 2+ , Sr 2+ and Ba 2+ [10]. Cortical granules were completely released from some oocytes.…”

“…The release of cortical granules is induced by the increased [Ca 2+ ] i caused by the spermatozoon. It is also induced by different treatments, such as electro-stimulation [7] and microinjection of Ca 2 + [9,10] in the pig, calcium ionophore in the hamster [1], and exposure to the medium containing Sr 2+ in the mouse [36]. In the present study, cortical granules were released within 6-8 h after electro-stimulation or microinjection of sperm extract, and the result was consistent with the previous report [11].…”

“…After microinjection of sperm extract or electrostimulation, oocytes were cultured in TL-PVA [10] for 6 h, then, mounted on slides, fixed in an acetic acidethanol (1:3, v/v) solution, and stained with 1% acetoorcein. Meiotic resumption of oocytes and their pronuclear formation were examined under a Nomarsky interference microscope.…”

“…Development of activated oocytes was examined in the manner described by Okada et al [10]. Briefly, oocytes after microinjection of sperm extract or electrostimulation were cultured for 4 h in NCSU23 medium [21] containing 5 µg/ml cytochalasin B (Sigma) and 4 mg/ml bovine serum albumin (BSA, Serologicals Co., Norcross, GA, USA), and further cultured in NCSU23 medium containing 4 mg/ml BSA.…”

“…In the pig, an electro-stimulation technique [5] has been used for oocyte activation [6][7][8]. [9,10] and soluble sperm extract [11]. In many species studied so far, such as the hamster [12], mouse [13], human [14], cow [15] and pig [11], injection of sperm extract activates oocytes with repetitive increases in intracellular Ca Moreover, it has been reported that oocytes injected with sperm extract develop to blastocysts in the mouse [16], cow [16] and pig [11].…”