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The Employment of Strontium to Activate Mouse Oocytes: Effects on Spermatid-Injection Outcome

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The Employment of Strontium to Activate Mouse Oocytes: Effects on Spermatid-Injection Outcome REPRODUCTIONRESEARCH The employment of strontium to activate mouse oocytes: effects on spermatid-injection outcome Jean Loren and Orly Lacham-Kaplan Monash Immunology and Stem Cell Laboratories, Level 3, STRIP 1- Building 75, Monash University, Wellington Rd., Clayton, Australia, 3800 Correspondence should be addressed to O Lacham-Kaplan; Email: [email protected] Abstract The present research investigated the effects of various strontium concentrations, in combination with different incubation periods, on mouse parthenogentic oocyte activation and blastocyst development. The results for blastocyst development showed a trend indicating that 10 mM strontium for 3 h was the optimal strontium protocol. Ethanol, an agent that incites oocyte activation via a monotonic rise in calcium, was employed as a control. The outcome of blastocyst formation arising from parthenogenic ethanol activation was significantly less (P < 0.001) than that achieved by the optimal strontium protocol. To assess the impact of strontium oocyte activation on embryo viability following fertilization with immature germ cells, the protocol of 10 mM strontium for 3 h was applied to oocytes injected with round spermatids and then compared with other protocols. The results indicate that following round-spermatid injection the benefits derived from strontium artificial oocyte activation are evident during both pre- and post-implantation development. However, in order to adjust the protocol to the most effective round-spermatid injection in relation to the oocyte cell cycle, injection was done 1.5 h after strontium acti- vation followed by another 1.5 h activation in strontium. The implementation of round-spermatid injection in combination with this oocyte-activation protocol led to live-birth outcomes not significantly different to those outcomes obtained by mature spermatozoa. Reproduction (2006) 131 259–267 Introduction intracellular calcium oscillates continuously throughout oocyte activation (Cuthbertson & Cobbold 1985, Miyazaki Oocyte activation is not a sole event but a series of occur- et al. 1992), continuing until pronucleus formation (Jones rences that initiate the progression of the oocyte from a et al. 1995). It has been suggested that calcium oscil- haploid fertilized cell to a zygote. Mammalian oocyte lations coordinate the many events that occur throughout activation, in nature triggered by fertilization, allows the oocyte to resume meiosis and proceed to embryonic fertilization (Ben-Yosef & Shalgi 2001) and that each cal- development (Carroll 2001). Meiosis is completed with cium oscillation incrementally propels the events of the extrusion of the second polar body and the resulting oocyte activation (Ducibella et al. 2002). Oocyte acti- haploid chromosomal state of the oocyte. vation can, however, occur with a single rise in calcium, Artificially recreating the events of oocyte activation has so the full role of calcium oscillations is unclear (Swann & become an important component of assisted reproduction Ozil 1994). An association has been identified between technology. To allow for the release of the oocyte from calcium oscillations and superior blastocyst composition metaphase II (MII) arrest, effective artificial oocyte acti- (Bos-Mikich et al. 1997). Additionally, the pattern, fre- vation is critical unto itself but it is also critical because of quency, and amplitude of early calcium oscillations the impact it has on later developmental events (Ozil & impact on post-implantation development (Ozil & Huneau Huneau 2001). Suboptimal artificial oocyte activation is 2001). Prevention of calcium oscillations completing their thought to contribute to disappointing outcomes experi- full course during fertilization will retard pronucleus for- enced by many procedures for assisted reproduction tech- mation (Lawrence et al. 1998) and calcium oscillations of nology (Alberio et al. 2001). abnormal frequencies will induce development arrest The earliest notable event in oocyte activation, in all (Gordo et al. 2002). studied species, is an increase in the level of intracellular The paucity of knowledge surrounding the events of calcium (Swann & Ozil 1994). In mammals, the levels of oocyte activation present some challenges for artificial q 2006 Society for Reproduction and Fertility DOI: 10.1530/rep.1.00894 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/25/2021 03:41:55AM via free access 260 J Loren and O Lacham-Kaplan oocyte activation. As calcium is recognized as the most the Australian National Health and Medical Research known fundamental factor of oocyte activation, many Council (NIH & MRC) code of practice for the care and approaches to artificially induce oocyte activation have use of animals for scientific purposes. focused on recreating increases in intracellular calcium. Methods to induce this include the direct injection of cal- Oocyte collection cium into the oocyte (Machaty et al. 1996), an electrical pulse that through phospholipid destabilization creates Hybrid F1 female mice (C57BL female £ CBA male) at 4–6 pores for the influx of extracellular calcium (Sasagawa & weeks of age were superovulated by subcutaneous Yanagimachi 1996), isolation and injection of the sperm injection of 10 IU pregnant mares’ serum gonadotrophin factor thought to be responsible for oocyte activation at (Intervet, Sydney, Australia) followed by subcutaneous fertilization (Fissore et al. 1998), employment of adeno- injection of 10 IU human chorionic gonadotrophin (Inter- vet) 48–50 h later. Female mice were killed by cervical dis- phostin A, an inositol trisphosphate (InsP3) receptor ago- nist, (Brind et al. 2000), promoting of extracelluar calcium location 13–14 h after injection of human chorionic entry via the use of ethanol (Cuthbertson & Cobbold gonadotrophin. The oocytes were released from the ovi- 1985), and inducing calcium oscillations by employing ducts into M2 handling medium containing 100 IU/ml hya- the divalent cation, strontium (Kline & Kline 1992). Alter- luronidase (type IV-S; Sigma Chemical Co., St Louis, MO, natively, protein inhibitors such as cycloheximide bypass USA) for no longer than 5 min to remove cumulus cells. an intracellular rise in calcium and directly induce meio- Morphologically normal MII oocytes were washed in and sis by undermining cyclin B and therefore maturation transferred to warm and equilibrated M16 culture medium promoting factor (MPF) (Bos-Mikich et al. 1995). While and placed into incubation under 5% CO2 in air at 37 8C these may be credible methods for re-enacting oocyte for 20 min before they were used for experiments. activation, none are able to achieve this with the effi- ciency provided by spermatozoa. Oocyte activation with strontium In the mouse, strontium has been successfully employed The amounts of SrCl (Sigma) that were required for differ- in many studies to induce artificial oocyte activation. The 2 ent-strength solutions were dissolved in sterile deionized ability of strontium to provoke calcium oscillations water (JDH Biosciences, Lenexa, KS, USA) to give 10 £ appears to be more physiologically sound than alternative stock solution. For each experiment, fresh strontium culture methods of oocyte activation that produce a monotonic medium was prepared. A volume of 0.1 ml prepared stock rise in calcium. Calcium oscillations induced by strontium solution was placed into an Eppendorf tube (Greiner lead to improved blastocyst composition (Bos-Mikich et al. Labortechnik, Frikenhaussen, Germany) containing 0.99 ml 1997) and superior pre- implantation development calcium-free M16. Microdroplets (20 ml) were arranged (Lacham-Kaplan et al. 2003). The aptitude of strontium onto a 35 mm Petri dish (Falcon, Franklin Lakes, NJ, USA), has lent itself to satisfactory outcomes in a range of mouse covered with mineral oil (Sigma) and incubated under 5% reproductive technologies; however, there is a paucity of CO in air at 37 8C for 30 min. For activation, oocytes were research that has investigated how strontium is best 2 washed twice, cultured in equilibrated strontium solution, applied. Therefore, the potential to exploit the benefits of and left for the allotted amount of time. Following the strontium may be thwarted by a lack of insight concerning oocyte activation period, oocytes were washed twice in its optimal employment. þ Ca2 /M16 where they remained for culture under mineral The aim of the present study was to compare various oil in an atmosphere of 5% CO in air at 37 8C. strontium protocols with the intention of revealing an opti- 2 mal strontium strength and incubation time combination. The research also intended to compare strontium acti- Oocyte activation with ethanol vation with ethanol, an agent that activates oocytes A50ml droplet of 8% ethanol (BDH, Poole, Dorset, through the production of a monotonic rise in calcium. England) in M2 handling medium was placed onto a The development of artificially activated oocytes was com- 35 mm Petri dish (Falcon) and covered with mineral oil. pared with that of in vitro-fertilized oocytes. In addition, Oocytes were transferred into 8% ethanol for 5 min exactly we examined the effects of these activation protocols on Once removed oocytes were washed three times in pre- pre- and post-implantation embryo development following equilibrated M16 in which they remained for culture under injection of immature male germ cells into MII oocytes. mineral oil in an atmosphere of 5% CO2 in air
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