Overcoming MIII Arrest from Spontaneous Activation in Cultured Rat Oocytes

Abstract

The rat oocyte spontaneously activates under a wide variety of conditions. This process progresses to MIII arrest that is not responsive to parthenogenetic activation and development. Insofar as activation involves extrusion of the second polar body (PBII), we set out to determine if preventing this step by inhibiting microfilaments would change the course of spontaneous activation (SA). In particular, how long does the effect of SA persist while retaining reversibility of PBII extrusion once inhibitors are removed? We wanted to determine if the eggs would be responsive to parthenogenetic activation and capable of resuming development once a permanent inhibition is achieved. We set out to determine whether SA would depend on the ovular age of oocytes. Inhibiting of PBII extrusion was achieved by affecting microtubules with demecolcine or nocodazole or actin filaments with cytochalasin B (CB) and cytochalasin D (CD). We found that all oocytes undergo SA and progression to MIII; however, the rapidity of spontaneous activation is a function of the ovular age of the oocyte. The resumption of the meiosis period changes dramatically from 20 to 180 min with decreasing ovular age. We established that suppression of PB formation can be effectively achieved in oocytes of younger ovular age, and that inhibition of PB extrusion became irreversible after 3.5 h of treatment. We established that drug-treated oocytes could undergo subsequent reactivation and in vitro development to blastocysts. The rate of in vitro development of cytochalasin-treated group was comparable to parthenogenetic controls, while nocodazole and demecolcine produced oocytes that developed at lower frequencies. Thus, the application of the microfilament inhibiting drugs helps to overcome the negative effect of SA that results in MIII arrest. Here we also show optimized parthenogenetic stimulation that resulted in development to the blastocyst stage at frequency comparable to development of fertilized embryos.

An insufficient increase of cytosolic free calcium level results postovulatory aging-induced abortive spontaneous egg activation in rat

“…However, rat is an interesting model because the process of SEA is abortive [30] and aged eggs are arrested at M-III like stage [24]. The freshly ovulated eggs (14 h post-hCG surge) are less likely to undergo abortive SEA as compare to aged eggs in vivo [28,31,33].…”

“…Postovulatory aging-induced abortive SEA deteriorates the egg quality and limits assisted reproductive technologies (ART) such as in vitro fertilization (IVF), intra-cytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT) during animal cloning [14,[17][18][19]. Several drugs such as proteasome inhibitor, MG-135 [20,21], melatonin [22], verapamil [16] and pyruvate [23] have been used to prevent postovulatory aging-induced abortive SEA in vitro [16,24].…”

“…After extrusions of PB-II, rat egg neither form pronuclei and nor proceed to interphase, instead gets arrested at metaphase-III (M-III) like stage [14,24,27,28]. Postovulatory aging is one of the important factors that induce abortive SEA in rat.…”

Production of Transgenic Rats

“…This prompted us to take a more detailed look into specifics of spontaneous activation. We found that spontaneous activation as measured by polar body (PB) extrusion times vary dramatically with ovular age of oocytes (Galat et al, 2007). For example, it is complete by 40 min in oocytes isolated 22 h post-hCG, whereas it lasts 1 h 30 min in oocytes isolated 18 h post-hCG and up to 3 h in oocytes isolated 14 h post-hCG.…”

“…However, other groups had difficulties reproducing the achievement, perhaps because of inherited developmental toxicity and strain dependency of this particular drug (Mizumoto et al, 2008(Mizumoto et al, , 2010Nakajima et al, 2008;Tomioka et al, 2007;Webb et al, 2010). In search of alternatives, we discovered that the activation of rat oocytes could be reversibly suppressed with microtubule and microfilament disruptive agents such as cytochalasin B, cytochalasin D, nocodazole, and demecolcyne (Galat et al, 2007). Although the activation suppression was successful, these treatments decreased the parthenogenetic development as compared to controls.…”