Evaluation of maturation competence of metaphase II
oocytes in mice based on the distance
between pericentriolar materials of meiotic spindle
Distance of PCM during oocyte maturation
Chizuka Sakai & Yumi Hoshino & Yusuke Sato &
Received: 21 July 2010 /Accepted: 13 October 2010 /Published online: 17 November 2010
# Springer Science+Business Media, LLC 2010
Purpose To ascertain whether metaphase II (MII) spindle
shape influences oocyte competence, we examined the
meiotic spindle organization in in vivo ovulated (IVO)
oocytes and in spontaneously matured or follicle stimulat-
ing hormone (FSH)-induced oocytes.
Methods FSH-induced oocytes matured in Waymouth’s
MB752/1 or human tubal fluid (HTF) media and oocytes
matured spontaneously in the basal medium were obtained,
and spindles were detected by immunofluorescence. To
evaluate the fertilization-associated differences in spindle
morphology, we performed in vitro fertilization and
analysed integrin mRNA expression.
Results The distance between the pericentriolar materials
(PCMs) in oocytes matured under all conditions was
initially more, but it reduced gradually and increased again
thereafter. Therefore, oocytes exhibiting a reduction in the
distance between PCMs had the highest development rate
to blastocyst in each condition.
Conclusion These results indicate that the ‘maturation
competence’ of MII oocytes can be evaluated on the basis
of the distance between PCMs.
Keywords In vitro maturation.Maturation competence.
Meiotic spindle.Mouse oocyte.Pericentriolar materials
vesicle breakdown (GVBD), is either a spontaneous or a
gonadotropin-induced response. The former occurs as a
consequence of oocyte removal from the inhibitory environ-
ment of the follicle , while the latter is induced by
gonadotropin action on cumulus cells that produce meiosis-
inducing signals capable of overriding meiotic arrest [2–4]. In
both the processes, a key step is the decrease in intracellular
cyclic adenosine monophosphate (cAMP) levels [5, 6] and the
subsequent activation of the M-phase promoting factor (MPF)
and the mitogen-activated protein kinase (MAPK) pathway
. Follicle stimulating hormone (FSH) induces complete
meiotic maturation, i.e., first polar body (PB1) emission, of
cultured cumulus-oocyte complexes (COCs). Hypoxanthine
maintainsmeiotic arrest inculturedCOCs,while FSH reverses
this inhibitory effect of hypoxanthine .
In vitro maturation (IVM) has been efficiently used to
obtain metaphase II (MII)-arrested mouse oocytes that are
competent to be fertilized and are capable of producing
viable embryos [7, 8]. The developmental competence of in
vitro matured (IVM) oocytes of mammals is likely to be
inferior to that of in vivo matured oocytes [9–12]. After it
was reported that mammalian oocytes can spontaneously
resume and complete meiosis upon removal from the
follicle [1, 13], much effort has been expended to modify
the culture conditions of IVM in order to obtain high-
quality oocytes for embryo production [14, 15]. In some
mammals, such as cattle, IVM represents the industry
standard and is routinely used for in vitro fertilization or
nuclear transfer and embryo production strategies, and this
method affords relatively high rates of blastocyst develop-
ment and implantation [16, 17]. Although live young
Capsule The ‘maturation competence’ of MII oocytes can be
evaluated on the basis of the distance between pericentriolar materials
(PCMs) of spindles.
C. Sakai (*):Y. Hoshino:Y. Sato:E. Sato
Laboratory of Animal Reproduction,
Graduate School of Agricultural Science, Tohoku University,
Sendai 981-8555, Japan
J Assist Reprod Genet (2011) 28:157–166
individuals have been successfully produced from IVM
oocytes of some mammals, including humans, the distin-
guishing features of in vivo ovulated (IVO) oocytes, which
confer a high developmental potential to these oocytes,
remain obscure . From a practical point of view, to
optimize IVM, it is necessary to achieve successful
embryonic development and identify oocyte markers that
predict successful nuclear and cytoplasmic maturation.
In oocytes, the meiotic spindle plays an important role in
chromosome alignment and separation during meiosis.
Typically, meiotic spindles of mouse oocytes are anastral;
however, the degree of spindle pole tapering and minus end
focusing varies widely between species  and with the
conditions under which meiotic maturation occurs .
Significant differences have been observed between IVO
and IVM oocytes of mice with regard to meiotic spindle
shape and size. IVO oocytes exhibit spindles with a focused
pole, whereas IVM oocytes typically display large barrel-
shaped anastral spindles that appear more pronounced after
different treatments [20, 21]. The significance of deviations
in the meiotic spindle shape and size with respect to the
quality of oocytes has not been fully investigated.
We aimed to identify the maturation conditions under
which the shape of the spindle in IVM oocytes was similar
to that of the spindle in IVO MII oocytes and to increase
the maturation competence of IVM oocytes. To this end, we
cultured mouse oocytes in the germinal-vesicle (GV) stage
under different conditions and evaluated the relationship
between spindle shape and ‘maturation competence’.
Materials and methods
ICR mice were purchased from Japan SLC Inc. (Shizuoka)
and bred in our laboratory. Immature 20- to 23-day-old
mice were used for all experiments. The experimental
procedures described in this report were performed in
accordance with the Guide for the Care and Use of
Laboratory Animals published by Tohoku University.
Collection of oocytes matured in vivo and in vitro
The maturation time under each maturation condition was
divided into phase I, II, and III (Fig. 1); phase I was
immediately after PB1 emission, phase II was the common
maturation time, and phase III was the prolonged matura-
tion time after PB1 emission. To obtain in vivo oocytes, we
first primed the mice with 5 IU of pregnant mare’s serum
gonadotropin (PMSG) (Teikoku Hormone MFG, Tokyo)
and then with 5 IU of human chorionic gonadotropin (hCG)
(Teikoku Hormone MFG) after 48 h. At 13 (phase I), 14
(phase II), and 18 h (phase III) after hCG treatment, MII-
Fig. 1 General timeline of mat-
uration in mice. Phase I, II, and
III in in vivo matured oocytes
are 13, 14, and 18 h, respec-
tively. Phase I, II, and III in
spontaneously matured oocytes
are 10, 12, and 18 h, respec-
tively. Phase I, II, and III in
FSH-induced matured oocytes
are 15, 18, and 24 h, respec-
tively. The arrows indicate the
maturation time reached inci-
dence rate to MII oocyte prateau
158 J Assist Reprod Genet (2011) 28:157–166
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