Original Article

*[FTQUVCVKE2TGUUWTG#ƑGEVUIn Vitro Maturation of Oocytes

and Follicles and Increases Granulosa Cell Death
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* Corresponding Address: P.O.Box: 6714967346, Department of Biology, Faculty of Basic Sciences, Razi University,
Kermanshah, Iran
Email: azadbakhtm_tmu@yahoo.com
5HFHLYHG-XO$FFHSWHG-DQ
Abstract
2EMHFWLYHThis study examines the effects of hydrostatic pressure on in vitro maturation
(IVM) of oocytes derived from in vitro grown follicles.
0DWHULDOVDQG0HWKRGV In this experimental study, preantral follicles were isolated from
12-day-old female NMRI mice. Each follicle was cultured individually in Alpha Minimal
(VVHQWLDO0HGLXPĮ0(0XQGHUPLQHUDORLOIRUGD\V7KHQIROOLFOHVZHUHLQGXFHGIRU
IVM and divided into two groups, control and experiment. In the experiment group follicles
were subjected to 20 mmHg pressure for 30 minutes and cultured for 24-48 hours. We as-
sessed for viability and IVM of the oocytes. The percentage of apoptosis in cumulus cells
was determined by the TUNEL assay. A comparison between groups was made using the
student’s t test.
5HVXOWV The percentage of metaphase II oocytes (MII) increased in hydrostatic pressure-
treated follicles compared to controls (p<0.05). Cumulus cell viability reduced in hydro-
static pressure-treated follicles compared to controls (p<0.05). Exposure of follicles to
pressure increased apoptosis in cumulus cells compared to controls (p<0.05).
&RQFOXVLRQHydrostatic pressure, by inducing apoptosis in cumulus cells, participates in
the cumulus oocyte coupled relationship with oocyte maturation.

Keywords: In vitro Maturation, Oocyte, Hydrostatic Pressure, Apoptosis, Mouse

Cell Journal(Yakhteh), Vol 15, No 4, Winter 2014, Pages: 282- 293

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Introduction
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In vitroPDWXUDWLRQ,90RIPDPPDOLDQRRF\WHV  JURZWK IDFWRUV  RSWLPL]DWLRQ RI FXOWXUH
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in vitro IHUWLOL]DWLRQ ,9) LV D URXWLQH SURFHGXUH WKHODWHUIDFWRUVKDYHDQLPSRUWDQWUROHLQWKHVXF
LQQXPHURXVLQIHUWLOLW\FOLQLFV6RPHZRPHQKRZ FHVVRI,903K\VLFDOIRUFHVDIIHFWIROOLFOHUXSWXUH
HYHUPD\IDLOWRUHVSRQGWRKRUPRQDOVWLPXODWLRQ DQG RYXODWLRQ E\ LQFUHDVLQJ IROOLFXODU ÀXLG SUHV
RUDUHDWULVNRIRYDULDQK\SHUVWLPXODWLRQ,90 sure due to an increase in hydrostatic pressure in
RIRRF\WHVRIIHUVDQDOWHUQDWLYHVWUDWHJ\WRREWDLQ WKHRYDULDQYDVFXODUV\VWHP$GHFUHDVHRIWHQVLOH
mature ooF\WHVLQWKHVHFDVHV7KHIHUWLOLW\ strength in the follicle wall and increase of the
rate from matured oocytes in vitro is much lower K\GURVWDWLFSUHVVXUHLQVLGHWKHIROOLFOHRUDFRP
than those of in vivoVWLPXODWLRQF\FOHVLQGLFDWLQJ ELQDWLRQ RI WKHVH HYHQWV LV QHHGHG IRU VXFFHVVIXO
WKDWLPSURYHPHQWLQ,90UHPDLQVDFKDOOHQJH IROOLFXODUUXSWXUH3K\VLFDOIRUFHVPD\FDXVH

CELL JOURNAL(Yakhteh), Vol 15, No 4, Winter 2014 282

Hydrostatic Pressure and Oocyte Maturation

tissue thinning and follicular rupture by elimina vitroJURZQIROOLFOHV,QWKHSUHVHQWVWXG\K\GUR

WLRQRIVHOHFWLYHFXPXOXVFHOOV2QWKHRWKHU
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tory process and the oocyte is freed into follicular
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lar wall and ruptures. The amount of cell death in
VWDWLFSUHVVXUHZDVXVHGWRLQYHVWLJDWHWKHLQYROYH
PHQW RI &2& FHOO GHDWK RQ WKH ,90 RI RRF\WHV
GHULYHGIURPin vitro grown follicles.
Materials and Methods
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WKHFXPXOXVRRF\WHFRPSOH[&2&VWKDWLPSDFWV FDO&R6W/RXLV0286$DQG*LEFR*UDQG
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LQÀXHQFHGE\YDULRXVDSRSWRWLFPHFKDQLVPV Ovarian follicle recovery and culture
The spatiotemporal pattern of apoptosis during
follicle growth and oocyte maturation is tightly 7KLV H[SHULPHQWDO VWXG\ ZDV UHYLHZHG DQG
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in the cumulus mass and between cumulus cells
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ity. The degree of apoptosis is correlated with de
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totic changes in the follicle may support or induce OLJKWFRQWUROOHG FRQGLWLRQV  KRXUV OLJKW 
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DQGJUDYLW\,WLQGHSHQGHQWRIVKDSHWRWDOPDVVRU to dissection medium that consisted of Alpha
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creased the incidence of cell death in cumulus cells
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were rinsed three times in dissection medium
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CELL JOURNAL(Yakhteh), Vol 15, No 4, Winter 2014 283

 Rashidi et al.

)DOFRQ)UDQFHDWÛ&8QGHUDQDWPRVSKHUH from in vitro grown follicles as well as the cell

RI  &22 LQ DLU IRU  GD\V ,Q DGGLWLRQ WKH death incidence and apoptosis in the cumulus
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each drop with fresh medium. and apoptosis.

Follicle monitoring and measurement In vitro maturation of oocytes

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WRUHGGDLO\XQGHUDQLQYHUWHGPLFURVFRSH2O\P ly described method with some modifications
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measurement of morphological features were FXOWXUH PHGLXP VXSSOHPHQWHG ZLWK  ,8PO
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two perpendicular diameters were measured us DWHGWKHRRF\WHVIRUQXFOHDUPDWXUDWLRQDQGYL
LQJ D FDOLEUDWHG RFXODU PLFURPHWHU 'LQR 'LJLWDO ability.
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RIîEHIRUHFXOWXUHDQGWKHQRQGD\V Evaluation of nuclear maturation
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originated from the follicle theca and attached to
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the dish were not included in the measurements. In
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with the longest length and widest perpendicular
Oocyte viability
width.
In this assessment the follicles were mechani
Experimental protocol FDOO\UXSWXUHGDQGWKHRRF\WHVZHUHGHQXGHGDIWHU
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which were defined as intact follicles with a
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central oocyte surrounded by a granulosa cell
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cated and placed in culture medium randomly
Hoechst/propidium iodide nuclear staining of
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cumulus oocyte complexes
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transferred to a pressure chamber according to +RHFKVWSURSLGLXP LRGLGH 3, QXFOHDU VWDLQ
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KRXUVZHDVVHVVHGWKH,90RIRRF\WHVGHULYHG cells were washed and immediately transferred

CELL JOURNAL(Yakhteh), Vol 15, No 4, Winter 2014 284

Hydrostatic Pressure and Oocyte Maturation

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blue and red fluorescence.
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were mounted in glycerol onto a slide and placed
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total cell number.
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Statistical analysis
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Follicle and oocyte measurement
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Table 1: Follicle and oocyte diameters during culture

N Day 1 Day 3 Day 6 Day 9 Day 12

Follicle diameter  “ “ “ “ “

Oocyte diameter  “ “ “ “ “

7ZRSHUSHQGLFXODUVZHUHPHDVXUHGDWîPDJQL¿FDWLRQDQGLVFDOFXODWHGEDVHGRQPLFURPHWHUV
Mean diameters ± SEM were calculated.

CELL JOURNAL(Yakhteh), Vol 15, No 4, Winter 2014 285

 Rashidi et al.

A B

C D

E F

Fig 1: Morphology of mouse follicle during in vitro growth.

The cultured isolated follicle on day 1 (A), day 3 (B), day 6 (C), day 9 (D) and day 12 (E, F).
; Theca cells monolayer, =RQDSHOOXFLGDQG $QWUDOFDYLW\6FDOHEDUȝP

CELL JOURNAL(Yakhteh), Vol 15, No 4, Winter 2014 286

Hydrostatic Pressure and Oocyte Maturation

Viability and in vitro maturation of oocytes &2&V IURP WKH H[SHULPHQW JURXS FRPSDUHG
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RI*9%'DQG0,,RRF\WHVLQFUHDVHGLQWKHH[SHUL H[SHULPHQWDO JURXS FRPSDUHG WR WKH FRQWURO
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JURXS S 7DEOH  &HOO PRUSKRORJ\
was scored as follows. Viable cells contained
EOXHVWDLQHG QRUPDO VPRRWK QXFOHL RU PXO
WLSOH EULJKW VSHFNV RI FKURPDWLQ 1RQYLDEOH
FHOOV FRQVLVWHG RI SLQNVWDLQHG QXFOHL ZLWK
S7KHSHUFHQWDJHVRI0,,RRF\WHVDQG3$HP HLWKHU PXOWLSOH EULJKW VSHFNV RI IUDJPHQWHG
EU\RVLQFUHDVHGLQWKHH[SHULPHQWJURXSFRPSDUHGWR chromatin which included discrete clusters of
WKHFRQWUROJURXSS7DEOH PHPEUDQHERXQGHG YHVLFOHV DQG RQH RU PRUH
spheres of condensed chromatin (significantly
Nuclear staining of the cumulus oocyte complexes more compact and smaller than normal nuclei)
7KHSHUFHQWDJHRIYLDEOHFHOOVZDVORZHULQ as seen in figure 2.

Table 2: Viability and IVM of oocytes derived from in vitro grown follicles
24 hours 48 hours
Groups N Viability GV GVBD MII PA GV GVBD MII PA

Control       22   
a a a a a a a a

Experiment          
a b b b b a b b

Control group; No pressure exposure and experiment group; Exposure to pressure.

GV; Germinal vesicle, GVB; Germinal vesicle breakdown, MII; Metaphase II and PA; Parthenogenetic embryo.
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Table 3: Cell death in COCs derived from in vitro grown follicles

0 hour 24 hours

Groups N Total cells Viable cells Non-viable cells Total cells Viable cells Non-viable cells

Control  “a    “a 

a a a a

Experiment  “a    “a 

b b b b

Control group; No pressure exposure and experiment group; Exposure to pressure.

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CELL JOURNAL(Yakhteh), Vol 15, No 4, Winter 2014 287

 Rashidi et al.

A B C

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A. Control group; without pressure exposure.
B. Experiment group; Exposure to pressure. Viable cells; Blue-stained smooth nuclei or multiple bright specks of condensed
chromatin, Dead cells; Pink-stained nuclei with multiple bright specks of fragmented chromatin or more spheres of condensed
chromatin and ; Dead cells.
C. Characteristics of viable and dead cell, ; Fragmented nucleus and ; Condensed nucleus.
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7KHDSRSWRWLFLQGH[ZDVKLJKHULQ&2&VIURP chromosomes counted as single nuclei; frag
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were smaller than 'healthy' interphase nuclei.
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displayed morphological characteristics of apo
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Table 4: Apoptosis in COCs derived from in vitro grown follicles

0 hour 24 hours

Groups N Total cells Apoptotic index Total cells Apoptotic index

Control  “a a “a a

Experiment  “a b “a b

Control group; No pressure exposure and experiment group; Exposure to pressure.

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CELL JOURNAL(Yakhteh), Vol 15, No 4, Winter 2014 288

Hydrostatic Pressure and Oocyte Maturation

A A'

B B'

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hours of pressure exposure.
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A. Phase contrast picture from control group. A'0DWFK¿JXUH781(/VWDLQLQJE\ÀXRUHVFHQFHPLFURVFRS\LQFRQWUROJURXS
that had no pressure exposure. B. Phase contrast picture from experiment group exposed to pressure. B'0DWK¿JXUH781(/
VWDLQLQJE\ÀXRUHVFHQFHPLFURVFRS\LQH[SHULPHQWJURXSH[SRVHGWRSUHVVXUH6FDOHEDU—P

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vitro LQFUHDVHG IROORZLQJ H[SRVXUH WR K\GURVWDWLF FODVV RI LQWDFW SUHDQWUDO IROOLFOHV UHWULHYHG IURP
pressure. The hydrostatic pressure increased the PLFH  2XU FXOWXUH V\VWHP LV EDVHG XSRQ WKH
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rate of oocytes. The YLDELOLW\ RI RRF\WHV GHULYHG
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,QWKHFXUUHQWH[SHULPHQWSUHDQWUDOIROOLFOHVLVR licular pressure was recorded. The main find
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reached a diameter similar to that of fully grown
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oocytes spontaneously underwent apoptosis dur
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PLGRYXODWRU\SKDVHDQG“PP+JDW cyte. The signals that produced by cumulus cells
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bits (20). These measurements were obtained ,QWKHGHDGFHOOVVRPHRIWKHQXFOHLZHUHIUDJ
by inserting a large micropipette into the fol mented and condensed. The percentage of these

CELL JOURNAL(Yakhteh), Vol 15, No 4, Winter 2014 290

Hydrostatic Pressure and Oocyte Maturation
types of nuclei in cumulus cells were increased in
K\GURVWDWLF SUHVVXUHWUHDWHG IROOLFOHV )UDJPHQWD
tion and condensation of nuclei are two mor
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latory process. According to the results of this
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the apoptosis rate of cumulus cells; the latter may
EH UHVSRQVLEOH IRU DQ LQFUHDVH LQ WKH 0,, RRF\WH
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pressure had a mild effect on the incidence of cell
death in cumulus cells but no aberrant effect on
RRF\WHYLDELOLW\
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sidered to be apoptotic cells that significantly
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Acknowledgements
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(PEU\RORJ\5HVHDUFK/DERUDWRU\DQG'HSDUWPHQW
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to hydrostatic pressure.
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OLNHWRWKDQN5D]L8QLYHUVLW\IRULWV¿QDQFLDOVXS
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in this article.
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VWXGLHV   2XU GDWD LQGLFDWHG WKDW WKH SHU
FHQWDJH RI SDUWKHQRJHQHWLF RRF\WHV VLJQL¿FDQWO\
LQFUHDVHGLQIROOLFOHVH[SRVHGWRK\GURVWDWLFSUHV
VXUHFRPSDUHGWRXQH[SRVHGIROOLFOHV
+\GURVWDWLF SUHVVXUH FDXVHG DQ LQFUHDVH LQ WKH
rate of cumulus cells that underwent apoptosis and
probablyEHUHVSRQVLEOHIRUWKHLQFUHDVHG0,,RR
F\WHUDWHDIWHU,90
7KHUH LV LQFUHDVLQJ HYLGHQFH WKDW K\GURVWDWLF
pressure plays important roles in cell shape and
VWUXFWXUH H[RF\WRVLV DQG JURZWK DQG GHDWK RI
DQLPDO FHOOV $OWKRXJK UHSURGXFWLYH ELRORJ\
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FKHPLFDOLQWHUDFWLRQVRYHUWKHSDVWFHQWXU\LWLV
WLPHWRIXUWKHUH[SORUHWKHPHFKDQLVPE\ZKLFK
PHFKDQLFDOIRUFHVFDQH[HUWWKHLUSRWHQWHIIHFWV
RQ JDPHWHV DQG HPEU\RV GXULQJ UHSURGXFWLRQ
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SLYRWDOUROHRIDSRSWRVLVLQRYXODWLRQSURPSWXV
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Conclusion
7KLV VWXG\ LPSOLFLWO\ H[SODLQV D PRGHO V\VWHP
WR GHYHORS DQ XQGHUVWDQGLQJ RI WKH OLQN EHWZHHQ
WKH SK\VLFDO FRQGLWLRQ RI D IROOLFOH DQG WKH RYX
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