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
LV DQ HI¿FLHQW PHWKRG WR SURGXFH PDWXUH RRF\WHV V\VWHPV   DQG HQYLURQPHQWDO DQG SK\VLFDO
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,QGXFWLRQRIRYXODWLRQWRREWDLQPDWXUHRRF\WHVIRU WRLQFUHDVHTXDOLW\RI,90RRF\WHV,QWKLVVHQVH
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 VWDWLFSUHVVXUHZDVXVHGWRLQYHVWLJDWHWKHLQYROYH
KDQG FXPXOXV FHOOV GLVVRFLDWH GXULQJ WKH RYXOD PHQW RI &2& FHOO GHDWK RQ WKH ,90 RI RRF\WHV
tory process and the oocyte is freed into follicular GHULYHGIURPin vitro grown follicles.
ÀXLG 3URJUDPPHG FHOO GHDWK SDUWLFLSDWHV LQ GH
JHQHUDWLRQRIIROOLFXODUFHOOVZHDNHQVWKHIROOLFX Materials and Methods
lar wall and ruptures. The amount of cell death in &KHPLFDOVZHUHSXUFKDVHGIURP6LJPD&KHPL
WKHFXPXOXVRRF\WHFRPSOH[&2&VWKDWLPSDFWV FDO&R6W/RXLV0286$DQG*LEFR*UDQG
RRF\WHGHYHORSPHQWSRWHQWLDOLVXQFOHDU'H ,VODQG1<86$XQOHVVRWKHUZLVHLQGLFDWHG
YHORSPHQW RI IROOLFOHV DQG WKHLU UHODWHG &2&V LV
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
UHJXODWHG 'LVUXSWLRQRI HLWKHUWLPLQJRUWKH DSSURYHGE\WKH/DERUDWRU\$QLPDO&DUH&RP
PDJQLWXGHRIDSRSWRVLVFDQDOWHUFHOOFRQQHFWLYLW\ PLWWHH RI WKH )DFXOW\ RI %DVLF 6FLHQFHV 5D]L
in the cumulus mass and between cumulus cells 8QLYHUVLW\.HUPDQVKDK,UDQ,QWKLVVWXG\
DQG WKH RRF\WH FDXVLQJ GH¿FLWV LQ RRF\WH TXDO  GD\ROG IHPDOH 105, PLFH ZHUH SUHSDUHG
ity. The degree of apoptosis is correlated with de IURP 5D]L 9DFFLQH DQG 6HUXP 5HVHDUFK ,QVWL
YHORSPHQWDO FRPSHWHQFH RI WKH HQFORVHG RRF\WHV WXWH ,UDQ $QLPDOV ZHUH PDLQWDLQHG DW D WHP
 ,W KDV EHHQ VXJJHVWHG WKDW PRGHUDWH DSRS SHUDWXUH RI Û& DQG  KXPLGLW\ XQGHU
totic changes in the follicle may support or induce OLJKWFRQWUROOHG FRQGLWLRQV  KRXUV OLJKW 
SUHPDWXUDWLRQOLNHFKDQJHVRIWKHRRF\WHZKLFKLV KRXUV GDUN DQG SURYLGHG ZLWK IRRG DQG ZDWHU
W\SLFDOIRUWKHLUSUHRYXODWRU\GHYHORSPHQW ad libitum.
+\GURVWDWLFSUHVVXUHLVWKHSUHVVXUHH[HUWHGE\D $IWHU WKH PLFH ZHUH VDFULILFHG WKHLU RYDULHV
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DQGJUDYLW\,WLQGHSHQGHQWRIVKDSHWRWDOPDVVRU to dissection medium that consisted of Alpha
VXUIDFHDUHDRIWKHÀXLG+\GURVWDWLFSUHVVXUH 0LQLPDO (VVHQWLDO 0HGLXP Į0(0 VXSSOH
DV D SK\VLFDO IRUFH SOD\V YDULRXV SK\VLRORJLFDO PHQWHG ZLWK  IHWDO ERYLQH VHUXP )%6
UROHVLQWKHUHSURGXFWLYHV\VWHP,QFUHDVHGLQWUD  ,8PO SHQLFLOOLQ * DQG  ȝJPO VWUHS
IROOLFXODU SUHVVXUH DQG VSRQWDQHRXV FRQWUDFWLRQV WRP\FLQ)ROOLFOHVZHUHLVRODWHGE\PHFKDQLFDO
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FRQVWDQW LQWUDIROOLFXODU SUHVVXUH EHWZHHQ  DQG VHOHFWHG DFFRUGLQJ WR WKH IROORZLQJ FULWHULD L
PP+JGXULQJWKHHQWLUHRYXODWRU\SURFHVV intact follicles with one or two layers of gran
 +\GURVWDWLF SUHVVXUH KDV EHHQ GHPRQVWUDWHG ulosa cells and some adhering theca cells; ii.
WRLQGXFHFHOOGHDWKLQGLIIHUHQWFHOOW\SHV YLVLEOH URXQG DQG FHQWUDO RRF\WH DQG LLL IRO
,QDSUHYLRXVVWXG\ZHKDYHVKRZQWKDWK\GURVWDWLF OLFOH GLDPHWHU EHWZHHQ  ȝP )ROOLFOHV
SUHVVXUH HQKDQFHG WKH ,90 RI WKH RRF\WHV IURP were rinsed three times in dissection medium
QRQYLWUL¿HGDQGYLWUL¿HGZDUPHGRYDULHVDQGLQ and transferred to culture medium that con
creased the incidence of cell death in cumulus cells sisted of dissection medium supplemented with
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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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ZH UHSODFHG  ȝO RI WKH FXOWXUH PHGLXP IURP ,90DQGILYHWLPHVIRUDVVHVVPHQWRIFHOOGHDWK
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
SXV -DSDQ )ROOLFOH DQG RRF\WH TXDQWLWDWLYH  )ROOLFOHV LQ ERWK JURXSV ZHUH FXOWXUHG LQ
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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To assess the rate of meiosis at the end of the
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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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(dUTP) nick-end labeling (TUNEL) were mounted in glycerol onto a slide and placed
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total cell number.
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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
$WDQGKRXUVYLDELOLW\RIWKHRRF\WHVZDVVLP WR WKH FRQWURO DW  DQG  KRXUV RI K\GUR
LODUEHWZHHQWKHH[SHULPHQWDQGFRQWUROJURXSV$W VWDWLF H[SRVXUH S 7KH SHUFHQWDJHV RI
KRXUVK\GURVWDWLFSUHVVXUHGLGQRWVLJQL¿FDQWO\DOWHU fragmented and condensed nuclear cumulus
WKHSHUFHQWDJHRI*9RRF\WHVZKHUHDVWKHSHUFHQWDJH FHOOV LQ QRQYLDEOH FHOOV ZHUH KLJKHU LQ WKH
RI*9%'DQG0,,RRF\WHVLQFUHDVHGLQWKHH[SHUL H[SHULPHQWDO JURXS FRPSDUHG WR WKH FRQWURO
PHQWJURXSFRPSDUHGWRWKHFRQWUROJURXSS JURXS S 7DEOH  &HOO PRUSKRORJ\
$WKRXUVWKHSHUFHQWDJHRI*9%'RRF\WHVZDV was scored as follows. Viable cells contained
VLPLODUEHWZHHQWKHH[SHULPHQWDQGFRQWUROJURXSV EOXHVWDLQHG QRUPDO VPRRWK QXFOHL RU PXO
ZKLOH WKH SHUFHQWDJH RI *9 RRF\WHV GHFUHDVHG LQ WLSOH EULJKW VSHFNV RI FKURPDWLQ 1RQYLDEOH
WKHH[SHULPHQWJURXSFRPSDUHGWRWKHFRQWUROJURXS FHOOV FRQVLVWHG RI SLQNVWDLQHG QXFOHL ZLWK
S7KHSHUFHQWDJHVRI0,,RRF\WHVDQG3$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.
'LIIHUHQWVXSHUVFULSWVLQGLFDWHVLJQL¿FDQWGLIIHUHQFHVS

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.

'LIIHUHQWVXSHUVFULSWVLQGLFDWHVLJQL¿FDQWGLIIHUHQFHVS

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

 Rashidi et al.

A B C

)LJ&HOOGHDWKLQ&2&VDVGHWHUPLQHGE\ÀXRUHVFHQFHPLFURVFRS\DIWHUKRXUVRISUHVVXUHH[SRVXUH
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.
6FDOH%DU$%ȝPDQG&ȝP

TUNEL labeling PHWDSKDVH RU DQDSKDVH VWDJHV ZLWK YLVLEOH

7KHDSRSWRWLFLQGH[ZDVKLJKHULQ&2&VIURP chromosomes counted as single nuclei; frag
WKH H[SHULPHQW JURXS FRPSDUHG ZLWK WKH FRQ PHQWHG QXFOHL ZKLFK LQFOXGHG GLVFUHWH FOXV
WUROJURXSDWDQGKRXUVRIK\GURVWDWLFH[ WHUV RI PHPEUDQHERXQGHG YHVLFOHV DQG FRQ
SRVXUHS7DEOH781(/UHDFWLRQZDV GHQVHG QXFOHL ZLWK LQWHQVH 3, VWDLQLQJ ZKLFK
DVVHVVHGE\WKHREVHUYDWLRQRIDGLVWLQFWEULJKW were smaller than 'healthy' interphase nuclei.
\HOORZVWDLQHGFKURPDWLQ1XFOHDUPRUSKRORJ\ $FFRUGLQJWRWKHDERYHFULWHULDWKHQXFOHLWKDW
ZDV DVVHVVHG RQ WKH EDVLV RI 3, VWDLQLQJ 7KH displayed morphological characteristics of apo
QXFOHL ZHUH FODVVL¿HG DFFRUGLQJ WR IRXU FOHDU ptosis (condensation and fragmentation) and bi
W\SHVRIPRUSKRORJ\KHDOWK\LQWHUSKDVHQXFOHL RFKHPLFDOFKDUDFWHULVWLFVRIDSRSWRVLV781(/
ZLWK XQLIRUP 3, VWDLQLQJ DQG D FOHDU RXWOLQH UHDFWLRQ SRVLWLYH ZHUH FRQVLGHUHG WR EH DSRS
PLWRVLV ZKLFK LQFOXGHG FHOOV DW WKH SURSKDVH WRWLFQXFOHL)LJ

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.

'LIIHUHQWVXSHUVFULSWVLQGLFDWHVLJQL¿FDQWGLIIHUHQFHS

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

Hydrostatic Pressure and Oocyte Maturation

A A'

B B'

)LJ,GHQWL¿FDWLRQRIDSRSWRVLVLQ&2&VIROORZLQJ781(/VWDLQLQJDQGFRXQWHUVWDLQLQJZLWKSURSLGLXPLRGLGH3,DIWHU
hours of pressure exposure.
$SRSWRWLFQXFOHLLGHQWL¿HGE\WKHREVHUYDWLRQRIDGLVWLQFWEULJKW\HOORZVWDLQHGFKURPDWLQ
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
PLOG FHOO GHDWK LQ FXPXOXV FHOOV LQ H[SHULPHQWDO OLTXLGSKDVHPRGHODVDQRSHQFXOWXUHV\VWHP
JURXSZLWKRXWDQ\DGYHUVHHIIHFWVRQWKHVXUYLYDO 1XWULHQWVKRUPRQHVDQGJDVHVDUHPRUHDYDLO
rate of oocytes. The YLDELOLW\ RI RRF\WHV GHULYHG DEOH LQ DQ RSHQ V\VWHP WKDQ LQ D FORVHG V\VWHP
from both groups was similar and independent of ZKLFKLQFUHDVHVRRF\WHVXUYLYDOUDWH)ROOLFOHVWKDW
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RI SDUWKHQRJHQHVLV LQFUHDVHG LQ RRF\WHV H[SRVHG OHQWWRDQWUDOIROOLFOHVLQGD\ROGPRXVHRYDULHV
to hydrostatic pressure. 7KLV WLPHSRLQW FRUUHVSRQGV WR WKH ¿UVW ZDYH RI

CELL JOURNAL(Yakhteh), Vol 15, No 4, Winter 2014 289

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meiotic maturation leadLQJWRRYXODWLRQ licular DQWUXPDIWHUZKLFKWKHSDVVLYHLQWUDIRO

,QWKHFXUUHQWH[SHULPHQWSUHDQWUDOIROOLFOHVLVR licular pressure was recorded. The main find
ODWHG IURP RYDULDQ WLVVXH JUHZ in vitro between ing of these studies showed that &2&V were
GD\VDQG)ROOLFOHJURZWKVORZHGVOLJKWO\DI H[SRVHG WR LQWUDIROOLFXODU SUHVVXUHV EHWZHHQ
WHUGD\EXWRRF\WHVFRQWLQXHGWRGHYHORSDQG  PP+J GXULQJ WKH HQWLUH RYXODWRU\ SUR
reached a diameter similar to that of fully grown FHVV,QDSUHYLRXVVWXG\ZHUHSRUWHGWKDW
oocytes in vivo. PP+JRIK\GURVWDWLFSUHVVXUHLQGXFHGPLOG
FHOOGHDWKLQFXPXOXVFHOOVGHFUHDVHGFHOOMXQF
5HVHDUFKHUVKDYHVXFFHVVIXOO\DFKLHYHG,90RI tions and waste paracrine correlation between
SUHDQWUDOIROOLFOHHQFORVHGRRF\WHVDQGRIRRF\WH FXPXOXVFHOOVDQGRRF\WHVDQGLQGXFHGPDWXUD
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EHHQSHUIRUPHGWRRSWLPL]Hin vitroFRQGLWLRQV
0,,RRF\WHVFRQVLGHUHGDVRRF\WHPDWXUDWLRQVLJ
which should imitate in vivoFRQGLWLRQV,Q
QL¿FDQWO\LQFUHDVHGLQIROOLFOHVH[SRVHGWRK\GUR
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K\GURVWDWLFSUHVVXUHV7KHPRVWLPSRU to hydrostatic pressure. These results indicated
tant biotic parameters are organic substances in WKDWK\GURVWDWLFSUHVVXUHLPSURYHGRRF\WHPDWXUD
PHGLD KRUPRQHV  DQG DFWLYDWRUV DQG LQ WLRQ&RQFRPLWDQWO\ZLWKLPSURYHGRRF\WHPDWX
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FRQWUDVW WR RWKHU SDUDPHWHUV DFWV LPPHGLDWHO\ &2&VGHULYHGIURPSUHRYXODWRU\IROOLFOHVin vitro.
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in vivo conditions. It has been reported that a FXPXOXVFHOOVGLVVRFLDWHGXULQJWKHRYXODWRU\SUR
ZHOOGHILQHG VXEOHWKDO KLJK K\GURVWDWLF SUHV FHVVUHOHDVLQJWKHRRF\WHLQWRWKHIROOLFXODUÀXLG
VXUHWUHDWPHQWRIIHUVDVROXWLRQWRLPSURYHWKH DQWUXP
RYHUDOO TXDOLW\ RI JDPHWHV DQG HPEU\RV IHUWL $ VWXG\ E\ ,NHGD HW DO  KDV GHPRQVWUDWHG
OL]LQJ DELOLW\ DQG GHYHORSPHQWDO FRPSHWHQFH WKDW FXPXOXV FHOOV LQ ERYLQH FXPXOXVHQFORVHG
 'X HW DO  KDYH VKRZQ WKDW SUHWUHDW
oocytes spontaneously underwent apoptosis dur
ment with a high hydrostatic pressure consid
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HUDEO\ LPSURYHG WKH ,93 RI SRUFLQH YLWULILHG
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oocytes.
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0DWRXVHN HW DO KDYH UHSRUWHG DQ LQFUHDVH LQ WRU\GHYHORSPHQW&XPXOXVFHOOVSOD\DQLP
LQWUDIROOLFXODU SUHVVXUH GXULQJ WKH RYXODWLQJ SRUWDQW UROH LQ RRF\WH PDWXUDWLRQ E\ NHHSLQJ WKH
SURFHVV  $ EDVDO LQWUDIROOLFXODU SUHVVXUH RRF\WHXQGHUPHLRWLFDUUHVWLQGXFLQJPHLRWLFUH
RI  “  PP+J ZDV UHSRUWHG DW WKH SUHR sumption and supporting cytoplasmic maturation
YXODWRU\ SKDVH  KRXUV DIWHU H&* ZKLFK LQ &XPXOXVFHOOVDQGRRF\WHVKDYHDUHODWLRQVKLS
FUHDVHGJUDGXDOO\WKURXJKRXWWKHRYXODWRU\SUR LQSUHRYXODWRU\IROOLFOHVWKDWGXHWRSDUDFULQHDQG
FHVVWR“PP+JDWKRXUVDIWHUK&* UHJXODWLRQ IDFWRUV FRQYHQLHQFH DYDLODEOH IRU RR
PLGRYXODWRU\SKDVHDQG“PP+JDW cyte. The signals that produced by cumulus cells
KRXUVDIWHUK&*7KHLQWUDIROOLFXODUSUHV HYHQZLWKZDVWHJDSMXQFWLRQFXPXOXVFHOOVDIIHFW
VXUHV KDYH EHHQ PHDVXUHG LQ WKH SUHRYXODWRU\ HGRQPDWXUDWLRQRIRRF\WH.
IROOLFOHV RI FRZV  KDPVWHUV  DQG UDE
bits (20). These measurements were obtained ,QWKHGHDGFHOOVVRPHRIWKHQXFOHLZHUHIUDJ
by inserting a large micropipette into the fol mented and condensed. The percentage of these

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Hydrostatic Pressure and Oocyte Maturation

types of nuclei in cumulus cells were increased in latory process. According to the results of this
K\GURVWDWLF SUHVVXUHWUHDWHG IROOLFOHV )UDJPHQWD VWXG\K\GURVWDWLFSUHVVXUHFDQEHXVHGWRLQFUHDVH
tion and condensation of nuclei are two mor the apoptosis rate of cumulus cells; the latter may
SKRORJLFDOIHDWXUHVRIDSRSWRWLFFHOOVWKHUHIRUH EH UHVSRQVLEOH IRU DQ LQFUHDVH LQ WKH 0,, RRF\WH
LQ WKH FXUUHQW VWXG\ WKH W\SH RI FHOO GHDWK RE UDWH DIWHU ,90 :H KDYH VKRZQ WKDW K\GURVWDWLF
VHUYHGLQFXPXOXVFHOOVZDVDSRSWRVLVDVFRQ pressure had a mild effect on the incidence of cell
ILUPHGE\781(/VWDLQLQJ,QYHVWLJDWLRQRIDS death in cumulus cells but no aberrant effect on
RSWRWLFFHOOGHDWKE\781(/VWDLQLQJKDVEHHQ RRF\WHYLDELOLW\
SHUIRUPHG LQ SUHYLRXV VWXGLHV   7KH
SHUFHQWDJH RI 781(/SRVLWLYH FHOOV ZDV FRQ Acknowledgements
sidered to be apoptotic cells that significantly :HZRXOGOLNHWRH[SUHVVRXUDSSUHFLDWLRQWRWKH
LQFUHDVHG LQ IROOLFOHV H[SRVHG WR K\GURVWDWLF (PEU\RORJ\5HVHDUFK/DERUDWRU\DQG'HSDUWPHQW
SUHVVXUH +\GURVWDWLF SUHVVXUH DV D FHOO GHDWK RI%LRORJ\DW5D]L8QLYHUVLW\7KHDXWKRUVZRXOG
LQGXFHUZLWKLQFUHDVLQJDSRSWRWLFFHOOV OLNHWRWKDQN5D]L8QLYHUVLW\IRULWV¿QDQFLDOVXS
LQ &2&V OHG WR LQFUHDVLQJ RRF\WH PDWXUDWLRQ SRUWRIWKLVSURMHFW7KHUHLVQRFRQÀLFWRILQWHUHVW
FRPSDUHGZLWKWKHJURXSWKDWKDGQRH[SRVXUH in this article.
to hydrostatic pressure.
+\GURVWDWLFSUHVVXUHLQFUHDVHGWKHSHUFHQWDJHRI References
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osmotic and hydrostatic pressure activates Dros- 365-369.
ophila oocytes in vitro in a calcium-dependent man-

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