*[FTQUVCVKE2TGUUWTG#ƑGEVU
In Vitro
Maturation of Oocytes
and Follicles and Increases Granulosa Cell Death
=DKUD5DVKLGL06F10HKUL$]DGEDNKW3K'1$OL$PLQL3K'1,VDF.DULPL3K'2
'HSDUWPHQWRI%LRORJ\)DFXOW\RI%DVLF6FLHQFHV5D]L8QLYHUVLW\.HUPDQVKDK,UDQ 'HSDUWPHQWRI%DVLF6FLHQFHV&ROOHJHRI9HWHULQDU\0HGLFLQH5D]L8QLYHUVLW\.HUPDQVKDK,UDQ
* Corresponding Address: P.O.Box: 6714967346, Department of Biology, Faculty of Basic Sciences, Razi University, Kermanshah, Iran
Email: azadbakhtm_tmu@yahoo.com
5HFHLYHG-XO$FFHSWHG-DQ
Abstract
2EMHFWLYHThis study examines the effects of hydrostatic pressure on in vitro maturation (IVM) of oocytes derived from in vitro grown follicles.
0DWHULDOVDQG0HWKRGV In this experimental study, preantral follicles were isolated from 12-day-old female NMRI mice. Each follicle was cultured individually in Alpha Minimal (VVHQWLDO0HGLXPĮ0(0XQGHUPLQHUDORLOIRUGD\V7KHQIROOLFOHVZHUHLQGXFHGIRU IVM and divided into two groups, control and experiment. In the experiment group follicles were subjected to 20 mmHg pressure for 30 minutes and cultured for 24-48 hours. We as-sessed for viability and IVM of the oocytes. The percentage of apoptosis in cumulus cells was determined by the TUNEL assay. A comparison between groups was made using the student’s t test.
5HVXOWV The percentage of metaphase II oocytes (MII) increased in hydrostatic pressure-treated follicles compared to controls (p<0.05). Cumulus cell viability reduced in hydro-static pressure-treated follicles compared to controls (p<0.05). Exposure of follicles to pressure increased apoptosis in cumulus cells compared to controls (p<0.05).
&RQFOXVLRQHydrostatic pressure, by inducing apoptosis in cumulus cells, participates in the cumulus oocyte coupled relationship with oocyte maturation.
Keywords:In vitro Maturation, Oocyte, Hydrostatic Pressure, Apoptosis, Mouse
Cell Journal(Yakhteh), Vol 15, No 4, Winter 2014, Pages: 282- 293
&LWDWLRQ5DVKLGL=$]DGEDNKW0$PLQL$.DULPL,+\GURVWDWLFSUHVVXUHDIIHFWVLQYLWURPDWXUDWLRQRIRRF\WHVDQG
IROOLFOHVDQGLQFUHDVHVJUDQXORVDFHOOGHDWK&HOO-Introduction
In vitroPDWXUDWLRQ,90RIPDPPDOLDQRRF\WHV LVDQHI¿FLHQWPHWKRGWRSURGXFHPDWXUHRRF\WHV IRUWKHLUXVHLQDVVLVWHGUHSURGXFWLYHWHFKQLTXHV ,QGXFWLRQRIRYXODWLRQWRREWDLQPDWXUHRRF\WHVIRU in vitro IHUWLOL]DWLRQ ,9) LV D URXWLQH SURFHGXUH LQQXPHURXVLQIHUWLOLW\FOLQLFV6RPHZRPHQKRZ HYHUPD\IDLOWRUHVSRQGWRKRUPRQDOVWLPXODWLRQ RUDUHDWULVNRIRYDULDQK\SHUVWLPXODWLRQ,90 RIRRF\WHVRIIHUVDQDOWHUQDWLYHVWUDWHJ\WRREWDLQ
mature ooF\WHVLQWKHVHFDVHV7KHIHUWLOLW\
rate from matured oocytes in vitro is much lower
than those of in vivoVWLPXODWLRQF\FOHVLQGLFDWLQJ
WKDWLPSURYHPHQWLQ,90UHPDLQVDFKDOOHQJH
6XSSOHPHQWDWLRQ RI WKH PHGLD ZLWK KRUPRQHV JURZWK IDFWRUV RSWLPL]DWLRQ RI FXOWXUH V\VWHPV DQG HQYLURQPHQWDO DQG SK\VLFDO FRQGLWLRQV RI IROOLFOHV KDYH EHHQ SURSRVHG WRLQFUHDVHTXDOLW\RI,90RRF\WHV,QWKLVVHQVH WKHODWHUIDFWRUVKDYHDQLPSRUWDQWUROHLQWKHVXF FHVVRI,903K\VLFDOIRUFHVDIIHFWIROOLFOHUXSWXUH DQGRYXODWLRQE\LQFUHDVLQJIROOLFXODUÀXLGSUHV
sure due to an increase in hydrostatic pressure in
WKHRYDULDQYDVFXODUV\VWHP$GHFUHDVHRIWHQVLOH
strength in the follicle wall and increase of the
tissue thinning and follicular rupture by elimina
WLRQRIVHOHFWLYHFXPXOXVFHOOV2QWKHRWKHU KDQG FXPXOXV FHOOV GLVVRFLDWH GXULQJ WKH RYXOD
tory process and the oocyte is freed into follicular
ÀXLG 3URJUDPPHG FHOO GHDWK SDUWLFLSDWHV LQ GH JHQHUDWLRQRIIROOLFXODUFHOOVZHDNHQVWKHIROOLFX
lar wall and ruptures. The amount of cell death in
WKHFXPXOXVRRF\WHFRPSOH[&2&VWKDWLPSDFWV RRF\WHGHYHORSPHQWSRWHQWLDOLVXQFOHDU'H YHORSPHQW RI IROOLFOHV DQG WKHLU UHODWHG &2&V LV LQÀXHQFHGE\YDULRXVDSRSWRWLFPHFKDQLVPV
The spatiotemporal pattern of apoptosis during follicle growth and oocyte maturation is tightly
UHJXODWHG'LVUXSWLRQRIHLWKHUWLPLQJRUWKH PDJQLWXGHRIDSRSWRVLVFDQDOWHUFHOOFRQQHFWLYLW\
in the cumulus mass and between cumulus cells
DQG WKH RRF\WH FDXVLQJ GH¿FLWV LQ RRF\WH TXDO
ity. The degree of apoptosis is correlated with de
YHORSPHQWDOFRPSHWHQFHRIWKHHQFORVHGRRF\WHV ,W KDV EHHQ VXJJHVWHG WKDW PRGHUDWH DSRS
totic changes in the follicle may support or induce
SUHPDWXUDWLRQOLNHFKDQJHVRIWKHRRF\WHZKLFKLV W\SLFDOIRUWKHLUSUHRYXODWRU\GHYHORSPHQW
+\GURVWDWLFSUHVVXUHLVWKHSUHVVXUHH[HUWHGE\D VWDWLFÀXLGWKDWGHSHQGVRQWKHÀXLG¶VGHSWKGHQVLW\ DQGJUDYLW\,WLQGHSHQGHQWRIVKDSHWRWDOPDVVRU VXUIDFHDUHDRIWKHÀXLG+\GURVWDWLFSUHVVXUH DV D SK\VLFDO IRUFH SOD\V YDULRXV SK\VLRORJLFDO UROHVLQWKHUHSURGXFWLYHV\VWHP,QFUHDVHGLQWUD IROOLFXODU SUHVVXUH DQG VSRQWDQHRXV FRQWUDFWLRQV WRJHWKHU ZLWK WKH HQ]\PDWLF GHJUDGDWLRQ RI WKH H[WUDFHOOXODUPDWUL[PD\EHLPSRUWDQWIRUUXSWXUH RIWKHIROOLFOHDWRYXODWLRQ7KHPDLQ¿QGLQJ RISUHYLRXVVWXGLHVLVWKHSUHVHQFHRIDUHODWLYHO\ FRQVWDQW LQWUDIROOLFXODUSUHVVXUH EHWZHHQ DQG PP+JGXULQJWKHHQWLUHRYXODWRU\SURFHVV +\GURVWDWLF SUHVVXUH KDV EHHQ GHPRQVWUDWHG WRLQGXFHFHOOGHDWKLQGLIIHUHQWFHOOW\SHV ,QDSUHYLRXVVWXG\ZHKDYHVKRZQWKDWK\GURVWDWLF SUHVVXUH HQKDQFHG WKH ,90 RI WKH RRF\WHV IURP QRQYLWUL¿HGDQGYLWUL¿HGZDUPHGRYDULHVDQGLQ
creased the incidence of cell death in cumulus cells
ZLWKRXWDVLJQRIFHOOGHDWKLQPRXVHRRF\WHV :HWRRNLQWRFRQVLGHUDWLRQWKHH[SRVXUHRI&2&V WRLQWUDIROOLFXODUSUHVVXUHLQWKHRYXODWLQJIROOLFOHV GXULQJWKHODWHRYXODWRU\SURFHVVDQGK\GURVWDWLF SUHVVXUHDVDFHOOGHDWKLQGXFHU7KXVZHGHVLJQHG WKHSUHVHQWVWXG\WRLQYHVWLJDWHWKHHIIHFWRIK\GUR VWDWLFSUHVVXUHRQLQGXFLQJFHOOGHDWKLQ&2&VDQG WRLPSURYHRRF\WH,90RIRRF\WHVGHULYHGIURPin
vitroJURZQIROOLFOHV,QWKHSUHVHQWVWXG\K\GUR VWDWLFSUHVVXUHZDVXVHGWRLQYHVWLJDWHWKHLQYROYH PHQW RI &2& FHOO GHDWK RQ WKH ,90 RI RRF\WHV GHULYHGIURPin vitro grown follicles.
Materials and Methods
&KHPLFDOVZHUHSXUFKDVHGIURP6LJPD&KHPL FDO&R6W/RXLV0286$DQG*LEFR*UDQG ,VODQG1<86$XQOHVVRWKHUZLVHLQGLFDWHG
Ovarian follicle recovery and culture
7KLV H[SHULPHQWDO VWXG\ ZDV UHYLHZHG DQG DSSURYHGE\WKH/DERUDWRU\$QLPDO&DUH&RP PLWWHH RI WKH )DFXOW\ RI %DVLF 6FLHQFHV 5D]L 8QLYHUVLW\.HUPDQVKDK,UDQ,QWKLVVWXG\ GD\ROG IHPDOH 105, PLFH ZHUH SUHSDUHG IURP 5D]L 9DFFLQH DQG 6HUXP 5HVHDUFK ,QVWL WXWH ,UDQ$QLPDOV ZHUH PDLQWDLQHG DW D WHP SHUDWXUH RI Û& DQG KXPLGLW\ XQGHU OLJKWFRQWUROOHG FRQGLWLRQV KRXUV OLJKW KRXUVGDUNDQGSURYLGHGZLWKIRRGDQGZDWHU
ad libitum.
$IWHU WKH PLFH ZHUH VDFULILFHG WKHLU RYDULHV ZHUH UHPRYHG DQG LPPHGLDWHO\ WUDQVIHUUHG
to dissection medium that consisted of Alpha
0LQLPDO (VVHQWLDO 0HGLXP Į0(0 VXSSOH PHQWHG ZLWK IHWDO ERYLQH VHUXP )%6 ,8PO SHQLFLOOLQ * DQG ȝJPO VWUHS WRP\FLQ)ROOLFOHVZHUHLVRODWHGE\PHFKDQLFDO GLVVHFWLRQ XQGHU D VWHUHRPLFURVFRSH 0RWLF 60= &KLQD DW [ PDJQLILFDWLRQ XVLQJ * VWHULOH QHHGOHV WR HQVXUH WKDW WKH IROOL FXODU VWUXFWXUH UHPDLQHG LQWDFW )ROOLFOHV ZHUH VHOHFWHG DFFRUGLQJ WR WKH IROORZLQJ FULWHULD L
intact follicles with one or two layers of gran
ulosa cells and some adhering theca cells; ii.
YLVLEOH URXQG DQG FHQWUDO RRF\WH DQG LLL IRO OLFOH GLDPHWHU EHWZHHQ ȝP )ROOLFOHV
were rinsed three times in dissection medium
and transferred to culture medium that con
sisted of dissection medium supplemented with
)DOFRQ)UDQFHDWÛ&8QGHUDQDWPRVSKHUH RI&22LQDLUIRUGD\V,QDGGLWLRQWKH PHGLXPZDVHTXLOLEUDWHGRYHUQLJKWSULRUWRWKH VWDUWRIFXOWXULQJ$WHYHU\KRXUVRIFXOWXUH ZHUHSODFHGȝORIWKHFXOWXUHPHGLXPIURP
each drop with fresh medium.
Follicle monitoring and measurement
'XULQJ WKH FXOWXUH SHULRG IROOLFOHV ZHUH PRQL WRUHGGDLO\XQGHUDQLQYHUWHGPLFURVFRSH2O\P SXV -DSDQ )ROOLFOH DQG RRF\WH TXDQWLWDWLYH
measurement of morphological features were
SHUIRUPHG DFFRUGLQJ WR D SUHYLRXVO\ GHVFULEHG PHWKRGZLWKPRGL¿FDWLRQV)RUHDFKIROOLFOH
two perpendicular diameters were measured us
LQJDFDOLEUDWHGRFXODUPLFURPHWHU'LQR'LJLWDO (\HSLHFH $0 7DLZDQ DW D PDJQL¿FDWLRQ RIîEHIRUHFXOWXUHDQGWKHQRQGD\V DQGRIFXOWXUH)ROOLFOHGLDPHWHUZDVUHFRUGHG LQPLFURPHWHUV6SLQGOHVKDSHGWKHFDFHOOVZKLFK
originated from the follicle theca and attached to the dish were not included in the measurements. In
DGGLWLRQ WKH VDPH PHDVXUHPHQW IRU HDFK RRF\WH ZDV FDOFXODWHG$IWHU WKH RRF\WHV ZHUH UHWULHYHG IURP WKH IROOLFOHV ZH UHFRUGHG WKHLU GLDPHWHUV IURPWKHRXWHUOD\HURIWKH]RQHRQRQHVLGHWRWKH RXWHUOD\HURIWKH]RQHRQWKHRSSRVLWHVLGHDORQJ
with the longest length and widest perpendicular width.
Experimental protocol
2Q GD\ ZH FKRVH JRRG TXDOLW\ IROOLFOHV
which were defined as intact follicles with a central oocyte surrounded by a granulosa cell
PDVVSHULSKHUDOVSLQGOHVKDSHGWKHFDFHOOPRQ ROD\HU YLVLEOH URXQG DQG FHQWUDO RRF\WH DQG IROOLFOHGLDPHWHUP)ROOLFOHVZHUHDOOR
cated and placed in culture medium randomly
DQG GLYLGHG LQWR WZR JURXSV H[SHULPHQW DQG FRQWURO,QWKHH[SHULPHQWJURXSIROOLFOHVZHUH
transferred to a pressure chamber according to
DQHVWDEOLVKHGPRGHODIWHUZKLFKWKH\ZHUH VXEMHFWHGWRPP+JK\GURVWDWLFSUHVVXUHIRU PLQXWHV)ROOLFOHVLQWKHFRQWUROJURXSZHUH WUDQVIHUUHGWRDVLPLODUSUHVVXUHFKDPEHUIRU PLQXWHVEXWZHUHQRWH[SRVHGWRDQ\K\GURVWDW LFSUHVVXUH$IWHUGHSUHVVXUL]DWLRQWKHFXOWXUH SODWHV ZHUH UHPRYHG IURP WKH SUHVVXUH FKDP EHU DQG FXOWXUHG IRU KRXUV$IWHU KRXUVZHDVVHVVHGWKH,90RIRRF\WHVGHULYHG
from in vitro grown follicles as well as the cell
death incidence and apoptosis in the cumulus
RRF\WHFRPSOH[HV&2&V7KHH[SHULPHQWZDV UHSHDWHG DW OHDVW QLQH WLPHV IRU HYDOXDWLRQ RI ,90DQGILYHWLPHVIRUDVVHVVPHQWRIFHOOGHDWK
and apoptosis.
In vitro maturation of oocytes
,90ZDVSHUIRUPHGDFFRUGLQJWRDSUHYLRXV
ly described method with some modifications
)ROOLFOHVLQERWKJURXSVZHUHFXOWXUHGLQ FXOWXUH PHGLXP VXSSOHPHQWHG ZLWK ,8PO KXPDQ FKRULRQLF JRQDGRWURSLQ K&* 6HUHQR ,QF$IWHUDQGKRXUVLQFXOWXUHZHHYDOX DWHGWKHRRF\WHVIRUQXFOHDUPDWXUDWLRQDQGYL
ability.
Evaluation of nuclear maturation
To assess the rate of meiosis at the end of the
PDWXUDWLRQ SHULRG IROOLFOHV ZHUH PHFKDQLFDOO\ UXSWXUHGRRF\WHVZHUHGHQXGHGDQGWKHLUQXFOHDU PDWXUDWLRQVWDWXVDVVHVVHGWRREVHUYHIRUWKHSUHV HQFHRIJHUPLQDOYHVLFOHV*9JHUPLQDOYHVLFOH EUHDNGRZQ*9%'PHWDSKDVH,,0,,DQGSDU WKHQRJHQHWLFHPEU\RV3$XQGHUDQLQYHUWHGPL FURVFRSH2O\PSXV,;-DSDQ
Oocyte viability
In this assessment the follicles were mechani
FDOO\UXSWXUHGDQGWKHRRF\WHVZHUHGHQXGHGDIWHU ZKLFKWKHLUYLDELOLW\ZDVDVVHVVHG2RF\WHVZHUH LQFXEDWHG LQ O RI JPO SURSLGLXP LR GLGH3,LQĮ0(0IRUVHFRQGV2RF\WHVZHUH ULQVHGLQ3%6DQGREVHUYHGXQGHULQYHUWHGPLFUR VFRSHHTXLSSHGZLWKDQXOWUDYLROHWODPSDWQP 2O\PSXV-DSDQ
Hoechst/propidium iodide nuclear staining of cumulus oocyte complexes
+RHFKVWSURSLGLXPLRGLGH3,QXFOHDUVWDLQ LQJ LV URXWLQHO\ XVHG IRU TXDQWLWDWLYH DQDO\VLV RI FHOO GHDWK 6XSUDYLWDO QXFOHDU VWDLQLQJ RI &2&V ZDV SHUIRUPHG DFFRUGLQJ WR WKH PHWK RG GHVFULEHG SUHYLRXVO\ ZLWK VOLJKW PRGLILFD WLRQV %ULHIO\ DW KRXUV DIWHU ,90 WKH &2&V ZHUH LQFXEDWHG ZLWK WKH FHOOSHUPHDQW G\H ELVEHQ]DPLGH +RHFKVW J PO LQ Į0(0 IRU PLQXWHV DW Û& 1H[W
LQWR WKH FHOOLPSHUPHDQW G\H 3, JPO LQ Į0(0 MXVW EHIRUH PLFURVFRS\ 6WDLQHG &2&V ZHUH VXEVHTXHQWO\ PRXQWHG LQ JO\FHURO JHQWO\ ÀDWWHQHGZLWKDFRYHUVOLSDQGYLVXDOL]HGIRUFHOO FRXQWLQJRQDÀXRUHVFHQFHPLFURVFRSH2O\PSXV $;-DSDQZLWKH[FLWDWLRQ¿OWHUVDWQPIRU
blue and red fluorescence.
Terminal deoxy-nucleotidyl transferase-mediated (dUTP) nick-end labeling (TUNEL)
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Û&7KHQ&2&VZHUHSHUPH DELOL]HG LQ ȝO GURSV RI YY 7ULWRQ ;WKDWFRQWDLQHGZY1DFLWUDWHLQ 3%6IRUPLQXWHVDWURRPWHPSHUDWXUH1H[W &2&V ZHUH ZDVKHG WKUHH WLPHV LQ 3%6 7KH &2&V ZHUH SODFHG LQ ȝO GURSV RI 781(/ UHDJHQW WKDW FRQWDLQHG ÀXRUHVFHLQ LVRWKLRF\ DQDWHFRQMXJDWHGG873DQGWKHHQ]\PHWHUPL QDO GHR[\QXFOHRWLG\O WUDQVIHUDVH DV SUHSDUHG E\ WKH PDQXIDFWXUHU WKHQ LQFXEDWHG LQ WKH GDUNIRUKRXUDWÛ&LQDKXPLGLILHGFKDP
EHU7KHQHJDWLYHFRQWUROVZHUHLQFXEDWHGLQWKH DEVHQFHRIWHUPLQDOGHR[\QXFOHRWLG\OWUDQVIHUDVH 7KHSRVLWLYHFRQWUROZDVLQFXEDWHGZLWK8 PO'1DVH,VROXWLRQIRUPLQXWHVDWÛ&WKHQ ULQVHGZLWK3%6&2&VZHUHZDVKHGLQ3%6393 DQGWUDQVIHUUHGWRȝOGURSVRIȝJPO3,LQ 3%6393 IRU PLQXWHV LQ D GDUN FKDPEHU DW URRP WHPSHUDWXUH7KH &2&V ZHUH ZDVKHG IRXU WLPHVLQ3%6393WRUHPRYHH[FHVV3,WKHQWKH\
were mounted in glycerol onto a slide and placed
XQGHUDFRYHUVOLS7KH&2&VZHUHREVHUYHGXQGHU DÀXRUHVFHQWPLFURVFRSH2O\PSXV$;-DSDQ 7KHDSRSWRWLFLQGH[RIWKH&2&VZDVFDOFXODWHG DVWKHSHUFHQWDJHRIDSRSWRWLFFHOOVUHODWLYHWRWKH
total cell number.
Statistical analysis
'DWDIRURRF\WHYLDELOLW\DQG,90WKHPHDQVRI &2&FHOOGHDWKDQGDSRSWRWLFLQGH[LQWKH&2&V ZHUHDQDO\]HGE\WKHVWXGHQW¶VWWHVW)RUWKHVWD WLVWLFDODQDO\VLVZHXWLOL]HG6366YHUVLRQVRIW ZDUH 'DWD ZHUH H[SUHVVHG DV PHDQ 6(0 DQG SZDVFRQVLGHUHGDVDPLQLPXPFULWHULRQIRU DVVLJQLQJVWDWLVWLFDOVLJQL¿FDQFH
Results
Follicle and oocyte measurement
3UHDQWUDOIROOLFXODUGLDPHWHULQFUHDVHGGXULQJin vitroFXOWXUH*UDQXORVDDQGWKHFDOFHOORXWJURZWK ZHUHSURPLQHQWDQGWKHDQWUDOFDYLWLHVZHUHYLVX DOL]HGDVFOHDUFDYLWLHVLQIROOLFOHVIURPGD\)LJ 7KHGLDPHWHUVRIWKHFXOWXUHGIROOLFOHVDWGLIIHU HQWGD\VRIFXOWXUHDQGDUHVKRZQLQ WDEOHS
Table 1: Follicle and oocyte diameters during culture
Day 12 Day 9
Day 6 Day 3
Day 1 N
Follicle diameter
Oocyte diameter
7ZRSHUSHQGLFXODUVZHUHPHDVXUHGDWîPDJQL¿FDWLRQDQGLVFDOFXODWHGEDVHGRQPLFURPHWHUV
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
B
C D
A
Viability and in vitro maturation of oocytes $WDQGKRXUVYLDELOLW\RIWKHRRF\WHVZDVVLP LODUEHWZHHQWKHH[SHULPHQWDQGFRQWUROJURXSV$W KRXUVK\GURVWDWLFSUHVVXUHGLGQRWVLJQL¿FDQWO\DOWHU WKHSHUFHQWDJHRI*9RRF\WHVZKHUHDVWKHSHUFHQWDJH RI*9%'DQG0,,RRF\WHVLQFUHDVHGLQWKHH[SHUL PHQWJURXSFRPSDUHGWRWKHFRQWUROJURXSS $WKRXUVWKHSHUFHQWDJHRI*9%'RRF\WHVZDV VLPLODUEHWZHHQWKHH[SHULPHQWDQGFRQWUROJURXSV ZKLOH WKH SHUFHQWDJH RI *9 RRF\WHV GHFUHDVHG LQ WKHH[SHULPHQWJURXSFRPSDUHGWRWKHFRQWUROJURXS S7KHSHUFHQWDJHVRI0,,RRF\WHVDQG3$HP EU\RVLQFUHDVHGLQWKHH[SHULPHQWJURXSFRPSDUHGWR WKHFRQWUROJURXSS7DEOH
Nuclear staining of the cumulus oocyte complexes
7KHSHUFHQWDJHRIYLDEOHFHOOVZDVORZHULQ
&2&V IURP WKH H[SHULPHQW JURXS FRPSDUHG WR WKH FRQWURO DW DQG KRXUV RI K\GUR VWDWLF H[SRVXUH S 7KH SHUFHQWDJHV RI
fragmented and condensed nuclear cumulus
FHOOV LQ QRQYLDEOH FHOOV ZHUH KLJKHU LQ WKH H[SHULPHQWDO JURXS FRPSDUHG WR WKH FRQWURO 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 HLWKHU PXOWLSOH EULJKW VSHFNV RI IUDJPHQWHG
chromatin which included discrete clusters of
PHPEUDQHERXQGHGYHVLFOHVDQGRQHRUPRUH
spheres of condensed chromatin (significantly more compact and smaller than normal nuclei) as seen in figure 2.
Table 2: Viability and IVM of oocytes derived from in vitro grown follicles
48 hours 24 hours PA MII GVBD GV PA MII GVBD GV Viability N Groups a a a 22 a a a a a Control b b a b b b b a Experiment
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
24 hours 0 hour Non-viable cells Viable cells Total cells Non-viable cells Viable cells Total cells N Groups a a a a a a Control b a b b b a Experiment
Control group; No pressure exposure and experiment group; Exposure to pressure.
)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
A B C
TUNEL labeling
7KHDSRSWRWLFLQGH[ZDVKLJKHULQ&2&VIURP WKH H[SHULPHQW JURXS FRPSDUHG ZLWK WKH FRQ WUROJURXSDWDQGKRXUVRIK\GURVWDWLFH[ SRVXUHS7DEOH781(/UHDFWLRQZDV DVVHVVHGE\WKHREVHUYDWLRQRIDGLVWLQFWEULJKW \HOORZVWDLQHGFKURPDWLQ1XFOHDUPRUSKRORJ\ ZDV DVVHVVHG RQ WKH EDVLV RI 3, VWDLQLQJ 7KH QXFOHL ZHUH FODVVL¿HG DFFRUGLQJ WR IRXU FOHDU W\SHVRIPRUSKRORJ\KHDOWK\LQWHUSKDVHQXFOHL ZLWK XQLIRUP 3, VWDLQLQJ DQG D FOHDU RXWOLQH PLWRVLV ZKLFK LQFOXGHG FHOOV DW WKH SURSKDVH
PHWDSKDVH RU DQDSKDVH VWDJHV ZLWK YLVLEOH
chromosomes counted as single nuclei; frag
PHQWHG QXFOHL ZKLFK LQFOXGHG GLVFUHWH FOXV WHUV RI PHPEUDQHERXQGHG YHVLFOHV DQG FRQ GHQVHG QXFOHL ZLWK LQWHQVH 3, VWDLQLQJ ZKLFK
were smaller than 'healthy' interphase nuclei.
$FFRUGLQJWRWKHDERYHFULWHULDWKHQXFOHLWKDW
displayed morphological characteristics of apo
ptosis (condensation and fragmentation) and bi
RFKHPLFDOFKDUDFWHULVWLFVRIDSRSWRVLV781(/ UHDFWLRQSRVLWLYHZHUHFRQVLGHUHGWREHDSRS WRWLFQXFOHL)LJ
Table 4: Apoptosis in COCs derived from in vitro grown follicles
24 hours 0 hour
Apoptotic index Total cells
Apoptotic index Total cells
N Groups
a
a
a
a
Control
b
a
b
a
Experiment
Control group; No pressure exposure and experiment group; Exposure to pressure.
)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[SRVHGWRSUHVVXUH6FDOHEDUP
A
B
A'
B'
Discussion
7KH SUHVHQW VWXG\ LQGLFDWHG WKDW WKH ,90 UDWH LQRRF\WHVGHULYHGIURPSUHRYXODWRU\IROOLFOHVin vitroLQFUHDVHGIROORZLQJH[SRVXUHWRK\GURVWDWLF
pressure. The hydrostatic pressure increased the
PLOGFHOOGHDWKLQFXPXOXVFHOOVLQH[SHULPHQWDO JURXSZLWKRXWDQ\DGYHUVHHIIHFWVRQWKHVXUYLYDO
rate of oocytes. The YLDELOLW\ RI RRF\WHV GHULYHG
from both groups was similar and independent of
H[SRVXUHWRK\GURVWDWLFSUHVVXUH7KHSHUFHQWDJH RI SDUWKHQRJHQHVLV LQFUHDVHG LQ RRF\WHV H[SRVHG
to hydrostatic pressure.
meiotic maturation leadLQJWRRYXODWLRQ
,QWKHFXUUHQWH[SHULPHQWSUHDQWUDOIROOLFOHVLVR ODWHG IURP RYDULDQ WLVVXH JUHZin vitro between
GD\VDQG)ROOLFOHJURZWKVORZHGVOLJKWO\DI WHUGD\EXWRRF\WHVFRQWLQXHGWRGHYHORSDQG
reached a diameter similar to that of fully grown
oocytes in vivo.
5HVHDUFKHUVKDYHVXFFHVVIXOO\DFKLHYHG,90RI SUHDQWUDOIROOLFOHHQFORVHGRRF\WHVDQGRIRRF\WH JUDQXORVDFHOOFRPSOH[HVIURPSUHDQWUDOIROOLFOHV REWDLQHGIURPPRXVHRYDULHV
3K\VLRORJLFDOO\ WKLV PDWXUDWLRQ SURFHVV LV GHSHQGHQW XSRQ VHYHUDO ELRWLFDO DQG DELRWL FDO SDUDPHWHUV 1XPHURXV H[SHULPHQWV KDYH EHHQSHUIRUPHGWRRSWLPL]Hin vitroFRQGLWLRQV
which should imitate in vivoFRQGLWLRQV,Q
WKHVHFLUFXPVWDQFHVDELRWLFSDUDPHWHUVFRQVLVW RIWHPSHUDWXUHS+DQGRVPRWLFDQG K\GURVWDWLFSUHVVXUHV7KHPRVWLPSRU
tant biotic parameters are organic substances in
PHGLD KRUPRQHV DQG DFWLYDWRUV DQG LQ KLELWRUV RI ,90 +\GURVWDWLF SUHVVXUH LQ FRQWUDVW WR RWKHU SDUDPHWHUV DFWV LPPHGLDWHO\
and uniformly at each point of the in vitro pro
GXFWLRQ,93,WFDQEHDSSOLHGZLWKWKHKLJKHVW SUHFLVLRQFRQVLVWHQF\DQGUHOLDELOLW\WRPLPLF in vivo conditions. It has been reported that a ZHOOGHILQHG VXEOHWKDO KLJK K\GURVWDWLF SUHV VXUHWUHDWPHQWRIIHUVDVROXWLRQWRLPSURYHWKH RYHUDOO TXDOLW\ RI JDPHWHV DQG HPEU\RV IHUWL OL]LQJ DELOLW\ DQG GHYHORSPHQWDO FRPSHWHQFH 'XHWDOKDYHVKRZQWKDWSUHWUHDW
ment with a high hydrostatic pressure consid
HUDEO\ LPSURYHG WKH ,93 RI SRUFLQH YLWULILHG
oocytes.
0DWRXVHNHWDOKDYHUHSRUWHGDQLQFUHDVHLQ LQWUDIROOLFXODU SUHVVXUH GXULQJ WKH RYXODWLQJ SURFHVV $ EDVDO LQWUDIROOLFXODU SUHVVXUH RIPP+JZDVUHSRUWHGDWWKHSUHR YXODWRU\SKDVHKRXUVDIWHUH&*ZKLFKLQ FUHDVHGJUDGXDOO\WKURXJKRXWWKHRYXODWRU\SUR FHVVWRPP+JDWKRXUVDIWHUK&* PLGRYXODWRU\SKDVHDQGPP+JDW KRXUVDIWHUK&*7KHLQWUDIROOLFXODUSUHV VXUHV KDYH EHHQ PHDVXUHG LQ WKH SUHRYXODWRU\ IROOLFOHV RI FRZV KDPVWHUV DQG UDE
bits (20). These measurements were obtained
by inserting a large micropipette into the fol
licular DQWUXPDIWHUZKLFKWKHSDVVLYHLQWUDIRO
licular pressure was recorded. The main find
ing of these studies showed that &2&V were
H[SRVHG WR LQWUDIROOLFXODU SUHVVXUHV EHWZHHQ PP+J GXULQJ WKH HQWLUH RYXODWRU\ SUR FHVV,QDSUHYLRXVVWXG\ZHUHSRUWHGWKDW PP+JRIK\GURVWDWLFSUHVVXUHLQGXFHGPLOG FHOOGHDWKLQFXPXOXVFHOOVGHFUHDVHGFHOOMXQF
tions and waste paracrine correlation between
FXPXOXVFHOOVDQGRRF\WHVDQGLQGXFHGPDWXUD WLRQ RI RRF\WHV GHULYHG IURP YLWULILHGZDUPHG PRXVHRYDULHV
$FFRUGLQJWRWKHUHVXOWVRIWKHDERYHPHQWLRQHG REVHUYDWLRQVZHVHOHFWHGDSUHVVXUHRIPP+J IRU WKH SUHVHQW LQYHVWLJDWLRQ 7KH SHUFHQWDJH RI 0,,RRF\WHVFRQVLGHUHGDVRRF\WHPDWXUDWLRQVLJ QL¿FDQWO\LQFUHDVHGLQIROOLFOHVH[SRVHGWRK\GUR VWDWLFSUHVVXUHFRPSDUHGWRWKRVHWKDWXQH[SRVHG
to hydrostatic pressure. These results indicated
WKDWK\GURVWDWLFSUHVVXUHLPSURYHGRRF\WHPDWXUD WLRQ&RQFRPLWDQWO\ZLWKLPSURYHGRRF\WHPDWX UDWLRQK\GURVWDWLFSUHVVXUHLQFUHDVHGFHOOGHDWKLQ &2&VGHULYHGIURPSUHRYXODWRU\IROOLFOHVin vitro. +\GURVWDWLF SUHVVXUH LQFUHDVHG FHOO GHDWK LQ FX PXOXVFHOOVZKLFKKDYHDFULWLFDOUROHLQRRF\WH PDWXUDWLRQ DQG IHUWLOL]DWLRQ 2Q WKH RWKHU KDQG FXPXOXVFHOOVGLVVRFLDWHGXULQJWKHRYXODWRU\SUR FHVVUHOHDVLQJWKHRRF\WHLQWRWKHIROOLFXODUÀXLG DQWUXP
$ VWXG\ E\ ,NHGD HW DO KDV GHPRQVWUDWHG WKDW FXPXOXV FHOOV LQ ERYLQH FXPXOXVHQFORVHG
oocytes spontaneously underwent apoptosis dur
LQJ,90$SRSWRWLFFKDQJHVLQWKHIROOLFOHSRVVL EO\VXSSRUWRULQGXFHSUHPDWXUDWLRQOLNHFKDQJHV WRWKHRRF\WHZKLFKLVW\SLFDOIRUWKHLUSUHRYXOD WRU\GHYHORSPHQW&XPXOXVFHOOVSOD\DQLP SRUWDQWUROHLQRRF\WHPDWXUDWLRQE\NHHSLQJWKH RRF\WHXQGHUPHLRWLFDUUHVWLQGXFLQJPHLRWLFUH
sumption and supporting cytoplasmic maturation
&XPXOXVFHOOVDQGRRF\WHVKDYHDUHODWLRQVKLS LQSUHRYXODWRU\IROOLFOHVWKDWGXHWRSDUDFULQHDQG UHJXODWLRQ IDFWRUV FRQYHQLHQFH DYDLODEOH IRU RR
cyte. The signals that produced by cumulus cells
HYHQZLWKZDVWHJDSMXQFWLRQFXPXOXVFHOOVDIIHFW
HGRQPDWXUDWLRQRIRRF\WH.
,QWKHGHDGFHOOVVRPHRIWKHQXFOHLZHUHIUDJ
types of nuclei in cumulus cells were increased in
K\GURVWDWLF SUHVVXUHWUHDWHG IROOLFOHV )UDJPHQWD
tion and condensation of nuclei are two mor
SKRORJLFDOIHDWXUHVRIDSRSWRWLFFHOOVWKHUHIRUH LQWKHFXUUHQWVWXG\WKHW\SHRIFHOOGHDWKRE VHUYHGLQFXPXOXVFHOOVZDVDSRSWRVLVDVFRQ ILUPHGE\781(/VWDLQLQJ,QYHVWLJDWLRQRIDS RSWRWLFFHOOGHDWKE\781(/VWDLQLQJKDVEHHQ SHUIRUPHG LQ SUHYLRXV VWXGLHV 7KH SHUFHQWDJH RI 781(/SRVLWLYH FHOOV ZDV FRQ
sidered to be apoptotic cells that significantly
LQFUHDVHG LQ IROOLFOHV H[SRVHG WR K\GURVWDWLF SUHVVXUH +\GURVWDWLF SUHVVXUH DV D FHOO GHDWK LQGXFHUZLWKLQFUHDVLQJDSRSWRWLFFHOOV LQ &2&V OHG WR LQFUHDVLQJ RRF\WH PDWXUDWLRQ FRPSDUHGZLWKWKHJURXSWKDWKDGQRH[SRVXUH
to hydrostatic pressure.
+\GURVWDWLFSUHVVXUHLQFUHDVHGWKHSHUFHQWDJHRI SDUWKHQRJHQHWLF RRF\WHV DV UHSRUWHG LQ SUHYLRXV VWXGLHV2XUGDWDLQGLFDWHGWKDWWKHSHU 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
7KHUHLVLQFUHDVLQJHYLGHQFHWKDWK\GURVWDWLF
pressure plays important roles in cell shape and
VWUXFWXUHH[RF\WRVLVDQGJURZWKDQGGHDWKRI DQLPDO FHOOV $OWKRXJK UHSURGXFWLYH ELRORJ\
has been dominated by a focus on genes and
FKHPLFDOLQWHUDFWLRQVRYHUWKHSDVWFHQWXU\LWLV WLPHWRIXUWKHUH[SORUHWKHPHFKDQLVPE\ZKLFK PHFKDQLFDOIRUFHVFDQH[HUWWKHLUSRWHQWHIIHFWV RQ JDPHWHV DQG HPEU\RV GXULQJ UHSURGXFWLRQ DV ZHOO DV WKURXJKRXW DGXOW OLIH 3URDS
optotic effects of hydrostatic pressure and the
SLYRWDOUROHRIDSRSWRVLVLQRYXODWLRQSURPSWXV WRLQYHVWLJDWHWKHHIIHFWVRIK\GURVWDWLFSUHVVXUH RQWKH,90RIPRXVHRRF\WHVDQGRQDSRSWRVLV LQ&2&VIURPRYDULDQIROOLFOHV
Conclusion
7KLV VWXG\ LPSOLFLWO\ H[SODLQV D PRGHO V\VWHP WRGHYHORSDQXQGHUVWDQGLQJRIWKHOLQNEHWZHHQ WKH SK\VLFDO FRQGLWLRQ RI D IROOLFOH DQG WKH RYX
latory process. According to the results of this
VWXG\K\GURVWDWLFSUHVVXUHFDQEHXVHGWRLQFUHDVH
the apoptosis rate of cumulus cells; the latter may
EHUHVSRQVLEOHIRUDQLQFUHDVHLQWKH0,,RRF\WH UDWH DIWHU ,90 :H KDYH VKRZQ WKDW K\GURVWDWLF
pressure had a mild effect on the incidence of cell death in cumulus cells but no aberrant effect on
RRF\WHYLDELOLW\
Acknowledgements
:HZRXOGOLNHWRH[SUHVVRXUDSSUHFLDWLRQWRWKH (PEU\RORJ\5HVHDUFK/DERUDWRU\DQG'HSDUWPHQW RI%LRORJ\DW5D]L8QLYHUVLW\7KHDXWKRUVZRXOG OLNHWRWKDQN5D]L8QLYHUVLW\IRULWV¿QDQFLDOVXS SRUWRIWKLVSURMHFW7KHUHLVQRFRQÀLFWRILQWHUHVW
in this article.
References
1. Mahmoudi R, Subhani A, Pasbakhsh P, Abolhasani F, Amiri I, Salehnia M, et al. The effects of cumulus cells on in vitro maturation of mouse germinal vesi-cle stage oocytes. Iran J Reprod Med. 2005; 3(2): 74-78.
2. Child TJ, Abdul-Jalil AK, Gulekli B, Tan SL. In vit-ro maturation and fertilization of oocytes fvit-rom un-stimulated normal ovaries, polycystic ovaries, and women with polycystic ovary syndrome. Fertil Steril. 2001; 76(5): 936-942.
3. Tavana S, Eimani H, Azarnia M, Shahverdi A, Eft-ekhari-Yazdi P. Effects of Saffron (Crocus sativus L.) Aqueous Extract on In vitro Maturation, Fertiliza-tion and Embryo Development of Mouse Oocytes. Cell J. 2012; 13(4): 259-264.
4. Hardy K, Wright CS, Franks S, Winston RM. In vitro maturation of oocytes. Br Med Bull. 2000; 56(3): 588-602.
5. Bos-Mikich A, Ferreira M, Höher M, Frantz G, Ol-iveira N, Dutra CG, et al. Fertilization outcome, em-bryo development and birth after unstimulated IVM. J Assist Reprod Genet. 2011; 28(2): 107-110. 6. Cortvrindt R, Smitz J, Van Steirteghem AC.
Assess-ment of the need for follicle stimulating hormone in early preantral mouse follicle culture in vitro. Hum Reprod. 1997; 12(4): 759-768.
7. Mao J, Smith MF, Rucker EB, Wu GM, McCauley TC, Cantley TC, et al. Effect of epidermal growth factor and insulin-like growth factor I on porcine preantral follicular growth, antrum formation, and stimulation of granulosal cell proliferation and sup-pression of apoptosis in vitro. J Anim Sci. 2004; 82(7): 1967-1975.
8. Itoh T, Hoshi H. Efficient isolation and long-term vi-ability of bovine small preantral follicles in vitro. In Vitro Cell Dev Biol Anim. 2000; 36(4): 235-240. 9. Telfer EE, McLaughlin M, Ding C, Thong KJ. A
the presence of activin. Hum Reprod. 2008; 23(5): 1151-1158.
10. Sirotkin AV. Effect of two types of stress (heat shock/ high temperature and malnutrition/serum depriva-tion) on porcine ovarian cell functions and their re-sponse to hormones. J Exp Biol. 2010; 213(Pt 12): 2125-2130.
11. Matousek M, Carati C, Gannon B, Brännström M. Novel method for intrafollicular pressure measure-ments in the rat ovary: increased intrafollicular pres-sure after hCG stimulation. Reproduction. 2001; 121(2): 307-314.
12. Murdoch WJ. Programmed cell death in preovula-tory ovine follicles. Biol Reprod. 1995; 53(1): 8-12. 13. Murdoch WJ, Gottsch ML. Proteolytic mechanisms
in the ovulatory folliculo-luteal transformation. Con-nect Tissue Res. 2003; 44(1): 50-57.
14. Tilly JL. Apoptosis and ovarian function. Rev Re-prod. 1996; 1(3): 162-172.
15. Ikeda S, Imai H, Yamada M. Apoptosis in cumulus cells during in vitro maturation of bovine cumulus-enclosed oocytes. Reproduction. 2003; 125(3): 369-376.
16. Hendriksen PJ, Vos PL, Steenweg WN, Bevers MM, Dieleman SJ. Bovine follicular development and its effect on the in vitro competence of oocytes. Theri-ogenology. 2000; 53(1): 11-20.
17. Zonia L, Munnik T. Life under pressure: hydrostatic pressure in cell growth and function. Trends Plant Sci. 2007; 12(3): 90-97.
18. Bronson RA, Bryant G, Balk MW, Emanuele N. In-trafollicular pressure within preovulatory follicles of the pig. Fertil Steril. 1979; 31(2): 205-213.
19. Talbot P. Intrafollicular pressure promotes partial evacuation of the antrum during hamster ovulation in vitro. J Exp Zool. 1983; 226(1): 129-135.
20. Espey LL, Lipner H. Measurements of intrafollicu-lar pressures in the rabbit ovary. Am Physiol. 1963; 205: 1067-1072.
21. Agar A, Yip SS, Hill MA, Coroneo MT. Pressure re-lated apoptosis in neuronal cell lines. J Neurosci Res. 2000; 60(4): 495-503.
22. Agar A, Li S, Agarwal N, Coroneo MT, Hill MA. Reti-nal ganglion cell line apoptosis induced by hydro-static pressure. Brain Res. 2006; 1086(1): 191-200. 23. Rashidi Z, Azadbakht M, Khazaei M. Hydrostatic
pressure improves in-vitro maturation of oocytes derived from vitrified-warmed mouse ovaries. Iran J Reprod Med. 2012; 10 (3): 257-264.
24. Pesty A, Miyara F, Debey P, Lefevre B, Poirot C. Multiparameter assessment of mouse oogenesis during follicular growth in vitro. Mol Hum Reprod. 2007; 13(1): 3-9.
25. Shacter E, Williams JA, Hinson RM, Sentürker S, Lee YJ. Oxidative stress interferes with cancer chemotherapy: inhibition of lymphoma cell apopto-sis and phagocytoapopto-sis. Blood. 2000; 96(1): 307-313. 26. Pocar P, Nestler D, Risch M, Fischer B. Apoptosis in
bovine cumulus-oocyte complexes after exposure to polychlorinated biphenyl mixtures during in vitro maturation. Reproduction. 2005; 130(6): 857-868. 27. Smitz JE, Cortvrindt RG. The earliest stages of
fol-liculogenesis in vitro. Reproduction. 2002; 123(2):
185-202.
28. Martins OG, Pesty A, Gouveia-Oliveira A, Cidadão AJ, Plancha CE, Lefevre B. Oocyte Ca2+ spike
ac-quisition during in vitro development of early prean-tral follicles: influence of age andhormonal supple-mentation. Zygote. 2002; 10(1): 59-64.
29. Cortvrindt R, Smitz J, Van Steirteghem AC. In-vitro maturation, fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system. Hum Reprod. 1996; 11(12): 2656-2666.
30. Demeestere I, Delbaere A, Gervy C, Van Den Bergh M, Devreker F, Englert Y. Effect of preantral follicle isolation technique on in-vitro follicular growth, oo-cyte maturation and embryo development in mice. Hum Reprod. 2002; 17(8): 2152-2159.
31. 6PLOMDNRYLü76UHWHQRYLü/M$OHNVLü6,QIOXHQFHRI abiotic and biotic factors on maturation of oocytes (mammalian eggs) in vitro conditions. Biotech Anim Husbandry. 2009; 25 (5-6): 505-522.
32. Sugiyama S, McGowan M, Phillips N, Kafi M, Young M. Effects of increased ambient temperature dur-ing IVM and/or IVF on the in vitro development of bovine zygotes. Reprod Domest Anim. 2007; 42(3): 271-274.
33. 6PLOMDNRYLü 7 -RVLSRYLF 6 .RVRYDF 2 'HOLF 1 Aleksic S, Petrovic MM. The role of pH values in porcine reproductive tracts of male and female in-dividuals. Biotech Anim Husbandry. 2008; 24(3-4): 101-108.
34. Van den Abbeel E, Schneider U, Liu J, Agca Y, Crit-ser JK, Van Steirteghem A. Osmotic responses and tolerance limits to changes in external osmolali-ties, and oolemma permeability characteristics, of human in vitro matured MII oocytes. Hum Reprod. 2007; 22(7): 1959-1972.
35. Du Y, Pribenszky CS, Molnár M, Zhang X, Yang H, Kuwayama M, et al. High hydrostatic pressure: a new way to improve in vitro developmental compe-tence of porcine matured oocytes after vitrification. Reproduction. 2008; 135(1): 13-17.
36. Reinthaller A, Kirchheimer JC, Deutinger J, Biegl-mayer C, Christ G, Binder BR. Plasminogen activa-tors, plasminogen activator inhibitor, and fibronectin in human granulosa cells and follicular fluid related to oocyte maturation and intrafollicular gonadotro-pin levels. Fertil Steril. 1990; 54(6): 1045-1051. 37. Pribenszky C, Du Y, Molnár M, Harnos A, Vajta G.
Increased stress tolerance of matured pig oocytes after high hydrostatic pressure treatment. Anim Re-prod Sci. 2008; 106(1-2): 200-207.
38. Kawamura K, Kawamura N, Mulders SM, Sollewijn Gelpke MD, Hsueh AJ. Ovarian brain-derived neu-rotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos. Proc Natl Acad Sci USA. 2005; 102(26): 9206-9211.
39. Kawamura K, Kumagai J, Sudo S, Chun SY, Pisar-ska M, Morita H, et al. Paracrine regulation of mammalian oocyte maturation and male germ cell survival. Proc Natl Acad Sci USA. 2004; 101(19): 7323-7328.
cells, but not in oocytes, may influence bovine em-bryonic developmental competence. Theriogenol-ogy. 2005; 63(8): 2147-2163.
41. Horner VL, Wolfner MF. Mechanical stimulation by osmotic and hydrostatic pressure activates Dros-ophila oocytes in vitro in a calcium-dependent
man-ner. Dev Biol. 2008; 316(1): 100-109.
