Hydrostatic Pressure Affects In Vitro Maturation of Oocytes and Follicles and Increases Granulosa Cell Death

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In 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

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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

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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

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sure due to an increase in hydrostatic pressure in

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strength in the follicle wall and increase of the

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tissue thinning and follicular rupture by elimina

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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

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The spatiotemporal pattern of apoptosis during follicle growth and oocyte maturation is tightly

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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

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creased the incidence of cell death in cumulus cells

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Materials and Methods

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Ovarian follicle recovery and culture

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ad libitum.

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to dissection medium that consisted of Alpha

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intact follicles with one or two layers of gran

ulosa cells and some adhering theca cells; ii.

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were rinsed three times in dissection medium

and transferred to culture medium that con

sisted of dissection medium supplemented with

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each drop with fresh medium.

Follicle monitoring and measurement

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measurement of morphological features were

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two perpendicular diameters were measured us

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originated from the follicle theca and attached to the dish were not included in the measurements. In

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with the longest length and widest perpendicular width.

Experimental protocol

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which were defined as intact follicles with a central oocyte surrounded by a granulosa cell

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cated and placed in culture medium randomly

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transferred to a pressure chamber according to

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from in vitro grown follicles as well as the cell

death incidence and apoptosis in the cumulus

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and apoptosis.

In vitro maturation of oocytes

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ly described method with some modifications

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ability.

Evaluation of nuclear maturation

To assess the rate of meiosis at the end of the

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Oocyte viability

In this assessment the follicles were mechani

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Hoechst/propidium iodide nuclear staining of cumulus oocyte complexes

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blue and red fluorescence.

Terminal deoxy-nucleotidyl transferase-mediated (dUTP) nick-end labeling (TUNEL)

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were mounted in glycerol onto a slide and placed

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total cell number.

Statistical analysis

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Results

Follicle and oocyte measurement

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Table 1: Follicle and oocyte diameters during culture

Day 12 Day 9

Day 6 Day 3

Day 1 N

“ “

“ “

“

Follicle diameter

“ “

“ “

“

Oocyte diameter

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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

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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

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fragmented and condensed nuclear cumulus

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was scored as follows. Viable cells contained

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chromatin which included discrete clusters of

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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.

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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.

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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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A B C

TUNEL labeling

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chromosomes counted as single nuclei; frag

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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.

(8)

)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

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.

(9)

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 RI“PP+JZDVUHSRUWHGDWWKHSUHR YXODWRU\SKDVHKRXUVDIWHUH&*ZKLFKLQ FUHDVHGJUDGXDOO\WKURXJKRXWWKHRYXODWRU\SUR FHVVWR“PP+JDWKRXUVDIWHUK&* PLGRYXODWRU\SKDVHDQG“PP+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

(10)

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.

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