Başlık: A COMPARISON OF TWO CO, CHAMBERS ON THE DEVELOPMENT OF MOUSE EMBRYOS IN CULTUREYazar(lar):KILIÇOĞLU, S. ÇetinCilt: 33 Sayı: 3 DOI: 10.1501/Vetfak_0000001040 Yayın Tarihi: 1986 PDF

A: V. Vet. Fak. Derg.

33 (3) :408 -415, 1986

A COMPARISON OF TWO CO, CHAMBERS ON THE DEVELOPMENT OF MOUSE EMBRYOS IN CULTURE

S. Çetin Kılıçoğlu

*

Fare embriyolarının kültüre edilmesinde ~'ararlanılan iki değişik CO, ortamı üzerinde çalışmalar

Özet: Fare embriyolannın in vitro kültüre edilmelerinde vasattaki

pH nın 7.2-7.4 de devamlTlrğınl sağlamak için kulla11llan değişik iki COı ortamının karşrlaştırması yaprlmıştır.

Bu ortalamalardan biri dört hareketli rafi ve bu. raflardan herbirinde altı ufak petri alabilecek gözleri olan, tabanda gazın içeri girebileceği tavanda da dışarı çıkabileceği iki kapakcığı bulunan, etüv içine oturtul-muş, transparent bir kabindir. Embriyoların gelişmesi için uygun ortam sağlıyabilmek amacıyla kontrollu bir pompa yardımıyla kabin içine 24 saat devamlı

%

Diğer ortam ise kontrollu gaz giriş-çıkışları olan ticari bir anaerobik ortamdır. Kültürleri içeren bu jar'a (kavanoz) 10 dakika süreyle

%

COı'li hava verilir daha sonra kapakcıklann sıkı/masıyla kapalr sistem haline gelen kavanoz 37 C derecedeki etüve konur. Embriyolarm incelen-mesi için vidalTkapağının her açllTşmda

%

Bu iki sistem içersinde toplam 286 embriyonun gelişmeleri sabah ve akşam olmak üzere (10.30-17.30) günde iki defa incelenmiştir. Mik-roskobik incelemelerden de anlaşılacağı üzere her iki ortam da emb,':jyo-ların gelişmelerinde yeterlidir ancak kul/anıınemb,':jyo-larında bazı ufak sak 111ca ve yarar/arın olduğu görülmüştür.

* Prof. Dr. Dept. of Obsıetrics and Diseases of Reproduction, Faculty of Veleri-nary Medicine, Ankara.

A. COMPARISON OF TWO CO, CHAMBERS ... 409

Summary: During the culture of mouse emhryos in medium at

pH 7.2-7.4 it is necessary to maintain a constant atmosphere of 5

%

the embryos in a cabinet with continuous flow of gas or by briefly gas-sing and sealing the cultures in an anaerobie jar. A total of 286 embryos were observed in this study. Embryonie development was assessed by mieroseopie examination until Day 6 (144 h) post eoitus. No signijicant dijJerenees in the development or in the proportion of mature blastocysts hatehing was observed fo 110IVing culture of one-eell mouse el11bryos in

this two systems. They were examined and scored for development at two t)mes (10.30-17.30) every day during survey. However both systems have some inherent handieaps and advantages.

Introduction

The inereasing interest in the eultivation of fertilized mammalian

embryos has urged seientists to determine the speeial nutritional

re-quirements of the embryo durİng the early cleavage stages. During the last 50 years eulture of the embryo has become the most popular model used in embryologieal research (2,3,4,6,8,12, i3, i5).

In 1949 Hammond (5) was the first worker to sueeessfully eulture

8 eell mouse eınbryos to blastoeysts. As a medium he employed a salt

solution eontaining sodium ehloride, potassium ehloride and

magne-sium ehloride with a glueose eoneentration of img supplemented with

about 5

%

(5) put i to 6 embryos in smail vessels in 2 to 3 ml of medium. Later

this method was used and further developed by many re search

wor-kers to sueeessfully eulture mouse and rabbit eınbryo smost of the

pre-implantation period (1,2,3,7,10,12,14, i6).

Sinee the mammalian embryo is highly adapted to the maternal

ehvironment the pH should be kept an optimal 7.4::1::0.5 by

equilib-ration with 5

~+-

%

%

concen-tration in the ineubator is thrrefore critica i and this report examines

the relative efficiency of two gassing systems on cleavage of the mouse embryo in a ehemieally define-d eulture medium (I ,2,4, 11,16).

Materials and Me~hods

One eell embryos were obtained from superovulated F2 hybrids

stan-110 ÇETİN KIUÇOGlU

dard labratory conditions at the AFRC Institute of Animal Physiology,

Animal Research Station, Cambridge. The yield of embryos was

inc-reased considerably by initial priming with gonadotrophins. An

intra-peritoneal injection of 5 LU. Pregnant Mare's Serum Gonadotrophin

(PMSG, FoIligon, Intervet) was followed 48 hours later by 5 LU. of

Human Chorionic Gonadotrophin (HCG, Chorulon, Intervet). After

HCG injection the females were paired with males. Mating was

confir-med the following morning (~O Day i) by the presence of a vaginal

plug.

During the morning of Day

ı

the oviducts were dissected and eovered with a drop of hyaluronidase in a sterile pet ri dish. The oviducts wcre examined under a stereo dis-seeting microseope and the eggs weıe loeated İn a cumulus cJot in the ampul1ary portİcn.

The cumulus eeli mass and rggs werc released from the ampulla using a fine needie (25 gau.) and watehmak-::r's foreeps. The eggs were

released from the cumulus eells faııowiag ineubatian at 370° C for 2-3

minutes in the enzyme solution.

The embryos were removed from the enzyme solution and washed

6 times with M i 6,- BSA and finally culturcd in M 16

+

Serum Albumin) under paraffin oil (6,8).

The embryos were then cultured in one of the following two

systems m~dntained in a eonstant 31' C incubator:

i. This system eonsistd of a transparent perspex cabinet with

re;novable shelves and abasal inlet and top outlet gas point (Fig. I).

A continioLIs flow of air generated by an aquatie aeration pump was

mİxed with COı from a controııable cylinder and va1ve attachment to

givc a measured air flow rate of 5 /~ CO2 in aif. Prior to entering the

culture cabinet the gas mixtur~ was passed through a cylinder of water to increase the humidity and also serve as 3 final indicator of the rıow

ratc.

2. A commereially available anaerobıc jar (Baird and Tatlock,

Ltd. London) with eontrollable inlet and outlet valves was used (Fig.2). The jar containing the cultures was gassed for 10 minutes from a cylin-der containing 5

%

A. COMPARISON OF TWO CO, CHAMBERS ..

J'ig.ı.The transparent c:T.briyo cu!turc cabinel. Sekil J. Transparent embriyo kültür kabini.

Fig. 2. The anacrobic jar.

Seki! 2. Anacrobik jar (Anaerobik kavanoz).

412 ÇETİN KILlÇOGLU

from the incubator and required regassing before returning to the

in-cubator.

A total of 286 mouse embryos were observed in this study. The

embryos were cultured up to stage of blastocyst hatching. They were

examined and scored for development at two times (I 0.30 and 17.30

hours) every day during the survey.

Results

No signifieant differences in the development or in the proportion

of mature blastocysts hatehing was observed following eulture of i

cell mouse embryos in the two system tested.

Embryonic development was assessed by microscopic

examina-tion until Day 6 (144 hours) post coitus. During this time 257 (% 90)

of the embryos had reached the blastocyst stage by the 4th day (96

hours) p.c. and 229 (89

%)

Thus the entire process of preimplantation development in vitro was

completed in 4-4 i /2 days (Fig. 3-4).

During use it was found more eonvenient to work with the

incu-bator installed cabined whieh also had alarger capacity for individual

eulture dishes (36 pet ri dishes in the cabinet but only 12 in the anaerobic jar). The jar also cooled markedly during the observation periods when

it was necessary to remove it from the incubator.

Although a continuous gas flow is required by the eabinet system

the COı cylinder used is mueh cheaper eompared with the 5

%

İn air cylinder used to gas the anaerobic jar cultures.

Discussion

The results demonstrated a consistently uniform rate of c1eavage

throughout and hatching of the majority of the blastoeysts.

Most workers agree that the entire process from i cell to blastocyst is eompleted in 4 - 4 1 /2 days (96 - 108 hours p.c.) and that was fully

confirmed in the present study irrcspective of the culture ehambers

used (6,8,9,17).

However both system s have so me inherent handicaps and

A. COMPARISON OF TWO COz CHAMBERS ...

Fig. 3. One-cell mouse cmbryos in Mı6 eulturc medilim. ıSOx Sekil 3. MI6 kültüründeki tek hüereli embriyolar.

Fig. 4. Hatehing of the mature blastocysts. 360x. Sekil 4. Açılım yapan olgun blastosüstler.

4H _{ÇETİN KILlÇOGLU}

Although the anaerobic jar required re!atİvely !ittlc gas com

pa-red with the continious flow system of the cabinet method, the gas for the jar was more expensive.

The mouse embryos developed well in both types of system, but the remova! of the jar from the incubator and the need for a fina! iO

minutes gassing period following examination of the cultures means

that an undesirabIe faıı in temperature must have occured which may

be critica! on culturİng more sensitiye embryos from another species.

The cabinet system offered greater capacity for embryo culture, was much easİer to use and is considered the system of ehoice.

I{cfCrCnlTS

I. Biggers, ,I.D., Whilten. "V.K., Whittirıgham, D.G. (1971): The m/tur" (ir moııse or" iıı ritra. LLL "V1cthods of Mammalian Eınbryology" Eti. J.e. Daniel .ir. hccıııarı San

Francisco.

2. Brinster, R.L. (1963): A method/iıı' iıı ritro eıılrimtioıı OrllWIlSe ol'a/i'Dm ;':'0 eell to blastocysr. Expl. Ccl!. Res. 32, 205.

3. Brinster, R.L.(ı965):Studies 011 the derelopmellt (!rmOIlSe emlır)'os iıı rirro. i V

liırerac-tioıı o/elll'rgy .wurees . .r. Rcprod. Fert. LO. 2"27.

4. Brinsle", R.L. (1969): Moııımalirm Emlı,.y,; Cıılrıırc. 1'1 "Ph" M~nı!ı~:ı1ian Oviduct'. Ed. E.S.E.Hafcz. RJ. BI;ırd"u. lln!v. of Ch'cago Press. 419.

5. Hammond, J. (1949): Reeore,.y aııd cııltııre of tl/bal moııse 0\"(/. Naturc Loııd. 168, 28.

6. Kılıçoğlu, S.ç. (1986): The iıı vitro cııltil'atioıı (!rmOI/Se Ol'aFom ollc cd/ to blastocl'st. A.Ü. Yet. Fak. Derg. 32(2) 301-310.

7. McLaren, A., Biggers, J.D. (1958): Sııccessl/jid derl'lopmellt aııd hi,.rh or /Ilice cultil'a-ted in ritro as early embryos. Naturc Lond. 182.877.

8. Rafferty, K.A. (1970): Met/1Ods iııEıpNimmtal Embryolog.ı' o/rhe Moııs('. The Jolıns Hopkins Pn:ss. Baltimore - London.

9. Streffer, c.,Yan Beuningen, D., Molls, M., Zamboglou, 1\., Schultz, S. (1980): Kiııe-tics Sfcel/ proliferatioıı in the preimplrll1ted ıııol/se eıııb,.yo iıı ri ro aııd iııI'irro. CeJl Tis-sue Kinet. 13, 135.

LO. Tarkowski, A.K. (1961): Mal/se chiıııaeras derelopl'd ji-olll fused I'ggs. ~ature Lond. 190, 857.

11. Wale s, R.G., Ouinn, P., Mkrdock, R.N. (1969): The fixatioıı of carbol/ dioxide by the eight eel! mouse embryos. J. Reprod. Fert. 20, 541.

A. COMPARISON OF TWO CO, CHAMBERS ... .115

13. Whitten, W.K. (197I): Nutrieııts requiremeıııs!;}r ıhe cu/lure of pre-imp/£lIltatiolı

emIJr-yas iıı vitra. In: "Advances in ıhe Bioscicnces." Vol. 6 P'~rg:ımon Press.

14. Whitten, W.K., Biggers, J.D. (1968): Comp/eıe del'elopmeııı iııI'ilro of ıhe pl'e-implaıı-tatioıı slages of the mouse iııa simp/e clıemically defined medium. J. Reprod. Fert. 17,399.

15. Whittingham, D.G. (1970): Biocheıııica/ aspecls of early gesıalion in: "Congeniıal Malformaıions". Proc. The Hauq~ I.C.S. Exccrpıa Med. Fdn. 204, ı19. 16. Whittingham, D.G. (1971): Cu/ture of mouse OH/. J. Reprod. Fert. Suppl. 14.7.

17. Zeilmaker, G.H. (1981): EmIJryo Iraıısfer iıı the mouse oıııl fııılıe ral. In: "Frozen Sto-rages of Laborotory Animals". Ed. G.H.7cilmaker. Gustav Fisher Verlag. Stuttgarı-New Yl'rk 1981.