Zuchthyg. 20, 1-4 (1985) 0 1985, Verlag Paul Parey, Berlin und Hamburg lSSN 0044-5371/InterCode: ZUCYAN

Aus der Klinik fur Geburtshilfe und Gynakologie des Rindes ( im Richard-Gotze-Haus), Abteilung fur experimentelle Fortpflanzungsbiologie der Tierarztlichen Hochschule Hannouer

Effect of polyvinylalcohol on in-vitro-culture of divided mouse zygotes

Von P. Agrawala* and J . Hahk

Contents: Mouse 4-cell stage zygotes, denuded and halved, were cultured in Whitten's medium to observe the effect of addition of PVA (polyuinylalcohol) and removal of the zona pellucida o n their in-vitro-development t o the blastocyst stage. Removal of the zona pellucida resulted in reduction in the number of cells f r o m 47.0 to 38.1 (p < 0.001) in medium without PVA and f r o m 51.1 to 43.9 ( p < 0,001) cells in medium with this supplement, Addition of P V A to the culture medium resulted in increased cleavage rate o f zygotes and thus the cell number of control, denuded and halved zy- gotes increased f r o m 47.0 to 51.1 ( p < 0.05), 38.1 to 43.9 ( p < 0.001) and 17.7 to 20.2 ( p < 0.05) respectively. Though the mode of action remains unknown, it is clear from this study that the addition of PVA has a positive influence on the cleavage rate during in-vitro-culture of mouse zygotes.

Inhalt: Einflui3 von Polyvinylalkohol auf die in vitro-Kultur von geteilten Mausezygo- ten In einem in-vitro-Kultivierungsversuch wurde erstens der EinfluJi der Entfernung der Zona peltucida und zweitens der Einfluj3 von PVA als Mediumzusatz auf die Zelltei- lungsrate von ganzen, zonafreicn und geteilten Mausezygoten untersucht. Die Entfer- nung der Zona pellucida fiihrte in Medium ohne P V A zu einer signfikanten Verminde- rung der Zellzahl von 47.0 bei den Kontrollen auf 38.1 bei den zonafreien Zygoten ( p < 0.001); in Medium mit PVA lagen die entsprechenden Werte bei51.1 bzw. 43.9 ( p < 0.001). Durch Zusatz von PVA z u m Kulturmedium kam es bei allen Zygoten zu einer Erhohung der Zellteilungsrate. Die Zellzahlen stiegen bei den Kontrollen uon 47.0 in Medium ohne PVA auf 51.1 in Medium mit PVA; beiden zonafreien Zygoten von 38.1 auf 43.9 und bei den geteilten Zygoten von 17.7 auf 20.2. Obwohl der ge- naue Wirkungsmechanismus des PVA nicht bekannt ist, zeigen die Ergebnisse deutlich den positiven Einfluj3 des P V A auf die Zellteilungsrate uon in-vitro-kultiuierten Mause- z ygo ten.

Introduction

The enormous value of monozygotic twins as experimental material for studying the cytoplasmic inheritance and effect of environmental conditions on characters of economic importance has already been well stressed. The artificial production of such twins and quadruplets has been attempted by several workers with different species and different amount of success. Mouse is until now the most commonly used animal for such experiments because of ethical and economical reasons. The rate of success,

* Stipendidt dcr Otto-Benecke-Stiftung, 5300 Bonn 1

US. Copyright Clearance Center Statement: 0044-5371 /85/2001-0001 $ 02.50/0

2 P. Agrawala and J. Hahn

however, has been very low except for one study by Moustafa and Hahn (1978). Short pre-implantation period and relatively low number of ICM cells have been suggested to be the possible reasons. Absence of zona pellucida could as well have a negative effect upon cell number of cultured divided zygotes, though it was not found t o be essential for pregnancy establishment after transfer of whole zona-free mouse zygotes t o the uterus (Rodrigues, 1981) . The number of ICM cells in control mouse blastocysts is of the order 13 t o 1 5 (Rossant, 1975) and halved zygotes are found to contain after cul- ture only about 37% the cell number of control zygotes (Fernandez and Izquierdo,

Recently polyvinylalcohol (PVA) has been reported t o be able to substitute for bovine serum albumin in medium for maintaining the viability of spermatozoa (Bavister, 1981) and improving the survival of frozen-thawed mouse zygotes (Creighton and Lin- dener, 1983). Kuzan et al. (1982) found the development of mouse 2-cell stage zy- gotes after 5 days of in-vitro-culture to be better after addition of PVA t o the culture medium. In this study we attempted to find out if addition of PVA t o Whitten's me- dium could also enhance the number of cells in cultured intact, zona-free and halved mouse zygotes.

1980).

Materials and Methods

Six to eight weeks old virgin CB6/F1 females were induced to superovulate with 5 I.U. of PMSG injected intraperitoneally. After 4 6 h they were given injections of 5 I.U. of HCG and mated to CB6 males. Females which were found to have mated 12 h later were used for experiments. 4-cell stage zygotes were collected 52 h after HCG-injec- tion by flushing the oviducts with culture medium in a Petri dish. The zona pellucida was weakened by treating the zygotes with 0.4% pronase solution (Calbiochem-Beh- ring, California) for two and a half minutes. Zygotes were washed three times in cul- ture medium before transferring them t o the Petri dishes containing culture medium with or without PVA supplement. Complete removal of zona pellucida and separation of blastomeres was accomplished by repeatedly sucking the zygotes in and out of a fine Pasteur pipette. Paires of blastomeres were placed singly in microdrops of culture medium under silicon oil and cultured a t 37 "C for 48 h under humidified atmosphere of 5% COz in air. Zygotes were observed after 20 and 48 h in culture. All morphologi- cally normal looking blastocysts a t second observation were fixed by the method of Tarkowski (1966), stained, and their cells were counted. Some zygotes were cultured intact after removal of zona pellucida forming the group of zonafree zygotes. Controls were placed in microdrops directly after flushing them out of the oviduct.

Results

The overall development of zygotes was observed t o be relatively good pointing the good quality of equipment and culture conditions. The rate of development t o the blastocyst stage of control (untreated), zona-free and halved zygotes was found to be similar in media with and without PVA supplement. All control zygotes developed to the blastocyst stage after culture for 48 h, whereas 83% of halved zygotes developed to the same stage. Percentage of zona-free zygotes reaching blastocyst stage was found t o be 92. Zona-free and halved zygotes which did not reach blastocyst stage were mostly degenerated. Data of all cell countings are given in Table 1. Cell counts were made from 49 intact, 52 zona-free and 5 1 halved zygotes cultured without PVA and 57 intact, 62 zona-free and 46 halved zygotes cultured with PVA supplement. Number of cells found in the blastocysts developed in the medium with PVA was 51.1, 43.9 and 20.2 in control, zona-free and halved zygotes respectively. A significantly lower number of cells i.e. 47.0 (p < 0.05), 38.1 (p < 0.001) and 17.7 (p < 0.05) in corresponding blastocysts

Effect of polyvinylalcohol on in-vitro-culture of divided mouse zygotes 3

Table 1. Number of cells in mouse blastocysts cultured in Whitten’s rnediunt with and without polyvinylalcohol supplement

Culture medium

Whitten’s medium + 0.3% BSA

Control zygotes Zona-free zygotes Halved zygotes Number Number of Number Number of Number of Number of of blasto- cells of blasto- cells blastocytes cells cytes Mean F s.e.m. cytes Mean i s.e.m. Mean i

s.e.m.

49 47.0 8.ga 52 38.1 7.2‘ 51 17.7 5.1e ~~ ~

Whitten’s I medium + 0.3% BSA I 57 51.1 10.Ob 62 43.9 7.1d 46 20.2 4.5f

I I +O.Ol%PVA 1 p (a, b) < 0.05 p (a, c) < 0.001 p (c, d ) < 0.001 p (e, f ) < 0.05

p (b, d ) < 0.001

was observed when zygotes were cultured without PVA. Removal of zona pellucida resulted in reduction in the number of cells from 47.0 to 38.1 (p < 0.001) in the me- dium without PVA and from 51.1 t o 43.9 (p < 0.001) cells in the medium with this supplement. There was no visible difference in the sizes of control and zona-free bla- stocysts in any of the treatments. Size of halved zygotes was nearly half the size of control zygotes after culture. Measurement of the size was not attempted.

Discussion

The experiments described in this report demonstrate that the absence of zona pelluci- da has little effect upon in-vitro-development of murine zygotes, which is in contrast to the observations of O’Brien et al. (1983). We have no explanation for the high per- centage of zona-free and halved zygotes (40 and 65 respectively), which d o not de- velop normally in their study; but our results are in agreement with the findings of different workers (Mintz, 1962; Edwards, 1964; Tarkowski and Wroblewska, 1967; Mochow and Olds, 1968; Trounson and Moore, 1974; Moustafa and Hahn, 1978; Ro- drigues, 1981; Niemann et al., 1983; Tsunoda and McLaren, 1983), who observed nor- mal development in a high percentage of zona-free and halved zygotes of different spe- cies in culture t o the late morula or blastocyst stage. However, the number of cells in blastocysts was considerably reduced by the removal of zona pellucida. Denuded bla- stocysts contained after culture with and without PVA respectively 85.9% and 81% of the cell number of control blastocysts, whereas halved zygotes contained 46% and 46.5% of the cell number of denuded blastocysts in respective media. These numbers are higher than 37% achieved by Fernandez and Izquierdo (1980) in halved zygotes at the morula-blastocyst transition stage. Addition of PVA to the culture medium was proved t o be highly effective in increasing the number of cells in blastocysts developed in vitro. The difference in the number of cells in blastocysts cultured with and without PVA was highly significant (p < 0.001) in case of zona-free, whole zygotes and significant (p < 0.05) in case of zona-free, halved and control zygotes. As it is still not clear how PVA exerts its positive action in the medium and there are no similar studies available, we can not give any argument to

4 P. Agrawdh and J . Hahn

support or compare our results. Our observance on the size of zona-free zygotes is in agreement with that of the Rottmann and Lampeter (1981), who did not notice any difference between the sizes of control and denuded mice zygotes.

References

Bavister, B.D., 1981: Substitution of a synthetic polymer for protein in a mammalian gamete cul- ture system. J. Exp. Zool. 217, 45-5 l . - Creighton, K.A. & G.M. Lindener, 1983: Effect of poly- vinyl alcohol on in vitro survival of frozen-thawed mouse embryos. Theriogenology 19, 120. - Ed- wards, R.G., 1964: Cleavage of one- and two-celled rabbit eggs in vitro after removal of the zona pellucida. J. Reprod. Fert. 7, 413. - Fernandez, M.S. & L. Izquierdo, 1980: Blastocoel formation in half and double mouse embryos. Anat. Embryol. 160, 77-81. - Kuzan, F.B., K.O. Pomeroy & G.E. Seidcl, Jr., 1982: Polyvinyl alcohol as a macromolecular substitute for bovine serum albumin in mouse embryo culture medium. Biol. Reprod. 26, suppl. 1 , 65A. - Mochow, C.R. & D. Olds, 1968: Effect of pronasc on subsequent development of mouse embryos. J. Anim. Sci. 27, 1194. - Moustafa, L.A. & J. Hahn, 1978: Experimentelk Erzeugung von identischen MBusezwillingen. Dtsch. Tierirztl. Wochenschr. 8 5 , 242-244. - Niemann, H., M.J. Illera & D. Smidt, 1983: In vitro development of pig morulae after enzymatic removal or microsurgical treatment of the zona pel- lucida. Theriogenology 19 , 142. - O’Brien, M.J., E.S. Critser & N.L. First, 1983: Developmental potential of isolated blastomeres from early murine embryos. Theriogenology 19, 143. - Rodri- gues, J.J., 1981 : Embryotransfrr bei drr Maus unter besonderer Berucksichtigung von zonafreien und geteilten Zygoten. Inaugural-Dissertation, Tieriirztliche Hochschule Hannover. - Rossant, J., 1975: Investigation of the determinative state of the mouse inner cell mass. J. Embryol. exp. Morph. 33, 979-990. - Tarkowski, A.K., 1966: An air-drying method for chromosome prepara- tion from mouse eggs. Cytogenetics 5, 394-600. - Tarkowski, A.K. & J. Wroblewska, 1967: De- velopment of blastomeres of mouse eggs isolated at the 4 and 8 cell stage. J. Embryol. exp. Mor- phol. 18, 155. - Trounson, A.O. & N.W. Moore, 1974: The survival and development of sheep eggs following complete or partial r e m o d of the zona pellucida. J . Reprod. Fert. 41,97-105. - Tsu- noda, Y. & A. McLaren, 1983: Effect of various procedures on the viability of mouse embryos containing half the normal number of blastomeres. J. Reprod. Fert. 69, 315-322.

Eingang der Arbeit am: 18.5.1984

Anschrift fur Sonderdrucke: Prof. Dr. J. Hahn, Klinik fur Geburtshilfe und Gynkkologie des Rindes der Tierarztlichen Hochschule, Bischofsholer Damm 15, D-3000 Hannover.