36. LAZÁR,L., DIETZOVÁ,Ilona: The effect of co-cultivation on the development of in vitro matured and fertilized cow oocytes. (Az együtt-tenyésztés hatása az in vitro érlelt és termékenyített szarvasmarha petesejt fejlõdésére)

36. Lazár

érk: 97.10.16.

UEVM, 04001 Kosice, Hlinkova 1/A, Slovak Republic

INTRODUCTION

Using the methods of maturation and in vitro fertilization, the embryo production as well as the selection and improvement of animal genetic potential can be considerably economized. At present, there is still little knowledge from the field of oocyte maturation and further embryo cultivation.
Bovine zygotes obtained by in vitro maturation and fertilization are considered to be more susceptible to conditions of manipulation than those obtained in vivo. Using the electron microscopy, ultrastructural differences between the both embryo types have been found. In vitro obtained embryos exhibit the higher degree of blastomer cytoplasm vacuolization, decreased cell compactness as well as more phagosomes than embryos obtained in vivo. These differences are likely to be responsible for their lower viability after freezing and thawing, for decreased rate of pregnancy following the transfer (S h a m s u d d i n et al., 1992; C s e h et al., 1996) as well as lower developmental potential in further cultivation under in vitro conditions (Van S o o m, K r u i f, 1992). I w a s a k i et al. (1990) and G o t o and I r i t a n i (1992) have found out that in vitro fertilized embryos had markedly smaller embryonic disc but also lower number of blastomers (C s e h et al.,1996) that may be one of the reasons of decreased viability.
At present, various types of somatic cells are in use for co-cultivation to improve quality and viability of embry os after in vitro fertilization. Recently, studies on possibility of using the addition of some growth factors into me dia have occurred. They are of great importance in the early stages of embryonic development, during getting over the de velopmental block and in normal development of embryos to the stage of blastocyst. During in vitro maturation, these compo nents may be responsible for subsequent improvement of bovine embryo viability. It was proved that both cells of oviducts and other somatic cells produce embryotrophic factors also supporting the normal development of embryos. To achieve as good results of cultivation as possible, the logical step is to analyze the early embryogenesis in great detail so that we are able to control the conditions of cultivation in connec tion with their needs.
The objective of this study was to test the developmental capacity of in vitro matured and fertilized cow oocytes on a monolayer of cumular cells and hamster ovarian cells.

MATERIAL AND METHODS

Ovaries of heifers and cows were collected at local abattoir and were transported to the laboratory at 30°C within 2 to 3 h of slaughter. The ovaries were washed 3 times in PBS supplemented with gentamycin. Bovine cumulus-oocyte complexes (COC) were obtained by aspiration the contents of follicles 2 -7mm using an 18 - gauge needle. For oocyte maturation, TCM 199 with Earle´s salts was modified by adition of sodium pyru-vate, calcium lactat, sodium bicarbonate, HEPES and gentamycin sulfate, and suplemented with 20% of oestral cow serum (pH 7,4). Oocytes were cultivated at 39°C, under parafine oil and concentration of 5% CO2 in air at maximum humidity 24-26 hour.
Frozen - thawed semen was used for fertilization. The spermatozoa were separated by layering (100l) semen under 1 ml calcium ion-free TALP medium containing 0,6% bovine serum albumine fraction V and allowing the spermatozoa to swimup during incubation for 1 h at 39°C. After incubation, the spermatozoa were washed with capacitation medium and centrifuged (900g for 10 min). Mature oocytes were washed 3 times with TALP fertilizing medium and placed into 400l TALP fertilization medium containing a PHE mixture (hypotaurine and epinephrine) heparin and 0,6% of bovine serum albumine fraction V under mineral oil. A sample of capacitated spermatozoa was then added to the oocytes for a final concentration approximately 1,5 x 106 sperm / ml. Gametes were incubated together for 17 to 20h at 39°C under 5% CO2 in air. After this time the oocyte with granulosa cells were gently 3 times washed and transfered into the TCM 199 Earl´s medium modified by adition 20% of oestral cow serum, sodium pyruvate, calcium lactate, sodium bicarbonate, HEPES and gentamycine sulfate. The oocytes of the first group were further cultured at 39°C on a monolayer that was grown from cumulus cells surrounding the oocytes. The oocytes of second group were denuded and cultivated without somatic cells. The third group were cultivated on the monolayer of hamster ovarial cells. During cultivation were control the cleavage rate of the embryos.

RESULTS

From 630 oocytes of first group were cleaved 314 oocytes (49,8%) 48 hours after fertilization. At other cultivation 141 (22,4%) of oocytes were developed to the stadium of blastocyst until day 8, after fertilization. Some of them were hatched (13/2,1%) until 10 day. From 117 oocytes of second group 57 (48,7%) were cleaved 48 hours after fertilization. From 89 oocytes of third group 27 (30,3%) were cleaved. None of fertilized oocytes from these groups were developed to the stadium of expanded blastocysta.

DISCUSSION

The results of our co-cultivation experiment revealed that cumular granulosa cells were helpful for further development of in vitro fertilized bovine embryos. The development of embryos on cumular cells obtained in our experiment is comparable with the results of other authors who use the same co-cultivation system. For instance, B r o u s s a r d et al. (1994) obtained 35 developed (20.6%) blastocysts of 170 fertilized oocytes on day 7 from the fertilization and 9 (5.3%) expanded blastocysts. On day 8, 53 (31.2%) blasto-cysts and 28 (16.5%) expanded blastocysts developed and one was hatched embryo from zona pellucida.
According to the latest knowledge, both mother reproductive tract and embryos alone produce factors that are able to improve the cell proliferation. In many animals, preimplantation embryo is able to develop in a simple medium containing only basic solu-tion and the energetic source. This fact points out to autocrine production of growth factors (H e y n e r et al., 1993). There is an effort to reduce differences in the devel-opmental capacity of in vivo and in vitro produced embryos. One of the ways is, to co-cultivate the embryos on co-cultures. Oviductal epithelial cells and granulosa cells are most frequently used (A o y a g i et al., 1990, S h a m s u d i n et al., 1994, R i e g e r et al., 1995). Good results have been obtained also by means of cohe rent BRL (Buffalo Rat liver) cell monolayer (V a n I z e r et al., 1992; H a w k , W a l l , 1994). In humane medicine, good results were obtained with MKE (Monkey kidney epithelial cells) and Vero cells (P l a c h o t et al., 1995). After the co-cultivation on Vero cells, even better pregnancy rates we re obtained than in co-cultivation on oviduct and granulosa cells (P l a c h o t et al., 1995) Although cultivation on cell cultures does not improve the embryo quality to the le vel of embryos obtained under in vivo conditions, in comparison with in vitro cultured embryos without co-cultivation, the morphology of embryos and their developmental capacity to the stage of blastocyst and hatched blastocyst greatly improves.
Cumular cells are of great importance in he nutrition and maturation of oocytes. During the preovulatory maturation, cumular cells undergo significant changes both in shape and orientation. This process includes breaking the connection between the oocyte and cumular cells as well as between cumular cells, with their subsequent ex pansion that plays primary role in the regulation of oocyte maturation (D e k e l , 1988). At the time of fertilization, cumular cells are still healthy and structurally well-developed (N o t t o l a et al., 1991) that also indica tes for the potential role of these cells in initial life activi-ties of oocytes (F u k u i et al.,). The expression was found out of mRNA transforming growth factor in human cumular cells immediately prior to the fertilization. It is assumed that this process influences the enhancement of the moving ability of capacitated sperms (T e s a r í k et al., 1990). In this way, the oocyte-cumular complex also exerts an indirect effect on the improvement of fertilization rate. The transfer of produced growth factors from the environment to the embryo is of great importance in cell differ-entiation and influences the viability of embryos. It was clearly proved that such growth factors are produced by the embryos (P e u r a, 1993). These factors act via specific receptors of embryonic cells whose exact mode of action still remains unclear.

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