ELSEVIER

EFFECT OF HIGH MOLECULAR WEIGHT FACTORS PRESENT IN BOVINE OVIDUCT- CONDITIONED MEDIUM ON IN VITRO BOVINE EMBRYO DEVELOPMENT

A. Vansteenbrugge. A. Van Langendonckt, I. Donnay, A. Massip and F. Dessy

UniversitC catholique de Louvain Unite des Sciences Veterinaires

Place Croix du Sud. 3, B- 1348 Louvain-la-Neuve, Belgium

Received for publication : December 13, 1995 Accepted : ApriZ 30, 1996

ABSTRACT

To investigate the presence of embryotrophic factors in bovine oviduct-conditioned medium (BOCM), the high molecular weight fraction (> 10 KDa) from BOCM was added to 3 chemically defined embryo culture media (TCM199, DMEM/F12 and modified synthetic oviduct fluid [mSOF]). Zygotes were obtained by in vitro maturation and fertilization of oocytes. Conditioning of TCM199 with oviduct cells increased both cleavage to the 5- to S-cell stage (59 vs 37%) and further development to the blastocyst stage (19 vs 4%). The low molecular weight fraction (< 10 KDa) of BOCM maintained development to the 5- to &cell stage but did not allow development to the blastocyst stage. Adding the high molecular weight fraction to the inactive low molecular weight fraction restored bovine embryo development up to the blastocyst stage. This embryotrophic effect of the high molecular weight fraction was not observed when this fraction was added to TCM199 or DMEM/F12 medium. Whereas adding this fraction to mSOF medium significantly (P<O.O5) increased embryo development up to the blastocyst stage (36%) in comparison with that of mSOF (15%) or BOCM (14% ).

These results show that BOCM contains high molecular weight factors promoting embryo development up to the blastocyst stage. Some chemically defined media mask the effect of these embryotrophic factors.

Key words: cattle, embryo development, in vitro fertilization, conditioned medium, oviduct, embryotrophic factors

INTRODUCTION

Several culture systems have been used (2) to overcome the 8 to 16-cell developmental block of cultured bovine embryos and to improve the yield of blastocysts. Complex media designed for cell culture such as Tissue Culture Medium 199 (TCM199) are routinely employed in co-culture (11, 14, 17) or after conditioning with a variety of somatic cells (11, 19, 42, 45). Other cell-free culture systems using simple media, supplemented or not with serum, have also been shown to support development up to the blastocyst stage (35, 38,40).

Acknowledgments This work was supported by a grant from the Fonds pour la Recherche Scientifique Medicale 3.4609.95, la Loterie Nationale 9.4555.9 1, the Minis&e de 1’Agriculture de la Region Wallonne (G. Lutgen), and by EU grant BI02-CT92-0067. The authors thank Dr. C. Boccart (Linalux, Belgium) for supplying frozen bull semen and la Societe des Abattoirs et Marches d’Anderlecht and la Societe Viangros for supplying cow ovaries.

Theriogenology 46%31-641, 1996 009%691W96/$15.00 0 1996 by Elsevier Saence Inc PII SOO93-691X(96)00214-X

632 Theriogenology

In co-culture, cells could contribute to embryo development either by removing embryotoxic components from the basic medium, by secreting embryotrophic factors or by doing both. Several lines of evidence support the detoxifying hypothesis (2). Some detrimental components such as hypoxantine, nicotinamide, glucose and phosphate could be removed or inactivated by cells (27, 35, 40, 43). Recently, Leppens et al. (23) have suggested that cells act only by changing the basic medium and not by secreting specific factors into the medium. However, an embryotrophic effect induced by cell-secreted factors can not be excluded (5, 6). Indeed, Heyman and MenCzo (17) as well as Minami et al. (30) have reported the existence of low molecular weight factors that enhance embryo development in medium conditioned either by bovine trophoblastic vesicles (17) or by mouse oviduct cells (17). As discussed by Bavister (3), a way to prove a beneficial effect of somatic cells is to demonstrate that secreted proteins isolated from cell cultures improve embryo development, i.e., by adding these factors to a conventional culture medium and by determining if these factors exert an embryotrophic effect.

A serum-free oviduct-conditioned medium supporting in vitro development of bovine embryo was developed in our laboratory (28). This bovine oviduct-conditioned medium without protein addition is helpful in the study of putative embryotrophic factors secreted by oviduct cells. Total protein content in this medium was about 30 pg ml-l, and most proteins were neosynthesized by cells and did not result from serum residues (44). In addition, it was demonstrated that serum-free oviduct-conditioned medium had a dual effect on bovine embryo development (29) that could be mediated by 2 different factors: one of low molecular mass (< 10 KDa) that is needed to reach the 5- to 8-cell stage, and another of higher molecular mass (> 10 KDa) that allows embryos to develop from the S-cell to the blastocyst stage.

The aim of our study was to go further into the investigation of the presence of embryotrophic factors in serum-free TCM199 conditioned by oviduct cells. The high molecular weight fraction of bovine oviduct-conditioned medium (BOCM) was isolated by ultrafiltration, and added to the low molecular weight fraction or to defined media, in order to test their effect on embryo development. Three defined media were chosen : TCM199, a mixture of equal parts of Dulbecco’s modified Eagle’s medium and Ham’s F12 (DMEM/F12), and modified synthetic oviduct fluid without protein (mSOF) (8). The TCM199 medium has been widely used in ruminant embryo culture either in co-culture or after being conditioned by oviduct cells (11). More recently, DMEM/F12 medium has been employed in our laboratory after being conditioned with Buffalo rat liver cells (45). mSOF supplemented with proteins allows the culture of bovine embryos without somatic cell support (40).

MATERIALS AND METHODS

Preparation of Bovine Oviductal Cell Conditioned Medium (BOCM)

Oviducts were recovered from slaughtered cows at a local abattoir regardless of the stage of their estrous cycle. Procedures for medium conditioning were previously described by Vansteenbrugge et al. (45). Briefly, epithelial layers were extruded from the oviduct by pressure on the external wall. Oviductal cells were resuspended in TCM199 (Gibco: cat. # 3 11.50). supplemented with 10% (v/v) heat-treated fetal calf serum (FCS) (Gibco, Paisley, Scotland) for seeding in culture flasks (Falcon, Erembodegem, Belgium). At confluence, the cell monolayers were washed with serum-free TCM199 medium and cultured in serum-free medium. The medium was renewed every 2 d (2 times). The first 3 harvests were pooled and stored at -80°C and were referred to as BOCM. Three batches of conditioned medium, previously tested for their embryotrophic activity, were used throughout the 3 experiments.

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Ultrafiltration of BOCM

The BOCM was ultrafiltered through a 10 KDa cut-off membrane (Centricon 10; Amicon, Beverly, US). For each experiment, 2 ml of BOCM were ultrafiltered by centrifugation for 1 h at 5000 x g and at 4°C. The filtrate (1900 ~1) containing low molecular weight factors (< 10 KDa) was collected, 2 ml of TCM199 were added to the retentate, then ultrafiltered once more to obtain a final retentate volume of 100 /.tl containing high molecular weight factors (> 10 KDa). Membrane integrity was checked after each ultrafiltration by quantifying the proteins in each fraction using the Coomassie blue method of Bradford (7), by analyzing the molecular weight of proteins from retentate by one-dimensional electrophoresis on a gradient polyacrylamide gel (10 to 18%; 20) and by ultrafiltration of cytochrome C (12KDa: Sigma Chemical Co., St.Louis, MO, USA; cat. # C-7752) solution (0.25 mg ml-l). The retentate (50 ~1) was added either to the filtrate or to 1 of the defined media to obtain a final volume of 1 ml. Filtrate and retentate added to filtrate or to defined medium were sterilized through a 0.22 pm filter (Millex GV; Millipore, Bedford, MA) and stored at -80°C.

Oocyte Maturation and Fertilization

Embryos were generated by in vitro maturation and fertilization of oocytes collected from abattoir ovaries according to the methods previously described by Vansteenbrugge et al. (45). All cultures were performed in a humidified atmosphere of 5% CO2 in air at 39°C.

After 24 h of maturation in medium TCM199 supplemented with 10% heat-treated FCS, oocytes were transferred into 500 ~1 of fertilization medium. The fertilization medium is a low bicarbonate (2 mg ml-l) Tyrode’s medium containing 6 mg fatty acid-free albumin fraction V ml-l, 4 mg sodium lactate ml-l, 0.11 mg sodium pyruvate ml-t (Sigma) with 10 pg heparin- sodium salt ml-l (167 U mg 1; Calbiochem, San Diego, CA, USA). Thawed semen was centrifuged on a Percoll (Pharmacia, Uppsala, Sweden) discontinuous density gradient (45/90%). Spermatozoa were added at a final concentration of 2 x lo6 ml-l. Oocytes and spermatozoa were co-incubated for 18 h.

Embryo Culture

Zygotes were vortexed to remove cumulus cells. Cultures were performed under mineral oil (Merck) in 40.~1 droplets containing about 40 completely degranulated zygotes, at 39’C and in the presence of 5% CO2 in humidified air. The cleavage rate (5to &cell stage) was evaluated on Day 2 of culture (66 h post insemination), and the number of embryos reaching the expanded blastocyst stage was determined on Day 8 (210 h post insemination).

The high molecular weight (MW) fraction (> 10 KDa) was added to the 3 different defined media in 3 separate experiments.

Experiment 1. The effect of adding the high MW fraction to low MW fraction (c 10 KDa) or to TCM199 medium was investigated. Zygotes were randomly allocated to 1) TCM199 (Gibco : cat. # 3 1 150), 2) BOCM, 3) filtrate (c 10 KDa), 4) filtrate supplemented with retentate (> 10 KDa) and 5) TCM199 supplemented with retentate. A total of 3 replicates was performed with 1 droplet per condition in each replicate.

Exoeriment 2. The effect of adding the high MW fraction to DMEM/Fl2 medium was investigated. Zygotes were randomly allocated to 1) BOCM, 2) filtrate (< 10 KDa), 3) filtrate supplemented with retentate (> 10 KDa), 4) DMEM/F12 medium (DMEM: Sigma: cat. # D-5523 and Ham’s F12: Sigma: cat. # N-6760) and 5) DMEM/Fl2 supplemented with retentate. A total of 3 replicates was performed with 1 droplet per condition in each replicate.

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Exoeriment 3. The effect of adding the high MW fraction to mSOF medium was investigated. This medium described by Takahashi and First (40) was supplemented with 20 ml 1-t essential amino acid mixture (Sigma: cat. # B6766), 10 ml 1-L non essential amino acids (Sigma: cat. # 7145) 1 mM glutamine and 3 mM Na pyruvate but without proteins such as serum albumin. To check the specificity of the putative effect of the factors present in the retentate, cytochrome C or ovalbumin were added to mSOF medium at the same protein concentration (25 jtg ml-l) as the retentate. Zygotes were cultured in 1) BOCM, 2) filtrate (< 10 KDa), 3) filtrate supplemented with retentate (> 10 KDa), 4) mSOF medium without protein supplementation, 5) mSOF supplemented with retentate, 6) mSOF supplemented with cytochrome C (Sigma: cat. # C-7752) and 7) mSOF supplemented with ovalbumin (Sigma: cat. # A-5503). A total of 4 replicates was realized with 1 droplet per condition in each replicate.

Statistical Analysis

Data were submitted to a two-way (culture conditions and replications) analysis of variance (ANOVA 2), and means were compared by orthogonal contrasts. When data were proportions, they were transformed by 2 X (arcsin [square-root]) before analysis, to normalize their distribution (10).

RESULTS

Ultrafiltration

Total protein content in BOCM was about 30 pg ml-l. After ultrafiltration, 90% of total proteins were in the retentate and 10% in the filtrate. A one-dimensional PAGE analysis of retentate proteins indicated that proteins present in the retentate had a molecular weight superior to 10 KDa.

Experiment 1

Table I. Effect on bovine embryo development of the high molecular weight fraction from BOCM (retentate) added to the low molecular weight fraction (filtrate) or to TCM199 medium

Conditions Zygotes (n)

5-8 cell n (%I

Blastocysts/ Zygotes n (%I

TCM199 103 38 (37)a 4 (4)a BOCM 106 63 (59)b 20 (19)b Filtrate 96 51 (53)b 7 (7)a Filtrate + Retentate 101 60 (59)b 21 (21)b TCM 199 + Retentate 104 38 (37)a 7 (7)a The results are the total of 3 replicates. No significant difference was observed between replications (P > 0.05). BOCM : bovine oviduct-conditioned medium; filtrate : low molecular weight fraction (< 10 KDa); retentate : high molecular weight fraction (> 10 KDa). a b Numbers within the same column with different superscripts are significantly different (P < 0.05).

Theriogenology 635

Conditioning of TCM199 with oviduct cells (Table 1) significantly increased both cleavage to the S- to &cell stage (59 vs 37%) and further development to the blastocyst stage (19 vs 4%). Filtration through a 10 KDa cut-off membrane (Filtrate) did not reduce cleavage to the 5- to S-cell stage (53 vs 59%) but did significantly decrease the percentage of blastocysts (7 vs 19%). Adding the retentate to the inactive filtrate restored the beneficial effect of the conditioning on blastocyst development (21 vs 7%). However, when added to TCM199, the retentate was not able to induce the same beneficial effect either to the 5- to S-cell stage or to the blastocyst stage. In the light of these results. we decided to add the retentate to another complex medium (DMEM/FlZ) and to a simple medium (mSOF).

Experiment 2

The results of adding the retentate to DMEM/Fl2 medium are presented in Table 2. As in Experiment 1, the filtrate lost the BOCM property of enhancing the blastocyst yield that was restored by adding the retentate. Both DMEM/F12 medium and BOCM had the same effect on early cleavages up to the 5- to &ceil stage, but no embryo developed to the blastocyst stage in DMEM/Fl2. No,difference was observed in blastocyst percentage when retentate was added to DMEM/Fl2 (0 to 1%).

Table 2. Effect on bovine embryo development of the high molecular weight fraction (retentate) from BOCM added to DMEM/Fl2 medium

Conditions Zygotes (n)

5-S cell n (%I

Blastocysts/ Zygotes

n (%)

BOCM 129 62 (48)a 30 (23)a

Filtrate 122 75 (6l)a 5 (4)b

Filtrate + Retentate 116 71 (61)a 20 (17)”

DMEM/F12 105 50 (48)a 0 (Qb

DMEIWF12 + Retentate 114 52 (46)a 1 (Ub

The results are the total of 3 replicates. No significant difference was observed between replications (P > 0.05). BOCM : bovine oviduct-conditioned medium; filtrate : low molecular weight fraction (< 10 KDa); retentate : high molecular weight fraction (> 10 KDa). a b Numbers within the same column with different superscripts are significantly different (P < 0.05).

Experiment 3

The results of Experiment 3 are presented in Table 3. A significant difference was observed between the replications, but there was no significant interaction between replications and culture media (P > 0.05). Moreover, no difference was noted at the 5- to 8-cell stage in any of the culture conditions. As observed in the first 2 experiments, the blastocyst percentage was significantly reduced in the filtrate (4%) comparison with BOCM (14%). Addition of the retentate to the filtrate restored activity (13%). Development to the blastocyst stage was not significantly different between BOCM (14%) and protein-free mSOF medium (15%). The addition of retentate to mSOF markedly improved embryo development up to the blastocyst stage (36%) compared with mSOF (15%) and with BOCM( 14%). Addition of cytochrome C or

636 Theriogenology

ovalbumin at the same protein concentration did not modify the ability of mSOF to promote blastocyst development.

Table 3. Effect on bovine embryo development of the high molecular weight fraction (retentate) from BOCM added to mSOF medium

Conditions Zygotes @I

5-8 cell n (%)

Blastocysts/ Zygotes

n (%I BOCM 176 94 (53)a 25 (14)a

Filtrate 178 94 (53)a 8 (4)b Filtrate -t Retentate 179 100 (56)” 24 (13)a

mSOF 168 96 (57)a 25 (15)a

mSOF + Retentate 176 108 (61)a 64 (36)c

mSOF + Cytochrome C 168 96 (57)a 26 (15)”

mSOF + Ovalbumin 169 101 (60)” 26 (15)”

The results are the total of 4 replicates. A significant difference was observed between replications (P < 0.05). BOCM : bovine oviduct-conditioned medium; filtrate : low molecular weight fraction (< 10 KDa); retentate : high molecular weight fraction (> 10 KDa). a b c Numbers within the same column with different superscripts are significantly different (P < 0.05).

DISCUSSION

Conditioning of TCM199 with bovine oviduct cells (BOCM) has a dual positive effect on bovine embryo development first up to the 5- to g-cell stage and second on blastocyst formation (29). Our results can be summarized in the following way: Removal of high molecular weight (MW) fraction from BOCM does not modify the effect of BOCM on the first 3 cleavages but significantly decreases the development up to the blastocyst stage. This development is restored by adding the high MW fraction to the low MW fraction. Furthermore, the addition of the high MW fraction to mSOF medium clearly improves the production of blastocysts compared with unfractionned BOCM or with protein-free mSOF. However, the addition of this same high MW fraction to TCM199 or to DMEM/F12 medium does not improve the rate of 5- to g-cell stage embryos or blastocysts in comparison with unsupplemented medium.

Development to the 5- to S-cell stage was poor in unconditioned TCM199 compared with the low MW fraction of BOCM. The poor effect of TCM199 compared with DMEM/F12 medium on early cleavage has also been shown (45). This detrimental effect of TCM199 on first cleavages could be accounted for by the absence or the low concentration of components that improve early cleavage such as glutamine (34). It is likely that several factors in TCM199 act in combination to reduce embryo cleavage. The beneficial effect of the low MW fraction of BOCM on early cleavage compared with that of unconditioned TCM199, could be explained by the modification of TCM199 by somatic cells which may modify the basal medium by removing, modifying embryotoxic substances or secreting low molecular weight embryotrophic factors such as amino acids (31) peptides and lipids (16, 18, 36), or by both. Further analysis of filtrate after conditioning is needed to understand the conditioning effect and nutritional requirements of early bovine embryos.

Theriogenology

Our study showed that removing high MW fraction from BOCM impairs bovine embryo development up to the blastocyst stage. Adding this fraction to the low MW fraction restores the embryotrophic activity up to the blastocyst stage. This demonstrates that high MW factors are necessary for bovine embryo development in BOCM through the 8- to l&cell stage up to the blastocyst stage. Similarly, Liu et al. (25) have recently demonstrated the embryotrophic effect of high MW factors produced by the human oviductal cells on mouse embryo development. This is in contrast with the studies of Minami et al. (30) on the mouse, showing that low MW oviductal factors are sufficient to allow for the development of embryos up to the blastocyst stage. Furthermore, our study showed that adding these high MW factors from BOCM to SOF medium markedly increases embryo development to the blastocyst stage. Since adding proteins such as cytochrome C or ovalbumin did not produce the same effect, we may conclude that the embryotrophic activity of the high MW factors is not due to a nonspecific protein effect. In our study we added BOCM high MW factors to a culture medium (mSOF) and demonstrate that factors secreted by oviduct cells improve embryo development.

The high MW fraction from BUCM coufd contain specific or nonspecific factors secreted by cells such as specific proteins, growth factors, antioxidants or chelators which could stimulate embryo development or/and detoxify the medium.

Several authors have studied the secretory activity of bovine oviduct cells. They secrete specific factors such as a 97 KDa estrus associated glycoprotein (4, 13), but his effect on early embryo development has not yet been clearly demonstrated. Nonspecific growth factors are also secreted by the oviduct, and could explain why different somatic cells are effective in supporting embryo development (46). Previous studies have reported a beneficial effect of such growth factors on bovine embryo development (21, 2241). Recently, Satoh et al. (39) have shown that a tissue inhibitor of metalloproteinase (TIMP- l), produced by bovine oviduct cells and present in our BOCM (unpublished data), could be involved in embryo development. However, this embryotrophic effect of TIMP-1 needs to be confirmed in futur studies

Other factors such as antioxidants or chelators could be secreted by cells and neutralize some embryotoxic components produced under the air oxygen concentration used in our study. Indeed, superoxide dismutase (SOD) activity has been demonstrated within rabbit and mouse oviductal fluid (9, 33), and transcripts for SOD have been detected in bovine embryo and in oviductal cells (15) . Moreover, SOD added to culture medium has been found to have a beneficial effect on mouse and rabbit embryo development up to the blastocyst stage (24, 33). However, it has also been reported that SOD in culture medium did not benefit embryonic development in the cow, mouse and sheep (26). These diverse results may be due to species differences or to differences in the culture system used. A previous report by Nasr et al. (32) demonstrated that chelators such as transferrin and apotransferrin improve mouse embryo culture efficiency by inhibiting free transition metal ions which catalyse the production of toxic hydroxy radicals.

It is also possible that several factors act synergistically to improve bovine embryo development up to the blastocyst stage. Indeed, Larson et al. (22) have observed a low embryotrophic effect of TGFa and bFGF alone; yet the combination of these 2 growth factors clearly improved bovine embryo development.

The effect of the addition of BOCM high MW fraction on embryo development depends on the medium. In our study, the high MW factors present in BOCM were efficient only when added to mSOF medium, but not to TCM199 or DMEM/F12 medium. In several laboratories (11, 19, 45) TCM199 and DMEM/F12 were ineffective in supporting embryo development up to the blastocyst stage without co-culture or conditioning, as we observed in Experiments I and 2 in our study. In contrast to TCM199 and DMEM/FlZ, mSOF medium without protein

638 Theriogenology

supplementation allows for the development of bovine embryos up to the blastocyst stage without somatic cell support. The blastocyst yield was similar in BOCM and in mSOF without addition of protein. The beneficial effect of mSOF without protein addition was also observed by Carolan et al. (8). However, mSOF is widely supplemented with proteins such as serum albumin or fetal calf serum (8,40) to improve bovine embryo development.

The embryotrophic effect of BOCM high MW factors observed after addition to mSOF and not to TCM199 or DMEM/F12 medium could be explained by the presence in these 2 last media of some ingredients detrimental to the development up to the blastocyst stage such as glucose, hypoxantine and nicotinamide. Indeed, these factors have been found to inhibit embryo development (1, 27, 40, 43) and are absent from mSOF. Besides the lack of an embryotrophic effect of high MW factors from BOCM after supplementation of TCM199, as opposed to the addition of low MW fraction to BOCM, supports the idea that cells could act during conditioning by changing the composition of basal medium (TCM199). Indeed, as discussed above, cells could modify TCM199 during conditioning not only by removing embryotoxic components but also by secreting embryotrophic factors. For example, the metabolism of glucose by cells reduces the concentration of glucose in the BOCM (37), but it also produces lactate, pyruvate and other substances promoting embryo development (40). In addition, cells could inhibit the effect of hypoxantine and nicotinamide during the conditioning. These modifications of TCM199 medium by cells could explain the beneficial effect of the low MW fraction when supplementing to the high MW fraction.

The fact that the embryotrophic effect of BOCM high MW factors in mSOF is significantly higher than in BOCM, low MW fraction could be explained as follows: BOCM low MW fractions could contain detrimental components present in basal TCM199 medium that are not completely removed or modified by cells such as nicotinamide, hypoxantine or glucose. Moreover, some metabolic byproducts produced by cells could also be detrimental. For example, ammonium ions resulting from deamination of amino acids during conditioning are present in our BOCM at a concentration of about 20 mg l-1 (unpublished results). This ammonium concentration could inhibit blastocyst development, as shown by Gardner et al. (12) in sheep embryos, and could reduce the effect of high MW embryotrophic factors secreted by cells.

We have shown that under the culture conditions of this study, oviduct cells play an active important role in sustaining in vitro bovine embryo development. Our data strongly suggest that these cells operate by detoxifying the basal medium and by secreting embryotrophic factors. Our bovine oviduct-conditioned medium contains high molecular weight factors that promote embryo development from the 5 to g-cell stage to the blastocyst stage. These factors added to a chemically defined medium (modified SOF medium) increase embryo development. It is likely that several factors act synergistically to improve embryo development. The isolation and identification of these factors could be of considerable benefit for the formulation of new culture media and for basic research into the regulation of embryo development.

REFERENCES

1. Bastia MC, McGee-Belser ST, Bryan SW, Vasquez JM. In vitro deleterious effect of hypoxantine in Ham’s nutrient mixture F-10 culture medium on human oocyte fertilization and early embryonic development. Fertil Steril 1993;60:876-880.

2. Bavister BD. Co-culture for embryo development : Is it really necessary? Hum Reprod 1992;7:1339-1341.

3. Bavister BD. Culture of preimplantation embryos : facts and artifacts. Hum Reprod Update 1995;1:91-148.

Theriogenology 639

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

1X.

19.

20.

21.

22.

23.

Boice ML, Geisert RD, Blair RM, Verhage HG. Identification and characterization of bovine oviductal glycoproteins synthesized at estrus. Biol Reprod 1990;43:457-465. Boice ML, Mavrogianis PA, Murphy CN, Prather RS, Day BN. Immunocytochemical analysis of the association of bovine oviduct-specific glycoproteins with early embryos. J Exp Zoo1 1992;263:225-229. Bongso A, Fong CY, Ng SC, Ratnam S. The search for improved in vitro systems should not be ignored: embryo co-culture may be one of them. Hum Reprod 1993;s: 1155-l 162. Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-254. Carolan C, Lonergan P, Van Langendonckt A, Mermillod P. Factors affecting bovine embryo development in synthetic oviduct fluid following oocyte maturation and fertilization in vitro. Theriogenology 1995;43: 1115- 1128. Chun YS, Kim JH, Lee HT, Chung KS. Effect of superoxide dismutase on the development of preimplantation mouse embryos. Theriogenology 1994;41:51 l-520. Dagnelie P. Les methodes de Iinference statistique. In : Dagnelie P (ed), Theorie et Methodes Statistiques. Gembloux Belgium: Gembloux Agronomic Press, 1986; 2:213- 240. Eyestone WH, First NL. Culture of early cattle embryos to the blastocyst stage with oviducal tissue or in conditioned medium. J Reprod Fertil 1989;85:715-720. Gardner DK, Lane M, Spitzer A, Batt PA. Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: Amino acids, vitamins, and culturing embryos in groups stimulate development. Biol Reprod 1994;50:390-400. Gerena RL, Killian GJ. Electrophoretic characterization of proteins in oviduct fluid of cows during the estrous cycle. J Exp Zoo1 1990;256: 113-120. Goto K, Iwai N, Takuma Y, Nakanishi Y. Co-culture of in vitro fertilized bovine embryos with different cell monolayers. J Anim Sci 1992;70: 1449-1453. Harvey MB, Arcellana-Panlilio MY, Zhang X, Schulz GA, Watson AJ. Expression of genes encoding antioxidants enzymes in preimplantation mouse and cow embryos and primary bovine oviduct cultures employed for embryo co-culture. Biol Reprod 1995;53:532-540. Henault MA, Killian GJ. Synthesis and secretion of lipids by bovine oviduct mucosal explants. J Reprod Fertil 1993;98:431-438. Heyman Y, MCnCzo Y. Interaction of trophoblastic vesicles with bovine embryos developing in vitro. In: Bavister BD (ed), The Mammalian Preimplantation. Embryo. Regulation of Growth and Differentiation In Vitro. New York: Plenum Press, 1987; 175- 191. Kane MT. Fatty acids as energy sources of culture of one-cell rabbit ova to viable morulae. Biol Reprod 1979;20:323-332. Kobayashi K. Takagi Y, Satoh T, Hoshi H, Oikawa T. Development of early bovine embryos to the blastocyst stage in serum-free conditioned medium from bovine granulosa cells. In Vitro Cell Dev Biol 1992;28A:255-259. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-685. Larson RC, Ignotz GG, Currie WB. Transforming growth factors l3 and basic fibroblast growth factor synergistically promote early bovine embryo development during the fourth cell cycle. Mol Reprod Dev 1992;33:432-435. Larson RC, Ignotz GG, Currie WB. Plateled derived growth factor (PDGF) stimulates development of bovine embryos during the fourth cell cycle. Development 1992;115:821-826. Leppens G, Sakkas D. Differential effect of epithelial cell-conditioned medium fractions on preimpiantation mouse embryo development. Hum Reprod 1995;lO: 1178-l 183.

640 Thenogenology

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

3.5.

36.

37.

38.

39.

40.

41.

42.

43.

Li J, Foote RH, Simkin M. Development of rabbit zygotes cultured in protein-free medium with catalase, taurine or superoxide dismutase. Biol Reprod 1993;48:33-37. Liu LPS, Chan STH, Ho PC, Yeung WSB. Human oviductal cells produce high molecular weight factor(s) that improves the development of mouse embryo. Hum Reprod 1995;10:2781-2786. Liu Z, Foote RH, Yang X. Development of early bovine embryos in co-culture with KSOM and taurine, superoxide dismutase or insulin. Theriogenology 1995;44:741-75 1. Loutradis DCH, Kiessling AA, Kallianidis K, Siskos K, Creatsas G, Michalas S, Aravantinos D. A preliminary trial of human zygote culture in Ham’s F-10 without hypoxantine. J Assist Reprod Genet 1993;10:271-275. Mermillod P, Mourmeaux JL, Wils C, Massip A, Dessy F. Protein-free oviduct-conditioned medium for complete bovine embryo development. Vet Ret 1992;130:13. Mermillod P, Vansteenbrugge A, Wils C, Mourmeaux J-L, Massip A, Dessy F. Characterization of the embryotrophic activity of exogenous protein-free oviduct- conditioned medium used in culture of cattle embryos. Biol Reprod 1993;49:582-587. Minami N, Utsumi K, Iritani A. Effect of low molecular weight oviductal factors on the development of mouse one-cell embryos in vitro. J Reprod Fertil 1992;96:735-745. Moore K, Bondioli KR. Glycine and alanine supplementation of culture medium enhances development of in vitro matured and fertilized cattle embryos. Biol Reprod 1993;48:833- 840. Nasr-Esfahani M, Johnson MH, Aitken RJ. The effect of iron and iron chelators on the in- vitro block to development of the mouse preimplantation embryo:BAT6 a new medium for improved culture of mouse embryos in vitro. Hum Reprod 1990;5:997-1003. Noda Y, Matsumoto H, Umaoka Y, Tatsumi K, Kishi J, Mori T. Involvement of superoxide radicals in the mouse two-cell block. Mol Reprod Dev 1991;28:356-360. Petters RM, Johnson BH, Reed ML, Archibong ML. Glucose, glutamine and inorganic phosphate in early development of the pig embryo in vitro. J Reprod Fertil 1990;89:269- 275. Pinyopummintr T, Bavister BD. In vitro-matured/in vitro-fertilized bovine oocytes can develop into morulaelblastocysts in chemically defined, protein-free culture media. Biol Reprod 1991;45:736-742. Quinn P, Whittingham DG. Effect of fatty acids on fertilization and development of mouse embryos. In Vitro J Androl 1982;3:440-444. Rieger D, Grisart B, Semple E, Van Langendonckt A, Betteridge KJ, Dessy F. Comparison of the effects of oviduct cell co-culture and oviductal cell-conditioned medium on the development and metabolic activity of cattle embryos. J Reprod Fertil 1995;105:91-98. Rosenkrans CF, Zeng GQ, Mcnamara GT, Schoff PK, First NL. Development of bovine embryos in vitro as affected by energy substrates. Biol Reprod 1993;49:459-462. Satoh T, Kobayashi K, Yamashita S, Kikuchi M, Sendai Y, Hishi H. Tissue inhibitor of metalloproteinases (TIMP-I) produced by granulosa and oviduct cells enhances in vitro development of bovine embryo. Biol Reprod 1994;50:835-844. Takahashi Y, First NL. In vitro development of bovine one-cell embryos: Influence of glucose, lactate, pyruvate, amino acids and vitamins. Theriogenology 1992;37:963-978. Thibodeaux JK, Del Vecchio RP, Hansel W. Role of platelet-derived growth factor in development of in vitro matured and in vitro fertilized bovine embryos. J Reprod Fertil 1993;98:61-66. Trounson A, Pushett D, Maclellan LJ, Lewis 1, Gardner DK. Current status of IVM/IVF and embryo culture in humans and farm animals. Theriogenology 1994;41:57-66. Tsai FCH, Gardner DK. Nicotinamide, a component of complex culture media, inhibits mouse embryo development in vitro and reduces subsequent developmental potential after transfert. Fertil Steril 1994:61:376-382.

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44. Van Langendonckt A, Vansteenbrugge A, Dessy-DoizC C, Flechon JE, Charpigny G, Mermillod P, Massip A, Dessy D. Characterization of bovine oviduct epithelial cell monolayers cultured under serum-free conditions. In Vitro Cell Dev Biol 1995;31:664- 670.

4.5. Vansteenbrugge A, Van Langendonckt A, Scutenaire C, Massip A, Dessy F. In vitro development of bovine embryos in Buffalo rat liver- or bovine oviduct-conditioned medium. Theriogenology 1994:42:93 l-940.

46. Watson AJ, Hogan A, Hahnel A, Wiemer KE, Schultz GA. Expression of growth factor ligand and receptor genes in the preimplantation bovine embryo. Mol Reprod Dev 1992;31:87-95.