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Biology of Reproduction 59, 1017-1022 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Activin A and Follistatin Regulate Developmental Competence of In Vitro-Produced Bovine Embryos1

Koji Yoshioka2,a, Chie Suzukia, , and Shokichi Iwamuraa

a Laboratory of Theriogenology, National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of activin A and/or follistatin on the development of bovine embryos were investigated. Presumptive zygotes matured and fertilized in vitro were cultured in a chemically defined medium (modified synthetic oviduct fluid medium; mSOF). Addition of 1–100 ng/ml of activin A to mSOF significantly increased the percentage of zygotes that developed to morulae and blastocysts (48–54% and 31–41%, respectively) compared with no addition (41% and 25%, respectively). In contrast, addition of 1–100 ng/ml follistatin significantly reduced the percentage of zygotes developing to morulae and blastocysts (29–31% and 17–20%, respectively) compared with no addition (41% and 28%, respectively). In a culture with 10 ng/ml of activin A, supplementation with the same concentration of follistatin neutralized the positive effect of activin A, while supplementation with 100 ng/ml of follistatin reduced the percentage of zygotes that developed. The total cell numbers in morulae and blastocysts were not affected by the addition of activin A and/or follistatin. The development-enhancing effects of activin A and the development-impeding effects of follistatin were observed when embryos were exposed to activin A or follistatin at a concentration of 10 ng/ml prior to the 9- to 16-cell stage. These results suggest that activin A and follistatin may affect bovine embryos until the third cell cycle and may play important roles in regulation of the developmental competence of bovine embryos.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Culture conditions for in vitro culture of bovine embryos have been the subject of many studies, both for understanding reproductive physiology and for application in the embryo transfer industry. A variety of embryo culture systems that include coculture with somatic cells [1–3], culture in conditioned media [4], and culture in defined media with or without serum/BSA [5–11] are presently employed. Chemically defined media for bovine embryo culture are especially useful for analyzing the physical action of substances such as inorganic compounds, energy substrates, hormones, cytokines, and vitamins on the development of preimplantation embryos [7, 9, 11, 12].

During mammalian development, a number of cytokines play a functional role in the process of cellular proliferation, differentiation, and morphogenesis in a spatial and temporal manner [13]. In early preimplantation embryos, it has been noticed that cytokines produced by both the female genital tract and the embryo itself act on embryonic cells as paracrine/autocrine factors [13, 14]. In cows, a variety of cytokines including transforming growth factor ß, basic fibroblast growth factor [15, 16], leukemia inhibitory factor [17], platelet-derived growth factor [15, 18], insulin-like growth factor-I [15], granulocyte-macrophage colony-stimulating factor [19], and activin A [12] have been reported to affect the development of preimplantation embryos.

In recent studies, addition of activin A to bovine [12] and murine [20] embryo culture media has increased the number of 1-cell embryos reaching the morula or blastocyst stage. Our previous study showed that recombinant human activin A stimulates development of bovine zygotes to the blastocyst stage when they are cultured in a chemically defined medium (modified synthetic oviduct fluid medium; mSOF) under a gas atmosphere of 5% CO2 in air [12]. However, mSOF is optimized under an atmosphere of 5% CO2:5% O2:90% N2 rather than 5% CO2 in air [6, 21, 22]. In our previous study, the percentages of zygotes that developed to the blastocyst stage were relatively low (6–19%) even when activin A was added to mSOF [12].

Follistatin is a glycosylated polypeptide, originally isolated from porcine follicular fluid, that inhibits FSH secretion [23, 24]. This protein has a high affinity for activin and is known as an activin-binding protein. Follistatin is expressed in diverse tissues, including gonads, pituitary, uterus, heart, lung, thymus, etc. [25], and neutralizes activin bioactivity in various systems, such as in the stimulation of FSH secretion in cultured pituitary cells [26] and in the differentiation of granulosa cells [27, 28]. However, it has not been determined whether follistatin neutralizes the development-enhancing effects of activin A in bovine embryos.

The objectives of this study were to confirm the development-enhancing effects of activin A in bovine embryos produced by in vitro maturation (IVM) and in vitro fertilization (IVF) using a chemically defined medium (mSOF) under a gas atmosphere of 5% CO2:5% O2:90% N2, and to determine the effects of follistatin or a combination of activin A and follistatin on embryo development. Furthermore, the developmental stages of embryos affected by these cytokines were examined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
IVM and IVF of Bovine Oocytes

Oocyte collection, IVM, and IVF were carried out as previously described [12]. Briefly, cumulus-oocyte complexes (COC) were aspirated from small antral follicles (2–5 mm in diameter) on bovine ovaries obtained from a slaughterhouse. After washing of COC with Hepes-buffered Tyrode's medium (TALP-Hepes [29]), only oocytes with 3–4 layers of intact and unexpanded cumulus cells were selected; these were then cultured for 22 h with maturation medium (Hepes-buffered TCM199 [Sigma Chemical Co., St. Louis, MO] + 10% heat-inactivated fetal bovine serum [Gibco Life Technologies, Grand Island, NY] supplemented with 0.2 mM sodium pyruvate, 0.02 U/ml porcine FSH [Sigma], 1 µg/ml estradiol-17ß [Sigma], and 50 µg/ml gentamicin sulfate [Sigma]) at 39°C in a humidified atmosphere containing 5% CO2 in air.

For IVF, motile spermatozoa were obtained by centrifugation (700 x g for 20 min) of frozen-thawed semen on a 45%/90% Percoll (Pharmacia Biotech, Uppsala, Sweden) gradient [12]. The sperm pellet was resuspended with modified Brackett and Oliphant's isotonic medium [30] without BSA but supplemented with 50 µg/ml gentamicin sulfate (mBO) and then washed by centrifugation at 500 x g for 5 min. COC were coincubated with spermatozoa in 100-µl droplets of mBO supplemented with 2.5 mM theophylline, 5 µg/ml heparin (Sigma), and 3 mg/ml fatty acid-free BSA (Sigma) for 20 h at 39°C in a humidified atmosphere containing 5% CO2 in air. The final concentration in fertilization drops was 5 x 106 spermatozoa/ml.

Culture of Embryos

After 20 h of coincubation with sperm, presumptive zygotes were stripped of cumulus cells by vortexing for 4 min in 1 ml of TALP-Hepes, then washed three times with culture medium. A chemically defined medium (mSOF) containing 3 mg/ml polyvinyl alcohol (Sigma) was used for the basal culture medium as previously described [12]. Presumptive zygotes were cultured in 30-µl droplets covered with paraffin oil at 39°C in a humidified atmosphere containing 5% CO2:5% O2:90% N2. Each droplet contained approximately 25 presumptive zygotes. At 120 h postinsemination, embryos were transferred to fresh medium containing 2.0 mM glucose and then cultured at 39°C in a humidified atmosphere containing 5% CO2:5% O2:90% N2.

Determination of Cell Number

Cell number was determined by an air-drying method as described previously [8]. Briefly, embryos were put into a hypotonic solution (0.9% sodium citrate supplemented with 0.3% fetal calf serum) for 15 min. Then they were treated with fixative I (methanol:acetic acid:distilled water, 10:3:7) and fixative II (methanol:acetic acid, 3:1). After staining with 2% Giemsa solution, the total cell numbers, including metaphase plates but excluding pyknotic nuclei, were counted under a brightfield microscope.

Experiment 1: Effects of Various Dosages of Activin A

The objective of experiment 1 was to determine the effects of various dosages of activin A on early embryonic development. After fertilization, presumptive zygotes were randomly placed in microdrops of mSOF containing recombinant human activin A (Austral Biologicals, San Ramon, CA) at concentrations of 0, 0.1, 1, 10, or 100 ng/ml and cultured for up to 220 h postinsemination. The percentages of presumptive zygotes that cleaved (>= 2-cell stage) were determined at 48 h postinsemination and that developed to morulae, blastocysts, and hatched blastocysts were assessed at 120, 175, and 220 h postinsemination, respectively, under a stereomicroscope.

Experiment 2: Effects of Various Dosages of Follistatin

The objective of experiment 2 was to determine the effects of various dosages of follistatin on early embryonic development. After fertilization, presumptive zygotes were randomly placed in microdrops of mSOF containing recombinant human follistatin (rhFS-288, lot no. B3904, NHPP, NIDDK [Rockville, MD], NICHD, USDA) at concentrations of 0, 0.1, 1, 10, or 100 ng/ml and cultured for up to 220 h postinsemination. The percentages of presumptive zygotes that cleaved (>= 2-cell stage) were determined at 48 h postinsemination and that developed to morulae, blastocysts, and hatched blastocysts were assessed at 120, 175, and 220 h postinsemination, respectively, under a stereomicroscope.

Experiment 3: Effects of a Combination of Activin A and Follistatin

Experiment 3 was conducted to determine the interaction of activin A and follistatin. At 20 h postinsemination, presumptive zygotes were randomly placed in microdrops of mSOF alone (control) and in mSOF containing 10 ng/ml activin A, 10 ng/ml follistatin, or both (10 ng/ml activin A + 10 ng/ml follistatin, 10 ng/ml activin A + 100 ng/ml follistatin). Embryos were cultured for up to 175 h postinsemination. The percentages of presumptive zygotes cleaved were determined at 48 h postinsemination and the percentages that developed to morulae and blastocysts were determined at 120 and 175 h postinsemination, respectively, under a stereomicroscope. Blastocysts that developed in each treatment were collected at 175 h postinsemination and their total number of cells was counted. In addition, morulae cultured in mSOF and in mSOF containing 10 ng/ml activin A and/or 10 ng/ml follistatin until 120 h postinsemination were also collected and their total numbers of cells were counted.

Experiment 4: Determination of the Developmental Stages of Embryos That Were Affected by Activin A or Follistatin

Experiment 4 was conducted to determine which developmental stages of embryos were affected by activin A and follistatin. After fertilization, presumptive zygotes were cultured in mSOF for 0, 12, 22, 40, 70, 100, or 150 h to obtain embryos at the 1-cell, 2-cell, 3- to 4-cell, 5- to 8-cell, 9- to 16-cell, morula, or blastocyst stages, respectively. Then embryos at each developmental stage were finally cultured in mSOF containing activin A or follistatin at concentrations of 10 ng/ml or no addition for up to 220 h postinsemination. The numbers of embryos that developed to the blastocyst and hatched blastocyst stages were recorded at 175 and 220 h postinsemination, respectively.

Statistical Analysis

Each experiment was replicated on several different days using a microdrop of presumptive zygotes or embryos per treatment on each day. Data were analyzed with the StatView (Abacus Concepts Inc., Berkeley, CA) software package. For analysis of development, each microdrop was considered to be an experimental unit, and the percentages of cleavage, morulae, blastocysts, and hatched blastocysts were calculated within each microdrop. Differences in the mean percentages of cleavage, morulae, blastocysts, and hatched blastocysts among the experimental groups were analyzed by one-way ANOVA. When ANOVA revealed a significant effect of the treatments, treatments were compared by Dunnett's procedure as a multiple comparison procedure. The total numbers of cells in morulae and blastocysts were subjected to logarithmic transformation and then assigned by one-way ANOVA. Differences of p < 0.05 were taken as significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Various Dosages of Activin A

Cleavage rates of presumptive zygotes cultured in mSOF with various concentrations of activin A (84–88%) were not significantly different. However, the percentages of zygotes that developed to the morula and blastocyst stages increased dose-dependently with addition of activin A from 0.1 to 10 ng/ml (Fig. 1). The percentages of zygotes that developed to morulae and blastocysts with addition of >= 1 ng/ml activin A (48–54% and 31–41%, respectively) were significantly higher (p < 0.05) than the percentages obtained with no addition of activin A (41% and 25%, respectively). In addition, hatching rates of embryos cultured in mSOF with >= 10 ng/ml activin A (17% for both dosages) were significantly higher (p < 0.05) than that for embryos cultured in mSOF with <= 1 ng/ml activin A (8–10%).



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  		FIG. 1. Effects of various dosages of recombinant human activin A on the development of bovine presumptive zygotes to the morula, blastocyst, and hatched blastocyst stages. The zygotes were cultured in mSOF up to 220 h postinsemination. Data are presented as means and SD in 6 replicates. Numbers in parentheses represent the numbers of presumptive zygotes tested per group. Different letters above the bars within the same category indicate significant differences (a vs. b, b vs. c: p < 0.05; a vs. c, a vs. d: p < 0.001).

Effects of Various Dosages of Follistatin

Cleavage rates of presumptive zygotes cultured in mSOF with various concentrations of follistatin (86–89%) were not significantly different. However, the percentages of embryos that developed to the morula and blastocyst stages were decreased dose-dependently by addition of follistatin from 0.1 to 10 ng/ml (Fig. 2). The percentages of zygotes that developed to morulae and blastocysts with addition of >= 1 ng/ml follistatin (29–31% and 17–20%, respectively) were significantly lower (p < 0.05) than the percentages obtained with no addition of follistatin (41% and 28%, respectively). There was no significant difference in hatching rate of embryos among the treatments (7–10%).



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  		FIG. 2. Effects of various dosages of recombinant human follistatin on the development of bovine presumptive zygotes to the morula, blastocyst, and hatched blastocyst stages. The zygotes were cultured in mSOF up to 220 h postinsemination. Data are presented as means and SD in 8 replicates. Numbers in parentheses represent the numbers of presumptive zygotes tested per group. Different letters above the bars within the same category indicate significant differences (a vs. b: p < 0.05; a vs. c: p < 0.01).

Effects of a Combination of Activin A and Follistatin

Cleavage rates of presumptive zygotes cultured in mSOF alone (control) and in mSOF containing 10 ng/ml activin A, 10 ng/ml follistatin, or both (10 ng/ml activin A + 10 ng/ml follistatin; 10 ng/ml activin A + 100 ng/ml follistatin) were all in the range 85–88% and were not significantly different. The percentages of zygotes that developed to the morula and blastocyst stages are shown in Figure 3. As was found in the previous experiment, the percentages of zygotes that developed to the morula and blastocyst stages with addition of 10 ng/ml activin A alone (46% and 32%, respectively) were significantly higher (p < 0.01) than the percentages obtained in the control (38% and 24%, respectively). Also, with addition of 10 ng/ml follistatin alone, these percentages (29% and 13%, respectively) were significantly impeded (p < 0.01). In the culture with 10 ng/ml of activin A, supplementation with the same concentration of follistatin reduced the percentages of embryos that developed to morulae and blastocysts to values (41% and 24%, respectively) that were similar to those for the control. Furthermore, the percentages of embryos that developed to morulae (29%) and blastocysts (18%) in the culture with a combination of 10 ng/ml activin A and 100 ng/ml follistatin were significantly lower (p < 0.01) than those in the control. The total numbers of cells in morulae and blastocysts obtained by culture with activin A and/or follistatin are shown in Table 1. There was no significant difference in the total number of cells in morulae or blastocysts among the treatments.



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  		FIG. 3. Effects of a combination of recombinant human activin A (ACT) and follistatin (FS) on the development of bovine presumptive zygotes to the morula and blastocyst stages. The zygotes were cultured in mSOF up to 175 h postinsemination. Data are presented as means and SD in 8 replicates. Numbers in parentheses represent the numbers of presumptive zygotes tested per group. Different letters above the bars within the same category indicate significant differences (a vs. b, a vs. c: p < 0.01; b vs. c: p < 0.001).


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  		TABLE 1. Effects of recombinant human activin A and follistatin on the total number of cells in morulae and blastocysts produced in in vitro culture.a

Effects of the Developmental Stages of Embryos Affected by Activin A or Follistatin

Figure 4 shows the effects of adding either 10 ng/ml activin A or 10 ng/ml follistatin at various developmental stages on the development in vitro of embryos to the blastocyst and hatched blastocyst stages. Addition of activin A to medium at the 1-cell, 2-cell, 3- to 4-cell, and 5- to 8-cell stages significantly increased (p < 0.05) the percentage of embryos that developed to blastocysts (32%, 43%, 45%, and 52%, respectively) compared with no addition (24%, 32%, 33%, and 39%, respectively) and compared with addition of follistatin (16%, 22%, 24%, and 30%, respectively) at each stage. Furthermore, the percentage of embryos at these stages that developed to blastocysts with addition of follistatin was significantly lower (p < 0.05) than the percentage obtained with no addition (Fig. 4, A–D). In contrast, addition of activin A or follistatin at the 9- to 16-cell and morula stages did not affect the percentage of embryos that developed to blastocysts (Fig. 4, E and F). There was no significant difference in hatching rate of embryos among the treatments (Fig. 4, B–G), except that the percentage of zygotes that hatched was significantly higher (p < 0.05) with addition of activin A than with addition of follistatin (Fig. 4A).



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  		FIG. 4. Effects of 10 ng/ml of recombinant human activin A (ACT) or 10 ng/ml of follistatin (FS), added at various developmental stages, on the development of bovine embryos to the blastocyst and hatched blastocyst stages. The 1-cell (A), 2-cell (B), 3- to 4-cell (C), 5- to 8-cell (D), and 9- to 16-cell embryos (E), morulae (F), and blastocysts (G) were cultured from 20, 32, 42, 60, 90, 120, and 170 h postinsemination, respectively, to 220 h postinsemination. Data are presented as means and SD in 8 replicates. Numbers in parentheses represent the numbers of embryos tested per group. Different letters above the bars within the same category indicate significant differences (a vs. b, a vs. c: p < 0.05; b vs. c: p < 0.001).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study clearly demonstrate that activin A enhanced the developmental competence of bovine embryos to the morula and blastocyst stages when embryos were cultured under a gas atmosphere of 5% CO2:5% O2:90% N2. In contrast, follistatin not only neutralized the development-enhancing effect of activin A added to medium but also impeded the developmental competence of embryos by itself. The development-enhancing effect of activin A and the development-impeding effect of follistatin were observed when embryos were exposed to these cytokines up to the third cell cycle of the embryos.

In our previous study, we found that activin A increased dose-dependently the percentages of zygotes that developed to blastocysts by 6–17% under a gas atmosphere of 5% CO2 in air [12]. However, mSOF is optimized in the culture of bovine embryos under a low oxygen condition (5% O2) rather than under a gas atmosphere of 5% CO2 in air (20% O2) [6, 21, 22]. When the oxygen tension was reduced to 5% in the present study, activin A increased dose-dependently the percentages of zygotes that developed to blastocysts by 25–41% (Fig. 1). Although the mechanism by which oxygen concentration affects embryo development in vitro is not fully understood, in part, low oxygen tension may suppress the production of reactive oxygen species such as O2- and H2O2 that cause lipid peroxidation and enzyme inactivation, resulting in cell damage in embryos [10, 22, 31]. The mechanism by which activin A enhances embryo development is not clear. However, the action of activin A may differ from that of low oxygen tension, since activin A enhanced embryo development at oxygen tensions of both of 20% and 5%.

One of the findings from this study is that follistatin affects bovine embryo development. Follistatin is an activin-binding protein [25], and it has antagonizing effects on the actions of activin in pituitary [26] and granulosa [27, 28] cells. Recently, Silva and Knight [32] reported that follistatin opposed the stimulatory effect of activin A on blastocyst yield when both activin A and follistatin were added to the medium during IVM of bovine COC. The results from the present study also show that follistatin neutralized the development-enhancing effect of activin A when both cytokines were added to medium during in vitro development at a concentration of 10 ng/ml (Fig. 3). Moreover, follistatin had a dose-dependent inhibitory effect on the development to morulae and blastocysts whether activin A was added or not (Figs. 2 and 3). There is a possibility that follistatin antagonizes the action of endogenous activin A produced by embryo itself. In mice, it has been reported that activin A is present in all cells of the embryos, from the 1-cell stage to the compacted morula stage [33–35]. Since transcripts of only the inhibin ßA subunit, and not transcripts of the {alpha} and ßB subunits, were detectable in bovine embryos from the 1-cell stage to morulae, at least the cleavage-stage embryos may produce activin A in cattle [36].

In recent studies it was shown that activin A during IVM enhanced postcleavage development of bovine oocytes but not the initial cleavage [32, 37]. The present finding that activin A and/or follistatin did not affect the cleavage rate when activin A and/or follistatin was added to mSOF after fertilization (Figs. 1–3) is in agreement with these reports. While the number of morulae and blastocysts was affected by addition of activin A and/or follistatin to medium (Fig. 3), the quality of morulae and blastocysts, as determined by evaluation of their cell number, seemed to be similar among the groups (Table 1). Activin A and follistatin had an effect on blastocyst yield when they were added to culture of bovine embryos prior to the 9- to 16-cell stage (Fig. 4, A–D). In contrast, addition of either cytokine at the 9- to 16-cell and morula stages did not affect the number of blastocysts (Fig. 4, E and F). Although addition of activin A to presumptive zygotes resulted in a higher hatching rate of embryos than did no addition (Fig. 1) or addition of follistatin (Fig. 4A), this seems to be the result of an increased number of embryos that developed to blastocysts. These results suggest that activin A does not generally support cell proliferation throughout the early development of bovine embryos but rather stimulates it for a limited period or at a limited developmental stage. Activin A may act on bovine embryos until the third cell cycle and may enhance subsequent development of embryos. The activation of embryonic transcription, which follows the transition from maternal to embryonic control of development, is known as zygotic gene activation (ZGA) [38]. In bovine embryos, this transition occurs at the 8-cell stage [39] or earlier [40]. The absence of appropriate ZGA leads to the failure of further embryo development beyond the early cleavage divisions [38]. Therefore, the development-enhancing effect of activin A may be associated with ZGA. In addition, bovine embryos normally develop to the 8- to 16-cell stage and are transported from the oviduct into uterus at 3–4 days after ovulation [41, 42]. The developmental period during which addition of activin A to medium affected bovine embryo development in vitro in the present study (prior to the 9- to 16-cell stage at 90 h postinsemination) seems to coincide with the period during which bovine embryos are present in the oviduct in vivo. Since activin A is produced by oviduct epithelial cells in mice [33] and cows [43], addition of activin A to embryo culture in vitro may reproduce the environment of the cleavage-stage embryos in the oviduct in vivo.

In conclusion, our results suggest that activin A and follistatin may play important roles in the regulation of the developmental competence of bovine embryos. Both activin A and follistatin are present in mammalian oviducts [33, 43, 44] and may physiologically regulate the development of early preimplantation embryos. Since addition of activin A to culture medium up to the third cell cycle of embryos is effective, identification of the mechanism by which activin A promotes embryo development may provide insights into the processes by which regulatory molecules control the specific developmental processes in a temporal manner.


   ACKNOWLEDGMENTS
 
The authors thank the National Hormone and Pituitary Program, NIDDK, NICHD, and USDA for providing recombinant human follistatin and T. Miura for helping with oocyte collection.


   FOOTNOTES
 
1 This work was supported by grants from the Ministry of Agriculture, Forestry and Fisheries of Japan. Back

2 Correspondence: Koji Yoshioka, Laboratory of Theriogenology, National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. FAX: 81 298 38 7880; kojiyos{at}niah.affrc.go.jp Back

Accepted: June 9, 1998.

Received: April 2, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Camous S, Heyman Y, Méziou W, Ménézo Y. Cleavage beyond the block stage and survival after transfer of early bovine embryos cultured with trophoblastic vesicles. J Reprod Fertil 1984; 72:479–485.[Abstract/Free Full Text]
  2. Eyestone WH, First NL. A study of 8- to 16-cell developmental block in bovine embryos cultured in vitro. Theriogenology 1986; 25:152 (abstract).[CrossRef]
  3. Kajihara Y, Goto K, Nakanishi Y, Ogawa K. In vitro fertilization of bovine follicular oocytes and their development up to hatched blastocysts in vitro. Jpn J Anim Reprod 1987; 33:173–180.
  4. Eyestone WH, First NL. Co-culture of early bovine embryos with oviductal tissue or in conditioned medium. J Reprod Fertil 1989; 85:715–720.[Abstract/Free Full Text]
  5. Tervit HR, Whittingham DG, Rowson LEA. Successful culture in vitro of sheep and cattle ova. J Reprod Fertil 1972; 30:493–497.[Abstract/Free Full Text]
  6. Fukui Y, McGowan LT, James RW, Pugh PA, Tervit HR. Factors affecting the in-vitro development to blastocysts of bovine oocytes matured and fertilized in vitro. J Reprod Fertil 1991; 92:59–64.[Abstract/Free Full Text]
  7. Pinyopummintr T, Bavister BD. In vitro matured/in vitro fertilized bovine oocytes can develop into morulae/blastocysts in chemically defined, protein-free culture media. Biol Reprod 1991; 45:736–742.[Abstract]
  8. 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.
  9. Kim JH, Niwa K, Lim JM, Okuda K. Effects of phosphate, energy substrates, and amino acids on development of in vitro-matured, in vitro-fertilized bovine oocytes in a chemically defined, protein-free culture medium. Biol Reprod 1993; 48:1320–1325.[Abstract]
  10. Nagao Y, Saeki K, Hoshi M, Kainuma H. Effects of oxygen concentration and oviductal epithelial tissue on the development of in vitro matured and fertilized bovine oocytes cultured in protein-free medium. Theriogenology 1994; 41:681–687.
  11. Keskintepe L, Burnley CA, Brackett BG. Production of viable bovine blastocysts in defined in vitro conditions. Biol Reprod 1995; 52:1410–1417.[Abstract]
  12. Yoshioka K, Kamomae H. Recombinant human activin A stimulates development of bovine one-cell embryos matured and fertilized in vitro. Mol Reprod Dev 1996; 45:151–156.[CrossRef][Medline]
  13. Heyner S, Shah N, Smith RM, Watson AJ, Schultz GA. The role of growth factors in embryo production. Theriogenology 1993; 39:151–161.
  14. Gandolfi F. Autocrine, paracrine and environmental factors influencing embryonic development from zygote to blastocyst. Theriogenology 1994; 41:95–100.[CrossRef]
  15. Larson RC, Ignotz GG, Currie WB. Platelet derived growth factor (PDGF) stimulates development of bovine embryos during the fourth cell cycle. Development 1992; 115:821–826.[Abstract]
  16. Larson RC, Ignotz GG, Currie WB. Transforming growth factor ß and basic fibroblast growth factor synergistically promote early bovine embryo development during the fourth cell cycle. Mol Reprod Dev 1992; 33:432–435.[CrossRef][Medline]
  17. Fukui Y, Matsuyama K. Development of in vitro matured and fertilized bovine embryos cultured in media containing human leukemia inhibitory factor. Theriogenology 1994; 42:663–673.
  18. Thibodeaux JK, Del Vecchio RP, Hansel W. Role of platelet-derived growth factor in development of in vitro matured and fertilized bovine embryos. J Reprod Fertil 1993; 98:61–66.[Abstract/Free Full Text]
  19. de Moraes AAS, Hansen PJ. Granulocyte-macrophage colony-stimulating factor promotes development of in vitro produced bovine embryos. Biol Reprod 1997; 57:1060–1065.[Abstract]
  20. Lu RZ, Shiota K, Toyoda Y, Takahashi M. Activin A (EDF) releases the "two-cell block" of mouse embryos in culture. Jpn J Anim Reprod 1990; 36:127–132.
  21. Watson AJ, Watson PH, Warnes D, Walker SK, Armstrong DT, Seamark RF. Preimplantation development of in vitro-matured and in vitro-fertilized ovine zygotes: comparison between coculture on oviduct epithelial cell monolayers and culture under low oxygen atmosphere. Biol Reprod 1994; 50:715–724.[Abstract]
  22. Takahashi Y, Hishinuma M, Matsui M, Tanaka H, Kanagawa H. Development of in vitro matured/fertilized bovine embryos in a chemically defined medium: influence of oxygen concentration in gas atmosphere. J Vet Med Sci 1996; 58:897–902.[Medline]
  23. Ueno N, Ling N, Ying SY, Esch F, Shimasaki S, Guillemin R. Isolation and partial characterization of follistatin: a single chain Mr 35,000 monomeric protein that inhibits the release of follicle-stimulating hormone. Proc Natl Acad Sci USA 1987; 84:8282–8286.[Abstract/Free Full Text]
  24. Shimasaki S, Koga M, Esch F, Cooksey K, Mercado M, Koba A, Ueno N, Ying SY, Ling N, Guillemin R. Primary structure of the human follistatin precursor and its genomic organization. Proc Natl Acad Sci USA 1988; 85:4218–4222.[Abstract/Free Full Text]
  25. DePaolo LV, Bicsak TA, Erickson GF, Shimasaki S, Ling N. Follistatin and activin: a potential intrinsic regulatory system within diverse tissues. Proc Soc Exp Biol Med 1991; 198:500–512.[CrossRef][Medline]
  26. Kogawa K, Nakamura N, Sugino K, Takio K, Titani K, Sugino H. Activin-binding protein is present in pituitary. Endocrinology 1991; 128:1434–1440.[Abstract/Free Full Text]
  27. Nakamura T, Sugino K, Titani K, Sugino H. Follistatin, an activin-binding protein, associates with heparan sulfate chain of proteoglycans on follicular granulosa cells. J Biol Chem 1991; 266:19432–19437.[Abstract/Free Full Text]
  28. Shukovski L, Findlay JK, Robertson DM. The effect of follicle-stimulating hormone-suppressing protein or follistatin on luteinizing bovine granulosa cells in vitro and its antagonistic effect on the action of activin. Endocrinology 1991; 129:3395–3402.[Abstract/Free Full Text]
  29. Bavister BD, Leibfried ML, Lieberman G. Development of preimplantation embryos of the golden hamster in a defined culture medium. Biol Reprod 1983; 28:235–247.[Abstract]
  30. Brackett BG, Oliphant G. Capacitation of rabbit spermatozoa in vitro. Biol Reprod 1975; 12:260–274.[Abstract]
  31. Nasr-Esfahani MH, Aitken JR, Johnson MH. Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro and in vivo. Development 1990; 109:501–507.[Abstract]
  32. Silva CC, Knight PG. Modulatory actions of activin-A and follistatin on the developmental competence of in vitro-matured bovine oocytes. Biol Reprod 1998; 58:558–565.[Abstract/Free Full Text]
  33. Lu RZ, Shiota K, Tachi C, Takahashi M. Histochemical demonstration of activin/inhibin ßA-, ßB- and {alpha}-subunits in early embryos and oviducts of different strains of mice. J Reprod Dev 1992; 38:79–90.
  34. Albano RM, Groome N, Smith JC. Activins are expressed in preimplantation mouse embryos and in ES and EC cells and are regulated on their differentiation. Development 1993; 117:711–723.[Abstract]
  35. Lu RZ, Matsuyama S, Nishihara M, Takahashi M. Developmental expression of activin/inhibin ßA, ßB, and {alpha} subunits, and activin receptor-IIB genes in preimplantation mouse embryos. Biol Reprod 1993; 49:1163–1169.[Abstract]
  36. Yoshioka K, Takata M, Taniguchi T, Yamanaka H, Sekikawa K. Gene expression of activin subunits, activin receptors and follistatin in preimplantation bovine embryos. Theriogenology 1997; 47:221 (abstract).
  37. Stock AE, Woodruff TK, Smith LC. Effects of inhibin A and activin A during in vitro maturation of bovine oocytes in hormone- and serum-free medium. Biol Reprod 1997; 56:1559–1564.[Abstract]
  38. De Sousa PA, Caveney A, Westhusin ME, Watson AJ. Temporal patterns of embryonic gene expression and their dependence on oogenetic factors. Theriogenology 1998; 49:115–128.[CrossRef][Medline]
  39. Barns FL, First NL. Embryonic transcription in in vitro cultured bovine embryos. Mol Reprod Dev 1991; 29:117–123.[CrossRef][Medline]
  40. Viuff D, Avery B, Greve T, King WA, Hyttel P. Transcriptional activity in in vitro produced bovine two- and four-cell embryos. Mol Reprod Dev 1996; 43:171–179.[CrossRef][Medline]
  41. Brinster RL. Embryo development. J Anim Sci 1974; 38:1003-1012.
  42. Bazer FW, Geisert RD, Zavy MT. Fertilization, cleavage, and implantation. In: Hafez ESE (ed)., Reproduction in Farm Animals, 6th edition. Philadelphia: Lea and Febiger; 1993: 188–212.
  43. Gandolfi F, Modina S, Brevini TAL, Passoni L, Artini P, Petraglia F, Lauria A. Activin ßA subunit is expressed in bovine oviduct. Mol Reprod Dev 1995; 40:286–291.[CrossRef][Medline]
  44. Kogawa K, Ogawa K, Hayashi Y, Nakamura T, Titani K, Sugino H. Immunohistochemical localization of follistatin in rat tissues. Endocrinol Jpn 1991; 38:383–391.[Medline]


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