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Attachment of porcine blastocysts to fibroblast monolayers in vitro
THERIOGENOLOGY
ATTACHMENT FIBROBLAST
OF PORCINE MONOLAYERS
BLASTOCYZTS IN YITRO ’ --
;50 ’
Frank B. Kuzan and Raymond Wd Wright, Jr. Washington State University, Pullman, WA 99164 Date Received: July 30, 1981
accepted: September 25, 1981 Abstract The objective of this study was to compare the ability of porcine blastocysts to attach to various cellular and non-cellular substrates h vitro. One hundred twenty-two hatched blastocysts were collected from 17 handmated gilts and sows at slaughter. Blastocysts were randomly assigned to one of four treatments: Minimal Essential Medium (MEM) supplemented with 10% (v/v) heat treated fetal calf serum (HTFCS), monolayers of bovine uterine fibroblasts in MEM + 10% HTFCS (Buf), monolayers of bovine testicular fibroblasts in MEM + 10% HTFCS (Etes), and MEM + 10% HTFCS exposed to uterine fibroblasts for 24 hr to condition the medium (cMEM). Embryos were cultured individually in 24 well Linbro culture plates at 37 C Embryos were observed at in a humidified gas atmosphere of 5% CO in air. 24 hr intervals by phase contrast micro&opy (100X) and measured with an ocular micrometer. Blastocyst attachment was greater (P < .Ol) in Buf (21/35) compared to MEM (7/32), cMEM (g/28), and Btes (l/27). Embryo diameter was greater (P < .05) 24 hr prior to, attachment in Buf compared to the other treatments. In addition, trophoblast monolayers continued to proliferate for 20 days when cocultured with uterine fibroblasts. These observations suggest that uterine fibroblasts provide a superior substratum for blastocyst attachment and the maintenance of swine trophoblast cells b vitro.
(a) (b) (c)
(d)
Scientific Paper No. 5986. College of Agricultural Research Center, Washington State University, Pullman. Project 0313. Supported in part by a grant from Select Sires, Inc. The authors wish to express their gratitude to the Washington State Pork Producers for contributions made to this study and to Dr. J. E. Evermann for the bovine testicular fibroblasts. Dept. Anim. Sci.
DECEMBER
198 1 VOL. 16 NO. 6
651
THERIOGENOLOGY
INTRODUCTION Several culture systems have been developed to investigate preimplantation development of swine embryos in vitro (1, 2 and 3). These studies did not describe the requirements for mzn=ce of the swine blastocyst & vitro. Shaffer and Wright (4) devised a culture system which would support the attachment of swine blastocysts and the proliferation of the trophoblast on a plastic or collagen substratum. Rob1 -et al. (5) reported that a modified Krebs-Ringer bicarbonate buffer supplemented with sheep serum would support the development of swine blastocysts for at least 48 hr -in vitro. Rob1 et al. failed to observe blastocyst attachment; however, the embryos remai~d~iable as determined bv an increase in cell number. Cole and Paul (6), Salomon-and Sherman (7), and Glass -et al. (8) have utilized cultured mouse and human cells (uterine. L-cells, JLS-VII cells, liver, and teratocarcinoma) to investigate the attachment-process of mouse blastocysts ti vitro. Blastocysts attached to the cellular monolayer in a manner similar to -- vivo attachment (7). Although in vivo mouse trophoblast cells are more invasive than those of swGeembryos, a coculture system may prove useful for the study of swine blastocyst attachment fi vitro. Therefore, the objectives of this study were to compare the ability of swine blastocysts to attach to fibroblast monolayers or a plastic substratum and to evaluate the invasive qualities of the porcine trophoblast -in vitro. MATERIALS
AND METHOOS
Animals and Embryo Collection. Blastocysts were collected at slaughter from 17 handmated gilts and sows (Yorkshire X Hampshire) 6 to 7 days after the onset of estrus (day 0). Embryos were flushed from the uteri with Minimal Essential Medium (MEM, Grand Island Biological Co.) as described by Shaffer and Wright (4). Blastocysts were washed three times in MEM supplemented with 10% (v/v) heat treated fetal calf serum (HTFCS, GIBCO) and placed randomly into one of four culture systems. Fibroblast Culture. Bovine uterine fibroblasts were obtained from Cubes of uterine endometrial tissue 4 to 5 davs after the onset of estrus. endometrial tissue, 1 to 2 mmJ, were-plated in 25 cmL culture flasks and maintained at 37 C in a humidified gas atmosphere of 5% CO2 in air. Following 3 days of culture fibroblast-like cells migrated from the tissue to produce cellular monolayers. At 3 day intervals, one-half of the Culture medium was replaced with fresh medium (MEM + 10% HTFCS). At 2 week intervals following the initiation of the cultuses, cells were trypsinized (.25% solution, GIBCO) and transferred to 75 cm culture flasks. Cells were plated in 24 well Linbro culture plates, at a density of 40,000 cells per Following 24 hr of incubation conditioned well, for use in embryo culture. medium was collected from uterine fibroblasts adjusted to pH 7.2, resterilized by filtration (.22 pm filter) and stored for as long as 3 days (4 C) prior to embryo culture. Bovine testicular fibroblasts were obtained from aseptically collected calf testes by the method of Younger (9). These cells were maintained in the same manner and used at the same cell density as uterine fibroblasts. Blastocysts were randomly assigned to one of four treatm. M M + 10% HTFCS (MEM), monolayers of bovine uterine fibroblasts in MEM + 10% HTFCS (Buf), monolayers of bovine testicular fibro-
652
DECEMBER
1981 VOL. 16 NO. 6
THERIOGENOLOGY
blasts in MEM + 10% HTFCS (Btes), and MEM + 10% HTFCS which had been exposed to uterine fibroblasts for 24 hr to condition the medium (cMEM). Culture plates were prepared so that each of the 4 treatments were represented by 6 culture wells. Blastocysts were cultured individually in .5 ml of culture medium at 37 C in a humidified gas atmosphere of 5% CO in air. One-half of the culture medium was replaced with fresh medium ever B third day. Embryos were observed at 24 hr intervals by phase contrast microscopy (100X), subjectively rated (attached/not attached), and measured with an ocular micrometer. Trophoblast monolavers were maintained in culture until obvious signs of cellular degeneration were observed. Treatment differences were evaluated by Chi-square analysis using the criteria of attachment vs. non-attachment. in embryo diameter Differences were tested by an one-way analysis of variance (10).
RESULTS
AND DISCUSSION
Attachment of swine blastocysts to the various substrata is presented in table 1. No difference was observed between MEM and cMEM in promoting blastocyst attachment to the plastic substratum (table 1). Bovine uterine fibroblasts were superior (P < .Ol) in promoting blastocyst attachment, while bovine testicular fibroblasts had an adverse effect (P < .05) on attachment (table 1).
TABLE
1.
Swine Blastocyst Cellular
and Non-cellular
Number Substrata
Attachment
of
Blastocysts
and Size at Attachment Substrata
Number of Blastocysts Attached
to
In Vitro --
Blastocyst Diameter 24 hr Prior to Attachment ii (urn)
SD
MEM
32
7a
259.4d
103.5
Buf
35
2lb
517.5e
147.7
Btes
28
lC
207.0d
0.0
cMEM
27
ga
193.zd
47.6
a,b,c,d,e Values with different b(P < _Ol>, c*e(P
DECEMBER
superscripts
are different.
< .05)
198 1 VOL. 16 NO. 6
653
THERIOGENOLOGY
Swine blastocysts collected 6 to 7 days after the onset of estrus were free of the zona pellucida (figure 1) with an average diameter of 314 pm. Mean embryo diameter did not differ (P > .lO) among treatments at the onset of culture. Blastocysts cocultured with uterine fibroblasts, however, were larger (P < .05) in diameter 24 hr prior to attachment (table 1).
Figure
1. Porcine blastocyst collected 6 days after mating. This blastocyst has recently emerged from the zona pellucida in utero (150 X). --
Blastocysts remained suspended in the culture medium for an average of 4 days (range 2 to 5 days) before attachment to the substratum (figure 2). blastocysts began If attachment failed to occur by 5 days, the free-floating to degenerate (figure 3). Following attachment the trophoblast grew as a
654
DECEMBER 1981 VOL. 16 NO. 6
THERIOGENOLOGY
Figurxe 2.
Figul-e 3.
DECEMBER
Swine blastocyst after 4 days of culture undergoing Note the slight trophoattachment to the substratum. outgrowth over the uterine fibroblasts 0-1.
Swine blastocyst after 4 days of culture undergoing de!genNote the loss of embryonic cells (E). (150 XI. eration.
1981 VOL. 16 NO. 6
655
THERIOGENOLOGY
monolayer (figure 4) and in coculture with fibroblasts (Buf or Btes) did not displace cells from the monolayer (figure 4). When grown in monolayer,
Figure 4.
Swine blastocyst 3 days after attachment monolayer. Note the shrunken appearance cell mass (E) (150 X).
to a uterine of the inner
swine trophoblast cells appeared to contain a greater amount of cytoplasm than bovine fibroblasts. The trophoblast cells were irregular, octagonal, in shape with prominent nuclei, while fibroblasts were spindle shaped containing small nuclei. Trophoblast cells, therefore, appeared much larger than fibroblast cells when viewed with phase contrast optics. In coculture with fibroblasts the trophoblast cells were not cell contact inhibited, which is characteristic of fibroblasts in vitro. The inner cell mass remained near the center of the trophobT%ticutgrowth (figure 2) and in all cases appeared to degenerate 3 days after trophoblast attachment (figure 4). The trophoblast of all attached embryos continued to prolifer$te for at least 20 days at which time the monolayer covered an area of 30 mm .
656
DECEMBER
1981 VOL. 16 NO. 6
THERIOGENOLOGY
Trophoblast cells of the porcine embryo undergo rapid prolifera_tion between days 9 and 10 of development with the embryo increasing in length to 14 mm by day 12 of development (11). A loose attachment between the embryo and uterine wall occurs as early as day 10 of development (11). Blastocyst elongation was not observed in this study, however, loose attachment of the blastocyst to the substratum was observed between days 8 and 12 of development. The process of swine blastocyst attachment and trophoblast outgrowth on non-cellular substrata has been described by Shaffer and Wright (4). Swine blastocysts cocultured with fibroblasts followed the same general developmental process with the following differences: blastocysts remained suspended in the medium for an average of 4 days compared to 24 hr, and trophoblast cells continued to proliferate for 20 days compared to 3 days. In the previous study (4) blastocysts were collected as late as day 9 of development, which may account for these differences. To investigate the possibility that uterine fibroblasts were releasing a "factor" which induced blastocyst attachment, conditioned medium was employed. The results of this study indicate that conditioned medium had no And furthermore, effect on blastocyst attachment to the plastic substratum. that uterine fibroblasts, or a surface protein of the fibroblasts, were required for enhanced -in vitro attachment. The use of conditioned medium, however, does not rule out the possibility that a labile substance released by the fibroblasts signals blastocyst attachment. Viability, as measured by embryo diameter, was greater in blast0 cysts Blastocysts cocultured with uterine cocultured with uterine fibroblasts. fibroblasts increased an average of 400 urn in diameter before attachment, while blastocysts in the other treatments decreased in size. This decrease in size was due to either cell loss (Btes) or the collapse of the blastocoele (MEM, cMEM). This observation suggests that the presence of uterine fibroblasts increased the viability of blastocysts in vitro, since cMEM had no effect on blastocyst diameter. The results of this study suggest that the coculture of porcine blastocysts with uterine fibroblasts provides a superior environment for the development of the trophoblast in vitro, and that the porcine trophoblast is non-invasive in vitro. Medium znd%?&ed by uterine fibroblasts did not enhance embryo attachment in-vitro, suggesting that physical presence of uterine fibroblasts was required. In addition, a coculture system has been described which may prove useful as a model to study early implantation in the pig.
DECEMBER
1981 VOL. 16 NO. 6
657
THERIOGENOLOGY
Literature
Cited
Polge, C. and C.L. Frederick. Culture and storage of fertilized pig Paris 1:211. eggs. VI Cong. Intern. Reprod. Anim. Insem. Artif. (Abstract). 1968. 2.
Pope, C.E. and B.N. Day. Culture of swine embryos -in vitro. J. Anim. Sci. 31:1035. (Abstract). 1970.
3.
Wright, R.W. Successful culture in vitro of swine embryos 1977. to the blastocyst stage. J.%im.ci. 44:854-858.
4.
Shaffer, S.J. and R.W. Wright. Attachment and trophoblastic outgrowth J. Anim. Sci. 46:1712-1717. 1978. of swine blastocysts -in vitro.
5.
Robl, J.M., J.L. Nelssen and D.L. Davis. Swine blastocyst development in vitro. J. Anim. Sci. 49(Suppl. 1):332. (Abstract). 1979. --
6.
Cole, R.J. and J. Paul. Properties of cultured preimplantation mouse In G.E.W. and rabbit embryos, and cell strains derived from them. Wolstenholm and M. O'Connor (Ed.) Preimplantation Stages 3 Pregnancy Little, Brown and Co., Boston. p. 82-122. 1965.
7.
Implantation and invasiveness of mouse Salomon, P.S. and M.I. Sherman. 1975. blastocysts on uterine monolayers. Exp. Cell Res. 90:261-218.
8.
Glass, R.H., A.I. Spindle and R.A. Pederson. Mouse embryo attachment to substratum and interactions of trophoblast with cultured cells. J. Exp. Zool. 208:327-335. 1979.
9.
Younger, J.S. Monolayer tissue cultures. I. preparation and standardization of suspensions of trypsin dispersed monkey kidney cells. Proc. Sot. Exp. Biol. Med. 85:202-205. 1954.
10.
Steele, R.G. and J.H. Torrie. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York. 1980.
11.
Anderson, L.L. Growth, protein content, and distribution Anat. Rec. 190:143-148. 1978. of early pig embryos.
658
DECEMBER
1981 VOL. 16 NO. 6
ATTACHMENT FIBROBLAST
OF PORCINE MONOLAYERS
BLASTOCYZTS IN YITRO ’ --
;50 ’
Frank B. Kuzan and Raymond Wd Wright, Jr. Washington State University, Pullman, WA 99164 Date Received: July 30, 1981
accepted: September 25, 1981 Abstract The objective of this study was to compare the ability of porcine blastocysts to attach to various cellular and non-cellular substrates h vitro. One hundred twenty-two hatched blastocysts were collected from 17 handmated gilts and sows at slaughter. Blastocysts were randomly assigned to one of four treatments: Minimal Essential Medium (MEM) supplemented with 10% (v/v) heat treated fetal calf serum (HTFCS), monolayers of bovine uterine fibroblasts in MEM + 10% HTFCS (Buf), monolayers of bovine testicular fibroblasts in MEM + 10% HTFCS (Etes), and MEM + 10% HTFCS exposed to uterine fibroblasts for 24 hr to condition the medium (cMEM). Embryos were cultured individually in 24 well Linbro culture plates at 37 C Embryos were observed at in a humidified gas atmosphere of 5% CO in air. 24 hr intervals by phase contrast micro&opy (100X) and measured with an ocular micrometer. Blastocyst attachment was greater (P < .Ol) in Buf (21/35) compared to MEM (7/32), cMEM (g/28), and Btes (l/27). Embryo diameter was greater (P < .05) 24 hr prior to, attachment in Buf compared to the other treatments. In addition, trophoblast monolayers continued to proliferate for 20 days when cocultured with uterine fibroblasts. These observations suggest that uterine fibroblasts provide a superior substratum for blastocyst attachment and the maintenance of swine trophoblast cells b vitro.
(a) (b) (c)
(d)
Scientific Paper No. 5986. College of Agricultural Research Center, Washington State University, Pullman. Project 0313. Supported in part by a grant from Select Sires, Inc. The authors wish to express their gratitude to the Washington State Pork Producers for contributions made to this study and to Dr. J. E. Evermann for the bovine testicular fibroblasts. Dept. Anim. Sci.
DECEMBER
198 1 VOL. 16 NO. 6
651
THERIOGENOLOGY
INTRODUCTION Several culture systems have been developed to investigate preimplantation development of swine embryos in vitro (1, 2 and 3). These studies did not describe the requirements for mzn=ce of the swine blastocyst & vitro. Shaffer and Wright (4) devised a culture system which would support the attachment of swine blastocysts and the proliferation of the trophoblast on a plastic or collagen substratum. Rob1 -et al. (5) reported that a modified Krebs-Ringer bicarbonate buffer supplemented with sheep serum would support the development of swine blastocysts for at least 48 hr -in vitro. Rob1 et al. failed to observe blastocyst attachment; however, the embryos remai~d~iable as determined bv an increase in cell number. Cole and Paul (6), Salomon-and Sherman (7), and Glass -et al. (8) have utilized cultured mouse and human cells (uterine. L-cells, JLS-VII cells, liver, and teratocarcinoma) to investigate the attachment-process of mouse blastocysts ti vitro. Blastocysts attached to the cellular monolayer in a manner similar to -- vivo attachment (7). Although in vivo mouse trophoblast cells are more invasive than those of swGeembryos, a coculture system may prove useful for the study of swine blastocyst attachment fi vitro. Therefore, the objectives of this study were to compare the ability of swine blastocysts to attach to fibroblast monolayers or a plastic substratum and to evaluate the invasive qualities of the porcine trophoblast -in vitro. MATERIALS
AND METHOOS
Animals and Embryo Collection. Blastocysts were collected at slaughter from 17 handmated gilts and sows (Yorkshire X Hampshire) 6 to 7 days after the onset of estrus (day 0). Embryos were flushed from the uteri with Minimal Essential Medium (MEM, Grand Island Biological Co.) as described by Shaffer and Wright (4). Blastocysts were washed three times in MEM supplemented with 10% (v/v) heat treated fetal calf serum (HTFCS, GIBCO) and placed randomly into one of four culture systems. Fibroblast Culture. Bovine uterine fibroblasts were obtained from Cubes of uterine endometrial tissue 4 to 5 davs after the onset of estrus. endometrial tissue, 1 to 2 mmJ, were-plated in 25 cmL culture flasks and maintained at 37 C in a humidified gas atmosphere of 5% CO2 in air. Following 3 days of culture fibroblast-like cells migrated from the tissue to produce cellular monolayers. At 3 day intervals, one-half of the Culture medium was replaced with fresh medium (MEM + 10% HTFCS). At 2 week intervals following the initiation of the cultuses, cells were trypsinized (.25% solution, GIBCO) and transferred to 75 cm culture flasks. Cells were plated in 24 well Linbro culture plates, at a density of 40,000 cells per Following 24 hr of incubation conditioned well, for use in embryo culture. medium was collected from uterine fibroblasts adjusted to pH 7.2, resterilized by filtration (.22 pm filter) and stored for as long as 3 days (4 C) prior to embryo culture. Bovine testicular fibroblasts were obtained from aseptically collected calf testes by the method of Younger (9). These cells were maintained in the same manner and used at the same cell density as uterine fibroblasts. Blastocysts were randomly assigned to one of four treatm. M M + 10% HTFCS (MEM), monolayers of bovine uterine fibroblasts in MEM + 10% HTFCS (Buf), monolayers of bovine testicular fibro-
652
DECEMBER
1981 VOL. 16 NO. 6
THERIOGENOLOGY
blasts in MEM + 10% HTFCS (Btes), and MEM + 10% HTFCS which had been exposed to uterine fibroblasts for 24 hr to condition the medium (cMEM). Culture plates were prepared so that each of the 4 treatments were represented by 6 culture wells. Blastocysts were cultured individually in .5 ml of culture medium at 37 C in a humidified gas atmosphere of 5% CO in air. One-half of the culture medium was replaced with fresh medium ever B third day. Embryos were observed at 24 hr intervals by phase contrast microscopy (100X), subjectively rated (attached/not attached), and measured with an ocular micrometer. Trophoblast monolavers were maintained in culture until obvious signs of cellular degeneration were observed. Treatment differences were evaluated by Chi-square analysis using the criteria of attachment vs. non-attachment. in embryo diameter Differences were tested by an one-way analysis of variance (10).
RESULTS
AND DISCUSSION
Attachment of swine blastocysts to the various substrata is presented in table 1. No difference was observed between MEM and cMEM in promoting blastocyst attachment to the plastic substratum (table 1). Bovine uterine fibroblasts were superior (P < .Ol) in promoting blastocyst attachment, while bovine testicular fibroblasts had an adverse effect (P < .05) on attachment (table 1).
TABLE
1.
Swine Blastocyst Cellular
and Non-cellular
Number Substrata
Attachment
of
Blastocysts
and Size at Attachment Substrata
Number of Blastocysts Attached
to
In Vitro --
Blastocyst Diameter 24 hr Prior to Attachment ii (urn)
SD
MEM
32
7a
259.4d
103.5
Buf
35
2lb
517.5e
147.7
Btes
28
lC
207.0d
0.0
cMEM
27
ga
193.zd
47.6
a,b,c,d,e Values with different b(P < _Ol>, c*e(P
DECEMBER
superscripts
are different.
< .05)
198 1 VOL. 16 NO. 6
653
THERIOGENOLOGY
Swine blastocysts collected 6 to 7 days after the onset of estrus were free of the zona pellucida (figure 1) with an average diameter of 314 pm. Mean embryo diameter did not differ (P > .lO) among treatments at the onset of culture. Blastocysts cocultured with uterine fibroblasts, however, were larger (P < .05) in diameter 24 hr prior to attachment (table 1).
Figure
1. Porcine blastocyst collected 6 days after mating. This blastocyst has recently emerged from the zona pellucida in utero (150 X). --
Blastocysts remained suspended in the culture medium for an average of 4 days (range 2 to 5 days) before attachment to the substratum (figure 2). blastocysts began If attachment failed to occur by 5 days, the free-floating to degenerate (figure 3). Following attachment the trophoblast grew as a
654
DECEMBER 1981 VOL. 16 NO. 6
THERIOGENOLOGY
Figurxe 2.
Figul-e 3.
DECEMBER
Swine blastocyst after 4 days of culture undergoing Note the slight trophoattachment to the substratum. outgrowth over the uterine fibroblasts 0-1.
Swine blastocyst after 4 days of culture undergoing de!genNote the loss of embryonic cells (E). (150 XI. eration.
1981 VOL. 16 NO. 6
655
THERIOGENOLOGY
monolayer (figure 4) and in coculture with fibroblasts (Buf or Btes) did not displace cells from the monolayer (figure 4). When grown in monolayer,
Figure 4.
Swine blastocyst 3 days after attachment monolayer. Note the shrunken appearance cell mass (E) (150 X).
to a uterine of the inner
swine trophoblast cells appeared to contain a greater amount of cytoplasm than bovine fibroblasts. The trophoblast cells were irregular, octagonal, in shape with prominent nuclei, while fibroblasts were spindle shaped containing small nuclei. Trophoblast cells, therefore, appeared much larger than fibroblast cells when viewed with phase contrast optics. In coculture with fibroblasts the trophoblast cells were not cell contact inhibited, which is characteristic of fibroblasts in vitro. The inner cell mass remained near the center of the trophobT%ticutgrowth (figure 2) and in all cases appeared to degenerate 3 days after trophoblast attachment (figure 4). The trophoblast of all attached embryos continued to prolifer$te for at least 20 days at which time the monolayer covered an area of 30 mm .
656
DECEMBER
1981 VOL. 16 NO. 6
THERIOGENOLOGY
Trophoblast cells of the porcine embryo undergo rapid prolifera_tion between days 9 and 10 of development with the embryo increasing in length to 14 mm by day 12 of development (11). A loose attachment between the embryo and uterine wall occurs as early as day 10 of development (11). Blastocyst elongation was not observed in this study, however, loose attachment of the blastocyst to the substratum was observed between days 8 and 12 of development. The process of swine blastocyst attachment and trophoblast outgrowth on non-cellular substrata has been described by Shaffer and Wright (4). Swine blastocysts cocultured with fibroblasts followed the same general developmental process with the following differences: blastocysts remained suspended in the medium for an average of 4 days compared to 24 hr, and trophoblast cells continued to proliferate for 20 days compared to 3 days. In the previous study (4) blastocysts were collected as late as day 9 of development, which may account for these differences. To investigate the possibility that uterine fibroblasts were releasing a "factor" which induced blastocyst attachment, conditioned medium was employed. The results of this study indicate that conditioned medium had no And furthermore, effect on blastocyst attachment to the plastic substratum. that uterine fibroblasts, or a surface protein of the fibroblasts, were required for enhanced -in vitro attachment. The use of conditioned medium, however, does not rule out the possibility that a labile substance released by the fibroblasts signals blastocyst attachment. Viability, as measured by embryo diameter, was greater in blast0 cysts Blastocysts cocultured with uterine cocultured with uterine fibroblasts. fibroblasts increased an average of 400 urn in diameter before attachment, while blastocysts in the other treatments decreased in size. This decrease in size was due to either cell loss (Btes) or the collapse of the blastocoele (MEM, cMEM). This observation suggests that the presence of uterine fibroblasts increased the viability of blastocysts in vitro, since cMEM had no effect on blastocyst diameter. The results of this study suggest that the coculture of porcine blastocysts with uterine fibroblasts provides a superior environment for the development of the trophoblast in vitro, and that the porcine trophoblast is non-invasive in vitro. Medium znd%?&ed by uterine fibroblasts did not enhance embryo attachment in-vitro, suggesting that physical presence of uterine fibroblasts was required. In addition, a coculture system has been described which may prove useful as a model to study early implantation in the pig.
DECEMBER
1981 VOL. 16 NO. 6
657
THERIOGENOLOGY
Literature
Cited
Polge, C. and C.L. Frederick. Culture and storage of fertilized pig Paris 1:211. eggs. VI Cong. Intern. Reprod. Anim. Insem. Artif. (Abstract). 1968. 2.
Pope, C.E. and B.N. Day. Culture of swine embryos -in vitro. J. Anim. Sci. 31:1035. (Abstract). 1970.
3.
Wright, R.W. Successful culture in vitro of swine embryos 1977. to the blastocyst stage. J.%im.ci. 44:854-858.
4.
Shaffer, S.J. and R.W. Wright. Attachment and trophoblastic outgrowth J. Anim. Sci. 46:1712-1717. 1978. of swine blastocysts -in vitro.
5.
Robl, J.M., J.L. Nelssen and D.L. Davis. Swine blastocyst development in vitro. J. Anim. Sci. 49(Suppl. 1):332. (Abstract). 1979. --
6.
Cole, R.J. and J. Paul. Properties of cultured preimplantation mouse In G.E.W. and rabbit embryos, and cell strains derived from them. Wolstenholm and M. O'Connor (Ed.) Preimplantation Stages 3 Pregnancy Little, Brown and Co., Boston. p. 82-122. 1965.
7.
Implantation and invasiveness of mouse Salomon, P.S. and M.I. Sherman. 1975. blastocysts on uterine monolayers. Exp. Cell Res. 90:261-218.
8.
Glass, R.H., A.I. Spindle and R.A. Pederson. Mouse embryo attachment to substratum and interactions of trophoblast with cultured cells. J. Exp. Zool. 208:327-335. 1979.
9.
Younger, J.S. Monolayer tissue cultures. I. preparation and standardization of suspensions of trypsin dispersed monkey kidney cells. Proc. Sot. Exp. Biol. Med. 85:202-205. 1954.
10.
Steele, R.G. and J.H. Torrie. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York. 1980.
11.
Anderson, L.L. Growth, protein content, and distribution Anat. Rec. 190:143-148. 1978. of early pig embryos.
658
DECEMBER
1981 VOL. 16 NO. 6