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In vitro culture and maintenance of trophoblastic vesicles: Observations on the development of contractile structures with concurrent vessel development
THERIOGENOLOGY
IN VITRO CULTURE AND MAINTENANCE OF TROPHCBLASTIC OBSERVATIONS ON THE DEVELOPMENT OF CONTRACTILE WITH CONCURRENT VESSEL DEVELOPMENT
J.P.
Received
VESICLES: STRUCTURES
Selgrath and R. W. Wright, Jr.* Department of Animal Sciences Washington State University Pullman, WA 99164-6332 for publication: Accepted:
April 25, 1987 November 11, 1987
ABSTRACT The use of trophoblastic vesicles as a model for the study of embryo development and cell to cell maternal recognition of pregnancy, In this report, we describe interaction has increased in recent years. the long-term culture of trophcblastic vesicles derived from enzymatically dispersed porcine blastocysts. Day 12 to 14 porcine embryos were enzymatically diSpersed to form single cell suspensions, then allowed tc plate in 25 cm Vesicles formed 3 to 14 d tissue-culture flasks. following plating and ranged in size from 0.1 to 4.Omm in diameter. Vesicles lamer than 1.5 mm at formation were able to float free in the medium for up to 65 days, at which time vesicles were histologically sectioned. Contractile elements formed in association with dense cell Movement of cells areas, and were found with tubular vascular vessels. that resembled eosinophilic cells was observed in the vessels and pulsed in synchrony with contractions of the primitive heart. Histological examination gave evidence of primitive blood cells and cardiac tissue, and no evidence of neural tissue was found, indicating the probability that the contractions were induced by permeable membrane depolarization. It is hypothesized from the observations reported in this paper that the cell differentiation required to form the contractile region and the vessel are under the control of the dense cell area. This indicates that trophoblastic vesicles formed by the enzymatic dispersal of porcine blastocysts is a possible model for the study of induced cell differentiation and formation of the cardiac organ system in vitro. Key words:
Porcine,
trophoblastic
vesicles,
culture,
development
INTRODUCTION Formation of vesicles derived mainly of trophoblastic cells has been accomplished by microdissection (l-4) by the selective killing of inner cell mass cells (5), or by the enzymatic dispersal of preimplantation Trophoblastic vesicles have allowed the determination of embryos (6,7). cell fates in developing embryos (8). Microinjection of inner cell mass cells into trophoblastic has been used to form intraspecific and interspecific chimeras, for the study of varied genetic effects on development (9). 1
Research supported and Home Economics 27725. Reprint requests.
vesicles allowing
by Washington State Universit College of Agriculture Research Center (Project 0313 r . Scientific Paper No.
APRIL 1988 VOL. 29 NO. 4
THERIOGENOLOGY
Information of trophoblastic effects on maternal recognition of pregnancy (3) and the use of trophoblastic cells in culture to bypass the block stage of development in vitro (2,4,10) have indicated that trophoblastic vesicles or trophoblastic cells can be used as models for the study of maternal-embryonic interaction and cell-to-cell interaction in farm animals. Enzymatic dispersal of preimplantation embryos was first described by Kuzan and Wright (6). Subsequent analysis described trilaminar tissue and eosinophilic development formation, contractile element presence, In this report we describe the morphological events of tissue (7). differentiation in trophoblastic vesicles. METHODS Handmated crossbred gilts and sows were slaughtered 12, 13, 14 and 15 d post breeding and the reproductive tracts were recovered. Corpora lutea (CL) were counted and filamentous embryos were recovered by flushing each uterine horn with Dulbecco's phcsthate buffered saline and 1% antibioticsolution (P#S) with 1% newborn calf serum (NCS) Embryos were washed three times in complete antimycotic (complete PBS). PRS. Embryos were then recovered from final rinse and transferred to a Five mls of 0.25% sterile petri dish with as little fluid as possible. trypsin in calcium and magnesium free Hanks balanced salt solution were Embryo cells were further added to enzymatically disperse the embryos. separated using agitation, accomplished by drawing media and cells through a sterile Pasteur pipet. Following dispersal, cells were transferred in equal volume to two Enzymatic bactivity was inhibited by the disposable centrifuqe tubes. (MEM! with, 10% olasma derived addition of 8 mls minimum essential media Ceil suspensions equine seruma (EqPDS) plus 1% antibiotic-antimycotic.D were washed by three-time centrifugation at 400 xg for 10 min, supernatant aspirated, and pellet resuspended in complete MEM. Dispersed cells were transferred to 25 cm tissue culture flasks and incubated under a 95% air, 5% CO2 atmosphere at 37°C. Trophoblastic
Vesicle
Formation
Dispersed embryonic cells were allowed to plate to tissue culture flasks for 3 d, after which flasks were poured off and floating nonplated cells were collected and pelleted by centrifugation at 400 xg, then they were resuspended and transferred to 25-cm tissue culture flasks. After 4 d, one-half of complete MEM was replaced daily in the flasks. Tissue culture flasks were observed daily for trophoblastic vesicles formation or for evidence of cell aggregation. Formed vesicles were transferred to tissue culture flasks with complete MEM. ZHyclone; Logan, Utah. Glbco Laboratories, Grand
874
Island,
New York.
APRIL
1988 VOL.
29 NO. 4
THERIOGENOLOGY
Observations Trophoblastic vesicles were measured daily by inverted microscope Plated cells and micrometer, and changes in size or shape were noted. examined daily for evidence of cellular aggregation. also were Contractile tissues were observed daily for rate and degree of contraction. Trophoblastic vesicles exhibiting contractile elements Sections were examined and stained with hemotoxylin-eosin. eosinophilic elements.
were sectioned for contractile,
RESULTS Time sequence of trophoblastic vesicle element activity is shown in Figure 1.
formation
and
contractile
Dispersed cells transferred tc tissue culture flasks became attached Vesicle expansion occurred and formed monolayers by 24 h posttransfer. from the cellular aggregations on the plate surface that were released to float free in the medium. In addition, floating cells were able to form vesicles by aggregation and continued vesicle expansion similar to the Vesicles morula to blastocyst transformation in the developing embryo. to form vesicles were formed by Gay 2 of culture, and plates continued for 14 d, at which time cultures were terminated. Initial cultures produced vesicles 0.2 mm to 4.0 mm in diameter. After 4 d, vesicles formed were 0.1 mm to 1.0 mm in diameter. Vesicles 4.0 mm in diameter were able to attain a maximum size of 1.5 cm by 20 d Maximum diameter varied according to initial size, with in culture. smaller vesicles exhibiting less expansion as compared with large Average growth rate obtained for all vesicles greater than 2.0 vesicles. mm in diameter at formation was 0.25 mm/day. Once maximum size was attained, vesicles began to form tissue type outarowths in an oraanized fashion (Fiaure 2). Localized tissue formation decrease in vesicle diameter. was accompanied by a Condensation of the main vesicle took place at the localized site of tissue formation. Large vesicles (greater than I..5 mm in diameter) were able to maintain free floating nature for 65 d, at which time culture was terminated and vesicles were histologically sectioned. Smaller vesicles (less than 1.5 mm in diameter) reattached to plates within 1 wk of ho new formation, and cells of the vesicles plated out in a monolayer. vesicles formed from the cells of reattached vesicles. Contractile elements formed as early as 7 d and as late as 34 d of culture. Formation of the contractile heart in the developing pig embryo Contractile elements and vasculature formed from occurs at 20 d (12). either plated cellular aggregations or from thickened areas of the All observed contractile regions were closely floating vesicles. associated with a very dense cellular region (Figure 3).
APRIL 1988 VOL. 29 NO. 4
875
Figure
1.
DAYS OF CULTURE
CONTRACTIONS
CONTINUE
vessel,
DEVELOPMENT
____1_-_____
TLSSUES CONTMUE
Time sequence of trophoblastic vesicle, contractile element, tubular tissue formation from enzymatically dispersed porcine blastocysts.
ELEMENT FORMATION
DISPERSED
CONTRACTILE
CELLS
CULWRES
TERMINATED
VESICLE
F’LATED CELL
FORMATION
TROPHOSLASTIC
FORMATXIN
TISSUE
DEFINED
VESSEL FORMATION
VESICLES
and defined
SECT IONEO
HISTOLOGCALLY
THERIOGENOLOGY
Figure
3.
Dense cellular region (arrow) in association contractile region of trophoblastic vesicle
APRIL 1988 VOL. 29 NO. 4
with (A, 400x).
877
THEFUOGENOLOGY
Contractions were rythmic and constant over two measurements 5 min Contraction rates of vesicles apart, consisting of up to 102 beats/min. changed daily, with no apparent pattern of change (Figure 4). Rate of contraction was not correlated to time in culture, change to fresh media, or change in temperature that occurred during the time of measurement Contractions continued until vesicles were (from 37°C to 30°C). sectioned for histological examination (30 to 45 d), or as in the case of plated cell contraction elements, until culture was terminated (7 d). Within 3 d of contractile element formation, tubular structures were identified in association with contractile areas (Figure 5). ISovement of within these areas was noted and was in synchrony with cells indicating the direct control of fluid and cell movement contractions, through the tubule by the contractile element. Examination of histological sections gave evidence of the formation elements with no of eosinophilic cells, blood islets, and-contractile Blood islets and evidence of a formina neural svstem (Fiaure 6). eosinophilic cells wet-6 associatei with the-contractile elements forming Contractile elements were positive an apparent vascular tissue matrix. for the formation presence of calcium activated ATP-ase activity, and calcium activated they unresponsive to acid reversal of were myo-fibriliary ATP-ase activity at pH 4.3 and pH 4.5, indicating a similarity to cardiac muscle fibers (7). DISCUSSION Our findings show that vesicles produced by enzymatically dispersed trophoblastic cells continue to develop for at least 6E. d in vitro. Development is indicated by outgrowths , or protrusions on the surface of the vesicle and by the formation and activity of a contracting region. Only vesicles larger than 1.5 mm in diameter continued to develop, indicating that in vitro survival and development could be a consequence of minimum cell number in the vesicle. were within vesicles always Contractal areas trophoblastic The ability of the associated with a region of dense cell mass. contracting region of the trophoblastic vesicles to move cells through a tubular structure (vessel) that is associated with the crrtracting region suggests the formation of a cardiac system in vitro. Due to the close association of the vessel and of the contracting region with the dense cellular area, it is hypothesized that the region controlling formation of the contractile element and the vessels is the dense cell mass. Histological evidence suggests no formation of a neural system association with areas of contraction. Since heart contraction rate partially controlled by perfusion of sodium through leaky membranes the sino-atria1 area (ll), possibly contraction in the vesicles controlled in a similar manner.
in is of is
This is the first report of the differentiation of an organ system formed from dispersed embryonic tissue in vitro. Further, the ability to produce large numbers of vesicles from dispersed embryo cells, and the evidence of a controlling region of cell differentiation that is
878
APRIL 1988 VOL. 29 NO. 4
THERIOGENOLOGY
80
60
40
20
0 0
5
10
15
20
25
30
DAY OF CONTRACTION Figure 4.
Contraction rates of primitive hearts in two normal vesicles. Day 1 is the initial day of contraction.
APRIL 1988 VOL. 29 NO. 4
879
THERIOGENOLOGY
880
Figure
5.
Tubular vessel (arrows) in association with primitive heart (A) and dense cellular region (B,400x).
Figure
6.
Ph tomicrograph of trophoblastic vesicle showing blood -3 et and eosinophilic 1s cells (A), and contractile region (B). Hemotoxylin-eosin stained (40x).
APRIL 1988 VOL. 29 NO. 4
THERIOGENOLOGY
morphologically visible, indicate that the excellent model system to study cell-to-cell regions and elements to cell differentiation.
trophoblastic interactions
vesicle is an and controlling
REFERENCES Advances
in
1.
Gardner, R. L. the Biosciences.
2.
Camous, S., Heyman, Y., Meziou, W. and Menezo, Y. Cleavage beyond the block stage and survival after transfer of early bovine embryos J. Reprod. Fertil. -72:479-485 cultured with trophoblastic vesicles. (1984).
3.
Heyman, Y., Camous, S., Fevre, J., Meziou, W. and Martal, J. Maintenance of the corpus luteum after uterine transfer of trophoblastic vesicles to cyclic cows and ewes. J. Reprod. Fertil. 70: 533-540 (1984).
4.
Heyman, Y., Menezo, Y., Chesne, P., Camous, S. and Garnier, V. In and ovine early embryos: improved vitro cleavage of bovine development with trophoblastic vesicles. using coculture Theriogenology z:59-68 (1987).
5.
Gardner, R. L. An investigation of inner cell their isolation from trophoblastic tissue following blastocyst. J. Embryol. Exp. Morph. g:279-312 (1972).
6.
Kuzan, F. B. and Wright, R.W., Jr. Allantochorion formation in vitro from enzymatically dispersed porcine blastocysts. Proc. West. Sect. Am. Sot. Anim. Sci. g:187-188 (1980).
7.
Allen, R. L. and Wright, R. W., Jr. Observations on the in vitro formation, development, and differentiation of trilaminar vesicles formed from enzymatically dispersed porcine blastocysts. Theriogenology 23:333-345 (1985).
8.
Rossant, J., and Papaicannou, The biology of emhryogenesis. V. E. &:Sherman, M. I. (ed.). Concepts in Mammalian Embryogenesis. MIT Press, Cambridge, 1977, pp l-36.
9.
Gardner, R. L. Analysis of determination and differentiation in the early mammalian embryo using intra- and interspecific chimaeras. In:Markert, C. L. Papaconstantinou, J. (eds.). The and i%velopmental Biology of Reproduction. Academic Press, New York, 1975, pp 207-238.
Manipulations on the blastocyst. Vol. 6, I971, pp. 279-296.
I&:
mass and the mouse
10.
Bunch, T. D., Foote, W. D., Call, J. W., Wright, R. W., Selgrath, J. P. and Foote, W. D. Long term culture of 2-B Submitted (1987). ovine embryos in various coculture systems.
11.
Guyton, y;z!c;:
12.
B. M. The Patton, Philadelphia, 1931,
APRIL
Jr., cell
A. C. &:Textbook Rhythmic excitation of the heart. Physiology, W. B. Saunders, Co., Philadelphia, 1981,
1988 VOL.
Embryology pp. 37-59.
29 NO. 4
of
the
Pig.
The
Blakiston
of pp
Co.,
881
IN VITRO CULTURE AND MAINTENANCE OF TROPHCBLASTIC OBSERVATIONS ON THE DEVELOPMENT OF CONTRACTILE WITH CONCURRENT VESSEL DEVELOPMENT
J.P.
Received
VESICLES: STRUCTURES
Selgrath and R. W. Wright, Jr.* Department of Animal Sciences Washington State University Pullman, WA 99164-6332 for publication: Accepted:
April 25, 1987 November 11, 1987
ABSTRACT The use of trophoblastic vesicles as a model for the study of embryo development and cell to cell maternal recognition of pregnancy, In this report, we describe interaction has increased in recent years. the long-term culture of trophcblastic vesicles derived from enzymatically dispersed porcine blastocysts. Day 12 to 14 porcine embryos were enzymatically diSpersed to form single cell suspensions, then allowed tc plate in 25 cm Vesicles formed 3 to 14 d tissue-culture flasks. following plating and ranged in size from 0.1 to 4.Omm in diameter. Vesicles lamer than 1.5 mm at formation were able to float free in the medium for up to 65 days, at which time vesicles were histologically sectioned. Contractile elements formed in association with dense cell Movement of cells areas, and were found with tubular vascular vessels. that resembled eosinophilic cells was observed in the vessels and pulsed in synchrony with contractions of the primitive heart. Histological examination gave evidence of primitive blood cells and cardiac tissue, and no evidence of neural tissue was found, indicating the probability that the contractions were induced by permeable membrane depolarization. It is hypothesized from the observations reported in this paper that the cell differentiation required to form the contractile region and the vessel are under the control of the dense cell area. This indicates that trophoblastic vesicles formed by the enzymatic dispersal of porcine blastocysts is a possible model for the study of induced cell differentiation and formation of the cardiac organ system in vitro. Key words:
Porcine,
trophoblastic
vesicles,
culture,
development
INTRODUCTION Formation of vesicles derived mainly of trophoblastic cells has been accomplished by microdissection (l-4) by the selective killing of inner cell mass cells (5), or by the enzymatic dispersal of preimplantation Trophoblastic vesicles have allowed the determination of embryos (6,7). cell fates in developing embryos (8). Microinjection of inner cell mass cells into trophoblastic has been used to form intraspecific and interspecific chimeras, for the study of varied genetic effects on development (9). 1
Research supported and Home Economics 27725. Reprint requests.
vesicles allowing
by Washington State Universit College of Agriculture Research Center (Project 0313 r . Scientific Paper No.
APRIL 1988 VOL. 29 NO. 4
THERIOGENOLOGY
Information of trophoblastic effects on maternal recognition of pregnancy (3) and the use of trophoblastic cells in culture to bypass the block stage of development in vitro (2,4,10) have indicated that trophoblastic vesicles or trophoblastic cells can be used as models for the study of maternal-embryonic interaction and cell-to-cell interaction in farm animals. Enzymatic dispersal of preimplantation embryos was first described by Kuzan and Wright (6). Subsequent analysis described trilaminar tissue and eosinophilic development formation, contractile element presence, In this report we describe the morphological events of tissue (7). differentiation in trophoblastic vesicles. METHODS Handmated crossbred gilts and sows were slaughtered 12, 13, 14 and 15 d post breeding and the reproductive tracts were recovered. Corpora lutea (CL) were counted and filamentous embryos were recovered by flushing each uterine horn with Dulbecco's phcsthate buffered saline and 1% antibioticsolution (P#S) with 1% newborn calf serum (NCS) Embryos were washed three times in complete antimycotic (complete PBS). PRS. Embryos were then recovered from final rinse and transferred to a Five mls of 0.25% sterile petri dish with as little fluid as possible. trypsin in calcium and magnesium free Hanks balanced salt solution were Embryo cells were further added to enzymatically disperse the embryos. separated using agitation, accomplished by drawing media and cells through a sterile Pasteur pipet. Following dispersal, cells were transferred in equal volume to two Enzymatic bactivity was inhibited by the disposable centrifuqe tubes. (MEM! with, 10% olasma derived addition of 8 mls minimum essential media Ceil suspensions equine seruma (EqPDS) plus 1% antibiotic-antimycotic.D were washed by three-time centrifugation at 400 xg for 10 min, supernatant aspirated, and pellet resuspended in complete MEM. Dispersed cells were transferred to 25 cm tissue culture flasks and incubated under a 95% air, 5% CO2 atmosphere at 37°C. Trophoblastic
Vesicle
Formation
Dispersed embryonic cells were allowed to plate to tissue culture flasks for 3 d, after which flasks were poured off and floating nonplated cells were collected and pelleted by centrifugation at 400 xg, then they were resuspended and transferred to 25-cm tissue culture flasks. After 4 d, one-half of complete MEM was replaced daily in the flasks. Tissue culture flasks were observed daily for trophoblastic vesicles formation or for evidence of cell aggregation. Formed vesicles were transferred to tissue culture flasks with complete MEM. ZHyclone; Logan, Utah. Glbco Laboratories, Grand
874
Island,
New York.
APRIL
1988 VOL.
29 NO. 4
THERIOGENOLOGY
Observations Trophoblastic vesicles were measured daily by inverted microscope Plated cells and micrometer, and changes in size or shape were noted. examined daily for evidence of cellular aggregation. also were Contractile tissues were observed daily for rate and degree of contraction. Trophoblastic vesicles exhibiting contractile elements Sections were examined and stained with hemotoxylin-eosin. eosinophilic elements.
were sectioned for contractile,
RESULTS Time sequence of trophoblastic vesicle element activity is shown in Figure 1.
formation
and
contractile
Dispersed cells transferred tc tissue culture flasks became attached Vesicle expansion occurred and formed monolayers by 24 h posttransfer. from the cellular aggregations on the plate surface that were released to float free in the medium. In addition, floating cells were able to form vesicles by aggregation and continued vesicle expansion similar to the Vesicles morula to blastocyst transformation in the developing embryo. to form vesicles were formed by Gay 2 of culture, and plates continued for 14 d, at which time cultures were terminated. Initial cultures produced vesicles 0.2 mm to 4.0 mm in diameter. After 4 d, vesicles formed were 0.1 mm to 1.0 mm in diameter. Vesicles 4.0 mm in diameter were able to attain a maximum size of 1.5 cm by 20 d Maximum diameter varied according to initial size, with in culture. smaller vesicles exhibiting less expansion as compared with large Average growth rate obtained for all vesicles greater than 2.0 vesicles. mm in diameter at formation was 0.25 mm/day. Once maximum size was attained, vesicles began to form tissue type outarowths in an oraanized fashion (Fiaure 2). Localized tissue formation decrease in vesicle diameter. was accompanied by a Condensation of the main vesicle took place at the localized site of tissue formation. Large vesicles (greater than I..5 mm in diameter) were able to maintain free floating nature for 65 d, at which time culture was terminated and vesicles were histologically sectioned. Smaller vesicles (less than 1.5 mm in diameter) reattached to plates within 1 wk of ho new formation, and cells of the vesicles plated out in a monolayer. vesicles formed from the cells of reattached vesicles. Contractile elements formed as early as 7 d and as late as 34 d of culture. Formation of the contractile heart in the developing pig embryo Contractile elements and vasculature formed from occurs at 20 d (12). either plated cellular aggregations or from thickened areas of the All observed contractile regions were closely floating vesicles. associated with a very dense cellular region (Figure 3).
APRIL 1988 VOL. 29 NO. 4
875
Figure
1.
DAYS OF CULTURE
CONTRACTIONS
CONTINUE
vessel,
DEVELOPMENT
____1_-_____
TLSSUES CONTMUE
Time sequence of trophoblastic vesicle, contractile element, tubular tissue formation from enzymatically dispersed porcine blastocysts.
ELEMENT FORMATION
DISPERSED
CONTRACTILE
CELLS
CULWRES
TERMINATED
VESICLE
F’LATED CELL
FORMATION
TROPHOSLASTIC
FORMATXIN
TISSUE
DEFINED
VESSEL FORMATION
VESICLES
and defined
SECT IONEO
HISTOLOGCALLY
THERIOGENOLOGY
Figure
3.
Dense cellular region (arrow) in association contractile region of trophoblastic vesicle
APRIL 1988 VOL. 29 NO. 4
with (A, 400x).
877
THEFUOGENOLOGY
Contractions were rythmic and constant over two measurements 5 min Contraction rates of vesicles apart, consisting of up to 102 beats/min. changed daily, with no apparent pattern of change (Figure 4). Rate of contraction was not correlated to time in culture, change to fresh media, or change in temperature that occurred during the time of measurement Contractions continued until vesicles were (from 37°C to 30°C). sectioned for histological examination (30 to 45 d), or as in the case of plated cell contraction elements, until culture was terminated (7 d). Within 3 d of contractile element formation, tubular structures were identified in association with contractile areas (Figure 5). ISovement of within these areas was noted and was in synchrony with cells indicating the direct control of fluid and cell movement contractions, through the tubule by the contractile element. Examination of histological sections gave evidence of the formation elements with no of eosinophilic cells, blood islets, and-contractile Blood islets and evidence of a formina neural svstem (Fiaure 6). eosinophilic cells wet-6 associatei with the-contractile elements forming Contractile elements were positive an apparent vascular tissue matrix. for the formation presence of calcium activated ATP-ase activity, and calcium activated they unresponsive to acid reversal of were myo-fibriliary ATP-ase activity at pH 4.3 and pH 4.5, indicating a similarity to cardiac muscle fibers (7). DISCUSSION Our findings show that vesicles produced by enzymatically dispersed trophoblastic cells continue to develop for at least 6E. d in vitro. Development is indicated by outgrowths , or protrusions on the surface of the vesicle and by the formation and activity of a contracting region. Only vesicles larger than 1.5 mm in diameter continued to develop, indicating that in vitro survival and development could be a consequence of minimum cell number in the vesicle. were within vesicles always Contractal areas trophoblastic The ability of the associated with a region of dense cell mass. contracting region of the trophoblastic vesicles to move cells through a tubular structure (vessel) that is associated with the crrtracting region suggests the formation of a cardiac system in vitro. Due to the close association of the vessel and of the contracting region with the dense cellular area, it is hypothesized that the region controlling formation of the contractile element and the vessels is the dense cell mass. Histological evidence suggests no formation of a neural system association with areas of contraction. Since heart contraction rate partially controlled by perfusion of sodium through leaky membranes the sino-atria1 area (ll), possibly contraction in the vesicles controlled in a similar manner.
in is of is
This is the first report of the differentiation of an organ system formed from dispersed embryonic tissue in vitro. Further, the ability to produce large numbers of vesicles from dispersed embryo cells, and the evidence of a controlling region of cell differentiation that is
878
APRIL 1988 VOL. 29 NO. 4
THERIOGENOLOGY
80
60
40
20
0 0
5
10
15
20
25
30
DAY OF CONTRACTION Figure 4.
Contraction rates of primitive hearts in two normal vesicles. Day 1 is the initial day of contraction.
APRIL 1988 VOL. 29 NO. 4
879
THERIOGENOLOGY
880
Figure
5.
Tubular vessel (arrows) in association with primitive heart (A) and dense cellular region (B,400x).
Figure
6.
Ph tomicrograph of trophoblastic vesicle showing blood -3 et and eosinophilic 1s cells (A), and contractile region (B). Hemotoxylin-eosin stained (40x).
APRIL 1988 VOL. 29 NO. 4
THERIOGENOLOGY
morphologically visible, indicate that the excellent model system to study cell-to-cell regions and elements to cell differentiation.
trophoblastic interactions
vesicle is an and controlling
REFERENCES Advances
in
1.
Gardner, R. L. the Biosciences.
2.
Camous, S., Heyman, Y., Meziou, W. and Menezo, Y. Cleavage beyond the block stage and survival after transfer of early bovine embryos J. Reprod. Fertil. -72:479-485 cultured with trophoblastic vesicles. (1984).
3.
Heyman, Y., Camous, S., Fevre, J., Meziou, W. and Martal, J. Maintenance of the corpus luteum after uterine transfer of trophoblastic vesicles to cyclic cows and ewes. J. Reprod. Fertil. 70: 533-540 (1984).
4.
Heyman, Y., Menezo, Y., Chesne, P., Camous, S. and Garnier, V. In and ovine early embryos: improved vitro cleavage of bovine development with trophoblastic vesicles. using coculture Theriogenology z:59-68 (1987).
5.
Gardner, R. L. An investigation of inner cell their isolation from trophoblastic tissue following blastocyst. J. Embryol. Exp. Morph. g:279-312 (1972).
6.
Kuzan, F. B. and Wright, R.W., Jr. Allantochorion formation in vitro from enzymatically dispersed porcine blastocysts. Proc. West. Sect. Am. Sot. Anim. Sci. g:187-188 (1980).
7.
Allen, R. L. and Wright, R. W., Jr. Observations on the in vitro formation, development, and differentiation of trilaminar vesicles formed from enzymatically dispersed porcine blastocysts. Theriogenology 23:333-345 (1985).
8.
Rossant, J., and Papaicannou, The biology of emhryogenesis. V. E. &:Sherman, M. I. (ed.). Concepts in Mammalian Embryogenesis. MIT Press, Cambridge, 1977, pp l-36.
9.
Gardner, R. L. Analysis of determination and differentiation in the early mammalian embryo using intra- and interspecific chimaeras. In:Markert, C. L. Papaconstantinou, J. (eds.). The and i%velopmental Biology of Reproduction. Academic Press, New York, 1975, pp 207-238.
Manipulations on the blastocyst. Vol. 6, I971, pp. 279-296.
I&:
mass and the mouse
10.
Bunch, T. D., Foote, W. D., Call, J. W., Wright, R. W., Selgrath, J. P. and Foote, W. D. Long term culture of 2-B Submitted (1987). ovine embryos in various coculture systems.
11.
Guyton, y;z!c;:
12.
B. M. The Patton, Philadelphia, 1931,
APRIL
Jr., cell
A. C. &:Textbook Rhythmic excitation of the heart. Physiology, W. B. Saunders, Co., Philadelphia, 1981,
1988 VOL.
Embryology pp. 37-59.
29 NO. 4
of
the
Pig.
The
Blakiston
of pp
Co.,
881