- Email: [email protected]
A three-dimensional culture system using alginate hydrogel prolongs hatched cattle embryo development in vitro
Accepted Manuscript A Three-Dimensional Culture System Using Alginate Hydrogel Prolongs Hatched Cattle Embryo Development in vitro Shuan Zhao, Zhen-Xing Liu, Hui Gao, Yi Wu, Yuan Fang, Shuai-Shuai Wu, Ming-Jie Li, Jia-Hua Bai, Yan Liu, Alexander Evans, Shen-Ming Zeng PII:
S0093-691X(15)00138-7
DOI:
10.1016/j.theriogenology.2015.03.011
Reference:
THE 13121
To appear in:
Theriogenology
Received Date: 13 October 2014 Revised Date:
9 March 2015
Accepted Date: 11 March 2015
Please cite this article as: Zhao S, Liu Z-X, Gao H, Wu Y, Fang Y, Wu S-S, Li M-J, Bai J-H, Liu Y, Evans A, Zeng S-M, A Three-Dimensional Culture System Using Alginate Hydrogel Prolongs Hatched Cattle Embryo Development in vitro, Theriogenology (2015), doi: 10.1016/j.theriogenology.2015.03.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Revised
1
A Three-Dimensional Culture System Using Alginate Hydrogel Prolongs
3
Hatched Cattle Embryo Development in vitro
4
Shuan Zhao a, Zhen-Xing Liu a, Hui Gao a, Yi Wu a, Yuan Fang a, Shuai-Shuai Wu
5
a
, Ming-Jie Li a, Jia-Hua Bai b, Yan Liu b, Alexander Evansc, Shen-Ming Zeng a,*
6
a
National Key Laboratory of Animal Nutrition, Laboratory of Animal Embryonic
7
Biotechnology; National Engineering Laboratory for Animal Breeding; Key
8
Laboratory of Animal Genetics, Breeding, and Reproduction of the Ministry of
9
Agriculture, College of Animal Science and Technology, China Agricultural
M AN U
SC
RI PT
2
10
University, Beijing, 100193, China
11
b
12
Agriculture and Forestry Sciences, Beijing, China.
13
c
14
*
15
West Road, Haidian District, Beijing 100193, China. Phone: 86-10-62733744; Fax:
16
86-10-62733744; E-mail: [email protected].
17
Additional footnotes: Shuan Zhao and Zhen-Xing Liu contributed equally to this
18
work.
19
TE D
Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of
School of Agriculture and Food Science in University College Dublin, Ireland,
AC C
EP
Corresponding author: Shenming Zeng, Dongke Building #251, No. 2 Yuanmingyuan
ACCEPTED MANUSCRIPT ABSTRACT: No successful method exists to maintain the three-dimensional
21
architecture of hatched embryos in vitro. Alginate, a linear polysaccharide derived
22
from brown algae, has characteristics that make it an ideal material as a
23
three-dimensional (3D) extracellular matrix (ECM) for in vitro cell, tissue or embryo
24
culture. In this study, alginate hydrogel was used for in vitro culture of post-hatched
25
bovine embryos in order to observe their development under the 3D system. In vitro
26
fertilized (IVF) and parthenogenetic activated (PA) post-hatched bovine blastocysts
27
were cultured in an alginate encapsulation culture system (AECS), an alginate overlay
28
culture system (AOCS), or control culture system. After 18 days of culture, the
29
survival rate of embryos cultured in AECS was higher than that in the control group
30
(p<0.05), and the embryos were expanded and elongated in AECS with the maximal
31
length of 1.125 mm. When the AECS shrinking embryos were taken out of the
32
alginate beads on Day 18 and cultured in the normal culture system, 9.09% of them
33
attached to the bottoms of the plastic wells and grew rapidly, with the largest area of
34
an attached embryo being 66.00 mm2 on Day 32. The embryos cultured in AOCS
35
developed mono- or multi-vesicular morphologies. Total cell number of the embryos
36
cultured in AECS on Day 19 was significantly higher than embryos on Day 8.
37
Additionally, AECS and AOCS supported differentiation of the embryonic cells.
38
Binuclear cells were visible in Day 26 adherent embryos, and the mRNA expression
39
patterns of Cdx2 and Oct4 in AOCS cultured embryos were similar to in vivo embryos
40
while IFNT and ISG15 mRNA were still expressed in Day 26 and Day 32
41
prolong-cultured embryos. In conclusion, AECS and AOCS did support cell
AC C
EP
TE D
M AN U
SC
RI PT
20
ACCEPTED MANUSCRIPT proliferation, elongation and differentiation of hatched bovine embryos during
43
prolonged in vitro culture. The culture system will be useful to further investigate the
44
molecular mechanisms controlling ruminant embryo elongation and implantation.
45
Key words: alginate, three-dimensional culture, embryo development, bovine
RI PT
42
AC C
EP
TE D
M AN U
SC
46
ACCEPTED MANUSCRIPT 47
48
1. Introduction The causes of early embryonic loss in ruminants are poorly understood and this is not helped by the lack of knowledge about the exact physiological mechanisms
50
underlining embryo elongation and implantation [1, 2]. In vitro models present ethical
51
and cost advantages over in vivo studies, which can help define the factors that
52
regulate early embryo development and cell differentiation. Three-dimensional (3D)
53
scaffolds for cell and tissue culture provide a potential model to study in vitro embryo
54
development, especially post-hatched embryos. In cattle, limited elongation of
55
post-hatch embryos in vitro via physical induction has been demonstrated using an
56
agarose gel tube system [1-3]. However, the embryos cultured in this system had
57
deficiencies in the development of their embryonic disks [1, 4].
M AN U
SC
RI PT
49
Alginate is a commonly used biomaterial and is often employed in tissue
59
engineering as an artificial extracellular matrix [5]. Alginate is derived from brown
60
algae and is composed of repeating units of β-D-mannuronic acid and α-L-guluronic
61
acid [6]. One of the favorable properties of alginate as a biomaterial is to form a
62
hydrogel by ionic cross-linking of the guluronic residues in the presence of a divalent
63
cation [7]. This permits the diffusion of nutrients and hormones through the 3D
64
scaffold that are essential for cell and tissue growth and development [6]. Alginate
65
hydrogel has been successfully used for supporting mouse ovarian follicle [8] and
66
granulosa cell-oocyte complexe (GOC) in vitro development. Moreover, the oocytes
67
recovered from the alginate encapsulated GOCs were able to resume meiosis,
68
undergo fertilization, and to produce viable offspring [9]. We hypothesize that bovine
69
embryos can survive long term and display morphological changes similar to in vivo
70
conditions if they are cultured in a proper biomechanical (3D) support system. The
AC C
EP
TE D
58
ACCEPTED MANUSCRIPT very early embryo can successfully grow in a traditional culture dish with the
72
protection of its zona pellucida (ZP). However, when the embryo hatches from its ZP,
73
it is virtually impossible for it to keep its normal morphology in vitro because the
74
embryonic cells attach to the bottom of culture dish, resulting in the disruption of
75
cell-to-cell interactions and its 3D architecture [10, 11]. Alternatively, we propose that
76
a 3D culture system can maintain the embryo architecture by providing a uniform
77
surrounding environment for the whole embryo, which mimics uterine conditions.
78
Our objective was to use alginate hydrogels to establish an in vitro culture system to
79
support bovine post-hatched embryo development. In the present study, in vitro
80
development of bovine embryos encapsulated in alginate hydrogels or cultured on top
81
of alginate hydrogels was evaluated by characterizing survival rate, cellular
82
proliferation, morphological changes and stage-specific gene expression.
M AN U
SC
RI PT
71
AC C
EP
TE D
83
ACCEPTED MANUSCRIPT 84
2. Materials and Methods
Unless otherwise indicated, all chemicals were obtained from Sigma Chemicals
86
Co. (St. Louis, MO). Similarly, all plastic-ware used was from Nunc-ware (Nunc;
87
NalgeNunc International, Roskilde, Denmark).
88
2.1 Bovine oocyte collection and in vitro maturation
RI PT
85
Bovine ovaries were collected immediately after slaughter and transported to our
90
laboratory in saline solution (0.9% NaCl) supplemented with penicillin G (100 IU/ml)
91
and streptomycin sulfate (100 μg/ml) at 35°C. Cumulus–oocyte complexes (COCs)
92
were aspirated from follicles with diameters from 2 to 8 mm. The COCs with
93
homogeneous cytoplasm and at least three intact layers of surrounding cumulus cells
94
were selected for in vitro maturation. After rinsing in pre-warmed maturation medium
95
consisting of TCM 199 with Earles’ salts (GIBCO BRL, Grand Island, NY, USA), 10%
96
(v/v) FBS (HyClone-Pierce, Shanghai, China), 10 µg/mL FSH (Bioniche, Canada), 10
97
µg/mL LH (Bioniche, Canada), 1 ng/mL EGF, and 0.1 mg/mL cysteine, groups of 20
98
COCs were cultured in 50 µL media droplets under mineral oil at 38.5 °C, in 5% CO2
99
in air with maximum humidity for 22 to 24 h.
101
M AN U
TE D
EP
AC C
100
SC
89
2.2 In Vitro Fertilization of oocytes After in vitro maturation, bovine COCs were washed three times and then placed
102
in 40 µL droplets of Brackett–Oliphant (BO) medium [12] containing 10 mg/mL BSA
103
under mineral oil for IVF as follows. Frozen semen (Beijing Dairy Cow Center,
104
Beijing, China) was thawed in a water bath at 37 °C for 30 s. Sperm were washed
105
twice in BO medium supplemented with 2.5 mM caffeine and then resuspended in BO
ACCEPTED MANUSCRIPT medium supplemented with 10 mg/mL BSA and 5 IU/mL heparin. The concentration
107
of spermatozoa was counted in haemocytometer and further diluted in BO medium to
108
a concentration of 2×106 /mL. An aliquot (50 µL) of this suspension was added into
109
each 50 µL droplet of BO medium containing 20 COCs under mineral oil for
110
insemination. In vitro fertilization was carried out in humidified air with 5% CO2 at
111
38.5 °C. After incubation for 6 to 8 h, loosely associated cumulus cells and
112
spermatozoa were removed from the oocytes by gentle vortexing. The presumptive
113
zygotes were then cultured in CR1aa medium [13] supplemented with essential or
114
non-essential amino acids and 3 mg/mL BSA for two days and continuously cultured
115
in CR1aa medium containing 10% FBS at 38.5 °C in a humidified atmosphere of 5%
116
CO2. The culture medium was refreshed every two days.
117
2.3 Parthenogenetic activation of oocytes
TE D
M AN U
SC
RI PT
106
After in vitro maturation, COCs were treated with 0.1% hyaluronidase to remove
119
cumulus cells. The parthenogenetic activation (PA) of the oocytes was carried out
120
according to the following protocol. Oocytes were rinsed three times in M199
121
containing 25 mM Hepes and 10% FBS and pre-treated in 5 µM ionomycin at 38.5 °C
122
for 5 min, then cultured in CR1aa supplemented with 2 mM 6-(Dimethylamino)
123
purine (6-DMAP) for 4 h. The oocytes were then first cultured in CR1aa medium with
124
3 mg/mL BSA for 2 days and continuously cultured in CR1aa medium with 10% FBS
125
for 4 days at 38.5 °C in a humidified atmosphere of 5% CO2. The culture medium was
126
refreshed every two days.
127
2.4 Alginate gel preparation
AC C
EP
118
ACCEPTED MANUSCRIPT The crosslinking solution used for gel formation was prepared by dissolving
129
calcium chloride (CaCl2) and sodium chloride (NaCl) in sterile MilliQ water to obtain
130
a 50 mM CaCl2 and 140 mM NaCl solution. Alginate solutions were prepared by
131
dissolving lyophilized alginate powder in sterile MilliQ water and were all 1.5%
132
alginate (w/v) concentrations.
133
2.5 Encapsulation and overlay procedure
RI PT
128
On Day 8 after IVF or PA, the hatched embryos were cultured in Alginate
135
Encapsulation Culture System (AECS) or Alginate Overlay Culture System (AOCS).
136
The encapsulation procedure was according to the method described previously [14]
137
but with some modifications. Briefly, the embryos were washed three times in
138
warmed PBS (Ca2+ and Mg2+ free, Invitrogen, Carlsbad, USA), and put into the
139
alginate solution. A total of 7 µL of the alginate solution containing one embryo was
140
slowly released to the crosslinking solution. After two minutes the beads containing
141
embryos were removed from the crosslinking solution, and washed thrice in CR1aa
142
containing 10% FBS for 15 min. The beads were then cultured individually in the
143
wells of 96-well plate with 100 µL CR1aa (10% FBS) under 50 µL mineral oil (Fig. 1)
144
at 38.5 °C in a humidified atmosphere of 5% CO2. One-half of the culture medium
145
was changed every two days.
M AN U
TE D
EP
AC C
146
SC
134
The Alginate Overlay Culture System was developed according to the following
147
protocol. A total of 60 µL alginate solution was added to one well of 96-well plate,
148
then 50 µL crosslinking solution was slowly added. After two minutes the alginate
149
overlay had formed on the bottom of the well. The well was filled with CR1aa
ACCEPTED MANUSCRIPT containing 10% FBS, and incubated under the conditions described above for three
151
hours. The medium was refreshed every hour. The embryos were individually cultured
152
in the wells of 96-well plate with 100 µL CR1aa (10% FBS) under 50 µL mineral oil
153
(Fig. 1) at 38.5 °C in a humidified atmosphere of 5% CO2. Half of the culture medium
154
was refreshed every two days. In the control group, the embryos were individually
155
cultured in the wells without any alginate under the same conditions (Fig. 1).
156
2.6 Blastocyst Cell Count
SC
RI PT
150
The nuclei of selected blastocysts were stained using Hoechst 33342 (10 ng/mL)
158
for 10 min, and washed thrice in DPBS. Embryos with labeled nuclei under UV
159
excitation were individually mounted on microscope slides in glycerol underneath a
160
cover slip and examination was carried out in whole mount.
161
2.7 Quantitative PCR on mRNA expression and statistical analysis
TE D
M AN U
157
Total cellular RNA (tcRNA) of Day 6 (n=100) embryos, Day 8 (n=100) early
163
blastocysts, Day 26 (n=15) and Day 32 (n=15) prolong-cultured embryos were
164
extracted using an RNAprep pure Tissue Kit (Thermo, USA). For each sample a
165
complete reaction, but without the reverse transcriptase (RT), was performed.
166
Amplification reactions were performed in 20 µL reaction volumes containing 1 µL
167
cDNA, 9 µL of 2 × Master SYBR Green mix (TIANGEN Biotech, Beijing, China), 9
168
µL sterile water and 0.5 µL each of forward and reverse gene specific primers (10 µM).
169
This mixture was denatured at 95 °C for 5 min; 45 cycles of PCR (95°C for 15 s,
170
60°C for 20 s, and 72°C for 50 s) in 7500 real-time PCR system (Applied Biosystems,
171
Foster City, CA, USA). The primers and Gene Bank source accessions for each gene
AC C
EP
162
ACCEPTED MANUSCRIPT 172
are shown in (Table 1). Relative expression of each gene was calculated by 2-△△Ct
173
[15].
174
2.8 Statistical analysis The survival rates of extend cultured embryos and the percentages of binuclear
176
cells in overall cell numbers were analyzed using a general linear model and one-way
177
post-hoc test using the MIXED procedure models of SAS (version 9.1, SAS Institute
178
Inc., Cary, NC). All data were expressed as mean ± S.E.M. The percentages were
179
subjected to arcsine transformation before analysis. Differences were considered to be
180
significant when P < 0.05.
AC C
EP
TE D
181
M AN U
SC
RI PT
175
ACCEPTED MANUSCRIPT 182
3. Results
183
3.1 Morphological changes of bovine embryos cultured in AECS After 18 days the morphologies of the embryos were completely different in
185
AECS from that in the control culture system (Fig. 2a). The embryos derived from PA
186
and IVF began elongation on Day 14, and the maximal length was 1.125 mm (Fig. 2a)
187
on Day 18. The ratio and average length of elongated embryos were not different
188
between IVF and PA groups (Table 2, P < 0.05). However, the survival rates in
189
AECS-PA and AECS-IVF were greater than that in the control group (Fig. 2b, P <
190
0.01). On Day 18, a total of 55 embryos that appeared to be shrinking from the 144
191
hatched blastocysts were removed from the gel and cultured in the normal culture
192
system. Interestingly, instead of death, 9.09% of these embryos quickly expanded
193
from Day 18 to 32 (Fig. 3), and the largest area was 66 mm2 (Fig. 3, Day 32).
194
3.2 Cell number of bovine embryos cultured in AECS
TE D
M AN U
SC
RI PT
184
The cell numbers in the embryos derived from PA and IVF reached tens of
196
thousands after 19 days of culture (Fig. 4a). The average total cell number of IVF
197
embryos was much more than in PA embryos (n=3, 42,608 ± 2,244 vs. 14,899 ± 4,919,
198
Fig. 4b, P < 0.01).
199
3.3 Morphological changes of bovine embryos cultured in AOCS
AC C
EP
195
200
Of the embryos cultured in AOCS, 67% (8/12) shrank or died by Day 16 (Fig.
201
5a). However, 25% (3/12) of the embryos grew for at least 32 days (Fig. 5b Fig. 6),
202
but none elongated in morphology.
203
3.4 Cell differentiation in the embryos cultured in AECS
ACCEPTED MANUSCRIPT 204
Hoechst 33342 staining revealed that binuclear cells were present in Day 26 adherent
embryos
(Fig.
7a),
which
was
further
confirmed
by
PAG
206
(Pregnancy-associated glycoprotein, a specific gene expressed in binuclear cells)
207
expression (Fig. 7b). The proportion of binuclear cells increased in response to
208
treatment with forskolin (Fig. 7a and 7c, P < 0.01).
209
3.5 Specific Gene expression in embryos cultured in AOCS
RI PT
205
High amounts of Oct4 mRNA and low levels of Cdx2 mRNA were detected in
211
both Day 6 and Day 8 blastocysts (Fig. 8a1). However, the transcription ratio of
212
Cdx2:Oct4 in blastocysts was 3.51 on Day 26 and this increased to 5.66 on Day 40
213
(Fig. 8). Moreover, both IFNT and ISG15 mRNA were still present in Day 26 and 32
214
embryos (Fig. 8b).
M AN U
SC
210
AC C
EP
TE D
215
ACCEPTED MANUSCRIPT 216
4. Discussion
Few studies have focused on post-hatching mammalian embryo development
218
although most pregnancy losses occur at this stage [16-20]. In order to better
219
characterize the development of bovine hatched-out embryos in vitro, alginate
220
hydrogel was employed for a 3D matrix. Previously, alginate hydrogels have been
221
used for an artificial zona pellucida for mouse morulae [21], for packaging ovarian
222
follicles in mice [9] and nonhuman primates [8], and to study early blastocysts
223
development in cattle [22] and in pigs [23]. Our report demonstrates for the first time
224
that alginate hydrogels can be successfully used for maintaining bovine post-hatched
225
embryo development in vitro.
M AN U
SC
RI PT
217
Bovine embryos cultured in AECS were able to undergo morphological changes
227
similar to in vivo, and its survival rate was also significantly higher than
228
non-encapsulated embryos, which were different from previous report in porcine [23].
229
This implies that three-dimension matrix support is necessary to maintain the
230
appropriate architecture for normal bovine embryos undergoing elongation.
EP
The embryos recovered from the alginate gel bead were able to attach and grow
AC C
231
TE D
226
232
rapidly at the bottom of the well (Fig. 3a), which rarely happens in normal culture
233
systems. This suggests that the embryos had more viable cells after 6 days culture in
234
AECS. This technology may be used for isolation of stem cells from early bovine
235
embryos. The binuclear cells, a subtype of trophoblast giant cells (TGCs), is the first
236
cell type to terminally differentiate during embryogenesis and is of vital importance
237
for implantation and modulation of post-implantation placentation [24]. After Hoechst
ACCEPTED MANUSCRIPT 33342 staining, we found some cells contained two enlarged nuclei similar to
239
previously characterized binucleate cells in BT-1 (bovine trophectoderm-1) cells [25].
240
Also, pregnancy associated glycoprotein (PAG), one of the genes specifically
241
expressed in binuclear cells, was detected in Day 26 attached embryonic cells
242
indicating their differentiation. The roles of binuclear trophoblast cells in implantation
243
have been extensively studied in a few ruminant species [26]. Forskolin, as an
244
activator of cAMP, can induce cell fusion to produce binuclear cells in human
245
choriocarcinoma (BeWo) cells [27]. In our study, the treatment also increased the
246
percentage of binuclear cells in the attached embryonic cells. This suggests that these
247
embryonic cells possess the ability to differentiate into placenta tissues.
M AN U
SC
RI PT
238
The embryos cultured in AOCS could survive for at least 32 days with mono- or
249
multi-vesicular cavities developing by Days 17 to 18 (Fig. 3a, 6b). The reason why
250
the embryos formed mono- or milti-vesicular cavities is unknown. Unlike mouse
251
embryos, both inner cell mass and trophectoderm cells express high levels of Oct4
252
mRNA in bovine blastocysts. The trophoblast cells derived from Day 7 in vitro
253
produced bovine embryos can differentiate into ICM cells untill Day 14 [28]. In this
254
study, the multi-vesicular embryos might be differentiated from pluripotent
255
trophoblast cells. The ratio of Cdx2:Oct4 mRNA in mouse late blastocysts was nearly
256
10-fold compared to bovine equivalent stage embryos, and the Cdx2 transcript levels
257
in bovine trophectoderm cells begin to exceed Oct4 levels at epiblast stages [28]. We
258
also found that the Cdx2:Oct4 transcription ratios in bovine embryos increased with
259
the culture time. On Day 8 the transcription ratio of Cdx2:Oct4 was 0.004, while on
AC C
EP
TE D
248
ACCEPTED MANUSCRIPT 260
Day 26 the ratio had increased to 3.51. This demonstrates that bovine embryos
261
cultured long-term in vitro displayed similar expression profiles of key developmental
262
marker genes to rapidly developing mouse embryos. IFNT is the pregnancy signal in ruminants, and IFNT mRNA and protein
264
expressions are relatively low at the blastocyst stage, but increase with the
265
morphological development of conceptuses [29, 30]. However, coincident with
266
trophectoderm attachment to the uterine lining, IFNT mRNA levels decline sharply by
267
Day 21 and cease by Day 24 of pregnancy in cattle [31]. In our results, IFNT mRNA
268
was still detectable on Day 26 and Day 32 extend-cultured IVF blastocysts, although
269
binucleate cells were differentiated. This suggests that normal IFNT expression might
270
be associated with complete elongation of embryos and/or other factors secreted from
271
the uterus.
TE D
M AN U
SC
RI PT
263
The AECS model used in this study did support moderate growth, elongation and
273
differentiation of bovine hatch blastocysts, but this system waits to be further
274
improved. Firstly, the arginine-glycine-aspartic acid (RGD) peptide sequence is the
275
most effective and commonly used peptide sequence to promote cell adhesion [32].
276
When covalently linked to alginate, RGD peptide sites could serve as ligands to
277
integrins in the embryonic cell membranes to mediate cellular migration and
278
embryonic adhesion. The AECS could be used to investige the mechanisms of
279
ruminant embryo elongation. Secondly, because embryo-maternal communication is
280
vital for bovine embryo development during the preimplantation stage of pregnancy
281
[33], the embryos could be co-cultured with uterine lumen and/or glandular epithelial
AC C
EP
272
ACCEPTED MANUSCRIPT cells with the alginate hydrogel to pursue the molecular communication pathways
283
during embryo implantation. Thirdly, as the energy demands of bovine embryos
284
increase they substantially enhance their glycolytic activity [34] and glucose uptake
285
[35] during compaction, blastocyst formation, expansion and elongation. Moreover,
286
IFNT is not only a pregnancy recognition signal but also a factor that stimulates the
287
proliferation of trophoblast cells and the development of early embryos [36]. However,
288
the exact molecular roles of the above factors in early embryo development and cell
289
differentiation need to be established. In the AECS these factors could be added to
290
defined culture medium to pursue their specific functions.
291
4.1 Conclusion
M AN U
SC
RI PT
282
The alginate encapsulation culture system (AECS) did support cell proliferation,
293
embryo elongation and differentiation of bovine hatch blastocysts. Whereas, the
294
embryos cultured in the alginate overlay culture system (AOCS) did not undergo
295
elongation, but did continuously develop with multi-vesicular changes in morphology.
296
We have established a model for long-term culture of bovine embryos that can be used
297
for investigating the molecular mechanisms of embryo elongation, cell differentiation
298
and implantation in farm animals.
300
EP
AC C
299
TE D
292
ACCEPTED MANUSCRIPT 301
302
Acknowledgments
This work was supported by a grant from the National Natural Science Foundation of China (Grant 31172209) and National Key Technologies R&D
304
Program (2011BAD19B03).
AC C
EP
TE D
M AN U
SC
RI PT
303
ACCEPTED MANUSCRIPT 305
306
Disclosure Statement
The authors of this article have no conflicts of interest to disclose.
AC C
EP
TE D
M AN U
SC
RI PT
307
ACCEPTED MANUSCRIPT 308
Reference:
309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349
[1] Brandao DO, Maddox-Hyttel P, Lovendahl P, Rumpf R, Stringfellow D, Callesen H. Post hatching development: a novel system for extended in vitro culture of bovine embryos. Biology of reproduction. 2004;71:2048-55. [2] Machado GM, Caixeta ES, Lucci CM, Rumpf R, Franco MM, Dode MA. Post-hatching development
RI PT
of bovine embryos in vitro: the effects of tunnel preparation and gender. Zygote. 2012;20:123-34. [3] Vajta G, Alexopoulos NI, Callesen H. Rapid growth and elongation of bovine blastocysts in vitro in a three-dimensional gel system. Theriogenology. 2004;62:1253-63.
[4] Vejlsted M, Du Y, Vajta G, Maddox-Hyttel P. Post-hatching development of the porcine and bovine embryo--defining criteria for expected development in vivo and in vitro. Theriogenology. 2006;65:153-65.
SC
[5] Lee KY, Mooney DJ. Hydrogels for tissue engineering. Chemical reviews. 2001;101:1869-79. [6] Amsden B, Turner N. Diffusion characteristics of calcium alginate gels. Biotechnology and bioengineering. 1999;65:605-10. 1998;31:267-85.
M AN U
[7] Wee S, Gombotz WR. Protein release from alginate matrices. Advanced drug delivery reviews. [8] Xu M, West-Farrell ER, Stouffer RL, Shea LD, Woodruff TK, Zelinski MB. Encapsulated three-dimensional culture supports development of nonhuman primate secondary follicles. Biology of reproduction. 2009;81:587-94.
[9] Xu M, Kreeger PK, Shea LD, Woodruff TK. Tissue-engineered follicles produce live, fertile offspring. Tissue engineering. 2006;12:2739-46.
TE D
[10] Kreeger PK, Deck JW, Woodruff TK, Shea LD. The in vitro regulation of ovarian follicle development using alginate-extracellular matrix gels. Biomaterials. 2006;27:714-23. [11] Smitz JE, Cortvrindt RG. The earliest stages of folliculogenesis in vitro. Reproduction. 2002;123:185-202.
[12] BRACKETT BG, OLIPHANT G. Capacitation of rabbit spermatozoa in vitro. Biology of reproduction.
EP
1975;12:260-74.
[13] Rosenkrans CF, Jr., Zeng GQ, GT MC, Schoff PK, First NL. Development of bovine embryos in vitro as affected by energy substrates. Biology of reproduction. 1993;49:459-62.
AC C
[14] Sargus-Patino CN, Wright EC, Plautz SA, Miles JR, Vallet JL, Pannier AK. In vitro development of preimplantation porcine embryos using alginate hydrogels as a three-dimensional extracellular matrix. Reproduction, fertility, and development. 2013. [15] Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2< sup>− ΔΔCT Method. methods. 2001;25:402-8. [16] Bertolini M, Beam SW, Shim H, Bertolini LR, Moyer AL, Famula TR, et al. Growth, development, and gene expression by in vivo- and in vitro-produced day 7 and 16 bovine embryos. Molecular reproduction and development. 2002;63:318-28. [17] Diskin MG, Morris DG. Embryonic and early foetal losses in cattle and other ruminants. Reproduction in domestic animals = Zuchthygiene. 2008;43 Suppl 2:260-7. [18] Alexopoulos NI, French AJ. The prevalence of embryonic remnants following the recovery of post-hatching bovine embryos produced in vitro or by somatic cell nuclear transfer. Animal reproduction science. 2009;114:43-53.
ACCEPTED MANUSCRIPT [19] Clemente M, Lopez-Vidriero I, O'Gaora P, Mehta JP, Forde N, Gutierrez-Adan A, et al. Transcriptome changes at the initiation of elongation in the bovine conceptus. Biology of reproduction. 2011;85:285-95. [20] Mamo S, Mehta JP, McGettigan P, Fair T, Spencer TE, Bazer FW, et al. RNA sequencing reveals novel gene clusters in bovine conceptuses associated with maternal recognition of pregnancy and implantation. Biology of reproduction. 2011;85:1143-51. [21] Krentz KJ, Nebel RL, Canseco RS, McGilliard ML. In vitro and in vivo development of mouse
RI PT
morulae encapsulated in 2% sodium alginate or 0.1% poly-l-lysine. Theriogenology. 1993;39:655-67.
[22] Yaniz JL, Santolaria P, Lopez-Gatius F. In vitro development of bovine embryos encapsulated in sodium alginate. Journal of veterinary medicine A, Physiology, pathology, clinical medicine. 2002;49:393-5.
[23] Sargus-Patino C. Alginate Hydrogel as a Three-dimensional Extracellular Matrix for In Vitro Models of Development. 2013.
SC
[24] Hu D, Cross JC. Development and function of trophoblast giant cells in the rodent placenta. The International journal of developmental biology. 2010;54:341-54.
[25] Nakano H, Shimada A, Imai K, Takahashi T, Hashizume K. The cytoplasmic expression of E-cadherin
M AN U
and beta-catenin in bovine trophoblasts during binucleate cell differentiation. Placenta. 2005;26:393-401.
[26] Igwebuike UM. Trophoblast cells of ruminant placentas—A minireview. Animal Reproduction Science. 2006;93:185-98.
[27] Wice B, Menton D, Geuze H, Schwartz AL. Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation< i> in vitro. Experimental cell research. 1990;186:306-16. [28] Berg DK, Smith CS, Pearton DJ, Wells DN, Broadhurst R, Donnison M, et al. Trophectoderm lineage
TE D
determination in cattle. Developmental cell. 2011;20:244-55.
[29] Kubisch HM, Larson MA, Roberts RM. Relationship between age of blastocyst formation and interferon-tau secretion by in vitro-derived bovine embryos. Molecular reproduction and development. 1998;49:254-60.
[30] Kubisch HM, Larson MA, Kiesling DO. Control of interferon-tau secretion by in vitro-derived
EP
bovine blastocysts during extended culture and outgrowth formation. Molecular reproduction and development. 2001;58:390-7.
[31] Ealy AD, Larson SF, Liu L, Alexenko AP, Winkelman GL, Kubisch HM, et al. Polymorphic forms of expressed bovine interferon-tau genes: relative transcript abundance during early placental
AC C
350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393
development, promoter sequences of genes and biological activity of protein products. Endocrinology. 2001;142:2906-15.
[32] Hersel U, Dahmen C, Kessler H. RGD modified polymers: biomaterials for stimulated cell adhesion and beyond. Biomaterials. 2003;24:4385-415. [33] Wolf E, Arnold GJ, Bauersachs S, Beier HM, Blum H, Einspanier R, et al. Embryo-Maternal Communication in Bovine – Strategies for Deciphering a Complex Cross-Talk. Reproduction in Domestic Animals. 2003;38:276-89. [34] Khurana NK, Niemann H. Energy metabolism in preimplantation bovine embryos derived in vitro or in vivo. Biol Reprod. 2000;62:847-56. [35] Gardner DK. Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture. Theriogenology. 1998;49:83-102.
ACCEPTED MANUSCRIPT 394 395 396
[36] Wang XL, Wang K, Han GC, Zeng SM. A potential autocrine role for interferon tau in ovine trophectoderm. Reproduction in domestic animals = Zuchthygiene. 2013;48:819-25.
AC C
EP
TE D
M AN U
SC
RI PT
397 398
ACCEPTED MANUSCRIPT 399
Tables:
400
Table 1. Primer pairs used for detection of mRNAs.
PCR primers (5’ -> 3’)
Fragment size
F, Forward; R, Reverse.
(base pairs)
accession number
328
DQ126156.1
129
DQ126146.1
254
NM_174411.2
F: GAGAGGCAACCTGGAGAG R: CAGAGCGGTGACAGACAC F: GCCACCATGTACGTGAGCTAC
Cdx2 R: ACATGGTATCCGCCGTAGTC F: ACGGCATCAACTACCCAGTG PAG
GAPDH
M AN U
R: GGAGTGCCCAAAGTGTGAGT
SC
Oct4
RI PT
Genes
F: CTCCCAACGTGTCTGTTGTG R: TGAGCTTGACAAAGTGGTCG
401
GeneBank
222
NM_001034034.2
Table 2. Percentage and mean (± s.e.m.) length of in vitro hatch-out embryos that
403
developed in aecs and control group on day 18. Blastocyst number
Rate of elongation (%)
Average length (mm)
PA IVF Control
20 144 158
8.89 ± 4.71 6.71 ± 1.45 0.00
1.06 ± 0.15 1.09 ± 0.08 0.00
EP
Groups
AC C
404 405
TE D
402
ACCEPTED MANUSCRIPT Figure legends:
407
Figure 1. Alginate encapsulation culture system (AECS), Alginate overlay culture
408
system (AOCS) and Control system.
409
Figure 2. Embryos cultured in alginate hydrogel embedded culture system (AECS).
410
(a) Morphological changes of bovine embryos cultured in AECS and Control on Days
411
14 to 18. AECS-PA: parthenogenetic activated (PA) embryos (n=20) cultured in
412
alginate encapsulation culture system. AECS-IVF: in vitro fertilized (IVF) embryos
413
(n=144) cultured in alginate encapsulation culture system. Control: in vitro fertilized
414
embryos (n=158) cultured in nonencapsulated culture condition. Bar, 500 µm. (b)
415
Survival rates of embryos cultured in AECS and Control groups. **, p < 0.01.
416
Figure 3. Morphologic changes of adherent embryos from Day 18 to 32 in culture.
417
After having been removed from the alginate beads on Day 18, the embryos attached
418
to the bottoms of culture wells and proliferated. Bar = 500 µm.
419
Figure 4. The cell number of bovine embryos cultured in AECS. (a) Light microscopy
420
images of PA- and IVF-derived Day 19 embryos with nuclei stained with Hoechst
421
33342. (b) Total cell number on Day 19 PA (n = 3) and IVF (n = 3) bovine embryos.
422
**p < 0.01.
423
Figure 5. Morphological changes of bovine IVF embryos cultured in AOCS during
424
Day 14 to Day 18 or Day 32. 1-8: the No. of each group. Bar, 200 µm.
425
Figure 6. Diameters of bovine embryos cultured in AOCS from Day 14 to 32 in
426
culture (n =3).
427
Figure 7. Identification of Binuclear cells according to morphological staining and
AC C
EP
TE D
M AN U
SC
RI PT
406
ACCEPTED MANUSCRIPT specific gene expression in Day 26 adherent embryos. (a) After Day 26,
429
trophectoderm cells were treated with or without 10 µM forskolin for 48 h. Binuclear
430
cells are marked by red circles. (b) PAG was expressed in Day 26 adherent embryos.
431
Expression of PAG was detected by RT-PCR, followed by agarose gel electrophoresis.
432
(c) Ratio of binuclear cells in total cell number of embryos. **p < 0.01, n = 3. All of
433
the experiments were repeated more than three times.
434
Figure 8. Expression of Cdx2, Oct4, IFNT and ISG15 in prolong-cultured bovine
435
embryos. Bovine embryos were collected on Days 6 and 8 after in vitro fertilization,
436
and Day 26 and Day 32 in AOCS. mRNA expression levels of Cdx2, Oct4, IFNT and
437
ISG15 were detected by RT-PCR, followed by agarose gel electrophoresis (a1, b).
438
Relative expression ratio changes of Cdx2 and Oct4 from Day 6 to 32 (a2). All of the
439
experiments were repeated more than three times.
SC
M AN U
TE D EP AC C
440
RI PT
428
ACCEPTED MANUSCRIPT
RI PT TE D
M AN U
SC
Fig. 1.
EP
442 443 444 445
Figures
AC C
441
EP
TE D
M AN U
Fig. 2.
AC C
446 447 448 449
SC
RI PT
ACCEPTED MANUSCRIPT
EP
TE D
Fig. 3.
AC C
450 451 452
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
EP
TE D
Fig. 4.
AC C
453 454 455
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
456 457 458
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig.5.
RI PT
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
Fig. 6.
AC C
460 461
SC
459
EP
Fig. 7.
AC C
462 463 464
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
Fig. 8.
AC C
465 466
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
1. Alginate hydrogel was used for in vitro culture of bovine post-hatched embryos. 2. Alginate encapsulation culture system and alginate overlay culture system could support cell proliferation, elongation and differentiation of hatched bovine embryos during prolonged in vitro culture.
S0093-691X(15)00138-7
DOI:
10.1016/j.theriogenology.2015.03.011
Reference:
THE 13121
To appear in:
Theriogenology
Received Date: 13 October 2014 Revised Date:
9 March 2015
Accepted Date: 11 March 2015
Please cite this article as: Zhao S, Liu Z-X, Gao H, Wu Y, Fang Y, Wu S-S, Li M-J, Bai J-H, Liu Y, Evans A, Zeng S-M, A Three-Dimensional Culture System Using Alginate Hydrogel Prolongs Hatched Cattle Embryo Development in vitro, Theriogenology (2015), doi: 10.1016/j.theriogenology.2015.03.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Revised
1
A Three-Dimensional Culture System Using Alginate Hydrogel Prolongs
3
Hatched Cattle Embryo Development in vitro
4
Shuan Zhao a, Zhen-Xing Liu a, Hui Gao a, Yi Wu a, Yuan Fang a, Shuai-Shuai Wu
5
a
, Ming-Jie Li a, Jia-Hua Bai b, Yan Liu b, Alexander Evansc, Shen-Ming Zeng a,*
6
a
National Key Laboratory of Animal Nutrition, Laboratory of Animal Embryonic
7
Biotechnology; National Engineering Laboratory for Animal Breeding; Key
8
Laboratory of Animal Genetics, Breeding, and Reproduction of the Ministry of
9
Agriculture, College of Animal Science and Technology, China Agricultural
M AN U
SC
RI PT
2
10
University, Beijing, 100193, China
11
b
12
Agriculture and Forestry Sciences, Beijing, China.
13
c
14
*
15
West Road, Haidian District, Beijing 100193, China. Phone: 86-10-62733744; Fax:
16
86-10-62733744; E-mail: [email protected].
17
Additional footnotes: Shuan Zhao and Zhen-Xing Liu contributed equally to this
18
work.
19
TE D
Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of
School of Agriculture and Food Science in University College Dublin, Ireland,
AC C
EP
Corresponding author: Shenming Zeng, Dongke Building #251, No. 2 Yuanmingyuan
ACCEPTED MANUSCRIPT ABSTRACT: No successful method exists to maintain the three-dimensional
21
architecture of hatched embryos in vitro. Alginate, a linear polysaccharide derived
22
from brown algae, has characteristics that make it an ideal material as a
23
three-dimensional (3D) extracellular matrix (ECM) for in vitro cell, tissue or embryo
24
culture. In this study, alginate hydrogel was used for in vitro culture of post-hatched
25
bovine embryos in order to observe their development under the 3D system. In vitro
26
fertilized (IVF) and parthenogenetic activated (PA) post-hatched bovine blastocysts
27
were cultured in an alginate encapsulation culture system (AECS), an alginate overlay
28
culture system (AOCS), or control culture system. After 18 days of culture, the
29
survival rate of embryos cultured in AECS was higher than that in the control group
30
(p<0.05), and the embryos were expanded and elongated in AECS with the maximal
31
length of 1.125 mm. When the AECS shrinking embryos were taken out of the
32
alginate beads on Day 18 and cultured in the normal culture system, 9.09% of them
33
attached to the bottoms of the plastic wells and grew rapidly, with the largest area of
34
an attached embryo being 66.00 mm2 on Day 32. The embryos cultured in AOCS
35
developed mono- or multi-vesicular morphologies. Total cell number of the embryos
36
cultured in AECS on Day 19 was significantly higher than embryos on Day 8.
37
Additionally, AECS and AOCS supported differentiation of the embryonic cells.
38
Binuclear cells were visible in Day 26 adherent embryos, and the mRNA expression
39
patterns of Cdx2 and Oct4 in AOCS cultured embryos were similar to in vivo embryos
40
while IFNT and ISG15 mRNA were still expressed in Day 26 and Day 32
41
prolong-cultured embryos. In conclusion, AECS and AOCS did support cell
AC C
EP
TE D
M AN U
SC
RI PT
20
ACCEPTED MANUSCRIPT proliferation, elongation and differentiation of hatched bovine embryos during
43
prolonged in vitro culture. The culture system will be useful to further investigate the
44
molecular mechanisms controlling ruminant embryo elongation and implantation.
45
Key words: alginate, three-dimensional culture, embryo development, bovine
RI PT
42
AC C
EP
TE D
M AN U
SC
46
ACCEPTED MANUSCRIPT 47
48
1. Introduction The causes of early embryonic loss in ruminants are poorly understood and this is not helped by the lack of knowledge about the exact physiological mechanisms
50
underlining embryo elongation and implantation [1, 2]. In vitro models present ethical
51
and cost advantages over in vivo studies, which can help define the factors that
52
regulate early embryo development and cell differentiation. Three-dimensional (3D)
53
scaffolds for cell and tissue culture provide a potential model to study in vitro embryo
54
development, especially post-hatched embryos. In cattle, limited elongation of
55
post-hatch embryos in vitro via physical induction has been demonstrated using an
56
agarose gel tube system [1-3]. However, the embryos cultured in this system had
57
deficiencies in the development of their embryonic disks [1, 4].
M AN U
SC
RI PT
49
Alginate is a commonly used biomaterial and is often employed in tissue
59
engineering as an artificial extracellular matrix [5]. Alginate is derived from brown
60
algae and is composed of repeating units of β-D-mannuronic acid and α-L-guluronic
61
acid [6]. One of the favorable properties of alginate as a biomaterial is to form a
62
hydrogel by ionic cross-linking of the guluronic residues in the presence of a divalent
63
cation [7]. This permits the diffusion of nutrients and hormones through the 3D
64
scaffold that are essential for cell and tissue growth and development [6]. Alginate
65
hydrogel has been successfully used for supporting mouse ovarian follicle [8] and
66
granulosa cell-oocyte complexe (GOC) in vitro development. Moreover, the oocytes
67
recovered from the alginate encapsulated GOCs were able to resume meiosis,
68
undergo fertilization, and to produce viable offspring [9]. We hypothesize that bovine
69
embryos can survive long term and display morphological changes similar to in vivo
70
conditions if they are cultured in a proper biomechanical (3D) support system. The
AC C
EP
TE D
58
ACCEPTED MANUSCRIPT very early embryo can successfully grow in a traditional culture dish with the
72
protection of its zona pellucida (ZP). However, when the embryo hatches from its ZP,
73
it is virtually impossible for it to keep its normal morphology in vitro because the
74
embryonic cells attach to the bottom of culture dish, resulting in the disruption of
75
cell-to-cell interactions and its 3D architecture [10, 11]. Alternatively, we propose that
76
a 3D culture system can maintain the embryo architecture by providing a uniform
77
surrounding environment for the whole embryo, which mimics uterine conditions.
78
Our objective was to use alginate hydrogels to establish an in vitro culture system to
79
support bovine post-hatched embryo development. In the present study, in vitro
80
development of bovine embryos encapsulated in alginate hydrogels or cultured on top
81
of alginate hydrogels was evaluated by characterizing survival rate, cellular
82
proliferation, morphological changes and stage-specific gene expression.
M AN U
SC
RI PT
71
AC C
EP
TE D
83
ACCEPTED MANUSCRIPT 84
2. Materials and Methods
Unless otherwise indicated, all chemicals were obtained from Sigma Chemicals
86
Co. (St. Louis, MO). Similarly, all plastic-ware used was from Nunc-ware (Nunc;
87
NalgeNunc International, Roskilde, Denmark).
88
2.1 Bovine oocyte collection and in vitro maturation
RI PT
85
Bovine ovaries were collected immediately after slaughter and transported to our
90
laboratory in saline solution (0.9% NaCl) supplemented with penicillin G (100 IU/ml)
91
and streptomycin sulfate (100 μg/ml) at 35°C. Cumulus–oocyte complexes (COCs)
92
were aspirated from follicles with diameters from 2 to 8 mm. The COCs with
93
homogeneous cytoplasm and at least three intact layers of surrounding cumulus cells
94
were selected for in vitro maturation. After rinsing in pre-warmed maturation medium
95
consisting of TCM 199 with Earles’ salts (GIBCO BRL, Grand Island, NY, USA), 10%
96
(v/v) FBS (HyClone-Pierce, Shanghai, China), 10 µg/mL FSH (Bioniche, Canada), 10
97
µg/mL LH (Bioniche, Canada), 1 ng/mL EGF, and 0.1 mg/mL cysteine, groups of 20
98
COCs were cultured in 50 µL media droplets under mineral oil at 38.5 °C, in 5% CO2
99
in air with maximum humidity for 22 to 24 h.
101
M AN U
TE D
EP
AC C
100
SC
89
2.2 In Vitro Fertilization of oocytes After in vitro maturation, bovine COCs were washed three times and then placed
102
in 40 µL droplets of Brackett–Oliphant (BO) medium [12] containing 10 mg/mL BSA
103
under mineral oil for IVF as follows. Frozen semen (Beijing Dairy Cow Center,
104
Beijing, China) was thawed in a water bath at 37 °C for 30 s. Sperm were washed
105
twice in BO medium supplemented with 2.5 mM caffeine and then resuspended in BO
ACCEPTED MANUSCRIPT medium supplemented with 10 mg/mL BSA and 5 IU/mL heparin. The concentration
107
of spermatozoa was counted in haemocytometer and further diluted in BO medium to
108
a concentration of 2×106 /mL. An aliquot (50 µL) of this suspension was added into
109
each 50 µL droplet of BO medium containing 20 COCs under mineral oil for
110
insemination. In vitro fertilization was carried out in humidified air with 5% CO2 at
111
38.5 °C. After incubation for 6 to 8 h, loosely associated cumulus cells and
112
spermatozoa were removed from the oocytes by gentle vortexing. The presumptive
113
zygotes were then cultured in CR1aa medium [13] supplemented with essential or
114
non-essential amino acids and 3 mg/mL BSA for two days and continuously cultured
115
in CR1aa medium containing 10% FBS at 38.5 °C in a humidified atmosphere of 5%
116
CO2. The culture medium was refreshed every two days.
117
2.3 Parthenogenetic activation of oocytes
TE D
M AN U
SC
RI PT
106
After in vitro maturation, COCs were treated with 0.1% hyaluronidase to remove
119
cumulus cells. The parthenogenetic activation (PA) of the oocytes was carried out
120
according to the following protocol. Oocytes were rinsed three times in M199
121
containing 25 mM Hepes and 10% FBS and pre-treated in 5 µM ionomycin at 38.5 °C
122
for 5 min, then cultured in CR1aa supplemented with 2 mM 6-(Dimethylamino)
123
purine (6-DMAP) for 4 h. The oocytes were then first cultured in CR1aa medium with
124
3 mg/mL BSA for 2 days and continuously cultured in CR1aa medium with 10% FBS
125
for 4 days at 38.5 °C in a humidified atmosphere of 5% CO2. The culture medium was
126
refreshed every two days.
127
2.4 Alginate gel preparation
AC C
EP
118
ACCEPTED MANUSCRIPT The crosslinking solution used for gel formation was prepared by dissolving
129
calcium chloride (CaCl2) and sodium chloride (NaCl) in sterile MilliQ water to obtain
130
a 50 mM CaCl2 and 140 mM NaCl solution. Alginate solutions were prepared by
131
dissolving lyophilized alginate powder in sterile MilliQ water and were all 1.5%
132
alginate (w/v) concentrations.
133
2.5 Encapsulation and overlay procedure
RI PT
128
On Day 8 after IVF or PA, the hatched embryos were cultured in Alginate
135
Encapsulation Culture System (AECS) or Alginate Overlay Culture System (AOCS).
136
The encapsulation procedure was according to the method described previously [14]
137
but with some modifications. Briefly, the embryos were washed three times in
138
warmed PBS (Ca2+ and Mg2+ free, Invitrogen, Carlsbad, USA), and put into the
139
alginate solution. A total of 7 µL of the alginate solution containing one embryo was
140
slowly released to the crosslinking solution. After two minutes the beads containing
141
embryos were removed from the crosslinking solution, and washed thrice in CR1aa
142
containing 10% FBS for 15 min. The beads were then cultured individually in the
143
wells of 96-well plate with 100 µL CR1aa (10% FBS) under 50 µL mineral oil (Fig. 1)
144
at 38.5 °C in a humidified atmosphere of 5% CO2. One-half of the culture medium
145
was changed every two days.
M AN U
TE D
EP
AC C
146
SC
134
The Alginate Overlay Culture System was developed according to the following
147
protocol. A total of 60 µL alginate solution was added to one well of 96-well plate,
148
then 50 µL crosslinking solution was slowly added. After two minutes the alginate
149
overlay had formed on the bottom of the well. The well was filled with CR1aa
ACCEPTED MANUSCRIPT containing 10% FBS, and incubated under the conditions described above for three
151
hours. The medium was refreshed every hour. The embryos were individually cultured
152
in the wells of 96-well plate with 100 µL CR1aa (10% FBS) under 50 µL mineral oil
153
(Fig. 1) at 38.5 °C in a humidified atmosphere of 5% CO2. Half of the culture medium
154
was refreshed every two days. In the control group, the embryos were individually
155
cultured in the wells without any alginate under the same conditions (Fig. 1).
156
2.6 Blastocyst Cell Count
SC
RI PT
150
The nuclei of selected blastocysts were stained using Hoechst 33342 (10 ng/mL)
158
for 10 min, and washed thrice in DPBS. Embryos with labeled nuclei under UV
159
excitation were individually mounted on microscope slides in glycerol underneath a
160
cover slip and examination was carried out in whole mount.
161
2.7 Quantitative PCR on mRNA expression and statistical analysis
TE D
M AN U
157
Total cellular RNA (tcRNA) of Day 6 (n=100) embryos, Day 8 (n=100) early
163
blastocysts, Day 26 (n=15) and Day 32 (n=15) prolong-cultured embryos were
164
extracted using an RNAprep pure Tissue Kit (Thermo, USA). For each sample a
165
complete reaction, but without the reverse transcriptase (RT), was performed.
166
Amplification reactions were performed in 20 µL reaction volumes containing 1 µL
167
cDNA, 9 µL of 2 × Master SYBR Green mix (TIANGEN Biotech, Beijing, China), 9
168
µL sterile water and 0.5 µL each of forward and reverse gene specific primers (10 µM).
169
This mixture was denatured at 95 °C for 5 min; 45 cycles of PCR (95°C for 15 s,
170
60°C for 20 s, and 72°C for 50 s) in 7500 real-time PCR system (Applied Biosystems,
171
Foster City, CA, USA). The primers and Gene Bank source accessions for each gene
AC C
EP
162
ACCEPTED MANUSCRIPT 172
are shown in (Table 1). Relative expression of each gene was calculated by 2-△△Ct
173
[15].
174
2.8 Statistical analysis The survival rates of extend cultured embryos and the percentages of binuclear
176
cells in overall cell numbers were analyzed using a general linear model and one-way
177
post-hoc test using the MIXED procedure models of SAS (version 9.1, SAS Institute
178
Inc., Cary, NC). All data were expressed as mean ± S.E.M. The percentages were
179
subjected to arcsine transformation before analysis. Differences were considered to be
180
significant when P < 0.05.
AC C
EP
TE D
181
M AN U
SC
RI PT
175
ACCEPTED MANUSCRIPT 182
3. Results
183
3.1 Morphological changes of bovine embryos cultured in AECS After 18 days the morphologies of the embryos were completely different in
185
AECS from that in the control culture system (Fig. 2a). The embryos derived from PA
186
and IVF began elongation on Day 14, and the maximal length was 1.125 mm (Fig. 2a)
187
on Day 18. The ratio and average length of elongated embryos were not different
188
between IVF and PA groups (Table 2, P < 0.05). However, the survival rates in
189
AECS-PA and AECS-IVF were greater than that in the control group (Fig. 2b, P <
190
0.01). On Day 18, a total of 55 embryos that appeared to be shrinking from the 144
191
hatched blastocysts were removed from the gel and cultured in the normal culture
192
system. Interestingly, instead of death, 9.09% of these embryos quickly expanded
193
from Day 18 to 32 (Fig. 3), and the largest area was 66 mm2 (Fig. 3, Day 32).
194
3.2 Cell number of bovine embryos cultured in AECS
TE D
M AN U
SC
RI PT
184
The cell numbers in the embryos derived from PA and IVF reached tens of
196
thousands after 19 days of culture (Fig. 4a). The average total cell number of IVF
197
embryos was much more than in PA embryos (n=3, 42,608 ± 2,244 vs. 14,899 ± 4,919,
198
Fig. 4b, P < 0.01).
199
3.3 Morphological changes of bovine embryos cultured in AOCS
AC C
EP
195
200
Of the embryos cultured in AOCS, 67% (8/12) shrank or died by Day 16 (Fig.
201
5a). However, 25% (3/12) of the embryos grew for at least 32 days (Fig. 5b Fig. 6),
202
but none elongated in morphology.
203
3.4 Cell differentiation in the embryos cultured in AECS
ACCEPTED MANUSCRIPT 204
Hoechst 33342 staining revealed that binuclear cells were present in Day 26 adherent
embryos
(Fig.
7a),
which
was
further
confirmed
by
PAG
206
(Pregnancy-associated glycoprotein, a specific gene expressed in binuclear cells)
207
expression (Fig. 7b). The proportion of binuclear cells increased in response to
208
treatment with forskolin (Fig. 7a and 7c, P < 0.01).
209
3.5 Specific Gene expression in embryos cultured in AOCS
RI PT
205
High amounts of Oct4 mRNA and low levels of Cdx2 mRNA were detected in
211
both Day 6 and Day 8 blastocysts (Fig. 8a1). However, the transcription ratio of
212
Cdx2:Oct4 in blastocysts was 3.51 on Day 26 and this increased to 5.66 on Day 40
213
(Fig. 8). Moreover, both IFNT and ISG15 mRNA were still present in Day 26 and 32
214
embryos (Fig. 8b).
M AN U
SC
210
AC C
EP
TE D
215
ACCEPTED MANUSCRIPT 216
4. Discussion
Few studies have focused on post-hatching mammalian embryo development
218
although most pregnancy losses occur at this stage [16-20]. In order to better
219
characterize the development of bovine hatched-out embryos in vitro, alginate
220
hydrogel was employed for a 3D matrix. Previously, alginate hydrogels have been
221
used for an artificial zona pellucida for mouse morulae [21], for packaging ovarian
222
follicles in mice [9] and nonhuman primates [8], and to study early blastocysts
223
development in cattle [22] and in pigs [23]. Our report demonstrates for the first time
224
that alginate hydrogels can be successfully used for maintaining bovine post-hatched
225
embryo development in vitro.
M AN U
SC
RI PT
217
Bovine embryos cultured in AECS were able to undergo morphological changes
227
similar to in vivo, and its survival rate was also significantly higher than
228
non-encapsulated embryos, which were different from previous report in porcine [23].
229
This implies that three-dimension matrix support is necessary to maintain the
230
appropriate architecture for normal bovine embryos undergoing elongation.
EP
The embryos recovered from the alginate gel bead were able to attach and grow
AC C
231
TE D
226
232
rapidly at the bottom of the well (Fig. 3a), which rarely happens in normal culture
233
systems. This suggests that the embryos had more viable cells after 6 days culture in
234
AECS. This technology may be used for isolation of stem cells from early bovine
235
embryos. The binuclear cells, a subtype of trophoblast giant cells (TGCs), is the first
236
cell type to terminally differentiate during embryogenesis and is of vital importance
237
for implantation and modulation of post-implantation placentation [24]. After Hoechst
ACCEPTED MANUSCRIPT 33342 staining, we found some cells contained two enlarged nuclei similar to
239
previously characterized binucleate cells in BT-1 (bovine trophectoderm-1) cells [25].
240
Also, pregnancy associated glycoprotein (PAG), one of the genes specifically
241
expressed in binuclear cells, was detected in Day 26 attached embryonic cells
242
indicating their differentiation. The roles of binuclear trophoblast cells in implantation
243
have been extensively studied in a few ruminant species [26]. Forskolin, as an
244
activator of cAMP, can induce cell fusion to produce binuclear cells in human
245
choriocarcinoma (BeWo) cells [27]. In our study, the treatment also increased the
246
percentage of binuclear cells in the attached embryonic cells. This suggests that these
247
embryonic cells possess the ability to differentiate into placenta tissues.
M AN U
SC
RI PT
238
The embryos cultured in AOCS could survive for at least 32 days with mono- or
249
multi-vesicular cavities developing by Days 17 to 18 (Fig. 3a, 6b). The reason why
250
the embryos formed mono- or milti-vesicular cavities is unknown. Unlike mouse
251
embryos, both inner cell mass and trophectoderm cells express high levels of Oct4
252
mRNA in bovine blastocysts. The trophoblast cells derived from Day 7 in vitro
253
produced bovine embryos can differentiate into ICM cells untill Day 14 [28]. In this
254
study, the multi-vesicular embryos might be differentiated from pluripotent
255
trophoblast cells. The ratio of Cdx2:Oct4 mRNA in mouse late blastocysts was nearly
256
10-fold compared to bovine equivalent stage embryos, and the Cdx2 transcript levels
257
in bovine trophectoderm cells begin to exceed Oct4 levels at epiblast stages [28]. We
258
also found that the Cdx2:Oct4 transcription ratios in bovine embryos increased with
259
the culture time. On Day 8 the transcription ratio of Cdx2:Oct4 was 0.004, while on
AC C
EP
TE D
248
ACCEPTED MANUSCRIPT 260
Day 26 the ratio had increased to 3.51. This demonstrates that bovine embryos
261
cultured long-term in vitro displayed similar expression profiles of key developmental
262
marker genes to rapidly developing mouse embryos. IFNT is the pregnancy signal in ruminants, and IFNT mRNA and protein
264
expressions are relatively low at the blastocyst stage, but increase with the
265
morphological development of conceptuses [29, 30]. However, coincident with
266
trophectoderm attachment to the uterine lining, IFNT mRNA levels decline sharply by
267
Day 21 and cease by Day 24 of pregnancy in cattle [31]. In our results, IFNT mRNA
268
was still detectable on Day 26 and Day 32 extend-cultured IVF blastocysts, although
269
binucleate cells were differentiated. This suggests that normal IFNT expression might
270
be associated with complete elongation of embryos and/or other factors secreted from
271
the uterus.
TE D
M AN U
SC
RI PT
263
The AECS model used in this study did support moderate growth, elongation and
273
differentiation of bovine hatch blastocysts, but this system waits to be further
274
improved. Firstly, the arginine-glycine-aspartic acid (RGD) peptide sequence is the
275
most effective and commonly used peptide sequence to promote cell adhesion [32].
276
When covalently linked to alginate, RGD peptide sites could serve as ligands to
277
integrins in the embryonic cell membranes to mediate cellular migration and
278
embryonic adhesion. The AECS could be used to investige the mechanisms of
279
ruminant embryo elongation. Secondly, because embryo-maternal communication is
280
vital for bovine embryo development during the preimplantation stage of pregnancy
281
[33], the embryos could be co-cultured with uterine lumen and/or glandular epithelial
AC C
EP
272
ACCEPTED MANUSCRIPT cells with the alginate hydrogel to pursue the molecular communication pathways
283
during embryo implantation. Thirdly, as the energy demands of bovine embryos
284
increase they substantially enhance their glycolytic activity [34] and glucose uptake
285
[35] during compaction, blastocyst formation, expansion and elongation. Moreover,
286
IFNT is not only a pregnancy recognition signal but also a factor that stimulates the
287
proliferation of trophoblast cells and the development of early embryos [36]. However,
288
the exact molecular roles of the above factors in early embryo development and cell
289
differentiation need to be established. In the AECS these factors could be added to
290
defined culture medium to pursue their specific functions.
291
4.1 Conclusion
M AN U
SC
RI PT
282
The alginate encapsulation culture system (AECS) did support cell proliferation,
293
embryo elongation and differentiation of bovine hatch blastocysts. Whereas, the
294
embryos cultured in the alginate overlay culture system (AOCS) did not undergo
295
elongation, but did continuously develop with multi-vesicular changes in morphology.
296
We have established a model for long-term culture of bovine embryos that can be used
297
for investigating the molecular mechanisms of embryo elongation, cell differentiation
298
and implantation in farm animals.
300
EP
AC C
299
TE D
292
ACCEPTED MANUSCRIPT 301
302
Acknowledgments
This work was supported by a grant from the National Natural Science Foundation of China (Grant 31172209) and National Key Technologies R&D
304
Program (2011BAD19B03).
AC C
EP
TE D
M AN U
SC
RI PT
303
ACCEPTED MANUSCRIPT 305
306
Disclosure Statement
The authors of this article have no conflicts of interest to disclose.
AC C
EP
TE D
M AN U
SC
RI PT
307
ACCEPTED MANUSCRIPT 308
Reference:
309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349
[1] Brandao DO, Maddox-Hyttel P, Lovendahl P, Rumpf R, Stringfellow D, Callesen H. Post hatching development: a novel system for extended in vitro culture of bovine embryos. Biology of reproduction. 2004;71:2048-55. [2] Machado GM, Caixeta ES, Lucci CM, Rumpf R, Franco MM, Dode MA. Post-hatching development
RI PT
of bovine embryos in vitro: the effects of tunnel preparation and gender. Zygote. 2012;20:123-34. [3] Vajta G, Alexopoulos NI, Callesen H. Rapid growth and elongation of bovine blastocysts in vitro in a three-dimensional gel system. Theriogenology. 2004;62:1253-63.
[4] Vejlsted M, Du Y, Vajta G, Maddox-Hyttel P. Post-hatching development of the porcine and bovine embryo--defining criteria for expected development in vivo and in vitro. Theriogenology. 2006;65:153-65.
SC
[5] Lee KY, Mooney DJ. Hydrogels for tissue engineering. Chemical reviews. 2001;101:1869-79. [6] Amsden B, Turner N. Diffusion characteristics of calcium alginate gels. Biotechnology and bioengineering. 1999;65:605-10. 1998;31:267-85.
M AN U
[7] Wee S, Gombotz WR. Protein release from alginate matrices. Advanced drug delivery reviews. [8] Xu M, West-Farrell ER, Stouffer RL, Shea LD, Woodruff TK, Zelinski MB. Encapsulated three-dimensional culture supports development of nonhuman primate secondary follicles. Biology of reproduction. 2009;81:587-94.
[9] Xu M, Kreeger PK, Shea LD, Woodruff TK. Tissue-engineered follicles produce live, fertile offspring. Tissue engineering. 2006;12:2739-46.
TE D
[10] Kreeger PK, Deck JW, Woodruff TK, Shea LD. The in vitro regulation of ovarian follicle development using alginate-extracellular matrix gels. Biomaterials. 2006;27:714-23. [11] Smitz JE, Cortvrindt RG. The earliest stages of folliculogenesis in vitro. Reproduction. 2002;123:185-202.
[12] BRACKETT BG, OLIPHANT G. Capacitation of rabbit spermatozoa in vitro. Biology of reproduction.
EP
1975;12:260-74.
[13] Rosenkrans CF, Jr., Zeng GQ, GT MC, Schoff PK, First NL. Development of bovine embryos in vitro as affected by energy substrates. Biology of reproduction. 1993;49:459-62.
AC C
[14] Sargus-Patino CN, Wright EC, Plautz SA, Miles JR, Vallet JL, Pannier AK. In vitro development of preimplantation porcine embryos using alginate hydrogels as a three-dimensional extracellular matrix. Reproduction, fertility, and development. 2013. [15] Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2< sup>− ΔΔCT Method. methods. 2001;25:402-8. [16] Bertolini M, Beam SW, Shim H, Bertolini LR, Moyer AL, Famula TR, et al. Growth, development, and gene expression by in vivo- and in vitro-produced day 7 and 16 bovine embryos. Molecular reproduction and development. 2002;63:318-28. [17] Diskin MG, Morris DG. Embryonic and early foetal losses in cattle and other ruminants. Reproduction in domestic animals = Zuchthygiene. 2008;43 Suppl 2:260-7. [18] Alexopoulos NI, French AJ. The prevalence of embryonic remnants following the recovery of post-hatching bovine embryos produced in vitro or by somatic cell nuclear transfer. Animal reproduction science. 2009;114:43-53.
ACCEPTED MANUSCRIPT [19] Clemente M, Lopez-Vidriero I, O'Gaora P, Mehta JP, Forde N, Gutierrez-Adan A, et al. Transcriptome changes at the initiation of elongation in the bovine conceptus. Biology of reproduction. 2011;85:285-95. [20] Mamo S, Mehta JP, McGettigan P, Fair T, Spencer TE, Bazer FW, et al. RNA sequencing reveals novel gene clusters in bovine conceptuses associated with maternal recognition of pregnancy and implantation. Biology of reproduction. 2011;85:1143-51. [21] Krentz KJ, Nebel RL, Canseco RS, McGilliard ML. In vitro and in vivo development of mouse
RI PT
morulae encapsulated in 2% sodium alginate or 0.1% poly-l-lysine. Theriogenology. 1993;39:655-67.
[22] Yaniz JL, Santolaria P, Lopez-Gatius F. In vitro development of bovine embryos encapsulated in sodium alginate. Journal of veterinary medicine A, Physiology, pathology, clinical medicine. 2002;49:393-5.
[23] Sargus-Patino C. Alginate Hydrogel as a Three-dimensional Extracellular Matrix for In Vitro Models of Development. 2013.
SC
[24] Hu D, Cross JC. Development and function of trophoblast giant cells in the rodent placenta. The International journal of developmental biology. 2010;54:341-54.
[25] Nakano H, Shimada A, Imai K, Takahashi T, Hashizume K. The cytoplasmic expression of E-cadherin
M AN U
and beta-catenin in bovine trophoblasts during binucleate cell differentiation. Placenta. 2005;26:393-401.
[26] Igwebuike UM. Trophoblast cells of ruminant placentas—A minireview. Animal Reproduction Science. 2006;93:185-98.
[27] Wice B, Menton D, Geuze H, Schwartz AL. Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation< i> in vitro. Experimental cell research. 1990;186:306-16. [28] Berg DK, Smith CS, Pearton DJ, Wells DN, Broadhurst R, Donnison M, et al. Trophectoderm lineage
TE D
determination in cattle. Developmental cell. 2011;20:244-55.
[29] Kubisch HM, Larson MA, Roberts RM. Relationship between age of blastocyst formation and interferon-tau secretion by in vitro-derived bovine embryos. Molecular reproduction and development. 1998;49:254-60.
[30] Kubisch HM, Larson MA, Kiesling DO. Control of interferon-tau secretion by in vitro-derived
EP
bovine blastocysts during extended culture and outgrowth formation. Molecular reproduction and development. 2001;58:390-7.
[31] Ealy AD, Larson SF, Liu L, Alexenko AP, Winkelman GL, Kubisch HM, et al. Polymorphic forms of expressed bovine interferon-tau genes: relative transcript abundance during early placental
AC C
350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393
development, promoter sequences of genes and biological activity of protein products. Endocrinology. 2001;142:2906-15.
[32] Hersel U, Dahmen C, Kessler H. RGD modified polymers: biomaterials for stimulated cell adhesion and beyond. Biomaterials. 2003;24:4385-415. [33] Wolf E, Arnold GJ, Bauersachs S, Beier HM, Blum H, Einspanier R, et al. Embryo-Maternal Communication in Bovine – Strategies for Deciphering a Complex Cross-Talk. Reproduction in Domestic Animals. 2003;38:276-89. [34] Khurana NK, Niemann H. Energy metabolism in preimplantation bovine embryos derived in vitro or in vivo. Biol Reprod. 2000;62:847-56. [35] Gardner DK. Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture. Theriogenology. 1998;49:83-102.
ACCEPTED MANUSCRIPT 394 395 396
[36] Wang XL, Wang K, Han GC, Zeng SM. A potential autocrine role for interferon tau in ovine trophectoderm. Reproduction in domestic animals = Zuchthygiene. 2013;48:819-25.
AC C
EP
TE D
M AN U
SC
RI PT
397 398
ACCEPTED MANUSCRIPT 399
Tables:
400
Table 1. Primer pairs used for detection of mRNAs.
PCR primers (5’ -> 3’)
Fragment size
F, Forward; R, Reverse.
(base pairs)
accession number
328
DQ126156.1
129
DQ126146.1
254
NM_174411.2
F: GAGAGGCAACCTGGAGAG R: CAGAGCGGTGACAGACAC F: GCCACCATGTACGTGAGCTAC
Cdx2 R: ACATGGTATCCGCCGTAGTC F: ACGGCATCAACTACCCAGTG PAG
GAPDH
M AN U
R: GGAGTGCCCAAAGTGTGAGT
SC
Oct4
RI PT
Genes
F: CTCCCAACGTGTCTGTTGTG R: TGAGCTTGACAAAGTGGTCG
401
GeneBank
222
NM_001034034.2
Table 2. Percentage and mean (± s.e.m.) length of in vitro hatch-out embryos that
403
developed in aecs and control group on day 18. Blastocyst number
Rate of elongation (%)
Average length (mm)
PA IVF Control
20 144 158
8.89 ± 4.71 6.71 ± 1.45 0.00
1.06 ± 0.15 1.09 ± 0.08 0.00
EP
Groups
AC C
404 405
TE D
402
ACCEPTED MANUSCRIPT Figure legends:
407
Figure 1. Alginate encapsulation culture system (AECS), Alginate overlay culture
408
system (AOCS) and Control system.
409
Figure 2. Embryos cultured in alginate hydrogel embedded culture system (AECS).
410
(a) Morphological changes of bovine embryos cultured in AECS and Control on Days
411
14 to 18. AECS-PA: parthenogenetic activated (PA) embryos (n=20) cultured in
412
alginate encapsulation culture system. AECS-IVF: in vitro fertilized (IVF) embryos
413
(n=144) cultured in alginate encapsulation culture system. Control: in vitro fertilized
414
embryos (n=158) cultured in nonencapsulated culture condition. Bar, 500 µm. (b)
415
Survival rates of embryos cultured in AECS and Control groups. **, p < 0.01.
416
Figure 3. Morphologic changes of adherent embryos from Day 18 to 32 in culture.
417
After having been removed from the alginate beads on Day 18, the embryos attached
418
to the bottoms of culture wells and proliferated. Bar = 500 µm.
419
Figure 4. The cell number of bovine embryos cultured in AECS. (a) Light microscopy
420
images of PA- and IVF-derived Day 19 embryos with nuclei stained with Hoechst
421
33342. (b) Total cell number on Day 19 PA (n = 3) and IVF (n = 3) bovine embryos.
422
**p < 0.01.
423
Figure 5. Morphological changes of bovine IVF embryos cultured in AOCS during
424
Day 14 to Day 18 or Day 32. 1-8: the No. of each group. Bar, 200 µm.
425
Figure 6. Diameters of bovine embryos cultured in AOCS from Day 14 to 32 in
426
culture (n =3).
427
Figure 7. Identification of Binuclear cells according to morphological staining and
AC C
EP
TE D
M AN U
SC
RI PT
406
ACCEPTED MANUSCRIPT specific gene expression in Day 26 adherent embryos. (a) After Day 26,
429
trophectoderm cells were treated with or without 10 µM forskolin for 48 h. Binuclear
430
cells are marked by red circles. (b) PAG was expressed in Day 26 adherent embryos.
431
Expression of PAG was detected by RT-PCR, followed by agarose gel electrophoresis.
432
(c) Ratio of binuclear cells in total cell number of embryos. **p < 0.01, n = 3. All of
433
the experiments were repeated more than three times.
434
Figure 8. Expression of Cdx2, Oct4, IFNT and ISG15 in prolong-cultured bovine
435
embryos. Bovine embryos were collected on Days 6 and 8 after in vitro fertilization,
436
and Day 26 and Day 32 in AOCS. mRNA expression levels of Cdx2, Oct4, IFNT and
437
ISG15 were detected by RT-PCR, followed by agarose gel electrophoresis (a1, b).
438
Relative expression ratio changes of Cdx2 and Oct4 from Day 6 to 32 (a2). All of the
439
experiments were repeated more than three times.
SC
M AN U
TE D EP AC C
440
RI PT
428
ACCEPTED MANUSCRIPT
RI PT TE D
M AN U
SC
Fig. 1.
EP
442 443 444 445
Figures
AC C
441
EP
TE D
M AN U
Fig. 2.
AC C
446 447 448 449
SC
RI PT
ACCEPTED MANUSCRIPT
EP
TE D
Fig. 3.
AC C
450 451 452
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
EP
TE D
Fig. 4.
AC C
453 454 455
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
456 457 458
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Fig.5.
RI PT
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
Fig. 6.
AC C
460 461
SC
459
EP
Fig. 7.
AC C
462 463 464
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
EP
TE D
M AN U
Fig. 8.
AC C
465 466
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
1. Alginate hydrogel was used for in vitro culture of bovine post-hatched embryos. 2. Alginate encapsulation culture system and alginate overlay culture system could support cell proliferation, elongation and differentiation of hatched bovine embryos during prolonged in vitro culture.