- Email: [email protected]
Evaluation of the simulated physiological oocyte maturation system for improving bovine in vitro embryo production
Accepted Manuscript Evaluation of the simulated physiological oocyte maturation system (SPOM) for improving bovine in vitro embryo production A.L.S. Guimarães, S.A. Pereira, L.O. Leme, M.A.N. Dode PII:
S0093-691X(14)00522-6
DOI:
10.1016/j.theriogenology.2014.07.042
Reference:
THE 12940
To appear in:
Theriogenology
Received Date: 29 April 2014 Revised Date:
20 June 2014
Accepted Date: 18 July 2014
Please cite this article as: Guimarães ALS, Pereira SA, Leme LO, Dode MAN, Evaluation of the simulated physiological oocyte maturation system (SPOM) for improving bovine in vitro embryo production, Theriogenology (2014), doi: 10.1016/j.theriogenology.2014.07.042. 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 1
REVISED
2 3
Evaluation of the simulated physiological oocyte maturation system (SPOM) for improving bovine in vitro embryo production
4
Guimarães, A. L. S.1; Pereira, S.A.2; Leme, L. O.1; Dode, M. A. N.1, 3*
5
1
6
Brazil; 2 Institute of Biology, University of Brasília, Brasília-DF, Brazil, 3Embrapa-
7
Genetic Resources and Biotechnology, Brasília-DF, Brazil.
8 9
*Corresponding author
RI PT
School of Agriculture and Veterinary Medicine, University of Brasilia, Brasília-DF,
Margot AlvesNunesDode
11
Researcher of Embrapa Genetic Resources and Biotechnology
12
E-mail: [email protected]
13
Address:
14
Parque Estação Biológica, Final Av. W5/N, Prédio PBI, 70770-900, Brasília- DF,
15
Brazil.
16
Tel.: +55 61 34484659
17
Fax: +55 61 3340 3658
18
E- mail address:
19
[email protected] (Dode, M. A. N.);
20
[email protected] (Guimarães, A. L. S.);
21
[email protected] (Pereira, S. A.)
22
[email protected] (Leme, L.O.)
25 26
M AN U
TE D
EP
AC C
23 24
SC
10
Abstract
The aim of this study was to test the SPOM-adapted system (simulated physiological oocyte maturation) during bovine oocyte maturation to improve
27
embryo development. Oocytes were obtained from follicles with a diameter of 3-8
28
mm that were aspirated from ovaries obtained from a slaughterhouse. To verify the
29
effect of the maturation system on in vitro embryo production, the cleavage,
30
blastocyst rates at D 7 and D 8, embryo size and total cell number were evaluated.
31
The resulting data on embryo development were analyzed by the Chi-Square test,
ACCEPTED MANUSCRIPT while data on embryo size and total cell number were analyzed by the Kruskal-
33
Wallis test. First, the SPOM system principle was tested in our IVM system, in
34
which 0.01 IU/ml of purified FSH (pFSH) and 10% of FCS were used during
35
maturation. However, the cleavage and blastocyst rates on D 7 and D 8 were
36
drastically reduced compared to those of the control group (P<0.05). Increasing the
37
dose of pFSH to 0.1 IU/ml in the SPOM-adapted system did not affect (P>0.05)
38
embryo production, which remained lower than that of the control. When less
39
competent oocytes obtained from 1-3 mm follicles were used, the SPOM-adapted
40
system was also unable to improve embryo production. To make the adapted system
41
as similar as possible to the reported system, recombinant FSH (FSHr) was
42
associated with BSA during maturation, and embryo culture was performed under
43
low oxygen tension conditions. Nevertheless, a reduction (P<0.05) in the blastocyst
44
rates was also observed, while the size and total cell number were similar to those of
45
the control group (P>0.05). It can be concluded that an SPOM-adapted system used
46
under different culture conditions does not improve in vitro embryo development.
48
SC
M AN U
TE D
Keywords: Cilostamide, maturation, SPOM, meiotic arrest.
EP
47
RI PT
32
1. Introduction
In assisted reproduction techniques (ARTs), oocytes used for in vitro maturation
50
(IVM) are obtained from follicles at different stages of development and therefore
51
constitute a heterogeneous population with different levels of competence [1]. When
52
these oocytes are removed from the follicular environment, they automatically resume
53
meiosis. Those that have not yet completed forming the cytoplasmic machinery to
54
support development are unable to become viable embryos [2]. Therefore, the
55
premature removal of oocytes from the follicles and their subsequent in vitro culture
AC C
49
ACCEPTED MANUSCRIPT 56
conditions are most likely the main factors responsible for the lower developmental
57
ability of oocytes that matured in vitro compared to oocytes that matured in vivo [3-5]. Although it seems difficult to influence the developmental response of oocytes
59
during their maturational period, changes in their basic maturation conditions have been
60
shown to improve oocyte competence, as reflected by the blastocyst yields after in vitro
61
fertilization (IVF) [6-9]. Because oocytes that are prematurely removed from their
62
follicles resume meiosis spontaneously via a mechanism different from that which
63
occurs in vivo, an alternative means of increasing the developmental potential of
64
oocytes in vitro would include simulating the events that occur in vivo during the
65
resumption of meiosis, as induced by the preovulatory LH surge.
M AN U
SC
RI PT
58
When LH reaches the follicle, it binds to its receptor on mural granulosa cells
67
and initiates signaling pathways involving cAMP and cGMP as second messengers [11-
68
13]. Initially, it induces an increase in the cAMP level, which stimulates the release of
69
EGF-like factor proteins [14], which ultimately will cause a decrease in intra-oocyte
70
cAMP levesl.
71
resulting in the degradation of cAMP [22]. The net reduction of cAMP within the
72
oocyte plays a critical role in the maintenance of meiotic arrest by activating the MPF
73
complex and thus promoting meiosis resumption from diplotene arrest [23, 24].
EP
LH also decreases cGMP and the subsequent activation of PDE 3A,
Instead, when oocytes are removed from their follicles, they undergo a rapid
AC C
74
TE D
66
75
decrease in their cAMP levels. This decrease leads immediately to the activation of
76
MPF and the automatic resumption of meiosis. Therefore, oocyte competence is
77
compromised, presumably due to an absence in the ovulatory cascade of events and a
78
reduced level of regulatory molecules and metabolites [25]. Aiming to improve the
79
developmental capacity of the oocytes used for IVM, a maturation system called SPOM
80
(Simulated physiological oocyte maturation) was recently proposed [6]. SPOM
ACCEPTED MANUSCRIPT simulates the physiological maturation that occurs in vivo and is based on the
82
physiological mechanisms of meiotic resumption. This system involves a short period
83
of pre-maturation, in the presence of adenylate cyclase (AC) activator, and an extended
84
maturation period, in which a phosphodiesterase inhibitors (PDE) and hormones to
85
overcome the effect of the inhibitor are used. The authors showed that the SPOM
86
system substantially improves oocyte developmental outcomes with increased
87
blastocyst rates and quality. Although the efficiency of the SPOM system has not yet
88
been confirmed by other laboratories due to its physiological approach, it represents an
89
interesting alternative to improve the competence and embryo production of bovine
90
oocytes. Here, we attempted to use the principles of the SPOM system under different
91
conditions to promote the in vitro maturation of bovine oocytes to improve embryonic
92
development.
M AN U
SC
RI PT
81
93
2. Materials and methods
TE D
94
Unless otherwise indicated, the reagents used in this research and any chemicals
96
used in the preparation of the maturation, fertilization and in vitro culture media were
97
purchased from Sigma (St. Louis, MO, USA).
99 100
AC C
98
EP
95
2.1 Oocyte recovery
Ovaries from crossbred females (Bos indicus X Bos taurus) were collected from
101
local abattoirs immediately after slaughter and transported in a solution of saline
102
(0.9%NaCl) supplemented with antibiotics (streptomycin -and100µg/mL penicillin G-
103
100IU/mL) at a temperature of 35-36°C. Cumulus-oocyte complexes (COCs) were
104
aspirated from follicles of 3-8 mm in diameter using an 18 gauge needle connected to a
105
vacuum system.
ACCEPTED MANUSCRIPT The recovery of oocytes from follicles of 1-3 mm in diameter was performed as
107
described previously [26]. Follicles were dissected from the ovarian cortex using
108
tweezers, scissors and a scalpel and were kept in TCM-199 (Invitrogen®, Carlsbad, CA,
109
USA) supplemented with 10% fetal calf serum (FCS; Invitrogen) and 0.075 mg/ mL
110
amikacin during the entire process. Once dissected, the follicles were measured using a
111
graduated ocular (eyepiece micrometerOSM-4 ®Olympus, Tokyo, Japan). To release
112
COCs, the follicles were ruptured; only those oocytes with at least four layers of
113
cumulus cells and a homogeneous cytoplasm were used for further work.
SC
RI PT
106
114
2.2 In vitro maturation (IVM)
116
Immediately after selection, the COCs were transferred to a basic maturation medium
117
consisting of TCM-199 supplemented with 10% FCS, 0.01 IU/mL purified FSH
118
(pFSH), 0.1 mg/mL glutamine and an antibiotic (0.075 mg/ mL amikacin). The COCS
119
were then matured for 22 hours at 39 ° C in 5 % CO2 in air.
TE D
120
M AN U
115
2.3 In vitro fertilization (IVF) and embryo culture (IVC)
122
Following maturation, groups of 25-30 COCs were transferred to 200-µL drops of
123
fertilization medium, which consisted of TALP [27] supplemented with penicillamine
124
(2 mM), hypotaurine (1 mM), epinephrine (250 mM) and heparin (10µg/mL−1). Frozen
125
semen from a bull of proven fertility that had been used for several years as the
126
reference bull for in vitro embryo production in our laboratory was used for all
127
treatments and replicates. Motile spermatozoa were obtained using the Percoll (GE®
128
Healthcare, Piscataway, NJ, USA) gradient method in microtubes [28] and were added
129
into the fertilization drop at a final concentration of 1×106 spermatozoa mL−1.
130
Spermatozoa and oocytes were co-incubated for 18 h at 39°C with 5% CO2 in air. The
131
day of in vitro insemination was considered to be day 0.
AC C
EP
121
ACCEPTED MANUSCRIPT Eighteen hours post insemination (pi), the presumptive zygotes were washed and
133
transferred to 200-µmL drops of synthetic oviduct fluid medium with amino acids,
134
citrate and inositol (SOFaaci [29]) supplemented with 5% FCS and incubated at 39 ◦C
135
in an atmosphere of 5% CO in air for 7 or 8 days. Embryos were evaluated on D 2 for
136
cleavage and on D 6, D 7 and D 8 for blastocyst rates.
137 138
2. Cell number
RI PT
132
On D 8, blastocysts were measured with a Motic camera (Moticam®Plus2.0,
140
Japan) and classified according to their diameter into three categories: 120-140, 140-160
141
and >160µm. Embryos with a diameter >160µm were used to evaluate the number of
142
cells. Embryos were exposed to Hoechst 33342 dye at a concentration of 1µg/mL for 5
143
minutes and were subsequently transferred to a slide and covered with a cover slip.
144
Slides were evaluated using an epifluorescence microscope (Zeiss Axiophot®,
145
Germanyfilter24 with a wavelength of 494/518 nm (excitation/emission), and the cell
146
nuclei were counted.
M AN U
TE D
147
SC
139
2.5 Experimental Design
149
2.5.1 Experiment 1: Evaluation of the SPOM-adapted system to a standard IVM system
150
to improve the developmental competence of oocytes
AC C
151
EP
148
In this experiment, we evaluated the SPOM principles as adapted to the IVM
152
system used in our laboratory. Initially 871 COCs were divided into two groups (control
153
and SPOM) in a total of seven replicate experiments. In the control group, COCs were
154
matured in basic medium for 22 hours. In the SPOM group, the oocytes were submitted
155
to the protocol described by Albuz et al. [6] with modifications. In this group,
156
immediately after selection, the oocytes were washed, transferred to prematuration
ACCEPTED MANUSCRIPT medium and cultured for a period of 2 hours at 39°C in 5% CO2 in air. Prematuration
158
medium consisted of TCM-199 with Earle's salts supplemented with 10% FCS, 0,075
159
mg/ ml of amikacin, 500µM of 3-Isobutil1–methylxanthine (IBMX), a non-specific
160
inhibitor of phosphodiesterase, and 100 µM of forskolin (FSK), an activator of
161
adenylate cyclase. After prematuration, the COCs were washed and transferred to a
162
200µL drop of maturation medium covered with silicone oil. The maturation medium
163
was the basal medium, to which 20µM of cilostamide, a potent inhibitor of type 3
164
phosphodiesterase (PDE 3A), was added. COCs were incubated at 39° C in 5% CO2 in
165
air for a period of 28 hours (extended maturation). After maturation, oocytes from both
166
groups were fertilized and cultured in vitro. On D 2, the cleavage rate of the embryos
167
was evaluated, and on D 7 and D 8, the blastocyst rate was evaluated.
M AN U
SC
RI PT
157
All the inhibitor used in the experiment FSK, IBMX and cilostamide were
169
prepared at 100X stock solution in dimethylsulfoxide (DMSO) to final concentrations of
170
100µM, 500 µM and 20 µM, respectively, and aliquoted in microtubes and stored at -20
171
° C until use The stock solutions were diluted fresh for each replica of experiments 1, 2
172
and 3.
173
2.5.2 Experiment 2: Evaluation of the SPOM-adapted system for the maturation of
174
bovine oocytes with different levels of competency
EP
Because in the previous experiment the SPOM-adapted system had a detrimental
AC C
175
TE D
168
176
effect on oocytes, we suspected that the FSH concentration may have been too low to
177
overcome the cilostamide inhibition. We therefore increased the concentration of the
178
pFSH in the maturation medium from 0.01IU/ mL to 0.1 IU/ML
179
This experiment aimed to evaluate if the SPOM-adapted system would improve
180
the developmental competence of less competent oocytes obtained from small follicles.
181
A total of 1040 were used in four experimental groups (G), G1) the 3-8 mm control
ACCEPTED MANUSCRIPT group, consisting of oocytes aspirated from follicles of 3-8 mm and matured in a
183
standard IVM system, G2) the 3-8 mm SPOM group, consisting of oocytes aspirated
184
from follicles of 3-8 mm and submitted to the SPOM-adapted system, G3) the 1-3 mm
185
control group, consisting of less competent oocytes obtained from follicles of 1-3 mm
186
and matured in a standard IVM system and G4) the 1-3 mm SPOM group, consisting of
187
less competent oocytes obtained from follicles of 1-3 mm and submitted to the SPOM-
188
adapted system. The maturation and SPOM system were the same as described in the
189
previous experiment, and the FSH concentration in the SPOM group was the only
190
difference between the two sets of conditions. After IVM, all groups underwent in vitro
191
fertilization and culture. Embryonic development was evaluated at D 2, D 7 and D 8.
M AN U
SC
RI PT
182
192 193
2.5.3 Experiment 3: Effects of the SPOM-adapted system under different culture
194
conditions on oocyte developmental competence.
In this experiment we tried to follow the SPOM protocol for oocyte maturation
196
described by Albuz et al. [6] as closely as possible. We tested the SPOM using
197
recombinant FSH [rFSH (Gonal-F®, Merck Serono, USA)] at a concentration of 0.1
198
UI/mL in the presence of BSA-FAF. A total of 452 oocytes were distributed into five
199
different culture conditions: G1) the control condition, in which oocytes underwent
200
standard IVM, IVF and IVC, in the presence of FCS and 5% CO2 in air (high O2); G2)
201
the SPOM-rFSH-BSA condition, with prematuration in BSA + FSK + IBMX,
202
maturation in rFSH + BSA + cilostamide and embryo culture in BSA under 5% O2 (low
203
O2); G3) the SPOM-pFSH-BSA condition, with prematuration in BSA + FSK + IBMX,
204
maturation in pFSH + BSA + cilostamide, embryo culture in BSA with 5% O2 (low O2);
205
G4) the SPOM-pFSH-FCS condition, with prematuration in BSA + FSK + IBMX,
206
maturation in pFSH + FCS + cilostamide and embryo culture in FCS under 5% CO2
AC C
EP
TE D
195
ACCEPTED MANUSCRIPT (high O2); and G5) the SPOM-rFSH-FCS FCS condition, with prematuration in BSA +
208
FSK + IBMX, maturation in rFSH + FCS + cilostamide and embryo culture with FCS
209
under 5% CO2 (low O2). After IVM, oocytes from all groups were fertilized and
210
cultured in vitro. Embryo development was evaluated at D 2, D 7 and D 8. D 8
211
blastocysts were measured, and those greater than 160µm were stained to determine the
212
total cell number.
RI PT
207
213
2.6 Statistical Analysis
SC
214
Data of embryo development rates were analyzed using the Chi-square test
216
(P<0.05). The embryo measurements and the cell number data were compared with the
217
Kruskal-Wallis test, using version 5.0 of the Prophet program (BBN Systems and
218
Technologies, 1996). 3. Results
TE D
219
M AN U
215
220
3.1 Experiment 1: Evaluation of the SPOM-adapted system to a standard IVM system to
221
improve oocyte developmental competence
In this experiment, the SPOM principle (prematuration for two hours in the
223
presence of forskolin and IBMX and prolonged maturation in presence of cilostamide)
224
was introduced in our protocol without any changes to our media composition. The
225
results showed that not only the cleavage but also the blastocyst rates at D 7 and D 8
226
were lower (P>0.05) in oocytes submitted to the SPOM-adapted system compared to
227
those submitted to the control system (Table 1).
228
3.2 Experiment 2: Evaluation of the SPOM-adapted system for the maturation of
229
oocytes with different levels of competency
AC C
EP
222
ACCEPTED MANUSCRIPT The cleavage (D 2) and blastocyst rates (D 7 and D 8) were higher in the control
231
group than in the other groups (Table 2). As in the previous experiment, the SPOM-
232
adapted system produced a deleterious effect (P<0.05) on embryonic development when
233
oocytes obtained from 3-8 mm follicles were used. For the less competent oocytes, the
234
use of the SPOM-adapted system was not able to improve their embryonic development
235
(Table 2).
236
3.3 Experiment 3: Effect of the SPOM-adapted system under different culture
237
conditions on oocyte developmental competence.
SC
M AN U
238
RI PT
230
The results obtained for the group submitted to the SPOM- adapted system used
240
in experiments 1 and 2 (pFSH, FCS and high O2 tension during embryo culture) were
241
consistent with those of previous experiments, showing lower cleavage and blastocyst
242
rates than the control group. However, when those same conditions were used but the
243
pFSH was replaced by rFSH, a further reduction in embryo development was observed
244
(Table 3). When the SPOM-adapted system, in which FCS was replaced by BSA,the
245
embryo culture was performed under low O2 tension and SOF was supplemented with
246
BSA and not FCS, the observed cleavage rates were similar to those of the control
247
group (Table 3). Although the blastocyst rates remained lower than those of the control
248
group, the size and total cell number of the embryos produced were similar to the size
249
and cell number of the control embryos (Table 4). In both conditions, the use of rFSH
250
affected embryo production and quality (Table 3 and 4).
AC C
EP
TE D
239
251
252
4. Discussion
ACCEPTED MANUSCRIPT The main factor determining the blastocyst rates following in vitro embryo
254
production techniques is the quality of the oocytes. Immature oocytes used for this
255
purpose usually have not acquired full developmental competence due to their
256
premature removal from the follicular environment. Thus, it is necessary to provide
257
appropriate conditions to allow oocytes to develop into viable embryos. When Albuz et
258
al. [6] proposed the SPOM system in an attempt to simulate the events that occur during
259
in vivo maturation, a new promising alternative emerged. This system involves two
260
steps. First, a short exposure to cAMP modulating agents causes a rapid increase in
261
intra-oocyte cAMP levels and mimics the action of LH in vivo. Subsequently, oocytes
262
are subjected to a prolonged IVM in the presence of cilostamide, an inhibitor of PDE
263
3A. This step retains meiosis, which is associated with FSH levels at a concentration
264
sufficient to overcome the effects of the inhibitor and to induce maturation.
M AN U
SC
RI PT
253
We attempted to adapt the SPOM principles to our standard IVM system, which
266
is similar to the system used in the majority of IVP laboratories. However, we found no
267
improvement in embryo production. On the contrary, we observed a substantial
268
decrease in blastocyst rates. Because we included SPOM principles but kept our
269
maturation media composition, we predicted that the negative effect of SPOM could be
270
due to the FSH concentrations. In the SPOM system, the oocytes do not resume meiosis
271
spontaneously, and a relatively high dose of FSH is needed to overcome the inhibitory
272
effect of cilostamide [6, 22]. In our IVM system, the concentration of FSH used was 10
273
times lower than the dose used in the SPOM protocol. In addition, we used purified
274
FSH, which is prepared by the extraction of the pituitary, which can lead to some
275
contamination and lower activity than the recombinant FSH used by Albuz et al. [6].
276
Therefore, it is possible that the FSH concentration in the media was not sufficient to
277
overcome the inhibitory effect of cilostamide, and many oocytes might not have
AC C
EP
TE D
265
ACCEPTED MANUSCRIPT 278
matured properly, thus leading to the low embryo development observed as early as the
279
cleavage state. In an attempt to overcome the presumptive low activity of FSH, we
280
increased the concentration of FSH in the second experiment by 10-fold. Albuz et al.[6] hypothesized that the SPOM system is able to improve the
282
developmental competence of oocytes from a heterogeneous population of bovine
283
ovaries, such as those from small antral follicles. Therefore, in the second experiment,
284
we also evaluated whether such a system could increase the quality of less competent
285
oocytes obtained from 1-3 mm dissected follicles. The increase in FSH concentration
286
did not affect the embryo production, nor did the SPOM system improve the quality of
287
the less competent oocytes. Similar results were reported for oocytes obtained from pre-
288
pubertal ewes, which are supposedly less competent. In these oocytes, no increases in
289
embryos production were observed when the SPOM system was used [22]. The lack of
290
data on the SPOM system in the literature makes it difficult to place our results in
291
context. There is no clear explanation for the discrepancy between our results and those
292
reported by Albuz et al.[6] in the bovine system, but the discrepancy may be due to
293
differences in the media, culture conditions and even the sub-species (Indicus x Taurus)
294
used in the two studies.
EP
TE D
M AN U
SC
RI PT
281
Finally, we changed the culture conditions to match our system as closely as
296
possible to that proposed by Albuz et al. [6]. The pFSH was replaced by rFSH, and the
297
FCS was replaced by BSA. Moreover, the O2 tension was decreased to 5% during the
298
embryo culture period. Nevertheless, we observed no positive effect of the SPOM-
299
adapted system on embryo production regardless of the culture conditions used.
300
However, it became very clear that the detrimental effect was minimized when low O2
301
tension was used during embryo culture in association with BSA, regardless of the FSH
302
source. The best results were observed when pFSH was used instead of rFSH. Although
AC C
295
ACCEPTED MANUSCRIPT 303
embryo production was lower when the SPOM system was used with BSA, the quality
304
of the embryos, as indicated by their size and the total cell number, was similar to that
305
of the control group. In contrast, the use of the adapted-SPOM system, in which FCS was present
307
during the maturation and culture, showed the worst results. Both the yield and quality
308
of the embryos were inferior to the control and the adapted-SPOM system with the BSA
309
groups. The possibility that the presence of FCS may have led an acceleration of
310
meiosis causing a premature aging of oocytes and, subsequently a reduced embryo
311
production cannot be ruled out. As mentioned earlier, there is a lack of studies using the
312
SPOM principle for the maturation of oocytes; however, some preliminary studies using
313
bovine oocytes have also reported a lower blastocyst rate in the adapted-SPOM than in
314
the control group [30].
M AN U
SC
RI PT
306
Interestingly, in the treatments in which rFSH was used, embryo production was
316
lower than in the groups to which pFSH was added during maturation, regardless of the
317
culture conditions. We have no explanation for this result. Studies in which the use of
318
rFSH and pFSH during IVM were compared have shown that embryo production was
319
similar between them. However, other studies demonstrated that the positive effect of
320
rFSH could only be detected when it was associated with other hormones [31]. Those
321
authors observed that the developmental competence of oocytes matured with r-FSH
322
and E2 was higher than that of oocytes matured with r-FSH alone, indicating a
323
synergistic effect between E2 and FSH. Moreover, no additive effect between E2 and
324
purified pFSH was observed, which the authors did not expect. This lack of effect might
325
be due to a differential effect of the recombinant product caused by a different
326
glycosylation patterns or by a contaminant to the purified pituitary FSH preparation.
327
Other studies on bovine oocytes [32], comparing rFSH and rLH addition and
AC C
EP
TE D
315
ACCEPTED MANUSCRIPT combinations of the two, noted that embryonic development was higher when oocytes
329
were matured with rFSH and rLH than with either rFSH or rLH alone. Those results
330
suggested that the effects of rFSH can change according to the presence of other factors
331
in the medium. In a study using ovine oocytes, in which the effect of rFSH on in vitro
332
maturation was evaluated, it was found that embryo development and viability were
333
lower when rFSH was used than when a commercial FSH obtained from the bovine
334
pituitary was used [33]. Curiously, the authors used the same r-FSH (Gonal-F) at the
335
same concentration (0.1 UI/mL) that we used and also had better results with the
336
pituitary FSH. It is well known that hypophysial gonadotrophins, unlike recombinant
337
hormones, can contain contaminants (i.e., growth factors) that can improve the
338
blastocyst yield [34, 35]. Another hypothesis that may explain the low levels of embryo
339
production when rFSH was used is that the required levels of this gonadotropin may
340
differ between species. Studies conducted with Brahman and Angus cows observed that
341
plasma concentrations of FSH were lower in Bos indicus than in Bos taurus, indicating
342
that Zebu cows may be more sensitive to the exposure of that gonadotropin [36, 37].
343
Although we are not sure whether this is the case, studies investigating the effects of
344
different concentrations of rFSH should be performed.
SC
M AN U
TE D
EP
Regardless the changes we made in the system, our data showed that the SPOM-
AC C
345
RI PT
328
346
adapted-system did not improve the production or quality of bovine embryos.
347
Acknowledgements
348
The authors thank EMBRAPA and CAPES for their financial support and the Qualimax
349
(Luziania-GO) slaughterhouse for providing the necessary biological materials for this
350
experiment.
351
Reference
ACCEPTED MANUSCRIPT [1] Jee BC, Chen HY, Chian RC. Effect of a phosphodiesterase type 3 inhibitor in
353
oocyte maturation medium on subsequent mouse embryo development. Fertility and
354
sterility. 2009;91:2037-42.
355
[2] Sirard MA, Richard F, Blondin P, Robert C. Contribution of the oocyte to embryo
356
quality. Theriogenology. 2006;65:126-36.
357
[3] van de Leemput E, Vos P, Zeinstra E, Bevers M, van der Weijden G, Dieleman S.
358
Improved in vitro embryo development using in vivo matured oocytes from heifers
359
superovulated with a controlled preovulatory LH surge. Theriogenology. 1999;52:335 -
360
49.
361
[4] Rizos D, Ward F, Duffy P, Boland M, Lonergan P. Consequences of bovine oocyte
362
maturation, fertilization or early embryo development in vitro versus in vivo:
363
implications for blastocyst yield and blastocyst quality. Mol Reprod Dev. 2002;61:234 -
364
48.
365
[5] Tesfaye D, Ghanem N, Carter F, Fair T, Sirard M, Hoelker M, et al. Gene
366
expression profile of cumulus cells derived from cumulus–oocyte complexes matured
367
either in vivo or in vitro. Reproduction, Fertility and Development. 2009;21:451-61.
368
[6] Albuz FK, Sasseville M, Lane M, Armstrong DT, Thompson JG, Gilchrist RB.
369
Simulated physiological oocyte maturation (SPOM): a novel in vitro maturation system
370
that substantially improves embryo yield and pregnancy outcomes. Human
371
reproduction. 2010;25:2999-3011.
372
[7] Luciano AM, Franciosi F, Modina SC, Lodde V. Gap junction-mediated
373
communications regulate chromatin remodeling during bovine oocyte growth and
374
differentiation through cAMP-dependent mechanism(s). Biology of reproduction.
375
2011;85:1252-9.
AC C
EP
TE D
M AN U
SC
RI PT
352
ACCEPTED MANUSCRIPT [8] Opiela J, Romanek J, Lipi, #x144, ski D, Smor, et al. Effect of Hyaluronan on
377
Developmental Competence and Quality of Oocytes and Obtained Blastocysts from In
378
Vitro Maturation of Bovine Oocytes. BioMed Research International. 2014;2014:8.
379
[9] Wang F, Tian X, Zhang L, He C, Ji P, Li Y, et al. Beneficial effect of resveratrol on
380
bovine oocyte maturation and subsequent embryonic development after in vitro
381
fertilization. Fertility and sterility. 2014;101:577-86.
382
[10] Shimada M, Ito J, Yamashita Y, Okazaki T, Isobe N. Phosphatidylinositol 3-kinase
383
in cumulus cells is responsible for both suppression of spontaneous maturation and
384
induction of gonadotropin-stimulated maturation of porcine oocytes. J Endocrinol.
385
2003;179:25 - 34.
386
[11] Tripathi A, Kumar KV, Chaube SK. Meiotic cell cycle arrest in mammalian
387
oocytes. Journal of cellular physiology. 2010;223:592-600.
388
[12] Shimada M, Nishibori M, Isobe N, Kawano N, Terada T. Luteinizing hormone
389
receptor formation in cumulus cells surrounding porcine oocytes and its role during
390
meiotic maturation of porcine oocytes. Biology of reproduction. 2003;68:1142-9.
391
[13] Shu YM, Zeng HT, Ren Z, Zhuang GL, Liang XY, Shen HW, et al. Effects of
392
cilostamide and forskolin on the meiotic resumption and embryonic development of
393
immature human oocytes. Human reproduction. 2008;23:504-13.
394
[14] Norris RP, Ratzan WJ, Freudzon M, Mehlmann LM, Krall J, Movsesian MA, et al.
395
Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in
396
the mouse oocyte. Development. 2009;136:1869-78.
397
[15] Assidi M, Sirard M-A. Oogenesis. Cumulus Cell Gene Expression as a Marker of
398
Oocyte Quality. 2013:231 - 52.
AC C
EP
TE D
M AN U
SC
RI PT
376
ACCEPTED MANUSCRIPT [16] Sela-Abramovich S, Chorev E, Galiani D, Dekel N. Mitogen-activated protein
400
kinase mediates luteinizing hormone-induced breakdown of communication and oocyte
401
maturation in rat ovarian follicles. Endocrinology. 2005;146:1236-44.
402
[17] Su YQ, Sugiura K, Eppig JJ. Mouse oocyte control of granulosa cell development
403
and function: paracrine regulation of cumulus cell metabolism. Seminars in
404
reproductive medicine. 2009;27:32-42.
405
[18] Dekel N. Regulation of Oocyte Maturation. Annals of the New York Academy of
406
Sciences. 1988;541:211-6.
407
[19] Zhang M, Ouyang H, Xia G. The signal pathway of gonadotrophins-induced
408
mammalian oocyte meiotic resumption. Molecular human reproduction. 2009;15:399-
409
409.
410
[20] Bu S, Xie H, Tao Y, Wang J, Xia G. Nitric oxide influences the maturation of
411
cumulus cell-enclosed mouse oocytes cultured in spontaneous maturation medium and
412
hypoxanthine-supplemented medium through different signaling pathways. Molecular
413
and cellular endocrinology. 2004;223:85-93.
414
[21] Zhang M, Tao Y, Xia G, Xie H, Hong H, Wang F, et al. Atrial natriuretic peptide
415
negatively regulates follicle-stimulating hormone-induced porcine oocyte maturation
416
and cumulus expansion via cGMP-dependent protein kinase pathway. Theriogenology.
417
2005;64:902-16.
418
[22] Rose RD, Gilchrist RB, Kelly JM, Thompson JG, Sutton-McDowall ML.
419
Regulation of sheep oocyte maturation using cAMP modulators. Theriogenology.
420
2013;79:142-8.
421
[23] Duckworth BC, Weaver JS, Ruderman JV. G2 arrest in Xenopus oocytes depends
422
on phosphorylation of cdc25 by protein kinase A. Proceedings of the National Academy
423
of Sciences of the United States of America. 2002;99:16794-9.
AC C
EP
TE D
M AN U
SC
RI PT
399
ACCEPTED MANUSCRIPT [24] Han SJ, Conti M. New Pathways from PKA to the Cdc2/cyclin B Complex in
425
Oocytes: Wee1B as a Potential PKA Substrate. Cell Cycle. 2006;5:227-31.
426
[25] Thomas RE, Thompson JG, Armstrong DT, Gilchrist RB. Effect of specific
427
phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on
428
meiotic and developmental capacity. Biology of reproduction. 2004;71:1142-9.
429
[26] Caixeta E, Ripamonte P, Franco M, Junior J, Dode M. Effect of follicle size on
430
mRNA expression in cumulus cells and oocytes of Bos indicus: an approach to identify
431
marker genes for developmental competence. Reprod Fertil Dev. 2009;21:655 - 64.
432
[27] Parrish JJ, Krogenaes A, Susko-Parrish JL. Effect of bovine sperm separation by
433
either swim-up or Percoll method on success of in vitro fertilization and early
434
embryonic development. Theriogenology. 1995;44:859-69.
435
[28] Machado GM, Carvalho JO, Filho ES, Caixeta ES, Franco MM, Rumpf R, et al.
436
Effect of Percoll volume, duration and force of centrifugation, on in vitro production
437
and sex ratio of bovine embryos. Theriogenology. 2009;71:1289-97.
438
[29] Holm P, Shukri NN, Vajta G, Booth P, Bendixen C, Callesen H. Developmental
439
kinetics of the first cell cycles of bovine in vitro produced embryos in relation to their in
440
vitro viability and sex. Theriogenology. 1998;50:1285-99.
441
[30] Ulloa SM, Heinzmann J, Herrmann D, Timmermann B, Baulain U, Grossfeld R, et
442
al. Effects of different oocyte retrieval and in vitro maturation systems on bovine
443
embryo development and quality. Zygote. 2014:1-11.
444
[31] Ali A, Sirard M. The effects of 17beta-estradiol and protein supplement on the
445
response to purified and recombinant follicle stimulating hormone in bovine oocytes.
446
Zygote. 2002;10:65 - 71.
447
[32] Anderiesz C, Ferraretti A, Magli C, Fiorentino A, Fortini D, Gianaroli L, et al.
448
Effect of recombinant human gonadotrophins on human, bovine and murine oocyte
AC C
EP
TE D
M AN U
SC
RI PT
424
ACCEPTED MANUSCRIPT meiosis, fertilization and embryonic development in vitro. Hum Reprod. 2000;15:1140 -
450
8.
451
[33] Accardo C, Dattena M, Pilichi S, Mara L, Chessa B, Cappai P. Effect of
452
recombinant human FSH and LH on in vitro maturation of sheep oocytes; embryo
453
development and viability. Animal Reproduction Science. 2004;81:77-86.
454
[34] Lonergan P, Carolan C, Van Langendonckt A, Donnay I, Khatir H, Mermillod P.
455
Role of epidermal growth factor in bovine oocyte maturation and preimplantation
456
embryo development in vitro. Biology of Reproduction. 1996;54:1420-9.
457
[35] Guler A, Poulin N, Mermillod P, Terqui M, Cognié Y. Effect of growth factors,
458
EGF and IGF-I, and estradiol on in vitro maturation of sheep oocytes. Theriogenology.
459
2000;54:209-18.
460
[36] Calder M, Caveney A, Westhusin M, Watson A. Cyclooxygenase-2 and
461
prostaglandin E2 receptor messenger RNAs are affected by bovine oocyte maturation
462
time and cumulus-oocyte complex quality, and PGE2 induces moderate expansion of
463
the bovine cumulus in vitro. Biol Reprod. 2001;65:135 - 40.
464
[37] Alvarez P, Spicer LJ, Chase CC, Payton ME, Hamilton TD, Stewart RE, et al.
465
Ovarian and endocrine characteristics during an estrous cycle in Angus, Brahman, and
466
Senepol cows in a subtropical environment. Journal of Animal Science. 2000;78:1291-
467
302.
469
SC
M AN U
TE D
EP
AC C
468
RI PT
449
ACCEPTED MANUSCRIPT
standard maturation system (Control).
Control
Oocyte number
Cleavage D 2 (%)
Blastocysts D 7 (%)
310
243 (78.4)a
142 (45.8)a
SC
Treatment
RI PT
Table 1. Embryonic development of bovine oocytes matured on an adapted system that simulates physiological oocyte maturation (SPOM) and a
AC C
EP
TE D
M AN U
SPOM 561 297 (52.9)b 69 (12.3)b a, b Different letters in the same column indicate significant difference by χ2 (P < 0.05).
Blastocysts D 8 (%) 155 (50.0)a 81 (14.4)b
ACCEPTED MANUSCRIPT
RI PT
Table 2. Embryonic development of more (3-8 mm) or less (1-3 mm) competent bovine oocytes matured on either an adapted system that simulates physiological oocyte maturation (SPOM) or a standard maturation system (Control). Treatment
Oocyte number
Cleavage D 2 (%) a
Blastocysts D 7 (%) a
Blastocysts D 8 (%) 297 (49.7)a
598
491 (82.1)
271 (45.3)
Spom 3-8 mm
321
175 (54.5)b
38 (11.8)b
38 (11.8)b
Control 1-3 mm
54
32 (59.3)b
11(20.4)b
11 (20.4)b
Spom 1-3 mm 67 34 (50.7)b 9 (13.4)b a, b Different letters in the same column indicate significant difference by χ2 (P < 0.05).
9 (13.4)b
AC C
EP
TE D
M AN U
SC
Control 3-8mm
ACCEPTED MANUSCRIPT
Table 3. Embryonic development of bovine oocytes matured on either an adapted system that simulates physiological oocyte maturation (SPOM)
RI PT
under different conditions using serum (FCS) or albumin (BSA), and purified (pFSH) or recombinant FSH (rFSH) during maturation and low O2 tension and BSA (↓ O2) or high O2 tension and FCS (↑ O2) during embryo culture, compared to a standard maturation system (Control). Cleavage D 2 (%)
Blastocysts D 7 (%)
Control
89
69 (77.5)a
29 (32.6)a
29 (32.6)a
Spom+rFSH+BSA↓O2
91
64 (70.3)a
17 (18.7)b
17 (18.7)b
Spom+pFSH+BSA↓O2
89
60 (67.4)a
20 (22.5)a, b
20 (22.5)a, b
Spom +pFSH+FCS↑O2
90
49 (54.5)b
6 (6.6)c
6 (6.6)c
M AN U
SC
Oocyte Number
Treatment
AC C
EP
TE D
Spom+rFSH+FCS↑O2 93 35 (37.7)c 2 (2.2)c a, b, c Different letters in the same column indicate significant difference by χ2 (P < 0,05).
Blastocysts D 8 (%)
2 (2.2)c
ACCEPTED MANUSCRIPT
RI PT
Table 4. Percentage, size and total cell number of D 8 embryos with diameters > 160 µm obtained from oocytes oocytes matured on either an adapted system that simulates physiological oocyte maturation (SPOM) under different conditions using serum (FCS) or albumin (BSA), and
Control SPOM+rFSH+BSA↓O2 SPOM+pFSH+BSA↓O2 SPOM+pFSH+FCS↑O2
Total embryos 29 20 17 6
Cell number of embryos > 160µm (mean±sd)
Size (µm) of Embryo > 160 µm (mean±sd)
150.8±26.7ª 139.6±35.6ª 137.9±51.6ª 125.0±41.0b
210.3±47.0a 210.1±57.6ª 218.5±56.5ª 163.4±63.9b
TE D
Treatment
M AN U
embryo culture, compared to a standard maturation system (Control).
SC
purified (pFSH) or recombinant FSH (rFSH) during maturation and low O2 tension and BSA (↓ O2) or high O2 tension and FCS (↑ O2) during
AC C
EP
SPOM+rFSH+ FCS↑O2 2 96.6±7.7c 171.0±41.0c a, b, c Different letters in the same column indicate significant difference (P < 0.05).
S0093-691X(14)00522-6
DOI:
10.1016/j.theriogenology.2014.07.042
Reference:
THE 12940
To appear in:
Theriogenology
Received Date: 29 April 2014 Revised Date:
20 June 2014
Accepted Date: 18 July 2014
Please cite this article as: Guimarães ALS, Pereira SA, Leme LO, Dode MAN, Evaluation of the simulated physiological oocyte maturation system (SPOM) for improving bovine in vitro embryo production, Theriogenology (2014), doi: 10.1016/j.theriogenology.2014.07.042. 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 1
REVISED
2 3
Evaluation of the simulated physiological oocyte maturation system (SPOM) for improving bovine in vitro embryo production
4
Guimarães, A. L. S.1; Pereira, S.A.2; Leme, L. O.1; Dode, M. A. N.1, 3*
5
1
6
Brazil; 2 Institute of Biology, University of Brasília, Brasília-DF, Brazil, 3Embrapa-
7
Genetic Resources and Biotechnology, Brasília-DF, Brazil.
8 9
*Corresponding author
RI PT
School of Agriculture and Veterinary Medicine, University of Brasilia, Brasília-DF,
Margot AlvesNunesDode
11
Researcher of Embrapa Genetic Resources and Biotechnology
12
E-mail: [email protected]
13
Address:
14
Parque Estação Biológica, Final Av. W5/N, Prédio PBI, 70770-900, Brasília- DF,
15
Brazil.
16
Tel.: +55 61 34484659
17
Fax: +55 61 3340 3658
18
E- mail address:
19
[email protected] (Dode, M. A. N.);
20
[email protected] (Guimarães, A. L. S.);
21
[email protected] (Pereira, S. A.)
22
[email protected] (Leme, L.O.)
25 26
M AN U
TE D
EP
AC C
23 24
SC
10
Abstract
The aim of this study was to test the SPOM-adapted system (simulated physiological oocyte maturation) during bovine oocyte maturation to improve
27
embryo development. Oocytes were obtained from follicles with a diameter of 3-8
28
mm that were aspirated from ovaries obtained from a slaughterhouse. To verify the
29
effect of the maturation system on in vitro embryo production, the cleavage,
30
blastocyst rates at D 7 and D 8, embryo size and total cell number were evaluated.
31
The resulting data on embryo development were analyzed by the Chi-Square test,
ACCEPTED MANUSCRIPT while data on embryo size and total cell number were analyzed by the Kruskal-
33
Wallis test. First, the SPOM system principle was tested in our IVM system, in
34
which 0.01 IU/ml of purified FSH (pFSH) and 10% of FCS were used during
35
maturation. However, the cleavage and blastocyst rates on D 7 and D 8 were
36
drastically reduced compared to those of the control group (P<0.05). Increasing the
37
dose of pFSH to 0.1 IU/ml in the SPOM-adapted system did not affect (P>0.05)
38
embryo production, which remained lower than that of the control. When less
39
competent oocytes obtained from 1-3 mm follicles were used, the SPOM-adapted
40
system was also unable to improve embryo production. To make the adapted system
41
as similar as possible to the reported system, recombinant FSH (FSHr) was
42
associated with BSA during maturation, and embryo culture was performed under
43
low oxygen tension conditions. Nevertheless, a reduction (P<0.05) in the blastocyst
44
rates was also observed, while the size and total cell number were similar to those of
45
the control group (P>0.05). It can be concluded that an SPOM-adapted system used
46
under different culture conditions does not improve in vitro embryo development.
48
SC
M AN U
TE D
Keywords: Cilostamide, maturation, SPOM, meiotic arrest.
EP
47
RI PT
32
1. Introduction
In assisted reproduction techniques (ARTs), oocytes used for in vitro maturation
50
(IVM) are obtained from follicles at different stages of development and therefore
51
constitute a heterogeneous population with different levels of competence [1]. When
52
these oocytes are removed from the follicular environment, they automatically resume
53
meiosis. Those that have not yet completed forming the cytoplasmic machinery to
54
support development are unable to become viable embryos [2]. Therefore, the
55
premature removal of oocytes from the follicles and their subsequent in vitro culture
AC C
49
ACCEPTED MANUSCRIPT 56
conditions are most likely the main factors responsible for the lower developmental
57
ability of oocytes that matured in vitro compared to oocytes that matured in vivo [3-5]. Although it seems difficult to influence the developmental response of oocytes
59
during their maturational period, changes in their basic maturation conditions have been
60
shown to improve oocyte competence, as reflected by the blastocyst yields after in vitro
61
fertilization (IVF) [6-9]. Because oocytes that are prematurely removed from their
62
follicles resume meiosis spontaneously via a mechanism different from that which
63
occurs in vivo, an alternative means of increasing the developmental potential of
64
oocytes in vitro would include simulating the events that occur in vivo during the
65
resumption of meiosis, as induced by the preovulatory LH surge.
M AN U
SC
RI PT
58
When LH reaches the follicle, it binds to its receptor on mural granulosa cells
67
and initiates signaling pathways involving cAMP and cGMP as second messengers [11-
68
13]. Initially, it induces an increase in the cAMP level, which stimulates the release of
69
EGF-like factor proteins [14], which ultimately will cause a decrease in intra-oocyte
70
cAMP levesl.
71
resulting in the degradation of cAMP [22]. The net reduction of cAMP within the
72
oocyte plays a critical role in the maintenance of meiotic arrest by activating the MPF
73
complex and thus promoting meiosis resumption from diplotene arrest [23, 24].
EP
LH also decreases cGMP and the subsequent activation of PDE 3A,
Instead, when oocytes are removed from their follicles, they undergo a rapid
AC C
74
TE D
66
75
decrease in their cAMP levels. This decrease leads immediately to the activation of
76
MPF and the automatic resumption of meiosis. Therefore, oocyte competence is
77
compromised, presumably due to an absence in the ovulatory cascade of events and a
78
reduced level of regulatory molecules and metabolites [25]. Aiming to improve the
79
developmental capacity of the oocytes used for IVM, a maturation system called SPOM
80
(Simulated physiological oocyte maturation) was recently proposed [6]. SPOM
ACCEPTED MANUSCRIPT simulates the physiological maturation that occurs in vivo and is based on the
82
physiological mechanisms of meiotic resumption. This system involves a short period
83
of pre-maturation, in the presence of adenylate cyclase (AC) activator, and an extended
84
maturation period, in which a phosphodiesterase inhibitors (PDE) and hormones to
85
overcome the effect of the inhibitor are used. The authors showed that the SPOM
86
system substantially improves oocyte developmental outcomes with increased
87
blastocyst rates and quality. Although the efficiency of the SPOM system has not yet
88
been confirmed by other laboratories due to its physiological approach, it represents an
89
interesting alternative to improve the competence and embryo production of bovine
90
oocytes. Here, we attempted to use the principles of the SPOM system under different
91
conditions to promote the in vitro maturation of bovine oocytes to improve embryonic
92
development.
M AN U
SC
RI PT
81
93
2. Materials and methods
TE D
94
Unless otherwise indicated, the reagents used in this research and any chemicals
96
used in the preparation of the maturation, fertilization and in vitro culture media were
97
purchased from Sigma (St. Louis, MO, USA).
99 100
AC C
98
EP
95
2.1 Oocyte recovery
Ovaries from crossbred females (Bos indicus X Bos taurus) were collected from
101
local abattoirs immediately after slaughter and transported in a solution of saline
102
(0.9%NaCl) supplemented with antibiotics (streptomycin -and100µg/mL penicillin G-
103
100IU/mL) at a temperature of 35-36°C. Cumulus-oocyte complexes (COCs) were
104
aspirated from follicles of 3-8 mm in diameter using an 18 gauge needle connected to a
105
vacuum system.
ACCEPTED MANUSCRIPT The recovery of oocytes from follicles of 1-3 mm in diameter was performed as
107
described previously [26]. Follicles were dissected from the ovarian cortex using
108
tweezers, scissors and a scalpel and were kept in TCM-199 (Invitrogen®, Carlsbad, CA,
109
USA) supplemented with 10% fetal calf serum (FCS; Invitrogen) and 0.075 mg/ mL
110
amikacin during the entire process. Once dissected, the follicles were measured using a
111
graduated ocular (eyepiece micrometerOSM-4 ®Olympus, Tokyo, Japan). To release
112
COCs, the follicles were ruptured; only those oocytes with at least four layers of
113
cumulus cells and a homogeneous cytoplasm were used for further work.
SC
RI PT
106
114
2.2 In vitro maturation (IVM)
116
Immediately after selection, the COCs were transferred to a basic maturation medium
117
consisting of TCM-199 supplemented with 10% FCS, 0.01 IU/mL purified FSH
118
(pFSH), 0.1 mg/mL glutamine and an antibiotic (0.075 mg/ mL amikacin). The COCS
119
were then matured for 22 hours at 39 ° C in 5 % CO2 in air.
TE D
120
M AN U
115
2.3 In vitro fertilization (IVF) and embryo culture (IVC)
122
Following maturation, groups of 25-30 COCs were transferred to 200-µL drops of
123
fertilization medium, which consisted of TALP [27] supplemented with penicillamine
124
(2 mM), hypotaurine (1 mM), epinephrine (250 mM) and heparin (10µg/mL−1). Frozen
125
semen from a bull of proven fertility that had been used for several years as the
126
reference bull for in vitro embryo production in our laboratory was used for all
127
treatments and replicates. Motile spermatozoa were obtained using the Percoll (GE®
128
Healthcare, Piscataway, NJ, USA) gradient method in microtubes [28] and were added
129
into the fertilization drop at a final concentration of 1×106 spermatozoa mL−1.
130
Spermatozoa and oocytes were co-incubated for 18 h at 39°C with 5% CO2 in air. The
131
day of in vitro insemination was considered to be day 0.
AC C
EP
121
ACCEPTED MANUSCRIPT Eighteen hours post insemination (pi), the presumptive zygotes were washed and
133
transferred to 200-µmL drops of synthetic oviduct fluid medium with amino acids,
134
citrate and inositol (SOFaaci [29]) supplemented with 5% FCS and incubated at 39 ◦C
135
in an atmosphere of 5% CO in air for 7 or 8 days. Embryos were evaluated on D 2 for
136
cleavage and on D 6, D 7 and D 8 for blastocyst rates.
137 138
2. Cell number
RI PT
132
On D 8, blastocysts were measured with a Motic camera (Moticam®Plus2.0,
140
Japan) and classified according to their diameter into three categories: 120-140, 140-160
141
and >160µm. Embryos with a diameter >160µm were used to evaluate the number of
142
cells. Embryos were exposed to Hoechst 33342 dye at a concentration of 1µg/mL for 5
143
minutes and were subsequently transferred to a slide and covered with a cover slip.
144
Slides were evaluated using an epifluorescence microscope (Zeiss Axiophot®,
145
Germanyfilter24 with a wavelength of 494/518 nm (excitation/emission), and the cell
146
nuclei were counted.
M AN U
TE D
147
SC
139
2.5 Experimental Design
149
2.5.1 Experiment 1: Evaluation of the SPOM-adapted system to a standard IVM system
150
to improve the developmental competence of oocytes
AC C
151
EP
148
In this experiment, we evaluated the SPOM principles as adapted to the IVM
152
system used in our laboratory. Initially 871 COCs were divided into two groups (control
153
and SPOM) in a total of seven replicate experiments. In the control group, COCs were
154
matured in basic medium for 22 hours. In the SPOM group, the oocytes were submitted
155
to the protocol described by Albuz et al. [6] with modifications. In this group,
156
immediately after selection, the oocytes were washed, transferred to prematuration
ACCEPTED MANUSCRIPT medium and cultured for a period of 2 hours at 39°C in 5% CO2 in air. Prematuration
158
medium consisted of TCM-199 with Earle's salts supplemented with 10% FCS, 0,075
159
mg/ ml of amikacin, 500µM of 3-Isobutil1–methylxanthine (IBMX), a non-specific
160
inhibitor of phosphodiesterase, and 100 µM of forskolin (FSK), an activator of
161
adenylate cyclase. After prematuration, the COCs were washed and transferred to a
162
200µL drop of maturation medium covered with silicone oil. The maturation medium
163
was the basal medium, to which 20µM of cilostamide, a potent inhibitor of type 3
164
phosphodiesterase (PDE 3A), was added. COCs were incubated at 39° C in 5% CO2 in
165
air for a period of 28 hours (extended maturation). After maturation, oocytes from both
166
groups were fertilized and cultured in vitro. On D 2, the cleavage rate of the embryos
167
was evaluated, and on D 7 and D 8, the blastocyst rate was evaluated.
M AN U
SC
RI PT
157
All the inhibitor used in the experiment FSK, IBMX and cilostamide were
169
prepared at 100X stock solution in dimethylsulfoxide (DMSO) to final concentrations of
170
100µM, 500 µM and 20 µM, respectively, and aliquoted in microtubes and stored at -20
171
° C until use The stock solutions were diluted fresh for each replica of experiments 1, 2
172
and 3.
173
2.5.2 Experiment 2: Evaluation of the SPOM-adapted system for the maturation of
174
bovine oocytes with different levels of competency
EP
Because in the previous experiment the SPOM-adapted system had a detrimental
AC C
175
TE D
168
176
effect on oocytes, we suspected that the FSH concentration may have been too low to
177
overcome the cilostamide inhibition. We therefore increased the concentration of the
178
pFSH in the maturation medium from 0.01IU/ mL to 0.1 IU/ML
179
This experiment aimed to evaluate if the SPOM-adapted system would improve
180
the developmental competence of less competent oocytes obtained from small follicles.
181
A total of 1040 were used in four experimental groups (G), G1) the 3-8 mm control
ACCEPTED MANUSCRIPT group, consisting of oocytes aspirated from follicles of 3-8 mm and matured in a
183
standard IVM system, G2) the 3-8 mm SPOM group, consisting of oocytes aspirated
184
from follicles of 3-8 mm and submitted to the SPOM-adapted system, G3) the 1-3 mm
185
control group, consisting of less competent oocytes obtained from follicles of 1-3 mm
186
and matured in a standard IVM system and G4) the 1-3 mm SPOM group, consisting of
187
less competent oocytes obtained from follicles of 1-3 mm and submitted to the SPOM-
188
adapted system. The maturation and SPOM system were the same as described in the
189
previous experiment, and the FSH concentration in the SPOM group was the only
190
difference between the two sets of conditions. After IVM, all groups underwent in vitro
191
fertilization and culture. Embryonic development was evaluated at D 2, D 7 and D 8.
M AN U
SC
RI PT
182
192 193
2.5.3 Experiment 3: Effects of the SPOM-adapted system under different culture
194
conditions on oocyte developmental competence.
In this experiment we tried to follow the SPOM protocol for oocyte maturation
196
described by Albuz et al. [6] as closely as possible. We tested the SPOM using
197
recombinant FSH [rFSH (Gonal-F®, Merck Serono, USA)] at a concentration of 0.1
198
UI/mL in the presence of BSA-FAF. A total of 452 oocytes were distributed into five
199
different culture conditions: G1) the control condition, in which oocytes underwent
200
standard IVM, IVF and IVC, in the presence of FCS and 5% CO2 in air (high O2); G2)
201
the SPOM-rFSH-BSA condition, with prematuration in BSA + FSK + IBMX,
202
maturation in rFSH + BSA + cilostamide and embryo culture in BSA under 5% O2 (low
203
O2); G3) the SPOM-pFSH-BSA condition, with prematuration in BSA + FSK + IBMX,
204
maturation in pFSH + BSA + cilostamide, embryo culture in BSA with 5% O2 (low O2);
205
G4) the SPOM-pFSH-FCS condition, with prematuration in BSA + FSK + IBMX,
206
maturation in pFSH + FCS + cilostamide and embryo culture in FCS under 5% CO2
AC C
EP
TE D
195
ACCEPTED MANUSCRIPT (high O2); and G5) the SPOM-rFSH-FCS FCS condition, with prematuration in BSA +
208
FSK + IBMX, maturation in rFSH + FCS + cilostamide and embryo culture with FCS
209
under 5% CO2 (low O2). After IVM, oocytes from all groups were fertilized and
210
cultured in vitro. Embryo development was evaluated at D 2, D 7 and D 8. D 8
211
blastocysts were measured, and those greater than 160µm were stained to determine the
212
total cell number.
RI PT
207
213
2.6 Statistical Analysis
SC
214
Data of embryo development rates were analyzed using the Chi-square test
216
(P<0.05). The embryo measurements and the cell number data were compared with the
217
Kruskal-Wallis test, using version 5.0 of the Prophet program (BBN Systems and
218
Technologies, 1996). 3. Results
TE D
219
M AN U
215
220
3.1 Experiment 1: Evaluation of the SPOM-adapted system to a standard IVM system to
221
improve oocyte developmental competence
In this experiment, the SPOM principle (prematuration for two hours in the
223
presence of forskolin and IBMX and prolonged maturation in presence of cilostamide)
224
was introduced in our protocol without any changes to our media composition. The
225
results showed that not only the cleavage but also the blastocyst rates at D 7 and D 8
226
were lower (P>0.05) in oocytes submitted to the SPOM-adapted system compared to
227
those submitted to the control system (Table 1).
228
3.2 Experiment 2: Evaluation of the SPOM-adapted system for the maturation of
229
oocytes with different levels of competency
AC C
EP
222
ACCEPTED MANUSCRIPT The cleavage (D 2) and blastocyst rates (D 7 and D 8) were higher in the control
231
group than in the other groups (Table 2). As in the previous experiment, the SPOM-
232
adapted system produced a deleterious effect (P<0.05) on embryonic development when
233
oocytes obtained from 3-8 mm follicles were used. For the less competent oocytes, the
234
use of the SPOM-adapted system was not able to improve their embryonic development
235
(Table 2).
236
3.3 Experiment 3: Effect of the SPOM-adapted system under different culture
237
conditions on oocyte developmental competence.
SC
M AN U
238
RI PT
230
The results obtained for the group submitted to the SPOM- adapted system used
240
in experiments 1 and 2 (pFSH, FCS and high O2 tension during embryo culture) were
241
consistent with those of previous experiments, showing lower cleavage and blastocyst
242
rates than the control group. However, when those same conditions were used but the
243
pFSH was replaced by rFSH, a further reduction in embryo development was observed
244
(Table 3). When the SPOM-adapted system, in which FCS was replaced by BSA,the
245
embryo culture was performed under low O2 tension and SOF was supplemented with
246
BSA and not FCS, the observed cleavage rates were similar to those of the control
247
group (Table 3). Although the blastocyst rates remained lower than those of the control
248
group, the size and total cell number of the embryos produced were similar to the size
249
and cell number of the control embryos (Table 4). In both conditions, the use of rFSH
250
affected embryo production and quality (Table 3 and 4).
AC C
EP
TE D
239
251
252
4. Discussion
ACCEPTED MANUSCRIPT The main factor determining the blastocyst rates following in vitro embryo
254
production techniques is the quality of the oocytes. Immature oocytes used for this
255
purpose usually have not acquired full developmental competence due to their
256
premature removal from the follicular environment. Thus, it is necessary to provide
257
appropriate conditions to allow oocytes to develop into viable embryos. When Albuz et
258
al. [6] proposed the SPOM system in an attempt to simulate the events that occur during
259
in vivo maturation, a new promising alternative emerged. This system involves two
260
steps. First, a short exposure to cAMP modulating agents causes a rapid increase in
261
intra-oocyte cAMP levels and mimics the action of LH in vivo. Subsequently, oocytes
262
are subjected to a prolonged IVM in the presence of cilostamide, an inhibitor of PDE
263
3A. This step retains meiosis, which is associated with FSH levels at a concentration
264
sufficient to overcome the effects of the inhibitor and to induce maturation.
M AN U
SC
RI PT
253
We attempted to adapt the SPOM principles to our standard IVM system, which
266
is similar to the system used in the majority of IVP laboratories. However, we found no
267
improvement in embryo production. On the contrary, we observed a substantial
268
decrease in blastocyst rates. Because we included SPOM principles but kept our
269
maturation media composition, we predicted that the negative effect of SPOM could be
270
due to the FSH concentrations. In the SPOM system, the oocytes do not resume meiosis
271
spontaneously, and a relatively high dose of FSH is needed to overcome the inhibitory
272
effect of cilostamide [6, 22]. In our IVM system, the concentration of FSH used was 10
273
times lower than the dose used in the SPOM protocol. In addition, we used purified
274
FSH, which is prepared by the extraction of the pituitary, which can lead to some
275
contamination and lower activity than the recombinant FSH used by Albuz et al. [6].
276
Therefore, it is possible that the FSH concentration in the media was not sufficient to
277
overcome the inhibitory effect of cilostamide, and many oocytes might not have
AC C
EP
TE D
265
ACCEPTED MANUSCRIPT 278
matured properly, thus leading to the low embryo development observed as early as the
279
cleavage state. In an attempt to overcome the presumptive low activity of FSH, we
280
increased the concentration of FSH in the second experiment by 10-fold. Albuz et al.[6] hypothesized that the SPOM system is able to improve the
282
developmental competence of oocytes from a heterogeneous population of bovine
283
ovaries, such as those from small antral follicles. Therefore, in the second experiment,
284
we also evaluated whether such a system could increase the quality of less competent
285
oocytes obtained from 1-3 mm dissected follicles. The increase in FSH concentration
286
did not affect the embryo production, nor did the SPOM system improve the quality of
287
the less competent oocytes. Similar results were reported for oocytes obtained from pre-
288
pubertal ewes, which are supposedly less competent. In these oocytes, no increases in
289
embryos production were observed when the SPOM system was used [22]. The lack of
290
data on the SPOM system in the literature makes it difficult to place our results in
291
context. There is no clear explanation for the discrepancy between our results and those
292
reported by Albuz et al.[6] in the bovine system, but the discrepancy may be due to
293
differences in the media, culture conditions and even the sub-species (Indicus x Taurus)
294
used in the two studies.
EP
TE D
M AN U
SC
RI PT
281
Finally, we changed the culture conditions to match our system as closely as
296
possible to that proposed by Albuz et al. [6]. The pFSH was replaced by rFSH, and the
297
FCS was replaced by BSA. Moreover, the O2 tension was decreased to 5% during the
298
embryo culture period. Nevertheless, we observed no positive effect of the SPOM-
299
adapted system on embryo production regardless of the culture conditions used.
300
However, it became very clear that the detrimental effect was minimized when low O2
301
tension was used during embryo culture in association with BSA, regardless of the FSH
302
source. The best results were observed when pFSH was used instead of rFSH. Although
AC C
295
ACCEPTED MANUSCRIPT 303
embryo production was lower when the SPOM system was used with BSA, the quality
304
of the embryos, as indicated by their size and the total cell number, was similar to that
305
of the control group. In contrast, the use of the adapted-SPOM system, in which FCS was present
307
during the maturation and culture, showed the worst results. Both the yield and quality
308
of the embryos were inferior to the control and the adapted-SPOM system with the BSA
309
groups. The possibility that the presence of FCS may have led an acceleration of
310
meiosis causing a premature aging of oocytes and, subsequently a reduced embryo
311
production cannot be ruled out. As mentioned earlier, there is a lack of studies using the
312
SPOM principle for the maturation of oocytes; however, some preliminary studies using
313
bovine oocytes have also reported a lower blastocyst rate in the adapted-SPOM than in
314
the control group [30].
M AN U
SC
RI PT
306
Interestingly, in the treatments in which rFSH was used, embryo production was
316
lower than in the groups to which pFSH was added during maturation, regardless of the
317
culture conditions. We have no explanation for this result. Studies in which the use of
318
rFSH and pFSH during IVM were compared have shown that embryo production was
319
similar between them. However, other studies demonstrated that the positive effect of
320
rFSH could only be detected when it was associated with other hormones [31]. Those
321
authors observed that the developmental competence of oocytes matured with r-FSH
322
and E2 was higher than that of oocytes matured with r-FSH alone, indicating a
323
synergistic effect between E2 and FSH. Moreover, no additive effect between E2 and
324
purified pFSH was observed, which the authors did not expect. This lack of effect might
325
be due to a differential effect of the recombinant product caused by a different
326
glycosylation patterns or by a contaminant to the purified pituitary FSH preparation.
327
Other studies on bovine oocytes [32], comparing rFSH and rLH addition and
AC C
EP
TE D
315
ACCEPTED MANUSCRIPT combinations of the two, noted that embryonic development was higher when oocytes
329
were matured with rFSH and rLH than with either rFSH or rLH alone. Those results
330
suggested that the effects of rFSH can change according to the presence of other factors
331
in the medium. In a study using ovine oocytes, in which the effect of rFSH on in vitro
332
maturation was evaluated, it was found that embryo development and viability were
333
lower when rFSH was used than when a commercial FSH obtained from the bovine
334
pituitary was used [33]. Curiously, the authors used the same r-FSH (Gonal-F) at the
335
same concentration (0.1 UI/mL) that we used and also had better results with the
336
pituitary FSH. It is well known that hypophysial gonadotrophins, unlike recombinant
337
hormones, can contain contaminants (i.e., growth factors) that can improve the
338
blastocyst yield [34, 35]. Another hypothesis that may explain the low levels of embryo
339
production when rFSH was used is that the required levels of this gonadotropin may
340
differ between species. Studies conducted with Brahman and Angus cows observed that
341
plasma concentrations of FSH were lower in Bos indicus than in Bos taurus, indicating
342
that Zebu cows may be more sensitive to the exposure of that gonadotropin [36, 37].
343
Although we are not sure whether this is the case, studies investigating the effects of
344
different concentrations of rFSH should be performed.
SC
M AN U
TE D
EP
Regardless the changes we made in the system, our data showed that the SPOM-
AC C
345
RI PT
328
346
adapted-system did not improve the production or quality of bovine embryos.
347
Acknowledgements
348
The authors thank EMBRAPA and CAPES for their financial support and the Qualimax
349
(Luziania-GO) slaughterhouse for providing the necessary biological materials for this
350
experiment.
351
Reference
ACCEPTED MANUSCRIPT [1] Jee BC, Chen HY, Chian RC. Effect of a phosphodiesterase type 3 inhibitor in
353
oocyte maturation medium on subsequent mouse embryo development. Fertility and
354
sterility. 2009;91:2037-42.
355
[2] Sirard MA, Richard F, Blondin P, Robert C. Contribution of the oocyte to embryo
356
quality. Theriogenology. 2006;65:126-36.
357
[3] van de Leemput E, Vos P, Zeinstra E, Bevers M, van der Weijden G, Dieleman S.
358
Improved in vitro embryo development using in vivo matured oocytes from heifers
359
superovulated with a controlled preovulatory LH surge. Theriogenology. 1999;52:335 -
360
49.
361
[4] Rizos D, Ward F, Duffy P, Boland M, Lonergan P. Consequences of bovine oocyte
362
maturation, fertilization or early embryo development in vitro versus in vivo:
363
implications for blastocyst yield and blastocyst quality. Mol Reprod Dev. 2002;61:234 -
364
48.
365
[5] Tesfaye D, Ghanem N, Carter F, Fair T, Sirard M, Hoelker M, et al. Gene
366
expression profile of cumulus cells derived from cumulus–oocyte complexes matured
367
either in vivo or in vitro. Reproduction, Fertility and Development. 2009;21:451-61.
368
[6] Albuz FK, Sasseville M, Lane M, Armstrong DT, Thompson JG, Gilchrist RB.
369
Simulated physiological oocyte maturation (SPOM): a novel in vitro maturation system
370
that substantially improves embryo yield and pregnancy outcomes. Human
371
reproduction. 2010;25:2999-3011.
372
[7] Luciano AM, Franciosi F, Modina SC, Lodde V. Gap junction-mediated
373
communications regulate chromatin remodeling during bovine oocyte growth and
374
differentiation through cAMP-dependent mechanism(s). Biology of reproduction.
375
2011;85:1252-9.
AC C
EP
TE D
M AN U
SC
RI PT
352
ACCEPTED MANUSCRIPT [8] Opiela J, Romanek J, Lipi, #x144, ski D, Smor, et al. Effect of Hyaluronan on
377
Developmental Competence and Quality of Oocytes and Obtained Blastocysts from In
378
Vitro Maturation of Bovine Oocytes. BioMed Research International. 2014;2014:8.
379
[9] Wang F, Tian X, Zhang L, He C, Ji P, Li Y, et al. Beneficial effect of resveratrol on
380
bovine oocyte maturation and subsequent embryonic development after in vitro
381
fertilization. Fertility and sterility. 2014;101:577-86.
382
[10] Shimada M, Ito J, Yamashita Y, Okazaki T, Isobe N. Phosphatidylinositol 3-kinase
383
in cumulus cells is responsible for both suppression of spontaneous maturation and
384
induction of gonadotropin-stimulated maturation of porcine oocytes. J Endocrinol.
385
2003;179:25 - 34.
386
[11] Tripathi A, Kumar KV, Chaube SK. Meiotic cell cycle arrest in mammalian
387
oocytes. Journal of cellular physiology. 2010;223:592-600.
388
[12] Shimada M, Nishibori M, Isobe N, Kawano N, Terada T. Luteinizing hormone
389
receptor formation in cumulus cells surrounding porcine oocytes and its role during
390
meiotic maturation of porcine oocytes. Biology of reproduction. 2003;68:1142-9.
391
[13] Shu YM, Zeng HT, Ren Z, Zhuang GL, Liang XY, Shen HW, et al. Effects of
392
cilostamide and forskolin on the meiotic resumption and embryonic development of
393
immature human oocytes. Human reproduction. 2008;23:504-13.
394
[14] Norris RP, Ratzan WJ, Freudzon M, Mehlmann LM, Krall J, Movsesian MA, et al.
395
Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in
396
the mouse oocyte. Development. 2009;136:1869-78.
397
[15] Assidi M, Sirard M-A. Oogenesis. Cumulus Cell Gene Expression as a Marker of
398
Oocyte Quality. 2013:231 - 52.
AC C
EP
TE D
M AN U
SC
RI PT
376
ACCEPTED MANUSCRIPT [16] Sela-Abramovich S, Chorev E, Galiani D, Dekel N. Mitogen-activated protein
400
kinase mediates luteinizing hormone-induced breakdown of communication and oocyte
401
maturation in rat ovarian follicles. Endocrinology. 2005;146:1236-44.
402
[17] Su YQ, Sugiura K, Eppig JJ. Mouse oocyte control of granulosa cell development
403
and function: paracrine regulation of cumulus cell metabolism. Seminars in
404
reproductive medicine. 2009;27:32-42.
405
[18] Dekel N. Regulation of Oocyte Maturation. Annals of the New York Academy of
406
Sciences. 1988;541:211-6.
407
[19] Zhang M, Ouyang H, Xia G. The signal pathway of gonadotrophins-induced
408
mammalian oocyte meiotic resumption. Molecular human reproduction. 2009;15:399-
409
409.
410
[20] Bu S, Xie H, Tao Y, Wang J, Xia G. Nitric oxide influences the maturation of
411
cumulus cell-enclosed mouse oocytes cultured in spontaneous maturation medium and
412
hypoxanthine-supplemented medium through different signaling pathways. Molecular
413
and cellular endocrinology. 2004;223:85-93.
414
[21] Zhang M, Tao Y, Xia G, Xie H, Hong H, Wang F, et al. Atrial natriuretic peptide
415
negatively regulates follicle-stimulating hormone-induced porcine oocyte maturation
416
and cumulus expansion via cGMP-dependent protein kinase pathway. Theriogenology.
417
2005;64:902-16.
418
[22] Rose RD, Gilchrist RB, Kelly JM, Thompson JG, Sutton-McDowall ML.
419
Regulation of sheep oocyte maturation using cAMP modulators. Theriogenology.
420
2013;79:142-8.
421
[23] Duckworth BC, Weaver JS, Ruderman JV. G2 arrest in Xenopus oocytes depends
422
on phosphorylation of cdc25 by protein kinase A. Proceedings of the National Academy
423
of Sciences of the United States of America. 2002;99:16794-9.
AC C
EP
TE D
M AN U
SC
RI PT
399
ACCEPTED MANUSCRIPT [24] Han SJ, Conti M. New Pathways from PKA to the Cdc2/cyclin B Complex in
425
Oocytes: Wee1B as a Potential PKA Substrate. Cell Cycle. 2006;5:227-31.
426
[25] Thomas RE, Thompson JG, Armstrong DT, Gilchrist RB. Effect of specific
427
phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on
428
meiotic and developmental capacity. Biology of reproduction. 2004;71:1142-9.
429
[26] Caixeta E, Ripamonte P, Franco M, Junior J, Dode M. Effect of follicle size on
430
mRNA expression in cumulus cells and oocytes of Bos indicus: an approach to identify
431
marker genes for developmental competence. Reprod Fertil Dev. 2009;21:655 - 64.
432
[27] Parrish JJ, Krogenaes A, Susko-Parrish JL. Effect of bovine sperm separation by
433
either swim-up or Percoll method on success of in vitro fertilization and early
434
embryonic development. Theriogenology. 1995;44:859-69.
435
[28] Machado GM, Carvalho JO, Filho ES, Caixeta ES, Franco MM, Rumpf R, et al.
436
Effect of Percoll volume, duration and force of centrifugation, on in vitro production
437
and sex ratio of bovine embryos. Theriogenology. 2009;71:1289-97.
438
[29] Holm P, Shukri NN, Vajta G, Booth P, Bendixen C, Callesen H. Developmental
439
kinetics of the first cell cycles of bovine in vitro produced embryos in relation to their in
440
vitro viability and sex. Theriogenology. 1998;50:1285-99.
441
[30] Ulloa SM, Heinzmann J, Herrmann D, Timmermann B, Baulain U, Grossfeld R, et
442
al. Effects of different oocyte retrieval and in vitro maturation systems on bovine
443
embryo development and quality. Zygote. 2014:1-11.
444
[31] Ali A, Sirard M. The effects of 17beta-estradiol and protein supplement on the
445
response to purified and recombinant follicle stimulating hormone in bovine oocytes.
446
Zygote. 2002;10:65 - 71.
447
[32] Anderiesz C, Ferraretti A, Magli C, Fiorentino A, Fortini D, Gianaroli L, et al.
448
Effect of recombinant human gonadotrophins on human, bovine and murine oocyte
AC C
EP
TE D
M AN U
SC
RI PT
424
ACCEPTED MANUSCRIPT meiosis, fertilization and embryonic development in vitro. Hum Reprod. 2000;15:1140 -
450
8.
451
[33] Accardo C, Dattena M, Pilichi S, Mara L, Chessa B, Cappai P. Effect of
452
recombinant human FSH and LH on in vitro maturation of sheep oocytes; embryo
453
development and viability. Animal Reproduction Science. 2004;81:77-86.
454
[34] Lonergan P, Carolan C, Van Langendonckt A, Donnay I, Khatir H, Mermillod P.
455
Role of epidermal growth factor in bovine oocyte maturation and preimplantation
456
embryo development in vitro. Biology of Reproduction. 1996;54:1420-9.
457
[35] Guler A, Poulin N, Mermillod P, Terqui M, Cognié Y. Effect of growth factors,
458
EGF and IGF-I, and estradiol on in vitro maturation of sheep oocytes. Theriogenology.
459
2000;54:209-18.
460
[36] Calder M, Caveney A, Westhusin M, Watson A. Cyclooxygenase-2 and
461
prostaglandin E2 receptor messenger RNAs are affected by bovine oocyte maturation
462
time and cumulus-oocyte complex quality, and PGE2 induces moderate expansion of
463
the bovine cumulus in vitro. Biol Reprod. 2001;65:135 - 40.
464
[37] Alvarez P, Spicer LJ, Chase CC, Payton ME, Hamilton TD, Stewart RE, et al.
465
Ovarian and endocrine characteristics during an estrous cycle in Angus, Brahman, and
466
Senepol cows in a subtropical environment. Journal of Animal Science. 2000;78:1291-
467
302.
469
SC
M AN U
TE D
EP
AC C
468
RI PT
449
ACCEPTED MANUSCRIPT
standard maturation system (Control).
Control
Oocyte number
Cleavage D 2 (%)
Blastocysts D 7 (%)
310
243 (78.4)a
142 (45.8)a
SC
Treatment
RI PT
Table 1. Embryonic development of bovine oocytes matured on an adapted system that simulates physiological oocyte maturation (SPOM) and a
AC C
EP
TE D
M AN U
SPOM 561 297 (52.9)b 69 (12.3)b a, b Different letters in the same column indicate significant difference by χ2 (P < 0.05).
Blastocysts D 8 (%) 155 (50.0)a 81 (14.4)b
ACCEPTED MANUSCRIPT
RI PT
Table 2. Embryonic development of more (3-8 mm) or less (1-3 mm) competent bovine oocytes matured on either an adapted system that simulates physiological oocyte maturation (SPOM) or a standard maturation system (Control). Treatment
Oocyte number
Cleavage D 2 (%) a
Blastocysts D 7 (%) a
Blastocysts D 8 (%) 297 (49.7)a
598
491 (82.1)
271 (45.3)
Spom 3-8 mm
321
175 (54.5)b
38 (11.8)b
38 (11.8)b
Control 1-3 mm
54
32 (59.3)b
11(20.4)b
11 (20.4)b
Spom 1-3 mm 67 34 (50.7)b 9 (13.4)b a, b Different letters in the same column indicate significant difference by χ2 (P < 0.05).
9 (13.4)b
AC C
EP
TE D
M AN U
SC
Control 3-8mm
ACCEPTED MANUSCRIPT
Table 3. Embryonic development of bovine oocytes matured on either an adapted system that simulates physiological oocyte maturation (SPOM)
RI PT
under different conditions using serum (FCS) or albumin (BSA), and purified (pFSH) or recombinant FSH (rFSH) during maturation and low O2 tension and BSA (↓ O2) or high O2 tension and FCS (↑ O2) during embryo culture, compared to a standard maturation system (Control). Cleavage D 2 (%)
Blastocysts D 7 (%)
Control
89
69 (77.5)a
29 (32.6)a
29 (32.6)a
Spom+rFSH+BSA↓O2
91
64 (70.3)a
17 (18.7)b
17 (18.7)b
Spom+pFSH+BSA↓O2
89
60 (67.4)a
20 (22.5)a, b
20 (22.5)a, b
Spom +pFSH+FCS↑O2
90
49 (54.5)b
6 (6.6)c
6 (6.6)c
M AN U
SC
Oocyte Number
Treatment
AC C
EP
TE D
Spom+rFSH+FCS↑O2 93 35 (37.7)c 2 (2.2)c a, b, c Different letters in the same column indicate significant difference by χ2 (P < 0,05).
Blastocysts D 8 (%)
2 (2.2)c
ACCEPTED MANUSCRIPT
RI PT
Table 4. Percentage, size and total cell number of D 8 embryos with diameters > 160 µm obtained from oocytes oocytes matured on either an adapted system that simulates physiological oocyte maturation (SPOM) under different conditions using serum (FCS) or albumin (BSA), and
Control SPOM+rFSH+BSA↓O2 SPOM+pFSH+BSA↓O2 SPOM+pFSH+FCS↑O2
Total embryos 29 20 17 6
Cell number of embryos > 160µm (mean±sd)
Size (µm) of Embryo > 160 µm (mean±sd)
150.8±26.7ª 139.6±35.6ª 137.9±51.6ª 125.0±41.0b
210.3±47.0a 210.1±57.6ª 218.5±56.5ª 163.4±63.9b
TE D
Treatment
M AN U
embryo culture, compared to a standard maturation system (Control).
SC
purified (pFSH) or recombinant FSH (rFSH) during maturation and low O2 tension and BSA (↓ O2) or high O2 tension and FCS (↑ O2) during
AC C
EP
SPOM+rFSH+ FCS↑O2 2 96.6±7.7c 171.0±41.0c a, b, c Different letters in the same column indicate significant difference (P < 0.05).