- Email: [email protected]
The effect of conspecific ampulla oviductal epithelial cells during in vitro maturation on oocyte developmental competence and maturation-promoting factor (MPF) activity in sheep
Accepted Manuscript The effect of conspecific ampulla oviductal epithelial cells during in vitro maturation on oocyte developmental competence and maturation-promoting factor (MPF) activity in sheep Navid Dadashpour Davachi, Hamid Kohram, Ahmad Zare Shahneh, Mahdi Zhandi, Rouzbeh Fallahi, Reza Masoudi, Ali Reza Yousefi, Pawel M. Bartlewski PII:
S0093-691X(16)30451-4
DOI:
10.1016/j.theriogenology.2016.09.034
Reference:
THE 13833
To appear in:
Theriogenology
Please cite this article as: Davachi ND, Kohram H, Shahneh AZ, Zhandi M, Fallahi R, Masoudi R, Yousefi AR, Bartlewski PM, The effect of conspecific ampulla oviductal epithelial cells during in vitro maturation on oocyte developmental competence and maturation-promoting factor (MPF) activity in sheep, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.09.034. 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
Title: The effect of conspecific ampulla oviductal epithelial cells during in vitro maturation on
3
oocyte developmental competence and maturation-promoting factor (MPF) activity in sheep
5
RI PT
4
Short title: Oviductal epithelial cells affect ovine oocyte maturation in vitro
6 1, 2, *
, Hamid Kohram 2, Ahmad Zare Shahneh 2, Mahdi
Authors: Navid Dadashpour Davachi
8
Zhandi2, Rouzbeh Fallahi 1, Reza Masoudi 2, Ali Reza Yousefi 1, 2, Pawel M. Bartlewski 3, *
9
1
SC
7
M AN U
Department of Research, Breeding and Production of Laboratory Animals, Razi Vaccine and
10
Serum Research Institute, Agricultural Research, Education and Extension Organization
11
(AREEO), Karaj, Iran
12
2
13
University of Tehran, Karaj, Iran
14
3
15
Guelph, ON, Canada.
TE D
Department of Animal Science, Faculty College of Agriculture and Natural Resources,
Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph,
EP
16
*Corresponding authors: 1Department of Research, Breeding and Production of Laboratory
18
Animals, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and
19
Extension Organization (AREEO), Karaj, Iran; Tel.: +98 912 344 6217; Facsimile: +98 26
20
34502805. E-mail: [email protected]; Department of Biomedical Sciences, Ontario
21
Veterinary College, University of Guelph, Veterinary College, University of Guelph, Guelph,
22
ON, Canada; Tel: +01 519 824 4120 (ext. 53330); Facsimile: +01 519 767 1548; E-mail:
23
[email protected]
AC C
17
1
ACCEPTED MANUSCRIPT
Abstract
25
The acquisition of fertilization ability by oocytes is one of the prerequisites for successful in
26
vitro embryo production (IVEP). In the present study, we examined the influence of conspecific
27
ampulla oviductal epithelial cells incubated with cumulus-oocyte complexes (COCs) throughout
28
the in vitro maturation (IVM) phase on the developmental competence and maturation-
29
promoting factor (MPF) activity of sheep oocytes. There were six experimental groups in this
30
study, namely four groups with and two groups without oviductal epithelial cells added to IVM
31
media: adult COCs matured in vitro with the ampulla oviductal epithelial cells obtained from
32
adult (AAE; G1) or prepubertal donors (PAE; G4), COCs obtained from prepubertal animals co-
33
cultured with AAE (G2) or PAE (G3), and adult (C1) and prepubertal sheep COCs (C2) matured
34
without oviductal epithelial cells. Co-incubation of oocytes retrieved from both adult and
35
sexually immature donors with AAE (G1 and G2) resulted in significantly improved rates of
36
metaphase-II (M-II) attainment and blastocyst formation (G1: 42.2±1.1 and G2: 21.2±1.0) as
37
well as blastocyst development (total cell count; G1: 130.3±7.8, G2: 70.2±3.5, C1: 94.34±4.1,
38
and C2: 49.67±2.0) compared with their respective controls (C1: 30.5±1.2 and C2: 8.3±1.0).
39
Prior to IVM, the activity of MPF was greater (P<0.05) for oocytes obtained from ewes (G1, G4
40
and C1) compared with those from ewe lambs (G2, G3 and C2). The greatest increment in MPF
41
activity was recorded in G2 (MPF activity before IVM/MPF activity after IVM=3.62) followed
42
by C2 and G3 (2.22 and 2.20, respectively), and then all remaining groups of oocytes (C1: 1.89,
43
G1: 1.87, and G4: 1.86). In summary, co-incubation with AAE during the 24-hr IVM had a
44
positive impact on ovine oocyte competence and ensuing IVEP efficiency. A significant increase
45
in MPF activity following IVM of G2 oocytes could be responsible, at least partly, for the
46
improved rate of blastocyst formation after in vitro fertilization of prepubertal sheep oocytes.
AC C
EP
TE D
M AN U
SC
RI PT
24
2
ACCEPTED MANUSCRIPT
Keywords: Sheep, oviductal epithelial cells, in vitro maturation, maturation promoting factor
48
1. Introduction
49
Oocyte source and quality pre-determine the outcome of in vitro embryo production (IVEP) in
50
mammalian species. The use of prepubertal animals as oocyte donors has invariably been
51
associated with a decrease in the efficiency of IVEP in several livestock species including cattle
52
[1], sheep [2], and pigs [3]. Abnormal cytoplasmic maturation and reduced ability to achieve the
53
blastocyst stage post-fertilization are the primary deficiencies observed during IVEP procedures
54
that utilize oocytes obtained from prepubertal animals as compared to those recovered from
55
sexually mature donors. Both the follicle size and oocyte diameter are associated with oocyte
56
competence and embryo development [4, 5]. However, the use of oocytes from prepubescent
57
donors for embryo production and transfer is highly beneficial as shortening the inter-generation
58
interval can maximize the genetic improvement of farmed animals [2, 6].
M AN U
SC
RI PT
47
Maturation-promoting factor (MPF) is a heterodimer protein composed of the catalytic
60
and regulatory subunits that plays a critical role during meiosis and mitosis [7]. Cyclin-
61
dependent kinase 1 (CDK1), also known as CDC2, is a member of the Ser/Thr protein kinase
62
family. This protein is a catalytic subunit of the highly conserved MPF complex, which is
63
essential for G1/S and G2/M phase transitions of the eukaryotic cell cycle. Mitotic cyclins bind
64
stably to the catalytic MPF subunit and function as regulatory subunits; the interaction between
65
these two subunits is necessary for the control of protein kinase activity. The accumulation and
66
destruction of the cyclins as well as their phosphorylation and dephosphorylation regulate the
67
kinase activity throughout the cell cycle. MPF activity rises initially just before germinal vesicle
68
breakdown (GVBD), increases until metaphase-I (M-I), decreases in the anaphase-telophase
69
stage, and reaches a maximum level at metaphase-II (M-II). MPF activity in oocytes obtained
AC C
EP
TE D
59
3
ACCEPTED MANUSCRIPT
from heifer calves and ewe lambs was significantly lower compared with that measured in cows’
71
and ewes’ oocytes [6] and, in contrast to mammalian species, MPF activity of prepubertal
72
oocytes was significantly higher than that of adult oocytes in mice [7]. Substantially higher MPF
73
activity and developmental competence of oocytes >135 µm in diameter was observed in
74
prepubertal goatsAnguita et al. [7]. From all these indications, MPF activity could be a pivotal
75
factor behind oocyte developmental competence.
RI PT
70
Oocyte competence is acquired during its growth when the synthesis and storage of RNA
77
and proteins take place [8]. Oocyte mRNA content is affected by several factors such as nutrition
78
[9], composition of IVM culture media [10], an array of in vivo and in vitro conditions [11],
79
oocyte recovery method [12, 13], and quality and quantity of the cumulus-oophorous cell layers
80
[14]. In a recent study [15], an application of the new co-culture strategy during the IVM stage
81
has led to improvements in several aspects of oocyte competence, zona pellucida (ZP)
82
maturation, reduction of polyspermy, and an overall IVEP yield.
TE D
M AN U
SC
76
The central role of the oviduct as the site of gamete storage, fertilization and early
84
embryogenesis is unequivocal. Oviducts consist of three major segments, namely the
85
infundibulum, ampulla and isthmus. The three oviductal regions play the specialized and critical
86
role in gamete physiology, fertilization and preimplantation embryo development. Oviductal
87
epithelial cells harvested from the ampullary region affect ZP hardening in vitro [15], whereas
88
the epithelial cells obtained from the isthmic segment mainly influence the rate of early embryo
89
development [16]. We hypothesized that a co-culture system using oocytes and ampulla
90
oviductal epithelial cells would lead to an improvement in IVM conditions. Such an
91
improvement may, in turn, pave the way to the increased IVEP efficiency, especially when
92
prepubertal animals are used as oocyte donors. Therefore, the primary aim of this study was to
AC C
EP
83
4
ACCEPTED MANUSCRIPT
evaluate and compare the effects of ampulla oviductal epithelial cells, from prepubescent and
94
sexually mature ewes, added to IVM media on MPF activity and developmental potential of
95
ovine oocytes, also obtained from prepubertal and sexually mature animals.
96
2. Materials and Methods
97
All chemicals were purchased from Sigma-Aldrich Chemical Co. unless otherwise stated. Adult
98
testes and ovaries were obtained from the slaughtered Lory-Bakhtiary sheep aged 2 to 3years,
99
and prepubertal ovaries were recovered from the slaughtered 2- to 3-month-old Lory-Bakhtiary
SC
RI PT
93
ewe lambs.
101
2. 1. Experimental design
102
There were six experimental groups in the present study design; four groups (G1-G4) were
103
considered the “treatment groups” and two were considered the “control groups” (C1 and C2). In
104
G1 and G4, the oocytes were recovered from adult donors and co-cultured with ampulla
105
oviductal epithelial cells obtained from adult (AAE) or prepubertal donors (PAE), respectively.
106
In G2 and G3, the oocytes were recovered from ewe lambs and then co-cultured with the
107
oviductal cells obtained from adult and prepubertal animals, respectively. In the two control
108
groups, the IVM was performed without the addition of oviductal cells; C1 and C2 oocytes were
109
obtained from adult and prepubertal donors, respectively. More details of the present
110
experimental design are given in Table 1.
111
2.2. Preparation of ovine ampulla oviductal epithelial cells
112
Adult (AAE) and prepubertal ampulla oviductal epithelial cells (PAE) were obtained as
113
previously described by Dadashpour Davachi et al. [15]. Briefly, the ovine reproductive tracts
114
were collected immediately after slaughter. In the ewes of the present study, the oviducts
115
ipsilateral to ovaries with visible corpora haemorrhagica (indicative of ovulations occurring
AC C
EP
TE D
M AN U
100
5
ACCEPTED MANUSCRIPT
within the preceding 24 h) were selected for harvesting AAE. Such oviducts were used in this
117
study because they contained epithelial cells that had been exposed to circulating estradiol and
118
due to ovulations were triggered to produce trophic oocyte-nourishing proteins. No ovulations
119
were seen in the 2- to 3-month−old ewe lambs and both oviducts were collected from all
120
prepubertal donors at slaughter. All of the connective tissues were trimmed off and, before
121
washing in phosphate buffered saline without Ca2+/Mg2+/(PBS-), both ends of the oviduct were
122
tightly tied up with sterile surgical sutures. Oviducts were then dipped in 70% ethanol and
123
transported on ice to the laboratory in PBS solution supplemented with 2% penicillin–
124
streptomycin. Following their arrival at the laboratory, the oviducts were dipped again in 70%
125
ethanol and washed in HEPES buffered tissue culture medium (TCM199), in a laminar flow
126
hood. The ampulla was divided into two–three segments and gently squeezed in a stripping
127
motion with forceps to recover epithelial cells. The identification of different segments of the
128
oviduct was based on differences in the oviducts' outer diameter [15]. A yellowish matter
129
containing epithelial cells was collected into the culture medium TCM-199, supplemented with
130
2% estrous cow serum (ECS) and 0.25 mg/mL gentamicin. The cell suspension was pipetted 10
131
to 15 times with a 1000-µL filtered tip (JET Biofil, Guangzhou, China) and then passed 5 times
132
through a 1-mL tuberculin syringe attached to a 22-gauge needle. Following the washing
133
procedure, the cells were evaluated under a stereomicroscope to determine the size of cell
134
clusters; the preferred size of oviductal cell clusters is approximately half of a typical cumulus-
135
oocyte complex (COC) with compact cumulus layers. Finally, thirty AEC/PEC clusters were
136
transferred to 250-µL drops of IVM medium [15].
137
2.3. Ovaries and oocyte recovery
AC C
EP
TE D
M AN U
SC
RI PT
116
6
ACCEPTED MANUSCRIPT
The ovaries were dissected immediately after the slaughter of lambs/ewes and placed in a thermo
139
insulated container filled with PBS, supplemented with penicillin–streptomycin 100 µg/mL
140
(Gibco; Grand Island, NY, USA), at 37°C. The ovaries were transported to the laboratory within
141
2 h of collection. Ovarian antral follicles <4 mm in diameter were aspirated using an aspiration
142
pump (MEDAP Sekretsauger P7040; Tilburg, The Netherlands) fitted with a disposable vacuum
143
line (length-35 cm, the internal diameter of 3 mm). The flow rate was set at 10 mL H2O/min
144
using an attached disposable 20-gauge needle [15]. Ovarian follicles from ewe lambs were
145
aspirated under a stereo microscope; all visible follicles were aspirated.
146
2.4. In vitro maturation (I VM) of oocytes
M AN U
SC
RI PT
138
The TCM199 medium used for IVM was supplemented with 10% heat-inactivated fetal
148
calf serum (FCS) (Cat. Number: F4135), 0.2 mM sodium pyruvate, 5 µg/mL of gentamicin, 10
149
µg/mL of ovine follicle-stimulating hormone (oFSH), and 1 µg/mL of estradiol (basic maturation
150
medium). In the control groups (C1 and C2), thirty COCs from adult or prepubertal donors were
151
cultured in basic maturation medium droplets. In the co-culture IVM system(G1 to G4), thirty
152
COCs from sexually mature or prepubescent donors were matured in a droplet of primary
153
maturation medium (250 µL) along with 30 AAE or PAE clusters. All the droplets were covered
154
with mineral oil and incubated for 24 h at 38.5°C, in 5% CO2 in the air and 95% humidity. The
155
detection of various stages of oocyte maturation was based on the position of polar bodies and
156
chromosome arrangement. The ocytes with circular arrangements of chromosomes and no polar
157
body were considered the M-I phas oocytes; the oocytes with completely extruded polar bodies
158
and parallel arrangement of chromosomes were considered as early M-II stage oocytes; if the
159
polar body did not extrude from cytoplasm and the chromosomes were arranged in a circular
160
aggregation, the oocytes were considered arrested at the M-II phase; and finally, if the polar
AC C
EP
TE D
147
7
ACCEPTED MANUSCRIPT
bodies were extruded from cytoplasm and the chromosomes arranged in circular aggregation, the
162
oocytes were considered the fully developed M-II oocytes.
163
2.5. Sperm preparation, in vitro fertilization (IVF) and in vitro culture (IVC), and staining
164
methods
165
Testicles from some 2- to 3-year-old Lory-Bakhtiary rams were removed at slaughter, placed in a
166
box set chilled to 5°C and transported to the laboratory. Subsequently, the testicles were washed
167
three times in PBS supplemented with 100 µL/mL gentamicin; this and a few following
168
procedures were performed at 5°C in a cold room. Blood and connective tissues were removed
169
aseptically prior to sperm recovery. A 1-mL syringe attached to a 22-gauge needle was inserted
170
into the vas deferens to gently aspirate its entire content, and the recovered spermatozoa were
171
diluted 1:100 in sperm-TALP. Semen was stored in the refrigerator for up to 24 h. On the day of
172
IVF, semen samples were evaluated for progressive motility under a microscope. Motile
173
spermatozoa were separated using the Percoll gradient (45% over 90%) centrifugation according
174
to the method described by Hansen and Ortega et al. [16]. A conical tube containing the semen
175
and Percoll gradient was placed into a tube holder pre-warmed to 38.5°C and centrifuged at 1000
176
× g for 10 min. After centrifugation, a sperm pellet was collected from the bottom of the tube,
177
transferred to a 15-mL conical tube containing 10 mL HEPPES-SOF, and centrifuged again for 5
178
min at 200 × g. The supernatant was then gently removed with a Pasteur pipette. In order to
179
adjust a final sperm concentration to 26 × 106/mL (for a final concentration of sperm in the
180
fertilization drop of 1 × 106/mL), 10 µL of sperm suspension was added to 90 µL water to kill
181
spermatozoa, then 10 µL of the diluted sample loaded onto a haemocytometer, sperm
182
enumerated in 5 squares and the result multiplied by by 500,000 to determine sperm
183
concentration per 1 mL. The spermatozoa were diluted in the fertilization medium that had been
AC C
EP
TE D
M AN U
SC
RI PT
161
8
ACCEPTED MANUSCRIPT
pre-equilibrated in the incubator [16] and contained 12 mM KCL, 25 mM NaHCO3, 90 mM
185
NaCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 10 mM sodium lactate, sodium pyruvate 0.018 g/100
186
mL, CaCl2 0.147 g/100 mL, 3 mg/mL BSA (fatty acid free), and 50 µg/mL gentamicin. At least
187
15 min before the addition of sperm, the oocytes were transferred to fertilization droplets. Lastly,
188
10 µL of pre-incubated sperm solution were added to 90 µL of fertilization medium containing
189
∼20 oocytes.
RI PT
184
Co-incubation of gametes was carried out for 18 h at 38.5°C, in 5% CO2 in the air and
191
95% humidity. After co-incubation, the spermatozoa attached to the ZP were removed by gentle
192
pipetting, and the fertilized oocytes were transferred to synthetic oviductal fluid supplemented
193
with 0.4 g EFAF BSA (SOF-BSA) for the ensuing 7-day incubation (IVC). At the end of the 7-
194
day period, the rate of embryo development to the blastocyst stage was determined. The embryos
195
were removed from the culture medium, washed twice in PBS-PVP (1 mg/mL), and fixed in a
196
100-µL drop of paraformaldehyde solution [4% (w/v) in PBS, pH 7.4] at room temperature.
197
Finally, the embryos were washed three times in PBS-PVP and stained for 15 min in 1 µg/mL
198
Hoechst 33342 before microscopic evaluation using a fluorescent microscope equipped with a
199
UV filter [15].
200
2.6. MPF activity of oocytes
201
Determination of MPF activity utilized the method developed by Catalá et al. [17], with some
202
minor modifications. Before and after IVM, the batches of 30 oocytes (three replicates) were
203
washed three times in PBS and placed in tubes containing 5 µL of lysis buffer (50 mM Tris–
204
HCl, pH 7.5, 0.5 M NaCl, 5 mM EDTA, 0.01% Brij35, 1 mM PMSF, 0.05 mg/mL leupeptin, 50
205
mM 2-mercaptoethanol, 25 mM β-glycerophosphate and 1 mM Na-orthovanadate). The samples
206
were frozen in liquid nitrogen and sonicated three times at 1°C for 25 s. Cell extracts were stored
AC C
EP
TE D
M AN U
SC
190
9
ACCEPTED MANUSCRIPT
at −80 °C until further analyses [17]. The activity of MPF activity was assessed by the CDC2
208
kinase activity assay using the MESACUP CDC2 kinase assay kit (MBL; Madrid, Spain)
209
according to the manufacturer's specifications. Oocyte extracts (5 µL) were mixed with 10×
210
CDC2 Reaction Buffer (25 mM Hepes buffer pH 7.5 and 10 mM MgCl2) and 10% biotinylated
211
MV Peptide (SLYSSPGGAYC). The phosphorylation reaction was initiated by adding 0.1 mM
212
ATP (Sigma–Aldrich) in a final volume of 50 µL. The mixture was incubated at 30°C for 30min.
213
The reaction was terminated by adding 200 µL of phosphorylation Stop Reagent (PBS
214
containing 50 mM EGTA). The phosphorylated MV peptide content was detected by the
215
colorimetric ELISA reaction at 492 nm and expressed as OD (optical density) units [17].
216
2. 7. Data analysis
217
The variables determined in this study were analyzed by one-way analysis of varaince (ANOVA)
218
and the Tukey test for comparisons of mean values with a significant main effect. All
219
percentages that did not meet the criteria of the binomial model of variable distribution and
220
uniformity were transformed by the arcsine transformation to achieve normal distribution prior to
221
ANOVA. P values <0.05 were considered statistically significant. Data are presented as mean ±
222
SEM, in Table 3 total number of blastomeres are presented as number.
223
3. Results
224
3.1. In vitro maturation
225
Ovine oocytes at different stages of in vitro maturation are shown in Fig. 1. Table 2 summarizes
226
the proportions of ovine oocytes attaining different stages of nuclear maturation after the 24-h
227
period of co-culture with or without ampulla oviductal epithelial cells. The percentage of oocytes
228
that reached metaphase II (M-II) was greater (P<0.05) for G1(ewe oocytes co-matured with
229
AAE) compared with all other groups.Oocytes obtained from ewe lambs and co-cultured with
AC C
EP
TE D
M AN U
SC
RI PT
207
10
ACCEPTED MANUSCRIPT
AAE (G2) had a significantly higher percentage of oocytes arrested at early M-II compared with
231
prepubertal oocytes co-incubated with PAE (G3). The percentages of oocytes arrested at
232
germinal vesicle (GV) and germinal vesicle breakdown (GVBD) were greater (P<0.05) in G3
233
compared with all other groups. Finally, groups G2, G3 and C2 (oocytes from ewe lambs
234
matured without oviductal epithelial cells) showed the greater (P<0.05) proportion of oocytes
235
arrested at metaphase I (M-I) compared with G1 and G4 (ewe oocytes + PAE).
236
3.2. Embryo development
237
The total number of embryos that reached the blastocyst stage was greatest (P<0.05) when adult
238
ewe oocytes were co-cultured during IVM with oviductal epithelial cells recovered from adult
239
ewe oviducts (P < 0.05). The percentage of cleaved zygotes as well as the proportion of embryos
240
that reached the blastocyst stage in G2 was significantly higher compared with G3 and C2. The
241
percentage of cleaved zygotes did not vary among the groups of oocytes retrieved from adult
242
donors (G1, G4, and C1). There were no significant differences in the percentage of embryos
243
reaching the blastocyst between in G4 and C1. Oocytes recovered from adult donors and co-
244
cultured with ampullary epithelial cells (G1) geve rise to blastocysts with a significantly (P<
245
0.05) higher number of cells than G4, G2, G3, C1 and C2 oocytes. In addition, the total cell
246
number was significantly greater for G2 compared with G3 and C2 blastocysts.
247
3. 3. MPF activity
248
At collection time, there were no differences in MPF activity between G1, G4 and C1 nor
249
between G2, G3 and C2 groups of oocytes. MPF activity in oocytes collected from ewes (G1, G4
250
and C1) was greater (P<0.05) compared with that in prepubescent oocytes (G2, G3 and C2).
251
Following the 24-h IVM period, there were no still on differences in MPF activity between G1,
AC C
EP
TE D
M AN U
SC
RI PT
230
11
ACCEPTED MANUSCRIPT
G4 and C1 and it was greater (P<0.05) than that in G2, G3 and C2. However, G2 oocytes
253
(incubated with AAE) exceeded other prepubertal groups of oocytes (G3 and C2) the activity.
254
4. Discussion
255
The present study was carried out to determine the potential benefits of the co-culture system
256
using ampulla oviductal epithelial cells during the IVM stage on the developmental competence
257
of ovine oocytes. Our results indicate that co-incubation of ovine oocytes collected from ewes or
258
ewe lambs with adult oviductal cells results in significantly greater numbers of oocytes reaching
259
the M-II stage and blastocysts formation. Additionally, the resultant blastocysts contained more
260
healthy blastomeres. However, no improvement was noted when oocytes obtained from ewe
261
lambs were matured with oviductal cells obtained also from prepubescent animals. The
262
ampullary epithelial cells from sexually mature ewes could produce specific trophic factors that
263
enhanced MPF activity during oocyte maturation. In a previous study from our
264
laboratoryDadashpour Davachi et al. [15], the addition of adult ampulla oviductal epithelial cells
265
to IVM media substantially improved ZP hardening, reduced the incidence of polyspermy and
266
enhanced the hatchability of in vitro produced blastocysts. Several earlier studies have shown
267
that oocytes recovered from prepubertal animals have reduced meiotic and developmental
268
competence [7, 18, 19], and it was confirmed in the present study. However, a large proportion
269
of oocytes obtained from prepubertal ewe lambs that had been co-cultured with adult oviductal
270
epithelial cells resumed meiosis and produced significantly more blastocysts compared with the
271
prepubertal oocytes matured without oviductal cell clusters. This betterment in the
272
developmental competence of oocytes from sexually immature donors was clearly associated
273
with a rise in MPF activity. Our results showed that the use of the co-culture system with
274
conspecific adult oviductal epithelial cells during IVM could improve the development of
AC C
EP
TE D
M AN U
SC
RI PT
252
12
ACCEPTED MANUSCRIPT
275
prepubertal ovine oocytes and boost the efficiency of IVEP utilizing oocytes from sexually
276
mature animals. Crozet et al. [20] reported a progressive increase in the proportion of blastocysts obtained
278
from oocytes recovered from follicles 2-3 mm (6%),3.1-5 mm (12%), >5 mm in diameter (26%),
279
and ovulated oocytes (41%) in sexually mature goats. In prepubertal goats, the greatest
280
percentage of blastocysts (12%) was obtained from oocytes larger than 135 µm in diameter. It
281
has been observed, however, that oocytes from prepubertal goats are recovered mostly from
282
follicles that are 2-3 mm in size [8]. Prepubertal goat oocytes had significantly diminished
283
developmental competence and MPF activity that increased after IVM only in the largest
284
oocytes; therefore it was concluded that oocyte competence was related primarily to their size.
285
The present results suggest that the physiological status of donor animals and IVM culture
286
conditions are both very important factors determining the efficacy of IVEP in sheep. Not only
287
had the addition of adult oviductal cells to IVM culture media increased the rate of blastocyst
288
formation in vitro but it also improved their quality (increased total cell numbers). Similar trend
289
was observed for adult and prepubertal sheep oocytes, although the most significant
290
improvement was seen in G2 group (prepubertal sheep oocytes co-incubated with adult oviductal
291
cells).
SC
M AN U
TE D
EP
Some previous studies concluded that GV-oocytes do not possess MPF activity [21].
AC C
292
RI PT
277
293
However, Catalá et al. [17] demonstrated that there existed MPF activity in oocytes prior to
294
IVM, similar to our present findings. Catalá et al. [17] proposed that the observed MPF activity
295
of oocytes before IVM might be due to the experimental procedure that caused a delay in
296
performing the MPA activity assay. Since the oocytes could restart meiosis within the 30-min
297
interval, it can be assumed that in the present experiment MPF activity of pre-incubated oocytes
13
ACCEPTED MANUSCRIPT
there was a 2.2-fold increase in MPF activity after IVM of oocytes obtained from ewe lambs and
299
that increase was seen also in the control group of oocytes (matured without oviductal cells) and
300
oocytes incubated with prepubertal sheep oviductal cells. However, an increment in MPF activity
301
following the IVM of lamb oocytes with adult ampulla oviductal epithelial cells was nearly 4-
302
fold. Based on these observations, it can be concluded that oviductal epithelial cells obtained
303
from ewe lambs had limited potential to support oocyte maturation during IVM. Adult sheep
304
oocytes exposed to adult ampulla oviductal cells all exhibited a similar increment in MPF
305
activity post-IVM (approximately 1.9-fold). This would suggestthat oocytes with elevated MPF
306
activity before IVM do not respond to “stimulation” by oviductal cells as they have likely
307
reached the optimal developmental competence.
M AN U
SC
RI PT
298
In summary, it is evident that IVM culture conditions (i.e., co-culture with somatic cells)
309
have a strong influence on oocyte development and fertilization potential, particularly for
310
oocytes obtained from ewe lambs. Beneficial effects of conspecific adult ampulla oviductal
311
epithelial on in vitro matured ovine oocytes may be employed to improve the efficiency of IVEP
312
in this species. A substantial increase in MPF activity post-IVM in prepubertal sheep oocytes co-
313
cultured with AAE could be responsible for the improved rate of blastocyst development.
314
Conflicts of interest
315
The authors have no conflict of interest to declare.
AC C
EP
TE D
308
14
ACCEPTED MANUSCRIPT
References [1] Revel F, Mermillod P, Peynot N, Renard JP, Heyman Y. Low developmental capacity of in
319
vitro matured and fertilized oocytes from calves compared with that of cows. J Reprod Fertil.
320
1995;103:115-20.
321
[2] O'Brien JK, Dwarte D, Ryan JP, Maxwell WM, Evans G. Developmental capacity, energy
322
metabolism and ultrastructure of mature oocytes from prepubertal and adult sheep. Reprod Fertil
323
Dev. 1996;8:1029-37.
324
[3] Peters JK, Milliken G, Davis DL. Development of porcine embryos in vitro: effects of culture
325
medium and donor age. J Anim Sci. 2001;79:1578-83.
326
[4] Barnes FL, Sirard MA. Oocyte maturation. Semin Reprod Med. 2000;18:123-31.
327
[5] Gilchrist RB, Nayudu PL, Nowshari MA, Hodges JK. Meiotic competence of marmoset
328
monkey oocytes is related to follicle size and oocyte-somatic cell associations. Biol Reprod.
329
1995;52:1234-43.
330
[6] Salamone DF, Damiani P, Fissore RA, Robl JM, Duby RT. Biochemical and developmental
331
evidence that ooplasmic maturation of prepubertal bovine oocytes is compromised. Biol Reprod.
332
2001;64:1761-8.
333
[7] Anguita B, Jimenez-Macedo AR, Izquierdo D, Mogas T, Paramio MT. Effect of oocyte
334
diameter on meiotic competence, embryo development, p34 (cdc2) expression and MPF activity
335
in prepubertal goat oocytes. Theriogenology. 2007;67:526-36.
336
[8] Brevini TA, Vassena R, Francisci C, Gandolfi F. Role of adenosine triphosphate, active
337
mitochondria, and microtubules in the acquisition of developmental competence of
338
parthenogenetically activated pig oocytes. Biol Reprod. 2005;72:1218-23.
AC C
EP
TE D
M AN U
SC
RI PT
316 317 318
15
ACCEPTED MANUSCRIPT
[9] Pisani LF, Antonini S, Pocar P, Ferrari S, Brevini TA, Rhind SM, et al. Effects of pre-mating
340
nutrition on mRNA levels of developmentally relevant genes in sheep oocytes and granulosa
341
cells. Reproduction. 2008;136:303-12.
342
[10] Salhab M, Tosca L, Cabau C, Papillier P, Perreau C, Dupont J, et al. Kinetics of gene
343
expression and signaling in bovine cumulus cells throughout IVM in different mediums in
344
relation to oocyte developmental competence, cumulus apoptosis and progesterone secretion.
345
Theriogenology. 2011;75:90-104.
346
[11] Wells D, Patrizio P. Gene expression profiling of human oocytes at different maturational
347
stages and after in vitro maturation. Am J Obstet Gynecol. 2008;198:455 e1-9; discussion e9-11.
348
[12] Dadashpour Davachi N, Zare Shahneh A, Kohram H, Zhandi M, Dashti S, Shamsi H, et al.
349
In vitro ovine embryo production: the study of seasonal and oocyte recovery method effects. Iran
350
Red Crescent Med J. 2014;16:e20749.
351
[13] Dadashpour Davachi N, Zeinoaldini S, Kohram H. A novel ovine oocyte recovery method
352
from slaughterhouse material. Small Ruminant Research. 2012;106:168-72.
353
[14] Dadashpour Davachi N, Kohram H, Zainoaldini S. Cumulus cell layers as a critical factor in
354
meiotic competence and cumulus expansion of ovine oocytes. Small Rumin Res. 2012;102:37-
355
42.
356
[15] Dadashpour Davachi N, Zare Shahneh A, Kohram H, Zhandi M, Shamsi H, Hajiyavand
357
AM, et al. Differential influence of ampullary and isthmic derived epithelial cells on zona
358
pellucida hardening and in vitro fertilization in ovine. Reprod Biol. 2016;16:61-9.
359
[16] Hansen PJ, Ortega SM. In Vitro Production Protocol. In: Hansen PJ, editor. In Vitro
360
Production of Bovine Embryos. Florida, USA: University of Florida; 2014.
361
[17] Catalá MG, Izquierdo D, Uzbekova S, Morató R, Roura M, Romaguera R, et al. Brilliant
362
Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-
AC C
EP
TE D
M AN U
SC
RI PT
339
16
ACCEPTED MANUSCRIPT
promoting factor activity and embryo developmental competence in prepubertal sheep.
364
Reproduction. 2011;142:517-27.
365
[18] Martino A, Mogas T, Palomo MJ, Paramio MT. Meiotic competence of prepubertal goat
366
oocytes. Theriogenology. 1994;41:969-80.
367
[19] Blondin P, Sirard MA. Oocyte and follicular morphology as determining characteristics for
368
developmental competence in bovine oocytes. Mol Reprod Dev. 1995;41:54-62.
369
[20] Crozet N, Ahmed-Ali M, Dubos MP. Developmental competence of goat oocytes from
370
follicles of different size categories following maturation, fertilization and culture in vitro. J
371
Reprod Fertil. 1995;103:293-8.
372
[21] Dedieu T, Gall L, Crozet N, Sevellec C, Ruffini S. Mitogen-activated protein kinase activity
373
during goat oocyte maturation and the acquisition of meiotic competence. Mol Reprod Dev.
374
1996;45:351-8.
375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391
Figure legends:
TE D
M AN U
SC
RI PT
363
AC C
EP
Figure 1. (A) A yellow circle shows germinal vesicle (GV); a red circle shows germinal vesicle break down (GVBD) of G3 oocyte; at the centre of the figure a fertilized egg that attained the blastocyst stage is depicted. Note the cell numbers and a small diameter of a blastocyst that is approximately the size of unfertilized G3 oocytes. (B) A white arrow indicates the metaphase-I (M-I) stage of the oocyte following the 24-h IVM period(G3). (C) A white arrow points at the chromosomes arrested at early metaphase-II (M-II) and a yellow arrow indicates the first extruded polar body (G3). (D) A green circle indicates the M-I stage, a white arrow indicates chromosomes arrested at early M-II and a yellow arrow shows the first extruded polar body(G3). (E) A red arrow indicates an M-II stage oocyte (nucleus) and a yellow arrow indicates an unextruded first polar body (G3). (F) An M-II stage oocyte (nucleus: red arrow, and first polar body: a yellow arrow, are seen in the ooplasm; G3). Figure 2. (A) A cleaved embryo 18 h post fertilization; yellow arrows denote the first and second polar bodies(G3). Blastocyst 7 days post-fertilization (B-C1, C-C2, D-G1 and E-G3; note the differences in the numbers of blastomeres among different group blastocysts.
17
ACCEPTED MANUSCRIPT
Table 1. Description of experimental groups based on the sources of ovine oocytes and ampulla oviductal epithelial cells incubated throughout the 24-h in vitro maturation period. Biological material/Group
Control 1 (C1)
Control 2 (C2)
-
-
2-3−year old LB ewes
2-3−month old LB ewe lambs
AC C
EP
TE D
M AN U
SC
RI PT
Group 1 Group 2 Group 3 Group 4 (G1) (G2) (G3) (G4) 2-3−year 2-3−year 2-3−month 2-3−month Oviducta old LB old LB old LB ewe old LB ewe ewes ewes lambs lambs 2-3−year 2-3−month 2-3−month 2-3−year Ovaryb old LB old LB ewe old LB ewe old LB ewes lambs lambs ewes LB -Lory-Bakhtiary breed a as the source of ampulla oviductal epithelial cells b as the source of oocytes
ACCEPTED MANUSCRIPT
RI PT
Table 2. Percentages of ovine oocytes attaining various stages of nuclear maturation during the 24-h in vitro maturation period. Abbreviations used are as follows-G1: Group 1 (adult ampulla oviductal epithelial cells (AAE) + adult ewe oocytes); G2:Group 2 (AAE + lamb oocytes); G3: Group 3 (prepubertal sheep ampulla oviductal epithelial cells (PAE) + lamb oocytes); G4: Group 4 (PAE + adult ewe oocytes; C1: Control 1 (AAE); C2: Control 2 (PAE); COCs: cumulusoophorus complexes; GV: germinal vesicle; GVBD: germinal vesicle breakdown; M-I: metaphase I; and M-II: metaphase-II.
No. of GVBD Arrested at early GV (%) M-I (%) M-II % COCs (%) M-II (% ) a a a a 101 2.0± 0.1 0.9±0.01 7.9±0.1 4.0±0.1 85.1±2.0a G1 102 9.8±1.0b 20.6±1.0b 13.7±1.0b 11.8±1.0b 40.2±1.3b G2 c c b c 99 36.4±1.1 33.3±1.1 14.1±1.0 0.0 16.2±0.9c G3 9.2±1.0a 5.1±0.9a 75.5±1.9d 98 3.1±0.09a 7.1±0.7d G4 a d a a 6.7±1.0 9.5±1.0 4.8±0.9 73.3±1.7d 105 1.9±0.0 C1 102 36.3±1.0c 34.3±1.4c 12.7±0.9b 0.0c 16.7±0.8c C2 a-d Different letter superscripts in each column denote significant differences between mean values
AC C
EP
TE D
M AN U
SC
Group/Variables
ACCEPTED MANUSCRIPT
Table 3.A summary of the embryo development data. G1: Group 1 (adult ampulla oviductal epithelial cells (AAE) + adult ewe oocytes); G2: Group 2 (AAE + lamb oocytes); G3: Group 3 (prepubertal sheep ampulla oviductal epithelial cells (PAE) + lamb oocytes); G4: Group 4 (PAE + adult ewe oocytes; C1: Control 1 (AAE); C2: Control 2 (PAE). Total number of Cleaved zygotes Blastocyst Inseminated (%) (%) blastomeres (N) COC a a 101 89.9±1.8 42.2±1.1 130.3±7.8a G1 b b 99 74.2±1.3 21.2±1.0 70.2±3.5b G2 c c 102 66.7±1.2 8.0±0.8 49.7±1.7c G3 98 81.6±1.1a 30.1±1.3d 98.9±4.6d G4 a d 103 80.8±1.4 30.5±1.2 94.34±4.1d C1 101 65.7±1.0c 8.3±1.0c 49.67±2.0c C2 a-d Different letter superscripts in each column denote significant differences between mean values
AC C
EP
TE D
M AN U
SC
RI PT
Group/Variables
ACCEPTED MANUSCRIPT
RI PT
Table 4. Maturation-promoting factor (MPF) activity (expressed as optical density units) before and after IVM of ovine oocytes (n=90 oocytes per group). G1: Group 1 (adult ampulla oviductal epithelial cells (AAE) + adult ewe oocytes); G2: Group 2 (AAE + lamb oocytes); G3: Group 3 (prepubertal sheep ampulla oviductal epithelial cells (PAE) + lamb oocytes); G4: Group 4 (PAE + adult ewe oocytes; C1: Control 1 (AAE); C2: Control 2 (PAE). Total MPF activity MPF activity MPF activity ratio COC before IVM after IVM (after/before IVM) 90 0.89±0.06a 1.67±0.10a 1.87 G1 90 0.35±0.04b 1. 27±0.08b 3.62 G2 90 0.34±0.05b 0.75±0.07c 2.20 G3 90 0.87±0.05 a 1.62±0.09a 1.86 G4 a a 90 0.85±0.05 1.61±0.09 1.89 C1 90 0.35±0.04 b 0.78±0.08c 2.22 C2 a-c Different letter superscripts in each column denote significant differences between mean values
AC C
EP
TE D
M AN U
SC
Group/Variables
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
1- This is the first study that led to improvement in the maturation promoting activity in oocytes obtained from prepubertal donors. 2- The coculture system during IVM, using ampullary epithelial cells has a great potential to improve oocyte competence.
AC C
EP
TE D
M AN U
SC
RI PT
3- Oocyte obtained from prepubertal donors showed a great improvement in terms of nuclear maturation, in vitro fertilization and early embryo development.
S0093-691X(16)30451-4
DOI:
10.1016/j.theriogenology.2016.09.034
Reference:
THE 13833
To appear in:
Theriogenology
Please cite this article as: Davachi ND, Kohram H, Shahneh AZ, Zhandi M, Fallahi R, Masoudi R, Yousefi AR, Bartlewski PM, The effect of conspecific ampulla oviductal epithelial cells during in vitro maturation on oocyte developmental competence and maturation-promoting factor (MPF) activity in sheep, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.09.034. 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
Title: The effect of conspecific ampulla oviductal epithelial cells during in vitro maturation on
3
oocyte developmental competence and maturation-promoting factor (MPF) activity in sheep
5
RI PT
4
Short title: Oviductal epithelial cells affect ovine oocyte maturation in vitro
6 1, 2, *
, Hamid Kohram 2, Ahmad Zare Shahneh 2, Mahdi
Authors: Navid Dadashpour Davachi
8
Zhandi2, Rouzbeh Fallahi 1, Reza Masoudi 2, Ali Reza Yousefi 1, 2, Pawel M. Bartlewski 3, *
9
1
SC
7
M AN U
Department of Research, Breeding and Production of Laboratory Animals, Razi Vaccine and
10
Serum Research Institute, Agricultural Research, Education and Extension Organization
11
(AREEO), Karaj, Iran
12
2
13
University of Tehran, Karaj, Iran
14
3
15
Guelph, ON, Canada.
TE D
Department of Animal Science, Faculty College of Agriculture and Natural Resources,
Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph,
EP
16
*Corresponding authors: 1Department of Research, Breeding and Production of Laboratory
18
Animals, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and
19
Extension Organization (AREEO), Karaj, Iran; Tel.: +98 912 344 6217; Facsimile: +98 26
20
34502805. E-mail: [email protected]; Department of Biomedical Sciences, Ontario
21
Veterinary College, University of Guelph, Veterinary College, University of Guelph, Guelph,
22
ON, Canada; Tel: +01 519 824 4120 (ext. 53330); Facsimile: +01 519 767 1548; E-mail:
23
[email protected]
AC C
17
1
ACCEPTED MANUSCRIPT
Abstract
25
The acquisition of fertilization ability by oocytes is one of the prerequisites for successful in
26
vitro embryo production (IVEP). In the present study, we examined the influence of conspecific
27
ampulla oviductal epithelial cells incubated with cumulus-oocyte complexes (COCs) throughout
28
the in vitro maturation (IVM) phase on the developmental competence and maturation-
29
promoting factor (MPF) activity of sheep oocytes. There were six experimental groups in this
30
study, namely four groups with and two groups without oviductal epithelial cells added to IVM
31
media: adult COCs matured in vitro with the ampulla oviductal epithelial cells obtained from
32
adult (AAE; G1) or prepubertal donors (PAE; G4), COCs obtained from prepubertal animals co-
33
cultured with AAE (G2) or PAE (G3), and adult (C1) and prepubertal sheep COCs (C2) matured
34
without oviductal epithelial cells. Co-incubation of oocytes retrieved from both adult and
35
sexually immature donors with AAE (G1 and G2) resulted in significantly improved rates of
36
metaphase-II (M-II) attainment and blastocyst formation (G1: 42.2±1.1 and G2: 21.2±1.0) as
37
well as blastocyst development (total cell count; G1: 130.3±7.8, G2: 70.2±3.5, C1: 94.34±4.1,
38
and C2: 49.67±2.0) compared with their respective controls (C1: 30.5±1.2 and C2: 8.3±1.0).
39
Prior to IVM, the activity of MPF was greater (P<0.05) for oocytes obtained from ewes (G1, G4
40
and C1) compared with those from ewe lambs (G2, G3 and C2). The greatest increment in MPF
41
activity was recorded in G2 (MPF activity before IVM/MPF activity after IVM=3.62) followed
42
by C2 and G3 (2.22 and 2.20, respectively), and then all remaining groups of oocytes (C1: 1.89,
43
G1: 1.87, and G4: 1.86). In summary, co-incubation with AAE during the 24-hr IVM had a
44
positive impact on ovine oocyte competence and ensuing IVEP efficiency. A significant increase
45
in MPF activity following IVM of G2 oocytes could be responsible, at least partly, for the
46
improved rate of blastocyst formation after in vitro fertilization of prepubertal sheep oocytes.
AC C
EP
TE D
M AN U
SC
RI PT
24
2
ACCEPTED MANUSCRIPT
Keywords: Sheep, oviductal epithelial cells, in vitro maturation, maturation promoting factor
48
1. Introduction
49
Oocyte source and quality pre-determine the outcome of in vitro embryo production (IVEP) in
50
mammalian species. The use of prepubertal animals as oocyte donors has invariably been
51
associated with a decrease in the efficiency of IVEP in several livestock species including cattle
52
[1], sheep [2], and pigs [3]. Abnormal cytoplasmic maturation and reduced ability to achieve the
53
blastocyst stage post-fertilization are the primary deficiencies observed during IVEP procedures
54
that utilize oocytes obtained from prepubertal animals as compared to those recovered from
55
sexually mature donors. Both the follicle size and oocyte diameter are associated with oocyte
56
competence and embryo development [4, 5]. However, the use of oocytes from prepubescent
57
donors for embryo production and transfer is highly beneficial as shortening the inter-generation
58
interval can maximize the genetic improvement of farmed animals [2, 6].
M AN U
SC
RI PT
47
Maturation-promoting factor (MPF) is a heterodimer protein composed of the catalytic
60
and regulatory subunits that plays a critical role during meiosis and mitosis [7]. Cyclin-
61
dependent kinase 1 (CDK1), also known as CDC2, is a member of the Ser/Thr protein kinase
62
family. This protein is a catalytic subunit of the highly conserved MPF complex, which is
63
essential for G1/S and G2/M phase transitions of the eukaryotic cell cycle. Mitotic cyclins bind
64
stably to the catalytic MPF subunit and function as regulatory subunits; the interaction between
65
these two subunits is necessary for the control of protein kinase activity. The accumulation and
66
destruction of the cyclins as well as their phosphorylation and dephosphorylation regulate the
67
kinase activity throughout the cell cycle. MPF activity rises initially just before germinal vesicle
68
breakdown (GVBD), increases until metaphase-I (M-I), decreases in the anaphase-telophase
69
stage, and reaches a maximum level at metaphase-II (M-II). MPF activity in oocytes obtained
AC C
EP
TE D
59
3
ACCEPTED MANUSCRIPT
from heifer calves and ewe lambs was significantly lower compared with that measured in cows’
71
and ewes’ oocytes [6] and, in contrast to mammalian species, MPF activity of prepubertal
72
oocytes was significantly higher than that of adult oocytes in mice [7]. Substantially higher MPF
73
activity and developmental competence of oocytes >135 µm in diameter was observed in
74
prepubertal goatsAnguita et al. [7]. From all these indications, MPF activity could be a pivotal
75
factor behind oocyte developmental competence.
RI PT
70
Oocyte competence is acquired during its growth when the synthesis and storage of RNA
77
and proteins take place [8]. Oocyte mRNA content is affected by several factors such as nutrition
78
[9], composition of IVM culture media [10], an array of in vivo and in vitro conditions [11],
79
oocyte recovery method [12, 13], and quality and quantity of the cumulus-oophorous cell layers
80
[14]. In a recent study [15], an application of the new co-culture strategy during the IVM stage
81
has led to improvements in several aspects of oocyte competence, zona pellucida (ZP)
82
maturation, reduction of polyspermy, and an overall IVEP yield.
TE D
M AN U
SC
76
The central role of the oviduct as the site of gamete storage, fertilization and early
84
embryogenesis is unequivocal. Oviducts consist of three major segments, namely the
85
infundibulum, ampulla and isthmus. The three oviductal regions play the specialized and critical
86
role in gamete physiology, fertilization and preimplantation embryo development. Oviductal
87
epithelial cells harvested from the ampullary region affect ZP hardening in vitro [15], whereas
88
the epithelial cells obtained from the isthmic segment mainly influence the rate of early embryo
89
development [16]. We hypothesized that a co-culture system using oocytes and ampulla
90
oviductal epithelial cells would lead to an improvement in IVM conditions. Such an
91
improvement may, in turn, pave the way to the increased IVEP efficiency, especially when
92
prepubertal animals are used as oocyte donors. Therefore, the primary aim of this study was to
AC C
EP
83
4
ACCEPTED MANUSCRIPT
evaluate and compare the effects of ampulla oviductal epithelial cells, from prepubescent and
94
sexually mature ewes, added to IVM media on MPF activity and developmental potential of
95
ovine oocytes, also obtained from prepubertal and sexually mature animals.
96
2. Materials and Methods
97
All chemicals were purchased from Sigma-Aldrich Chemical Co. unless otherwise stated. Adult
98
testes and ovaries were obtained from the slaughtered Lory-Bakhtiary sheep aged 2 to 3years,
99
and prepubertal ovaries were recovered from the slaughtered 2- to 3-month-old Lory-Bakhtiary
SC
RI PT
93
ewe lambs.
101
2. 1. Experimental design
102
There were six experimental groups in the present study design; four groups (G1-G4) were
103
considered the “treatment groups” and two were considered the “control groups” (C1 and C2). In
104
G1 and G4, the oocytes were recovered from adult donors and co-cultured with ampulla
105
oviductal epithelial cells obtained from adult (AAE) or prepubertal donors (PAE), respectively.
106
In G2 and G3, the oocytes were recovered from ewe lambs and then co-cultured with the
107
oviductal cells obtained from adult and prepubertal animals, respectively. In the two control
108
groups, the IVM was performed without the addition of oviductal cells; C1 and C2 oocytes were
109
obtained from adult and prepubertal donors, respectively. More details of the present
110
experimental design are given in Table 1.
111
2.2. Preparation of ovine ampulla oviductal epithelial cells
112
Adult (AAE) and prepubertal ampulla oviductal epithelial cells (PAE) were obtained as
113
previously described by Dadashpour Davachi et al. [15]. Briefly, the ovine reproductive tracts
114
were collected immediately after slaughter. In the ewes of the present study, the oviducts
115
ipsilateral to ovaries with visible corpora haemorrhagica (indicative of ovulations occurring
AC C
EP
TE D
M AN U
100
5
ACCEPTED MANUSCRIPT
within the preceding 24 h) were selected for harvesting AAE. Such oviducts were used in this
117
study because they contained epithelial cells that had been exposed to circulating estradiol and
118
due to ovulations were triggered to produce trophic oocyte-nourishing proteins. No ovulations
119
were seen in the 2- to 3-month−old ewe lambs and both oviducts were collected from all
120
prepubertal donors at slaughter. All of the connective tissues were trimmed off and, before
121
washing in phosphate buffered saline without Ca2+/Mg2+/(PBS-), both ends of the oviduct were
122
tightly tied up with sterile surgical sutures. Oviducts were then dipped in 70% ethanol and
123
transported on ice to the laboratory in PBS solution supplemented with 2% penicillin–
124
streptomycin. Following their arrival at the laboratory, the oviducts were dipped again in 70%
125
ethanol and washed in HEPES buffered tissue culture medium (TCM199), in a laminar flow
126
hood. The ampulla was divided into two–three segments and gently squeezed in a stripping
127
motion with forceps to recover epithelial cells. The identification of different segments of the
128
oviduct was based on differences in the oviducts' outer diameter [15]. A yellowish matter
129
containing epithelial cells was collected into the culture medium TCM-199, supplemented with
130
2% estrous cow serum (ECS) and 0.25 mg/mL gentamicin. The cell suspension was pipetted 10
131
to 15 times with a 1000-µL filtered tip (JET Biofil, Guangzhou, China) and then passed 5 times
132
through a 1-mL tuberculin syringe attached to a 22-gauge needle. Following the washing
133
procedure, the cells were evaluated under a stereomicroscope to determine the size of cell
134
clusters; the preferred size of oviductal cell clusters is approximately half of a typical cumulus-
135
oocyte complex (COC) with compact cumulus layers. Finally, thirty AEC/PEC clusters were
136
transferred to 250-µL drops of IVM medium [15].
137
2.3. Ovaries and oocyte recovery
AC C
EP
TE D
M AN U
SC
RI PT
116
6
ACCEPTED MANUSCRIPT
The ovaries were dissected immediately after the slaughter of lambs/ewes and placed in a thermo
139
insulated container filled with PBS, supplemented with penicillin–streptomycin 100 µg/mL
140
(Gibco; Grand Island, NY, USA), at 37°C. The ovaries were transported to the laboratory within
141
2 h of collection. Ovarian antral follicles <4 mm in diameter were aspirated using an aspiration
142
pump (MEDAP Sekretsauger P7040; Tilburg, The Netherlands) fitted with a disposable vacuum
143
line (length-35 cm, the internal diameter of 3 mm). The flow rate was set at 10 mL H2O/min
144
using an attached disposable 20-gauge needle [15]. Ovarian follicles from ewe lambs were
145
aspirated under a stereo microscope; all visible follicles were aspirated.
146
2.4. In vitro maturation (I VM) of oocytes
M AN U
SC
RI PT
138
The TCM199 medium used for IVM was supplemented with 10% heat-inactivated fetal
148
calf serum (FCS) (Cat. Number: F4135), 0.2 mM sodium pyruvate, 5 µg/mL of gentamicin, 10
149
µg/mL of ovine follicle-stimulating hormone (oFSH), and 1 µg/mL of estradiol (basic maturation
150
medium). In the control groups (C1 and C2), thirty COCs from adult or prepubertal donors were
151
cultured in basic maturation medium droplets. In the co-culture IVM system(G1 to G4), thirty
152
COCs from sexually mature or prepubescent donors were matured in a droplet of primary
153
maturation medium (250 µL) along with 30 AAE or PAE clusters. All the droplets were covered
154
with mineral oil and incubated for 24 h at 38.5°C, in 5% CO2 in the air and 95% humidity. The
155
detection of various stages of oocyte maturation was based on the position of polar bodies and
156
chromosome arrangement. The ocytes with circular arrangements of chromosomes and no polar
157
body were considered the M-I phas oocytes; the oocytes with completely extruded polar bodies
158
and parallel arrangement of chromosomes were considered as early M-II stage oocytes; if the
159
polar body did not extrude from cytoplasm and the chromosomes were arranged in a circular
160
aggregation, the oocytes were considered arrested at the M-II phase; and finally, if the polar
AC C
EP
TE D
147
7
ACCEPTED MANUSCRIPT
bodies were extruded from cytoplasm and the chromosomes arranged in circular aggregation, the
162
oocytes were considered the fully developed M-II oocytes.
163
2.5. Sperm preparation, in vitro fertilization (IVF) and in vitro culture (IVC), and staining
164
methods
165
Testicles from some 2- to 3-year-old Lory-Bakhtiary rams were removed at slaughter, placed in a
166
box set chilled to 5°C and transported to the laboratory. Subsequently, the testicles were washed
167
three times in PBS supplemented with 100 µL/mL gentamicin; this and a few following
168
procedures were performed at 5°C in a cold room. Blood and connective tissues were removed
169
aseptically prior to sperm recovery. A 1-mL syringe attached to a 22-gauge needle was inserted
170
into the vas deferens to gently aspirate its entire content, and the recovered spermatozoa were
171
diluted 1:100 in sperm-TALP. Semen was stored in the refrigerator for up to 24 h. On the day of
172
IVF, semen samples were evaluated for progressive motility under a microscope. Motile
173
spermatozoa were separated using the Percoll gradient (45% over 90%) centrifugation according
174
to the method described by Hansen and Ortega et al. [16]. A conical tube containing the semen
175
and Percoll gradient was placed into a tube holder pre-warmed to 38.5°C and centrifuged at 1000
176
× g for 10 min. After centrifugation, a sperm pellet was collected from the bottom of the tube,
177
transferred to a 15-mL conical tube containing 10 mL HEPPES-SOF, and centrifuged again for 5
178
min at 200 × g. The supernatant was then gently removed with a Pasteur pipette. In order to
179
adjust a final sperm concentration to 26 × 106/mL (for a final concentration of sperm in the
180
fertilization drop of 1 × 106/mL), 10 µL of sperm suspension was added to 90 µL water to kill
181
spermatozoa, then 10 µL of the diluted sample loaded onto a haemocytometer, sperm
182
enumerated in 5 squares and the result multiplied by by 500,000 to determine sperm
183
concentration per 1 mL. The spermatozoa were diluted in the fertilization medium that had been
AC C
EP
TE D
M AN U
SC
RI PT
161
8
ACCEPTED MANUSCRIPT
pre-equilibrated in the incubator [16] and contained 12 mM KCL, 25 mM NaHCO3, 90 mM
185
NaCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 10 mM sodium lactate, sodium pyruvate 0.018 g/100
186
mL, CaCl2 0.147 g/100 mL, 3 mg/mL BSA (fatty acid free), and 50 µg/mL gentamicin. At least
187
15 min before the addition of sperm, the oocytes were transferred to fertilization droplets. Lastly,
188
10 µL of pre-incubated sperm solution were added to 90 µL of fertilization medium containing
189
∼20 oocytes.
RI PT
184
Co-incubation of gametes was carried out for 18 h at 38.5°C, in 5% CO2 in the air and
191
95% humidity. After co-incubation, the spermatozoa attached to the ZP were removed by gentle
192
pipetting, and the fertilized oocytes were transferred to synthetic oviductal fluid supplemented
193
with 0.4 g EFAF BSA (SOF-BSA) for the ensuing 7-day incubation (IVC). At the end of the 7-
194
day period, the rate of embryo development to the blastocyst stage was determined. The embryos
195
were removed from the culture medium, washed twice in PBS-PVP (1 mg/mL), and fixed in a
196
100-µL drop of paraformaldehyde solution [4% (w/v) in PBS, pH 7.4] at room temperature.
197
Finally, the embryos were washed three times in PBS-PVP and stained for 15 min in 1 µg/mL
198
Hoechst 33342 before microscopic evaluation using a fluorescent microscope equipped with a
199
UV filter [15].
200
2.6. MPF activity of oocytes
201
Determination of MPF activity utilized the method developed by Catalá et al. [17], with some
202
minor modifications. Before and after IVM, the batches of 30 oocytes (three replicates) were
203
washed three times in PBS and placed in tubes containing 5 µL of lysis buffer (50 mM Tris–
204
HCl, pH 7.5, 0.5 M NaCl, 5 mM EDTA, 0.01% Brij35, 1 mM PMSF, 0.05 mg/mL leupeptin, 50
205
mM 2-mercaptoethanol, 25 mM β-glycerophosphate and 1 mM Na-orthovanadate). The samples
206
were frozen in liquid nitrogen and sonicated three times at 1°C for 25 s. Cell extracts were stored
AC C
EP
TE D
M AN U
SC
190
9
ACCEPTED MANUSCRIPT
at −80 °C until further analyses [17]. The activity of MPF activity was assessed by the CDC2
208
kinase activity assay using the MESACUP CDC2 kinase assay kit (MBL; Madrid, Spain)
209
according to the manufacturer's specifications. Oocyte extracts (5 µL) were mixed with 10×
210
CDC2 Reaction Buffer (25 mM Hepes buffer pH 7.5 and 10 mM MgCl2) and 10% biotinylated
211
MV Peptide (SLYSSPGGAYC). The phosphorylation reaction was initiated by adding 0.1 mM
212
ATP (Sigma–Aldrich) in a final volume of 50 µL. The mixture was incubated at 30°C for 30min.
213
The reaction was terminated by adding 200 µL of phosphorylation Stop Reagent (PBS
214
containing 50 mM EGTA). The phosphorylated MV peptide content was detected by the
215
colorimetric ELISA reaction at 492 nm and expressed as OD (optical density) units [17].
216
2. 7. Data analysis
217
The variables determined in this study were analyzed by one-way analysis of varaince (ANOVA)
218
and the Tukey test for comparisons of mean values with a significant main effect. All
219
percentages that did not meet the criteria of the binomial model of variable distribution and
220
uniformity were transformed by the arcsine transformation to achieve normal distribution prior to
221
ANOVA. P values <0.05 were considered statistically significant. Data are presented as mean ±
222
SEM, in Table 3 total number of blastomeres are presented as number.
223
3. Results
224
3.1. In vitro maturation
225
Ovine oocytes at different stages of in vitro maturation are shown in Fig. 1. Table 2 summarizes
226
the proportions of ovine oocytes attaining different stages of nuclear maturation after the 24-h
227
period of co-culture with or without ampulla oviductal epithelial cells. The percentage of oocytes
228
that reached metaphase II (M-II) was greater (P<0.05) for G1(ewe oocytes co-matured with
229
AAE) compared with all other groups.Oocytes obtained from ewe lambs and co-cultured with
AC C
EP
TE D
M AN U
SC
RI PT
207
10
ACCEPTED MANUSCRIPT
AAE (G2) had a significantly higher percentage of oocytes arrested at early M-II compared with
231
prepubertal oocytes co-incubated with PAE (G3). The percentages of oocytes arrested at
232
germinal vesicle (GV) and germinal vesicle breakdown (GVBD) were greater (P<0.05) in G3
233
compared with all other groups. Finally, groups G2, G3 and C2 (oocytes from ewe lambs
234
matured without oviductal epithelial cells) showed the greater (P<0.05) proportion of oocytes
235
arrested at metaphase I (M-I) compared with G1 and G4 (ewe oocytes + PAE).
236
3.2. Embryo development
237
The total number of embryos that reached the blastocyst stage was greatest (P<0.05) when adult
238
ewe oocytes were co-cultured during IVM with oviductal epithelial cells recovered from adult
239
ewe oviducts (P < 0.05). The percentage of cleaved zygotes as well as the proportion of embryos
240
that reached the blastocyst stage in G2 was significantly higher compared with G3 and C2. The
241
percentage of cleaved zygotes did not vary among the groups of oocytes retrieved from adult
242
donors (G1, G4, and C1). There were no significant differences in the percentage of embryos
243
reaching the blastocyst between in G4 and C1. Oocytes recovered from adult donors and co-
244
cultured with ampullary epithelial cells (G1) geve rise to blastocysts with a significantly (P<
245
0.05) higher number of cells than G4, G2, G3, C1 and C2 oocytes. In addition, the total cell
246
number was significantly greater for G2 compared with G3 and C2 blastocysts.
247
3. 3. MPF activity
248
At collection time, there were no differences in MPF activity between G1, G4 and C1 nor
249
between G2, G3 and C2 groups of oocytes. MPF activity in oocytes collected from ewes (G1, G4
250
and C1) was greater (P<0.05) compared with that in prepubescent oocytes (G2, G3 and C2).
251
Following the 24-h IVM period, there were no still on differences in MPF activity between G1,
AC C
EP
TE D
M AN U
SC
RI PT
230
11
ACCEPTED MANUSCRIPT
G4 and C1 and it was greater (P<0.05) than that in G2, G3 and C2. However, G2 oocytes
253
(incubated with AAE) exceeded other prepubertal groups of oocytes (G3 and C2) the activity.
254
4. Discussion
255
The present study was carried out to determine the potential benefits of the co-culture system
256
using ampulla oviductal epithelial cells during the IVM stage on the developmental competence
257
of ovine oocytes. Our results indicate that co-incubation of ovine oocytes collected from ewes or
258
ewe lambs with adult oviductal cells results in significantly greater numbers of oocytes reaching
259
the M-II stage and blastocysts formation. Additionally, the resultant blastocysts contained more
260
healthy blastomeres. However, no improvement was noted when oocytes obtained from ewe
261
lambs were matured with oviductal cells obtained also from prepubescent animals. The
262
ampullary epithelial cells from sexually mature ewes could produce specific trophic factors that
263
enhanced MPF activity during oocyte maturation. In a previous study from our
264
laboratoryDadashpour Davachi et al. [15], the addition of adult ampulla oviductal epithelial cells
265
to IVM media substantially improved ZP hardening, reduced the incidence of polyspermy and
266
enhanced the hatchability of in vitro produced blastocysts. Several earlier studies have shown
267
that oocytes recovered from prepubertal animals have reduced meiotic and developmental
268
competence [7, 18, 19], and it was confirmed in the present study. However, a large proportion
269
of oocytes obtained from prepubertal ewe lambs that had been co-cultured with adult oviductal
270
epithelial cells resumed meiosis and produced significantly more blastocysts compared with the
271
prepubertal oocytes matured without oviductal cell clusters. This betterment in the
272
developmental competence of oocytes from sexually immature donors was clearly associated
273
with a rise in MPF activity. Our results showed that the use of the co-culture system with
274
conspecific adult oviductal epithelial cells during IVM could improve the development of
AC C
EP
TE D
M AN U
SC
RI PT
252
12
ACCEPTED MANUSCRIPT
275
prepubertal ovine oocytes and boost the efficiency of IVEP utilizing oocytes from sexually
276
mature animals. Crozet et al. [20] reported a progressive increase in the proportion of blastocysts obtained
278
from oocytes recovered from follicles 2-3 mm (6%),3.1-5 mm (12%), >5 mm in diameter (26%),
279
and ovulated oocytes (41%) in sexually mature goats. In prepubertal goats, the greatest
280
percentage of blastocysts (12%) was obtained from oocytes larger than 135 µm in diameter. It
281
has been observed, however, that oocytes from prepubertal goats are recovered mostly from
282
follicles that are 2-3 mm in size [8]. Prepubertal goat oocytes had significantly diminished
283
developmental competence and MPF activity that increased after IVM only in the largest
284
oocytes; therefore it was concluded that oocyte competence was related primarily to their size.
285
The present results suggest that the physiological status of donor animals and IVM culture
286
conditions are both very important factors determining the efficacy of IVEP in sheep. Not only
287
had the addition of adult oviductal cells to IVM culture media increased the rate of blastocyst
288
formation in vitro but it also improved their quality (increased total cell numbers). Similar trend
289
was observed for adult and prepubertal sheep oocytes, although the most significant
290
improvement was seen in G2 group (prepubertal sheep oocytes co-incubated with adult oviductal
291
cells).
SC
M AN U
TE D
EP
Some previous studies concluded that GV-oocytes do not possess MPF activity [21].
AC C
292
RI PT
277
293
However, Catalá et al. [17] demonstrated that there existed MPF activity in oocytes prior to
294
IVM, similar to our present findings. Catalá et al. [17] proposed that the observed MPF activity
295
of oocytes before IVM might be due to the experimental procedure that caused a delay in
296
performing the MPA activity assay. Since the oocytes could restart meiosis within the 30-min
297
interval, it can be assumed that in the present experiment MPF activity of pre-incubated oocytes
13
ACCEPTED MANUSCRIPT
there was a 2.2-fold increase in MPF activity after IVM of oocytes obtained from ewe lambs and
299
that increase was seen also in the control group of oocytes (matured without oviductal cells) and
300
oocytes incubated with prepubertal sheep oviductal cells. However, an increment in MPF activity
301
following the IVM of lamb oocytes with adult ampulla oviductal epithelial cells was nearly 4-
302
fold. Based on these observations, it can be concluded that oviductal epithelial cells obtained
303
from ewe lambs had limited potential to support oocyte maturation during IVM. Adult sheep
304
oocytes exposed to adult ampulla oviductal cells all exhibited a similar increment in MPF
305
activity post-IVM (approximately 1.9-fold). This would suggestthat oocytes with elevated MPF
306
activity before IVM do not respond to “stimulation” by oviductal cells as they have likely
307
reached the optimal developmental competence.
M AN U
SC
RI PT
298
In summary, it is evident that IVM culture conditions (i.e., co-culture with somatic cells)
309
have a strong influence on oocyte development and fertilization potential, particularly for
310
oocytes obtained from ewe lambs. Beneficial effects of conspecific adult ampulla oviductal
311
epithelial on in vitro matured ovine oocytes may be employed to improve the efficiency of IVEP
312
in this species. A substantial increase in MPF activity post-IVM in prepubertal sheep oocytes co-
313
cultured with AAE could be responsible for the improved rate of blastocyst development.
314
Conflicts of interest
315
The authors have no conflict of interest to declare.
AC C
EP
TE D
308
14
ACCEPTED MANUSCRIPT
References [1] Revel F, Mermillod P, Peynot N, Renard JP, Heyman Y. Low developmental capacity of in
319
vitro matured and fertilized oocytes from calves compared with that of cows. J Reprod Fertil.
320
1995;103:115-20.
321
[2] O'Brien JK, Dwarte D, Ryan JP, Maxwell WM, Evans G. Developmental capacity, energy
322
metabolism and ultrastructure of mature oocytes from prepubertal and adult sheep. Reprod Fertil
323
Dev. 1996;8:1029-37.
324
[3] Peters JK, Milliken G, Davis DL. Development of porcine embryos in vitro: effects of culture
325
medium and donor age. J Anim Sci. 2001;79:1578-83.
326
[4] Barnes FL, Sirard MA. Oocyte maturation. Semin Reprod Med. 2000;18:123-31.
327
[5] Gilchrist RB, Nayudu PL, Nowshari MA, Hodges JK. Meiotic competence of marmoset
328
monkey oocytes is related to follicle size and oocyte-somatic cell associations. Biol Reprod.
329
1995;52:1234-43.
330
[6] Salamone DF, Damiani P, Fissore RA, Robl JM, Duby RT. Biochemical and developmental
331
evidence that ooplasmic maturation of prepubertal bovine oocytes is compromised. Biol Reprod.
332
2001;64:1761-8.
333
[7] Anguita B, Jimenez-Macedo AR, Izquierdo D, Mogas T, Paramio MT. Effect of oocyte
334
diameter on meiotic competence, embryo development, p34 (cdc2) expression and MPF activity
335
in prepubertal goat oocytes. Theriogenology. 2007;67:526-36.
336
[8] Brevini TA, Vassena R, Francisci C, Gandolfi F. Role of adenosine triphosphate, active
337
mitochondria, and microtubules in the acquisition of developmental competence of
338
parthenogenetically activated pig oocytes. Biol Reprod. 2005;72:1218-23.
AC C
EP
TE D
M AN U
SC
RI PT
316 317 318
15
ACCEPTED MANUSCRIPT
[9] Pisani LF, Antonini S, Pocar P, Ferrari S, Brevini TA, Rhind SM, et al. Effects of pre-mating
340
nutrition on mRNA levels of developmentally relevant genes in sheep oocytes and granulosa
341
cells. Reproduction. 2008;136:303-12.
342
[10] Salhab M, Tosca L, Cabau C, Papillier P, Perreau C, Dupont J, et al. Kinetics of gene
343
expression and signaling in bovine cumulus cells throughout IVM in different mediums in
344
relation to oocyte developmental competence, cumulus apoptosis and progesterone secretion.
345
Theriogenology. 2011;75:90-104.
346
[11] Wells D, Patrizio P. Gene expression profiling of human oocytes at different maturational
347
stages and after in vitro maturation. Am J Obstet Gynecol. 2008;198:455 e1-9; discussion e9-11.
348
[12] Dadashpour Davachi N, Zare Shahneh A, Kohram H, Zhandi M, Dashti S, Shamsi H, et al.
349
In vitro ovine embryo production: the study of seasonal and oocyte recovery method effects. Iran
350
Red Crescent Med J. 2014;16:e20749.
351
[13] Dadashpour Davachi N, Zeinoaldini S, Kohram H. A novel ovine oocyte recovery method
352
from slaughterhouse material. Small Ruminant Research. 2012;106:168-72.
353
[14] Dadashpour Davachi N, Kohram H, Zainoaldini S. Cumulus cell layers as a critical factor in
354
meiotic competence and cumulus expansion of ovine oocytes. Small Rumin Res. 2012;102:37-
355
42.
356
[15] Dadashpour Davachi N, Zare Shahneh A, Kohram H, Zhandi M, Shamsi H, Hajiyavand
357
AM, et al. Differential influence of ampullary and isthmic derived epithelial cells on zona
358
pellucida hardening and in vitro fertilization in ovine. Reprod Biol. 2016;16:61-9.
359
[16] Hansen PJ, Ortega SM. In Vitro Production Protocol. In: Hansen PJ, editor. In Vitro
360
Production of Bovine Embryos. Florida, USA: University of Florida; 2014.
361
[17] Catalá MG, Izquierdo D, Uzbekova S, Morató R, Roura M, Romaguera R, et al. Brilliant
362
Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-
AC C
EP
TE D
M AN U
SC
RI PT
339
16
ACCEPTED MANUSCRIPT
promoting factor activity and embryo developmental competence in prepubertal sheep.
364
Reproduction. 2011;142:517-27.
365
[18] Martino A, Mogas T, Palomo MJ, Paramio MT. Meiotic competence of prepubertal goat
366
oocytes. Theriogenology. 1994;41:969-80.
367
[19] Blondin P, Sirard MA. Oocyte and follicular morphology as determining characteristics for
368
developmental competence in bovine oocytes. Mol Reprod Dev. 1995;41:54-62.
369
[20] Crozet N, Ahmed-Ali M, Dubos MP. Developmental competence of goat oocytes from
370
follicles of different size categories following maturation, fertilization and culture in vitro. J
371
Reprod Fertil. 1995;103:293-8.
372
[21] Dedieu T, Gall L, Crozet N, Sevellec C, Ruffini S. Mitogen-activated protein kinase activity
373
during goat oocyte maturation and the acquisition of meiotic competence. Mol Reprod Dev.
374
1996;45:351-8.
375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391
Figure legends:
TE D
M AN U
SC
RI PT
363
AC C
EP
Figure 1. (A) A yellow circle shows germinal vesicle (GV); a red circle shows germinal vesicle break down (GVBD) of G3 oocyte; at the centre of the figure a fertilized egg that attained the blastocyst stage is depicted. Note the cell numbers and a small diameter of a blastocyst that is approximately the size of unfertilized G3 oocytes. (B) A white arrow indicates the metaphase-I (M-I) stage of the oocyte following the 24-h IVM period(G3). (C) A white arrow points at the chromosomes arrested at early metaphase-II (M-II) and a yellow arrow indicates the first extruded polar body (G3). (D) A green circle indicates the M-I stage, a white arrow indicates chromosomes arrested at early M-II and a yellow arrow shows the first extruded polar body(G3). (E) A red arrow indicates an M-II stage oocyte (nucleus) and a yellow arrow indicates an unextruded first polar body (G3). (F) An M-II stage oocyte (nucleus: red arrow, and first polar body: a yellow arrow, are seen in the ooplasm; G3). Figure 2. (A) A cleaved embryo 18 h post fertilization; yellow arrows denote the first and second polar bodies(G3). Blastocyst 7 days post-fertilization (B-C1, C-C2, D-G1 and E-G3; note the differences in the numbers of blastomeres among different group blastocysts.
17
ACCEPTED MANUSCRIPT
Table 1. Description of experimental groups based on the sources of ovine oocytes and ampulla oviductal epithelial cells incubated throughout the 24-h in vitro maturation period. Biological material/Group
Control 1 (C1)
Control 2 (C2)
-
-
2-3−year old LB ewes
2-3−month old LB ewe lambs
AC C
EP
TE D
M AN U
SC
RI PT
Group 1 Group 2 Group 3 Group 4 (G1) (G2) (G3) (G4) 2-3−year 2-3−year 2-3−month 2-3−month Oviducta old LB old LB old LB ewe old LB ewe ewes ewes lambs lambs 2-3−year 2-3−month 2-3−month 2-3−year Ovaryb old LB old LB ewe old LB ewe old LB ewes lambs lambs ewes LB -Lory-Bakhtiary breed a as the source of ampulla oviductal epithelial cells b as the source of oocytes
ACCEPTED MANUSCRIPT
RI PT
Table 2. Percentages of ovine oocytes attaining various stages of nuclear maturation during the 24-h in vitro maturation period. Abbreviations used are as follows-G1: Group 1 (adult ampulla oviductal epithelial cells (AAE) + adult ewe oocytes); G2:Group 2 (AAE + lamb oocytes); G3: Group 3 (prepubertal sheep ampulla oviductal epithelial cells (PAE) + lamb oocytes); G4: Group 4 (PAE + adult ewe oocytes; C1: Control 1 (AAE); C2: Control 2 (PAE); COCs: cumulusoophorus complexes; GV: germinal vesicle; GVBD: germinal vesicle breakdown; M-I: metaphase I; and M-II: metaphase-II.
No. of GVBD Arrested at early GV (%) M-I (%) M-II % COCs (%) M-II (% ) a a a a 101 2.0± 0.1 0.9±0.01 7.9±0.1 4.0±0.1 85.1±2.0a G1 102 9.8±1.0b 20.6±1.0b 13.7±1.0b 11.8±1.0b 40.2±1.3b G2 c c b c 99 36.4±1.1 33.3±1.1 14.1±1.0 0.0 16.2±0.9c G3 9.2±1.0a 5.1±0.9a 75.5±1.9d 98 3.1±0.09a 7.1±0.7d G4 a d a a 6.7±1.0 9.5±1.0 4.8±0.9 73.3±1.7d 105 1.9±0.0 C1 102 36.3±1.0c 34.3±1.4c 12.7±0.9b 0.0c 16.7±0.8c C2 a-d Different letter superscripts in each column denote significant differences between mean values
AC C
EP
TE D
M AN U
SC
Group/Variables
ACCEPTED MANUSCRIPT
Table 3.A summary of the embryo development data. G1: Group 1 (adult ampulla oviductal epithelial cells (AAE) + adult ewe oocytes); G2: Group 2 (AAE + lamb oocytes); G3: Group 3 (prepubertal sheep ampulla oviductal epithelial cells (PAE) + lamb oocytes); G4: Group 4 (PAE + adult ewe oocytes; C1: Control 1 (AAE); C2: Control 2 (PAE). Total number of Cleaved zygotes Blastocyst Inseminated (%) (%) blastomeres (N) COC a a 101 89.9±1.8 42.2±1.1 130.3±7.8a G1 b b 99 74.2±1.3 21.2±1.0 70.2±3.5b G2 c c 102 66.7±1.2 8.0±0.8 49.7±1.7c G3 98 81.6±1.1a 30.1±1.3d 98.9±4.6d G4 a d 103 80.8±1.4 30.5±1.2 94.34±4.1d C1 101 65.7±1.0c 8.3±1.0c 49.67±2.0c C2 a-d Different letter superscripts in each column denote significant differences between mean values
AC C
EP
TE D
M AN U
SC
RI PT
Group/Variables
ACCEPTED MANUSCRIPT
RI PT
Table 4. Maturation-promoting factor (MPF) activity (expressed as optical density units) before and after IVM of ovine oocytes (n=90 oocytes per group). G1: Group 1 (adult ampulla oviductal epithelial cells (AAE) + adult ewe oocytes); G2: Group 2 (AAE + lamb oocytes); G3: Group 3 (prepubertal sheep ampulla oviductal epithelial cells (PAE) + lamb oocytes); G4: Group 4 (PAE + adult ewe oocytes; C1: Control 1 (AAE); C2: Control 2 (PAE). Total MPF activity MPF activity MPF activity ratio COC before IVM after IVM (after/before IVM) 90 0.89±0.06a 1.67±0.10a 1.87 G1 90 0.35±0.04b 1. 27±0.08b 3.62 G2 90 0.34±0.05b 0.75±0.07c 2.20 G3 90 0.87±0.05 a 1.62±0.09a 1.86 G4 a a 90 0.85±0.05 1.61±0.09 1.89 C1 90 0.35±0.04 b 0.78±0.08c 2.22 C2 a-c Different letter superscripts in each column denote significant differences between mean values
AC C
EP
TE D
M AN U
SC
Group/Variables
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
1- This is the first study that led to improvement in the maturation promoting activity in oocytes obtained from prepubertal donors. 2- The coculture system during IVM, using ampullary epithelial cells has a great potential to improve oocyte competence.
AC C
EP
TE D
M AN U
SC
RI PT
3- Oocyte obtained from prepubertal donors showed a great improvement in terms of nuclear maturation, in vitro fertilization and early embryo development.