Effects of co-incubation with conspecific ampulla oviductal epithelial cells and media composition on cryotolerance and developmental competence of in vitro matured sheep oocytes

Theriogenology 120 (2018) 10e15

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Effects of co-incubation with conspecific ampulla oviductal epithelial cells and media composition on cryotolerance and developmental competence of in vitro matured sheep oocytes Navid Dadashpour Davachi a, *, Roozbeh Fallahi a, Essa Dirandeh b, Xinyu Liu c, Pawel M. Bartlewski d a Department of Research, Breeding and Production of Laboratory Animals, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran b Department of Animal Science, Sari Agricultural Sciences and Natural Resources University, Sari, Mazandaran, P.O.BOX:578, Iran c 204 Shenyang Hospital, Shenyang, Liaoning, PR China d Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 May 2018 Received in revised form 4 July 2018 Accepted 27 July 2018 Available online 29 July 2018

Developmental potential of cryopreserved in vitro matured oocytes is very low in nearly all mammalian species studied to date. Despite relatively high cleavage rates, the vitrified/warmed metaphase II oocytes have a decreased rate of blastocyst formation, which can be attributed to the elevated cytoplasmic lipid content and lipid droplet fragmentation. Secretory products of ampulla oviductal epithelial cells (AECs) at the periovulatory stage of the ovarian cycle enhance the viability of in vitro matured oocytes. The present study was undertaken to determine if co-culture of cumulus-oophorus complexes (COCs) with conspecific AECs or reducing the lipid content of in vitro matured ovine oocytes would improve their cryotolerance and ensuing developmental competence. Ovine COCs aspirated from the slaughterhouse ovaries were matured in the following media or culture conditions: TCM199 þ FBS þ AECs (T1); TCM199 þ FBS (T2); TCM199 þ BSA (T3); TCM199 þ 0.6 mg/mL of L-carnitine (T4); TCM199þ Lcarnitine þ FBS (T5), or TCM199 only (Ctr). Subsequently, the oocytes were vitrified and used for in vitro fertilization (IVF). The lowest degree of zona pellucida (ZP) hardening following vitrification of in vitro matured sheep oocytes was observed in T1 and T5 (P < 0.05). Cleavage, blastocyst formation and ensuing development (i.e., total cell numbers) as well as blastocyst hatching rates were all greater (P < 0.05) in T1 compared with the remaining groups; in vitro matured COCs in T4 and Ctr did not develop beyond the cleavage stage. The inner cell mass: trophectoderm cell ratio in T1 (1:3.29) was significantly greater compared with T2 (1:3.39), T3 (1:3.40) and T5 (1:3.44). The present results indicate that the ovine COCs/ AECs co-culture system had the most positive influence on cryotolerance, ZP hardening, and developmental competence of in vitro matured oocytes. © 2018 Elsevier Inc. All rights reserved.

Keywords: Oocytes Vitrification Oviductal epithelial cells Zona pellucida in vitro embryo production Sheep

1. Introduction The prospect of irreversibly losing ovarian function (women) and the genetic gain or salvage due to long-term storage and transfer (domestic livestock and endangered animal species) appear to be the most compelling reasons for oocyte freezing [1,2]. Even though a vast majority of women undergoing elective egg

* Corresponding author. E-mail addresses: [email protected], (N. Dadashpour Davachi). https://doi.org/10.1016/j.theriogenology.2018.07.032 0093-691X/© 2018 Elsevier Inc. All rights reserved.

[email protected]

freezing do not use them later on, there appears to be a steady increase in the number of female millennials and younger generation X women using egg-freezing services [3]. Oocyte preservation in animals permits the indefinite storage of unfertilized eggs usually obtained from hormonally super stimulated donor females or post mortem, and the transfer or exchange of genetic material without the necessity of animal transportation. However, mammalian oocytes are highly sensitive to chilling and freezing. Despite numerous efforts to improve oocyte cryopreservation protocols, low survival rates of vitrified oocytes remain the major limitation in gamete cryobiology. Vitrification of both in vitro

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matured or ovulated oocytes is associated with abnormal spindle formation leading to chromosomal aberrations and/or mitotic arrest, altered distribution of cortical granules and increased polyspermy or, on the contrary, the premature cortical granule exocytosis resulting in zona pellucida (ZP) hardening [4]. Lipid accumulation and cytoplasmic fragmentation in oocytes during in vitro maturation (IVM) may exert adverse effects on later stages of in vitro embryo production (IVP) and vitrification/warming procedures [5]. Fetal calf serum (FBS) is commonly added to IVM media to provide COCs with growth factors and other bioactive molecules that are essential for proper and timely oocyte maturation; FBS is also the major source of fatty acids utilized by oocytes during the IVM procedure. Several studies have shown that using lipid reducing agents in IVP media may effectively prevent the formation of cytoplasmic lipid droplets [6,7]. The elimination of lipid droplets or reducing the lipid content of oocytes with various chemical reagents (e.g., L-carnitine promoting the transport of fatty acids from the cytosol to mitochondria for beta-oxidation [6e9]; can improve their cryotolerance [8]. One of the problems associated with oocyte freezing is the induction of a primary activation event, namely the zona pellucida (ZP) hardening, which significantly impedes ensuing fertilization and may affect implantation of resultant blastocysts [10,11]. The ZP hardening is evoked by a fusion of cortical granules with the plasma membrane, and the release of their content into the ZP layers. The membrane fusion event is Ca2þ-dependent and it is normally triggered by an increase in intracellular Ca2þlevels initiated by the sperm-egg fusion [10,11]. Cytokines secreted by epithelial cells of female tubular genitalia may also influence the viability of gametes and pre-implantation embryos [12]. There are two major classes of cytokines produced by the female reproductive tract: cytokines acting as survival agents [granulocyte macrophage colony-stimulating factor (GMCSF), leukemia inhibitory factor (LIF), heparin-binding epidermal growth factor (HB-EGF), and insulin-like growth factor (IGFII)] and apoptosis-inducing cytokines [tumor necrosis factor alpha (TNFa), TNF-related apoptosis-inducing ligand (TRAIL) and interferon gamma (IFNg)]. The balance between these two types of cytokines regulates the competence of germ cells and progression of early embryonic development. However, the IVP media currently used do not contain cytokines in spite of numerous publications supporting their positive influence around fertilization and in early pregnancy [12]. Therefore, in vitro matured oocytes are unlikely to possess similar properties to those of ovulated oocytes [13,14]. Recently, we have developed and evaluated a new co-culture system of ovine cumulus-oophorus complexes (COCs) with ampulla oviductal epithelial cells (AECs) collected during the periovulatory stage of the interovulatory interval [15,16]. The results of those studies showed that oocytes matured in the co-culture system were significantly more competent compared with those incubated in a standard culture medium or the conventional (monolayer) co-culture system, wherein the epithelial cells are exposed to enzymatic detachment and the attainment of sufficient confluency prior to their use in co-culture systems takes several days; all those factors significantly reduce the biopotency of the incubated cells. These observations are indicative of the beneficial effects exerted by the secretory products of AECs on oocyte fertilizing ability and ensuing development of cultured embryos. Similar studies do not exist for vitrified oocytes used for in vitro fertilization. Considering all the facts above, we hypothesized that the ovarian cycle-stage specific co-culture system (i.e., aspirated oocytes matured with conspecific AECs from cyclic females) and/or the reduction of lipid content of IVM media would improve cryotolerance of ovine oocytes. The present experiment set out to

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determine if: i. co-incubation of COCs with epithelial cells obtained from the ampullary segment of the oviduct of sexually mature females in metestrus and/or ii. the modification of IVM media composition aimed to reduce the amount of bioavailable fatty acids would affect cryotolerance of in vitro matured sheep oocytes. To evaluate the effects of modified IVM conditions, we followed the oocyte and early embryo development from warming to in vitro fertilization (IVF) and culture (IVC) procedures, and assessed oocyte nuclear maturation, ZP hardening and blastocyst differentiation. 2. Materials and methods 2.1. Chemicals and reagents All chemicals and media were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise indicated. 2.2. Animals and oocyte recovery The present study utilized clinically healthy cyclic ewes (SeptembereFebruary; longitude-35.69 N, latitude 51.39 E) of the LoriBakhtiary breed, aged 2e3-years. The ovaries (n ¼ 730) were collected immediately after slaughter, placed in an insulated container filled with physiologic saline solution (0.9% NaCl) supplemented with penicillin-streptomycin (100 mg/mL; Gibco, Grand Island, NY, USA), and transported to the laboratory at 37  C within 2 h of collection. COCs were aspirated from transparent antral follicles measuring 2e6 mm in diameter using an aspiration pump (MEDAP Sekretsauger P7040, Tilburg, NL), fitted with a disposable vacuum line (length: 35 cm, internal diameter: 3 mm) and set at the constant flow rate of 10 mL H2O/min and a disposable 20-gauge needle [17,18]. The medium used for oocyte recovery was HEPESTCM supplemented with 10% (FBS), 0.2 mM of sodium pyruvate, 5 mg/mL of gentamicin, 100 ml/mL heparin. The oocytes with at least three layers of cumulus granulosa cells and with uniform granulated cytoplasm were used for subsequent experimental procedures. 2.3. Preparation of ampulla oviductal epithelial cells (AECs) Complete ovine reproductive tracts were collected immediately after slaughter and the oviducts ipsilateral to the ovaries with corpora heamorrhagica (i.e., ovarian antral follicles that ruptured within the last 24 h) were used for harvesting AECs. Such oviducts were used in this study because they contain epithelial cells that had been exposed to elevated(pre-ovulatory) estradiol concentrations and other secretory products from follicular fluid, which trigger the production of the trophic, oocyte-nourishing proteins [19]. Before washing the oviducts in phosphate buffered saline without Ca2þ/Mg2þ (PBS), both ends of the oviduct were tied up with sterile surgical sutures. The oviducts were then dipped in 70% ethanol and transported on ice to the laboratory in PBS solution supplemented with 2% penicillin/streptomycin. After the arrival to the laboratory, the oviducts were dipped again in 70% ethanol and washed in HEPES-buffered tissue culture medium (TCM-199), in a laminar flow hood (BioLAF, Milano, Italy). Identification of the ampulla and the isthmus was based primarily on a difference in their outer diameter and transparency [16]. The ampulla was then divided into two or three 3e4cm segments that were gently squeezed, in a stripping motion, with forceps to recover epithelial cells [15,19,20]. A yellowish matter containing epithelial cells was collected into the culture medium TCM199, supplemented with 2% estrous cow serum and 0.25 mg/mL of gentamicin. The cell suspension was pipetted 10e15 times using a 1000-mL filtered tip (JET Biofil, Guangzhou, China) and then passed 5 times through a 1-mL

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sucrose in HEPES-buffered medium 199, respectively, and supplemented with 20% FBS. Finally, the oocytes were washed twice in fertilization medium (Minitube, Tiefenbach, Germany) supplemented with 20% FBS, and then cultured for 1 h before being transferred into fertilization media supplemented with EFAF BSA. The oocyte survival rate was evaluated on the basis of visual inspection of ZP and membrane integrity, and alterations in the appearance of cytoplasm; all gametes that were demonstrating white or colorless, contained dispersed cytoplasm and were characterized by an absence of sharp demarcations between the cell membrane and ZP were classified as dead [9].

tuberculin syringe attached to a 22-gauge needle. Following the washing procedure, the cells were evaluated under a stereomicroscope to determine the size of cell clusters as previously described by Ref. [16]; the optimal size of oviductal cell clusters is approximately half of that for a typical cumulus-oocyte complex (COC) with compact cumulus layers. 2.4. In vitro maturation (IVM) The medium used for IVM was TCM199 supplemented with 10% FBS, 0.2 mM of sodium pyruvate, 5 mg/mL of gentamicin, 10 mg/mL of follicle-stimulating hormone from sheep pituitary (FSH), and 1 mg/mL of estradiol [17,18]. There were six experimental groups in the present study design; five groups (T1-T5) were considered the “treatment groups” and one was considered the “control group (Ctr; Table 1). In T1, ovine COCs were matured with ampulla oviductal epithelial cells (AECs) in the presence of fetal bovine serum (FBS). In the remaining groups, the COCs were matured in the simple culture system, without the addition of AECs, and media containing 10% FBS (T2), 10% fatty acid-free bovine serum albumin (FAFBSA; T3), L-carnitine (T4; 0.6 mg/ mL [8]; or L-carnitine and FBS (T5). The sixth group served as a control (Ctr: COCs matured without AECs and in the supplement-free IVM medium). In all groups, thirty COCs were cultured in the drops of the maturation medium (250 mL). In T1, thirty clusters of previously recovered AECs had been added to each drop. The drops were covered with mineral oil and COCs were incubated for 24 h at 38.5  C and 95% humidity in 5% CO2. Subsequently, the cumulus cells were trimmed by gentle repeated pipetting of in vitro matured COCs in Hepes TCM-199 supplemented with 1 mg/mL of hyaluronidase through a fine glass pipette (Hilgenberg Pasteur pipette, Münnerstadt, Germany). Denuded oocytes were washed twice in Hepes TCM-199 with 10% FBS and placed individually in the drops of pre-warmed (38.5  C) wash medium under mineral oil to determine the presence of the first polar body, using an inverted microscope (200 image magnification; Nikon, Tokyo, Japan). The oocytes with a polar body were classified as mature (metaphase-II) and the oocytes without a polar body as immature [9].

2.6. Evaluation of zona pellucida (ZP) hardening, in vitro fertilization (IVF) procedures and in vitro embryo culture (IVC) ZP hardening was then assessed by monitoring the time taken for ZP digestion of the vitrified/warmed in vitro matured oocytes in a drop of 50 mL of 0.5% (w/v) pronase solution in PBS [16]. Testicles from some 2- to 3-year-old Lory-Bakhtiary rams were obtained after slaughter, placed in a box set chilled to 5  C, and transported to the laboratory. The testicles were washed three times in PBS supplemented with 100 m$props_value{literPattern}/mL of gentamicin; this and a few following procedures were performed at 5  C in a cold room. Blood and connective tissue were removed aseptically before sperm recovery. A 1-mL syringe attached to a 22gauge needle was inserted into the vas deferens to gently aspirate its entire content, and the recovered spermatozoa were diluted 1:100 in sperm-TALP. Semen was stored in a refrigerator for up to 24 h. On the day of IVF, semen samples were evaluated for progressive motility under a microscope. Motile spermatozoa were separated using the Percoll gradient (45% over 90%) centrifugation [15,20]. A conical tube containing semen samples and Percoll gradient was placed into a tube holder pre-warmed to 38.5  C and centrifuged at  1000 g for 10 min. After centrifugation, a sperm pellet was collected from the bottom of the tube, transferred to a 15-mL conical tube containing 10 mL of HEPES-SOF, and centrifuged again for 5 min at  200 g. The supernatant was then gently removed with a Pasteur pipette. To adjust a final sperm concentration to 26  106/mL (for a final concentration of sperm in the fertilization drop of 1  106/mL), 10 mL of spermcontaining suspension was added to 90 mL of water to kill spermatozoa, then 10 mL of the diluted sample loaded onto a hemocytometer, sperm enumerated in five squares, and the result multiplied by 500,000 to determine sperm concentration per 1 mL. The spermatozoa were diluted in the fertilization medium that had been preequilibrated in the incubator [13,15,20] and contained 12 mM KCL, 25 mM NaHCO3, 90 mMNaCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 10 mM sodium lactate, sodium pyruvate (0.018 g/100 mL), CaCl2 (0.147 g/100 mL), 3 mg/mL of BSA (fatty acid free), and 50 mg/mL of gentamicin. Mature vitrified/warmed oocytes were washed with a fertilization medium supplemented with 10% bovine albumin serum (BSA, Sigma-Aldrich), and 3 IU of heparin. The oocytes were transferred to fertilization droplets at least 15 min before the addition of sperm. Finally, 10 mL of the pre-incubated sperm solution were added to 90 mL of fertilization medium containing ~20 oocytes and co-

2.5. Vitrification and warming of oocytes The oocytes were vitrified using a commercial vitrification device (Kitazato, Tokyo, Japan). They were incubated in the first vitrification solution [7.5% DMSO and 7.5% ethylene glycol (EG), in HEPES-buffered medium 199 with 20% FBS] for 10e12 min and then transferred to the second vitrification solution [15% DMSO, 15% EG and 0.5 M sucrose in Hepes-buffered medium 199 with 20% FBS] for ~60 s. Subsequently, the oocytes were loaded onto cryotop and submerged in liquid nitrogen for storage [9]. Immediately after removing the cryotop from a liquid nitrogen tank, the entire device was submerged in 3 mL of pre-warmed (38  C) HEPES-buffered medium 199 supplemented with 20% FBS and 1 M sucrose; the detached oocytes were left in the medium for 1 min. Then, the oocytes were transferred to the second (3 min) and third (5 min) warming solutions containing 0.5 M and 0.25 M

Table 1 Experimental groups based on the presence (þ) or absence () of ampulla oviductal epithelial cells (AECs) and various media components during in vitro maturation of ovine oocytes. Biological material/Group

Treatment 1 (T1)

Treatment 2 (T2)

Treatment 3 (T3)

Treatment 4 (T4)

Treatment 5 (T5)

Control (Ctr)

AECs TCM199 FBS BSA L-carnitine

þ þ þ e e

e þ þ e e

e þ e þ e

e þ e e þ

e þ þ e þ

e þ e e e

N. Dadashpour Davachi et al. / Theriogenology 120 (2018) 10e15

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Table 2 Oocyte maturation and zona pellucida (ZP) hardening following vitrification and warming of in vitro matured sheep oocytes.

**

Groups

No. of COCs

Oocytes reaching metaphase-II stage (%)

No.*

Oocytes with non-digested ZP (%)

T1 T2 T3 T4 T5 Ctr

408 420 416 420 412 401

92.7 ± 1.1a 81.1 ± 1.1b 70.2 ± 1.2c 50.1 ± 1.1d 81.1 ± 1.2be 52.0 ± 1.0df

189 170 146 105 167 105

77.1 ± 1.2a 86.0 ± 1.0b 92.8 ± 1.4c 92.2 ± 1.1c 76.4 ± 1.5a 91.3 ± 1.5c

Number of vitrified/warmed in vitro matured sheep oocytes randomly selected for the assessment of ZP hardening. Within columns, values with different letter superscripts differ significantly (P < 0.05).

a-f

incubated for 18 h at 38.5  C, in 5% CO2 in the air and 95% humidity. After the completion of the IVF procedure, all spermatozoa that were attached to ZP were removed by gentle pipetting, and the fertilized oocytes were transferred to synthetic oviductal fluid supplemented with 0.4 g of EFAF BSA (SOF-BSA) for the ensuing 7dayincubation (IVC). At the end of the 7-day period, the rate of embryo development to the blastocyst stage was determined and quality of resultant blastocysts evaluated microscopically. To determine the number of cells in the inner cell mass (ICM) and trophectoderm (TE), the expanded blastocysts were subjected to differential staining according to the method described by Thouas, Korfiatis [21], Briefly, Day 7 blastocysts (expanded/hatched) were washed in HTCM þ 5 mg/mL of BSA. Then, the blastocysts were exposed to 0.5% Triton X-100 and subsequently to 90 mg/mL of propidium iodide (PI) for 15 and 25 s, respectively. Subsequently, embryos were fixed and counter stained with Hoechst (10 mg/mL) in cold (4  C) ethanol solution overnight. Fluorescently labeled embryos were mounted on a glass microscopic slide in a droplet of glycerol and sealed with a coverslip. The embryos were examined under fluorescent microscope (ZEISS LSM 880, Germany), and all visible ICM and TE cells were counted. The ICM was defined as a group of embryonic cells that were stained with only Hoechst 33342 (blue), while the TE cells were stained by both Hoechst 33342 and propidium iodide. 2.7. Statistical analyses All percentages were transformed according to the binomial model of variables and arcsine transformation to achieve normal distribution of the raw data sets. The results were analyzed by oneway analysis of variance (ANOVA) and the Tukey test for comparison of individual mean values (SAS, Inc., Cary, NC, USA); P value < 0.05 denoted statistical significance. Data are given as mean ± standard error of the mean (SEM). 3. Results 3.1. In vitro maturation and zona pellucida (ZP) hardening The percentage of oocytes that reached metaphase-II (M  II)

was greater (P < 0.05) for T1 oocytes (co-cultured with AECs in TCM199 þ FBS) compared with all other groups of ovine oocytes (Table 2). The proportion of in vitro matured oocytes in T2 (TCM199 þ FBS) and T5(TCM199 þ L-carnitine þ FBS) groups that reached M  II was significantly greater than in T3 (serum substituted with fatty acid-free BSA), T4 (TCM199 þ L-carnitine) and Ctr (TCM199) groups. The proportion of vitrified/warmed in vitro matured oocytes with non-digested ZP was greater (P < 0.05) in T3, T4 and Ctr compared with T1, T2 and T5 groups, and it was greater in T2 compared with T1 and T5 oocytes. 3.2. Early embryonic development The cleavage and blastocyst formation rate (Table 3) were both greater (P < 0.05) when oocytes were matured in AOCs (T1) compared with all other groups. The percentage of cleaved oocytes in T3, T4 and Ctr was significantly lower (P < 0.05) compared with those of in T2 and T5. No cleaved oocytes attained the blastocyst stage in groups T4 and Ctr, and the percentage of expanded blastocysts was less (P < 0.05) in T3 compared with T2 and T5 groups. Lastly, the T1 group of oocytes was the only one that contained hatching or hatched blastocysts on Day 7 of the IVC. The total, inner cell mass (ICM) and trophoblast (TE) cell numbers as well as the ICM:TE cell number ratio were all greater (P < 0.05) for Day 7 blastocysts developing from T1 oocytes compared with those derived from T2, T3 and T5 treatment groups (Table 3) but there were no differences (P > 0.05) in the ICM:TE ratio among the four subsets of blastocysts. 4. Discussion Several previous studies, mainly in small laboratory rodents, have shown that ZP of the vitrified in vitro matured oocytes undergoes premature cortical granule reaction and hardening [22,23], resulting in a drastic reduction in the IVF success rate. Lipids in the vitrified oocytes tend to form a more packed configuration under the ZP [10], which is linked to premature ZP hardening. In addition, vitrified oocytes exhibit a low level of lipid unsaturation, indicative of oxidative damages, which leads to a dramatic decrease in oocyte competence. Due to the reversible nature of post-vitrification lipid

Table 3 Developmental competence of vitrified/warmed in vitro matured sheep oocytes.

*

Groups

No.*

Cleavage rates (%)

Expanded blastocysts (%)

Hatching/hatched blastocysts on Day 7 of IVC (%)

T1 T2 T3 T4 T5 Ctr

189 170 146 105 167 104

60.1 ± 1.0a 45.3 ± 1.1 b 30.5 ± 1.4c 29.0 ± 1.6c 43.3 ± 1.1b 30.1 ± 1.2c

22.5 ± 9.7a 10.1 ± 0.8b 4.0 ± 0.2c 0d 10.3 ± 0.6b 0d

66.6 ± 1.6 0 0 0 0 0

Number of vitrified/warmed in vitro matured sheep oocytes randomly selected for IVF/IVC. Within columns, values with different letter superscripts differ significantly (P < 0.05).

a-d

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alterations in oocytes, and the possibility of restoring the lipid content and configuration in vitrified oocytes to resemble those in non-vitrified gametes [10], we hypothesized that modifying certain IVM culture condition could enhance development of vitrified oocytes. We have recently demonstrated that co-incubation of immature ovine oocytes with ampulla oviductal epithelial cells collected at the specific stage of the estrous cycle (i.e., metestrus) reduces the incidence of premature ZP hardening and exerts beneficial effects on the cleavage and blastocyst formation rates [13,15,16]. Therefore, this study was designed to determine if IVM of vitrified/warmed ovine oocytes in the presence of AECs collected at metestrusor reducing the lipid content of ovine IVM oocytes would prevent the premature ZP hardening and improve ensuing fertilization rates and blastocyst formation. The present experiment revealed that the rate of ZP hardening in T1 (co-culture with ampullary epithelial cells in TCM199 þ FBS) and T5 oocytes (TCM199 þ L-carnitine þ FBS) was significantly lower compared with T2 oocytes (TCM199 þ FBS). Clearly, the effect of exposure to AECs on ZP configuration was similar to that produced by the lipid reducing agent, L-carnitine. Several earlier studies have shown that the use of Ca2þ-free media was associated with a significant increase in the viability of in vitro matured vitrified oocytes [11,24]. However, Larman et al. [11] and Fujiwara et al. [24] demonstrated that when dimethyl sulfoxide (DMSO) was used as a cryoprotective agent, the elimination of extracellular calcium from the vitrification medium did not prevent ZP hardening after vitrification/warming of in vitro matured oocytes. This observation could be explained by the mechanism of DMSOdependent calcium accumulation; DMSO induces calcium release from intracellular calcium reservoirs whereas other cryoprotectants (e.g., ethylene glycol) cause an influx of calcium from the incubation medium across the plasma membrane. In the current study, a combination of DMSO and EG was used as the cryoprotective agent. Therefore, any reduction in the extracellular content of Ca2þwould result in reduced ZP hardening. The specific mechanism(s) whereby oviductal epithelial cells and their products would influence the lipid content/distribution and/or Ca2þ bioavailability in maturing oocytes remains to be elucidated. A plausible mechanism of the improvement in ZP quality of T1 oocytes is the action of an unknown factor(s) secreted by AECs during IVM on lipid transport and intracellular calcium storage. Such a mechanism could involve the alteration in gene expression and/or blocking the calcium channels. However, FBS added to incubation media in T1 has some chelating effects on the free extracellular ions (i.e., a type of bonding of molecules to the metal ions; [25,26], which may have led to a significant reduction in Ca2þ bioavailability and the degree of ZP hardening in T2 and T5 groups compared with T3 (in T3 AECs were not added and FBS was replaced with EFAFBSA). However, even though there were no significant differences in ZP hardening betweenT1 and T5 oocytes, the fertilization and hatching rate were still greater in T1 compared with all other groups. Dadashpour Davachi et al. [16] showed that the addition of conspecific ampulla oviductal epithelial cells during IVM significantly improved the ovine IVP outcomes. This effect of AECs can be attributed to both the improvement in ZP hardening and a rise in maturation promoting factor activity in freshly collected nonpreserved oocytes. The present results indicate that similar beneficiary effects of AECs could be observed after vitrification of sheep oocytes. Moreover, the results of those earlier and our present study are supportive of the existence of strong growth promoting effects of cytokines/growth factors secreted by oviductal epithelial cells during the IVM procedure. The combined effects of these factors on oocyte developmental competence, cryotolerance and fertilizing ability may be mediated by subtle alterations in gene expression before and after vitrification (i.e., pre-vitrification

control of aquaporin expression associated with cryotolerance of gametes and embryonic genome activation post-fertilization). These hypotheses warrant further studies. In summary, the results of the present experiment showed that IVM of oocytes in the presence of AECs in the incubation medium had more beneficial effects on ZP hardening (comparable to those obtained with L-carnitine) as well as cryotolerance and developmental potential of sheep oocytes than all chemical constituents of the media used. It would now be interesting and justified to design a series of experiments to identify and further corroborate the effects of AECs secretory products on the calcium release and transport, lipid saturation and distribution and gene expression in in vitro matured oocytes. Authors’ contributions Navid Dadashpour Davachi designed the present study and played an instrumental role during data acquisition and analysis as well as interpretation of the results. Roozbeh Fallahi and Essa Dirandeh have participated in manuscript drafting. Xinyu Liu and Pawel M. Bartlewski have participated in drafting the article, revising it critically for important intellectual content, and final approval of the version to be submitted for peer review. Conflicts of interest None of the authors has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this manuscript. Acknowledgements The authors would like to acknowledge the financial support of Razi Vaccine & Serum Research Institute under the projects no. 218-18-038-960595 and no. 1328/250. References [1] Konc J, Kanyo K, Kriston R, Somosk B, Cseh S. Cryopreservation of embryos and oocytes in human assisted reproduction. BioMed Res Int 2014;2014:9. [2] Mandawala AA, Harvey SC, Roy TK, Fowler KE. Cryopreservation of animal oocytes and embryos: current progress and future prospects. Theriogenology 2016;86:1637e44. [3] Greenwood EA, Pasch L, Huddleston H. Reproductive planning in women undergoing elective egg freeze. Fertil Steril.108:e191. [4] Pereira RM, Marques CC. Animal oocyte and embryo cryopreservation. Cell Tissue Bank 2008;9:267e77. [5] Held-Hoelker E, Klein SL, Rings F, Salilew-Wondim D, Saeed-Zidane M, Neuhoff C, et al. Cryosurvival of in vitro produced bovine embryos supplemented with l-Carnitine and concurrent reduction of fatty acids. Theriogenology 2017;96:145e52. [6] Rizos D, Gutierrez-Adan A, Perez-Garnelo S, De La Fuente J, Boland MP, Lonergan P. Bovine embryo culture in the presence or absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression. Biol Reprod 2003;68:236e43. [7] Seidel Jr GE. Modifying oocytes and embryos to improve their cryopreservation. Theriogenology 2006;65:228e35. [8] Chankitisakul V, Somfai T, Inaba Y, Techakumphu M, Nagai T. Supplementation of maturation medium with L-carnitine improves cryo-tolerance of bovine in vitro matured oocytes. Theriogenology 2013;79:590e8. [9] Wiesak T, Wasielak M, Zlotkowska A, Milewski R. Effect of vitrification on the zona pellucida hardening and follistatin and cathepsin B genes expression and developmental competence of in vitro matured bovine oocytes. Cryobiology 2017;76:18e23. [10] Rusciano G, De Canditiis C, Zito G, Rubessa M, Roca MS, Carotenuto R, et al. Raman-microscopy investigation of vitrification-induced structural damages in mature bovine oocytes. PLoS One 2017;12. e0177677. [11] Larman MG, Sheehan CB, Gardner DK. Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes. Reproduction 2006;131:53e61. [12] Richter KS. The importance of growth factors for preimplantation embryo development and in-vitro culture. Curr Opin Obstet Gynecol 2008;20: 292e304.

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