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Bovine uterus-derived exosomes improve developmental competence of somatic cell nuclear transfer embryos
Accepted Manuscript Bovine uterus-derived exosomes improve developmental competence of somatic cell nuclear transfer embryos Fang Qiao, Hui Ge, Xiaonan Ma, Ying Zhang, Zhenzi Zuo, Mengyun Wang, Yong Zhang, Yongsheng Wang PII:
S0093-691X(18)30111-0
DOI:
10.1016/j.theriogenology.2018.03.027
Reference:
THE 14489
To appear in:
Theriogenology
Received Date: 2 January 2018 Revised Date:
13 March 2018
Accepted Date: 16 March 2018
Please cite this article as: Qiao F, Ge H, Ma X, Zhang Y, Zuo Z, Wang M, Zhang Y, Wang Y, Bovine uterus-derived exosomes improve developmental competence of somatic cell nuclear transfer embryos, Theriogenology (2018), doi: 10.1016/j.theriogenology.2018.03.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Revised
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Bovine uterus-derived exosomes improve developmental competence
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of somatic cell nuclear transfer embryos
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Fang Qiao a, Hui Ge a, Xiaonan Ma a, Ying Zhang a, Zhenzi Zuo a, Mengyun Wang a, Yong Zhang a,*, Yongsheng Wang a,*
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a College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, PR
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China 2 Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F
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University, Yangling, Shaanxi, 712100, PR China
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* Corresponding author: [email protected] (Yongsheng Wang); [email protected] (Yong Zhang)
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Fax: 086-029-87080092
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Abstract Exosomes widely exist in various tissues and body fluids, including blood, tissue fluid, and
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urine. In the present study, exosomes were first isolated from the early luteal phase uterus and
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confirmed through morphological examination, immunofluorescence (IF) staining of special
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membrane antigen, and Western blot. The effects of exosomes on the developmental competence
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of somatic cell nuclear transfer (SCNT) embryos were investigated. Transmission electron
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microscopy results showed that the isolated exsomes were spherical particles with a 50 nm to 150
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nm diameter. Immunostaining showed that the surface of these isolated particles were CD9
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postive, which was confirmed using Western blot. Supplementing SCNT embryos with these
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isolated exsomes on day 4 of culture significantly increased the blastocyst formation rate (31% vs.
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34%, 40.3%, and 34.3%) and hatching rate (30.3% vs. 33.3%, 40.7%, and 35%) in comparison
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with the non-supplementation (control),
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Blastocysts
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mass/trophectoderm cell ratio (48% vs 37.9%) and lower apoptosis index (2.1% vs 6.5%) than the
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control group. The gene expression analysis of the blastocysts also showed that the exsomes
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supplementation significantly enhanced the expression levels of IFNT and acrogranin and
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decreased the expression levels of HSP70, BAX and BIP. In conclusion, the present study
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indicated that the early luteal phase uterus secretes exosomes, which might play important roles in
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the development of SCNT embryos.
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Keywords：uterine exosomes, SCNT embryo, developmental potential, bovine.
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1. Introduction In vitro culture (IVC) is a key step in producing in vitro fertilized (IVF) and somatic cell
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nuclear transfer (SCNT) embryos. However, the developmental potential of preimplantation
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embryo cultured in vitro is inferior to that cultured in vivo [1]. Moreover, SCNT embryos are
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more sensitive to culture conditions than fertilized or parthenogenetic (PA) embryos. The effect of
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the culture environment on in vitro-produced (IVP) embryos has been well documented in terms
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of aberrant gene expression profiles [2], altered inner cell mass/trophectoderm cell (ICM/TE) ratio
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[3], decreased intercellular communication [4], increased apoptosis index and cryosensitivity [5].
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Therefore, improving the IVC conditions might be a crucial factor in enhancing enhance the
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developmental efficiency of IVP embryos, especially SCNT embryos. Exosomes are a small
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membrane nanovesicles with 30-100 nm diameter and widely present in almost all tissue fluid,
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including blood, and urine, the oviduct, and the uterus [6]. Exosomes contain proteins, lipids,
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mRNAs, miRNAs, and DNA cargoes and are essential for intercellular communications [7-10].
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The Exosome-mediated transfer of these compounds into neighboring cells are indispensable for
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early embryo development [8, 11], implantation [12, 13] and pregnancy [6, 14]. The existence and
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importance of exosomes have been confirmed in regular embryo development; however, their
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effects on the development of SCNT embryos need to be elucidated.
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Initiation of mammal pregnancy requires synergistic interactions through embryonic-maternal
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cross-talk
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membrane-derived vesicles, particularly exosomes, and provided new dimensions in intercellular
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signaling [16]. Exsomes released by the endometrial epithelium into the uterine cavity are
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involved in the transfer of signaling proteins, miRNAs, and mRNAs to either the embryo or the
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adjacent endometrium. This transfer of materials affects endometrial receptivity, embryonic
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development, and implantation [17]. Our previous study showed that the addition of exsomes
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isolated from the conditioned medium of PA embryos increased the cleavage rate and blastocyst
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formation in cloned embryos [18], indicating that exogenous exosomes play crucial roles on
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embryonic development. Other studies also confirmed that bovine oviduct-derived exosomes
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improved the quality of PA embryos [6].
Studies
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essential
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This study was conducted to investigate the effect of exosomes derived from bovine uterine
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luminal fluid (ULF) on the development of SCNT embryos. This study is the first to report the
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effects of uterus-derived components on early embryonic development. This research may
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contribute to the improvement of cloning efficiency in mammals and may provide new insights on
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assisted-reproduction technologies.
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2. Materals and methods All chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA)
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unless otherwise noted. Disposable, sterile plasticware were purchased from Nunclon (Roskilde,
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Denmark). All procedures in this experiment were approved by the Animal Care and Use
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Committee of Northwest A & F University and were performed in accordance with animal
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welfare and ethics.
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2.1 Exosomes isolation
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Ten uteri were obtained from the slaughteredhouse and were transported to the laboratory on
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ice. The uteri were in early luteal phase,which corresponds to days 4-8 of the estrous cycle (corpus
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luteum diameter: 12 mm to 18mm) [19], The uteri were then washed thrice in cold sterile PBS,
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trimmed free of tissue on a cooled surface, and gently flushed with PBS. After the first
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centrifugation at 300 × g for 10 minutes, the ULF was clarified by two sequential centrifugations
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at 10,000 × g for 60 min and 100,000 × g for 120 min. All procedures were performed at 4 ºC, and
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the exosomes were isolated and purified following a previously published protocol with minor
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modifications [20]. Briefly, after the endometrial epithelium was removed, the ULF supernatant
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was filtered through a 0.2 µm nylon filter and centrifuged again at 100,000 × g for another 60 min,
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The resulting pellet was suspended in 20 µL of PBS (pH 7.2) and stored at −80 °C.
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2.2 Identification of exosomes through transmission electron microscopy, IF,and and Western blot
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After gradient centrifugation, 7.5 µL of the pellet suspension was top loaded on 300-mesh grids
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and allowed to settle for 20 min at room temperature. The grids were then stained in 2% uranyl
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acetate, dried overnight at room temperature, and visualized with an energy-filtering transmission
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electron microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) at 120 KV.
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Immunofluorescence was conducted according to published protocols with slight modifications
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[17]. Briefly, 7.5 µL of purified pellet suspension was incubated with 5 µL of 4 µm
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aldehyde/sulfate latex beads (Life Technologies Corp., Grand Island, NY, USA) in a 30 µL final
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volume of PBS at room temperature for 15 min. PBS (170 µL) was then added, and the
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suspension was incubated in a tube rotator for 2.5 h at room temperature. Subsequently, 22 µL of
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1M glycine/PBS was added and mixed gently to block the unbound sites of the latex beads, and
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the mixture was allowed to stand on the bench for 30 min at room temperature.Afterwards, the
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beads were pelleted by centrifugation at 1,500 × g for 3 min at room temperature, and the pellets
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were washed twice with 1 mL of PBS/0.5% BSA. The exosome–bead complexes were incubated
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with anti-CD9 primary antibody (MEM-61, Thermo Fisher Scientific, Rockford, IL, USA) and
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immunoglobulin G (IgG) conjugated to fluorescein isothiocyanate isomer 1. A negative control
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antibody reaction was performed using normal mouse IgG untreated with anti-CD9 primary
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antibody. The labeled exosome–bead complexes were again pelleted, and the pellets were washed
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twice and resuspended in 20 µL of PBS/0.5% BSA. The final complexes (10 µL) were spread on a
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microscope slide with a drop of DakoCytomation fluorescent mounting medium (Dako,
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Carpinteria, CA, USA), air-dried, cover-slipped, and sealed with nail polish. Finally, the samples
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were visualized using a HAL 100 fluorescence microscope (Carl Zeiss Microscopy GmbH). The
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experiments were repeated in triplicate.
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For the Western blot assay, purified exosome precipitates were lysed in non-reducing Laemmli
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loading buffer and resolved in a 12% SDS-PAGE gel. The proteins were transferred to a PVDF
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membrane, and blocked with 10% skimmed milk for 4 h at room temperature. Primary mouse
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anti-GAPDH antibodie (1:2500 dilution, Sigma) and mouse anti-CD9 antibodies (1:1000 dilution;
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MEM-61, Thermo Fisher Scientific, Rockford, IL, USA) were mixed in TBST buffer (0.1%
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Tween 20 and 150 mM NaCl in 10 mM Tris-HCL, pH 7.4) with 5% skimmed milk and incubated
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with the membrane for 12 h at 4°C. After incubation with the primary antibodies, the blotting
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membrane was washed thrice for 15 min with TBST. The secondary anti-mouse antibody (1:1000
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dilution, Sigma) was mixed with TBST with 5% milk powder and incubated with the blotting
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membrane for 2 h at room temperature. The negative control group (NC) was not treated with
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anti-CD9 and anti-GAPDH primary antibodies. After incubation with the secondary antibodies,
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the blotting membrane was washed thrice for 15 min with TBST and visualized via
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chemiluminescence by using the ImageQuant LAS4000 biomolecular imager (GE Life Sciences).
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2.3 Oocyte collection and in vitro maturation (IVM)
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Ovaries were obtained from a local slaughterhouse and were transported to the laboratory in
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0.9% NaCl at 20–22°C. Immature cumulus oocyte complexes (COCs) were obtained by aspirating
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the follicles (2–8mm) from the ovaries. The COCs with more than three layers of compact
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cumulus cells and uniform cytoplasm were selected and used for IVM. The elected COCs were
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allowed to mature in vitro in a basic maturation medium (TCM-199, Gibco, BRL, Grand Island,
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NY, USA) supplemented with 10% (v/v) FBS, 1 µg/mL 17β-estradiol, 10 ng/ml epidermal growth
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factor, and 0.075 IU/mL human menopausal gonadotropin in humidified air with 5% CO2 at
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38.5°C for 22 h.
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2.4 Production of SCNT embryos and IVC The production of SCNT embryos was performed as previously described [21]. In brief, the in
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vitro-matured oocytes were enucleated using a 20 µm diameter aspiration pipette, microinjected
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into the perivitelline space of a female fetal fibroblast, and fused through a double electrical pulse
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stimulation. Successfully reconstructed embryos were maintained in mSOF containing 5 µg/ml
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cytochalasin B for 2 h and activated in 5 µM ionomycin for 4 min. Subsequently, the embryos
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were exposure to 1.9 mM dimethynopyridine in mSOF for 4 h. The embryos were cultured in the
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mSOF medium supplemented with or without exosomes from the luteal-phase uteri in accordance
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with the experimental design in a humidified atmosphere with 5% CO2 at 38.5°C.
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2.5 IF
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Immunofluorescence was conducted as described previously [22]. The embryos were fixed in
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4% (v/v) paraformaldehyde at 4 °C overnight, permeabilized with 0.2% (v/v) Triton X-100 in
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PBS for 20 min at room temperature, blocked with a blocking solution (Beyotime, P0102)
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overnight at 4 °C, and incubated overnight with primary anti-CDX2 mouse monoclonal antibody
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(BioGenex, Inc., San Ramon, CA) at 1:200 dilution in blocking buffer. After incubation, the
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embryos were washed in 0.1% PBS–PVA and treated with secondary Alexa Fluor 555-labeled
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goat anti-mouse IgG (1:500 dilution; Beyotime, A0459) in a dilution solution (Beyotime, P0108)
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for 2 h at room temperature. After the embryos were washed thrice in 0.1% PBS–PVA for 5 min
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per wash, nuclear labeling was performed using 4,6-diamidino-2-phenylindole hydrochloride
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(DAPI, Vysis Inc., Downers Grove, USA) for 3 min. After washing and mounting, the slides were
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examined by epifluorescence using a Nikon Eclipse Ti-S microscope (Nikon, Tokyo, Japan). All
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images were captured using the Nikon DS-Ri1 digital camera and saved in TIFF format. All the
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embryo cell nuclei were identified by their blue fluorescence, whereas the trophectoderm cell
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nuclei (CDX2 positive) exhibited red fluorescence.
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2.6 Apoptosis detection
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Apoptosis was detected using the DeadEnd Fluorometric TUNEL System (Promega, Madison,
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WI). Briefly, The blastocysts were fixed in 4% paraformaldehyde at 4 °C overnight,
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permeabilized in 0.2% Triton X-100 for 20 min, and incubated with FITC-conjugated dUTP and
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terminal deoxynucleotidyl transferase at 37 °C for 1 h in the dark. The tailing reaction was
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terminated in 2× SSC (SSC: 0.15 mol/L sodium chloride and 0.015 mol/L sodium citrate) for 15
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min. The embryos were then incubated in PBS containing 25 µg/mL RNase A for 30 min. After
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washing and DAPI staining, the embryos were mounted on slides for confocal microscopy and
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observed under Carl Zeiss LSM 510 laser confocal scanning microscope.
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2.7 Quantitative real-time PCR (q-PCR) Total RNA was extracted from day 7 blastocysts using the Cells-to-Signal™ kit (Invitrogen,
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USA) according to the manufacturer’s protocol. The cDNA was synthesized using
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PrimeScript™ RT reagent kit (TaKaRa, Japan) with a total volume of 20 µL (10 µL of 2 ×
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reaction mix, 1 µL of RT enzyme mix, 1 µL of oligo dT primer, 1 µL of random primers, 1 µL of
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DNA remover, 1 µg of RNA, and up to 20 µL of RNase-free dH2O). Reactions were performed in
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a low tube strip (Bio-Rad, GB). Each reaction mixture (20 µL) contained 2 µL (approximately
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100 ng) of cDNA template, 10 µL of SYBR® Premix Ex TII (2×), 0.8 µL of each PCR forward
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primer and reverse primer (10 µM), and 7.2 µL of dH2O. Quantitative PCR was performed using
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the CFX96 q-PCR detection system (Bio-Rad) with SYBR Premix Ex Taq™ II (TaKaRa, Japan).
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The thermal cycling conditions were as follows: 95°C for 30 s and 40 PCR cycles for 5 s at 95°C
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for DNA denaturation, 30 s at 60 °C for primer annealing, and 30 s at 72 °C for extension. The
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transcripts of the examined genes were quantified in triplicate and calculated relative to the
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housekeeping genes GAPDH and H2A.2. The specificity of the qPCR reaction was confirmed by
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the single peaks in the melt curves and by gel electrophoresis. The cDNA was replaced with water
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as negative control. The experiments were repeated at least thrice. All primers were designed
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using
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(http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The comparative cycle threshold method was
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used to quantify the expression levels. The primer sequences used for the PCR and qPCR are all
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listed in Table 1.
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2.8 Experimental design
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The experiments were divided into two parts. First, bovine exosomes were isolated from uteri
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in the early luteal phase, which corresponds to days 4–8 of the oestrous cycle (corpus luteum
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diameter: 12 mm to 18mm) by using differential ultracentrifugation protocol and identified using
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morphological examination, IF, and Western-blot. Second, the effects of the uterine exosomes on
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the development of SCNT embryos were assessed in terms of blastocysts formation rate, hatching
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rate,
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development-related genes. Between days3 and 4, the embryo develops into an 8-to 16-cell and
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embryo and moves from the oviduct to the uterus. To mimic the in vivo environment, we added
the
first
cellular
differentiation,
apoptosis
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index,
and
expression
of
several
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the uterine exosomes into the culture medium on day 3 (72h), 4 (96h), and 5 (120h) of culture at
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3 × 105 exosomes/mL after the SCNT embryos activatied [6, 23].
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2.9 Statistical analysis The embryo cleavage rate, blastocyst yield, and relative mRNA abundance for the candidate
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genes were analyzed using one-way ANOVA (p<0.05). All analyses were performed using
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SigmaStat software package (Jandel Scientific, San Rafael, CA). For the total cell number and
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apoptosis index, 30 blastocysts were randomly selected from each medium group and compared
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using one-way ANOVA. Statistical significance was considered at P < 0.05 .
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3. Results
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3.1 Isolation and identification of exosomes from ULF
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As shown in Figs. 1A and 1B, exosomes were isolated from the ULF by ultracentrifugation at
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two different centrifugal forces (10,000 and 100,000 × g). Transmission electron microscopy
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showed that the isolated exosomes were spherical particles with a 50 nm to 150 nm diameter.
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These spheres were further characterized by immunostaining with CD9, a specific antibody for
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exosomal membranes. As shown in Fig. 2, CD9 was strongly laterally localized and was clearly
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visualized on the surface of the exosomes (Fig 2A and 2B). No signal was observed in the control
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group (Figs. 2C and 2D), This result indicated that our observed signal was specific. The Western
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blot also showed that the exosomal protein CD9 existed in the sample (Fig. 3). These results
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demonstrated the presence of exosomes in ULF.
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3.2 Effects of uterus-derived exosomes on the development of SCNT embryos
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The exosomes extracted from the ten uteri were diluted in 400 µL of mSOF supplemented with
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8 mg/ml BSA and added into a fresh medium at days 3(72 h), 4(96 h), and 5(120 h) after SCNT
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embryo activation. Photographs of the SCNT blastocysts are presented in Fig. 4. As shown in
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Table 2, uterine exosome supplementation on day 4 of culture significantly increased the
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blastocyst formation rate (40.3% vs. 34%, 31%, and 34.3%, respectively, P<0.05) and hatching
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rate (40.7% vs. 33.3%, 30.3%, and 35%, respectively, P<0.05) in comparison with the
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non-supplementation group (control group), day 3 and day 5 supplementation groups.
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3.3 Effects of uterus-derived exosomes on SCNT embryo quality
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Based on the findings that exosome supplementation on day 4 of culture significantly enhanced
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the blastocyst formation and hatching rate, the quality of the blastocyst from the day 4 exsome
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supplementation group were further investigated using TUNEL assay (Fig. 5), The rate of
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ICM/TE differentiation (Fig. 6), and the expression levels of several development-related genes of
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the preimplantation embryos (Fig. 7) were also examined. The exsome supplementation
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significantly reduced the apoptotic index (2.1% vs. 6.5%, P<0.05; Fig. 5), and increased the
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ICM/TE ratio (48% vs. 37.9%, P<0.05; Fig. 6) of the SCNT blastocysts compared with the NC
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group. The expression levels of IFNT and acrogranin were significantly high, and those of BAX
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(pro-apoptosis marker), HSP70 and BIP were significantly low, The expression level of PMSB5
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showed no difference in the exosome supplementation day4 blastocyst group compared with that
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in the NC group (Fig. 7).
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4. Discussion
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Uterus-derived exosomes were first found and isolated from human ULF by Ng et al. [17] and
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were later confirmed to exist in various other animals [24]. In the present study, exosomes were
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successfully isolated from fresh luteal phase uterus by ultracentrifugation and were identified
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through morphologic examination, IF, and Western blot. Previous studies suggested that bovine
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exosomes exhibit a mean size of approximately 148 nm [24]. Similarly, our results showed that
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the exosomes were mainly 100 to 150 nm in diameter. The IF and Western blot confirmed that
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CD9, an exosomal marker protein, existed in the ULF. All these parameters were consistent with
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those of previous studies, indicating the successfully isolation of the exosomes from bovine ULF
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in the present study.
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Exosomes released by the endometrial epithelium are involved in the transfer of signaling
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miRNAs and mRNAs to either the blastocyst or the adjacent endometrium. This transfer of
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materials influences endometrial receptivity and implantation success. Many components of the
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ULF are required for conceptus development [25]. The endometrium secretes substances,
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collectively termed histotroph, that govern the growth and elongation of the conceptus via its
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effects on trophectoderm proliferation, migration, attachment and adhesion to the endometrial
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luminal epithelium [26]. These uterus-derived exosomes contain various materials, such as
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proteins, miRNAs and pluripotency genes mRNAs (Oct4, Sox2, Klf4, c-Myc, and Nanog), which
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change dynamically throughout pregnancy [27]. Although we did not performend experiments to
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examine the substances in the uterus-derived exosomes, the results showing the time-dependent
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effect of the exosomes on embryonic development indicated that the exosomes might contain
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different substances at different physiological period of pregnancy.
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Despite the efforts to elucidate the function of EVs during reproductive events, the role of
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bovine uterus-derived exosomes in early embryonic development remains unknown. Our results
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were the first to show that exosomes can be successfully isolated from bovine early luteal phase
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uterus and can successfully improve the development capacity of SCNT embryos in vitro. In the
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present study, the effects of the uterus-derived exosomes on the quality of SCNT embryos were
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assessed. Our results (Table 2) showed that the group with exosome supplementation on Day 4
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exhibited a significant improvement in blastocyst formation and hatching rate compared with the
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control group. In addition, the blastocyst quality in the day4 supplementation group was
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significantly improved as assessed by the total cell number, ICM/TE ratio(Fig. 6), and apoptotic
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index (Fig. 5). The day4 nuclear transfer embryo (NTE) group showed a higher ICM/TE ratio
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(48% vs. 37.9%, P<0.05; Fig. 6) than the control group. In the literature, the ICM: TE ratio is 44%
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–50% and 17% in blastocysts derived in vivo, and in NTE, respectively [28, 29]. Placental
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abnormalities or early fetal loss might be caused by the aberrant allocation of ICM and TE cells in
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the NTE during the preimplantation stages. The structural integrity of blastocyst composed of
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ICM and TE cells, might influence postimplantation development in mammals [30, 31]. The
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exosome supplementation on the day 4 of culture improved the blastocyst structural integrity, and
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such improvement may influence the postimplantation development.
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Apoptosis is a normal physiological process in which cells die after exposure to normal or
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pathologic stimuli in vivo. Embryos produced in vitro usually exhibit a high apoptosis index,
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probably because of suboptimal culture conditions [32]. The incidence of cell death in the human
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blastocyst is correlated with the cell number and embryo quality [33, 34]. The TUNEL assay (Fig.
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5), showed a significantly lower apoptosis index in the day 4 groupthean in the control group
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(2.1% vs. 6.5%, P<0.05; Fig. 5). This result suggested that the exosome supplementation on day 4
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of culture provided a better culture condition for the bovine SCNT embryos, The incidence of
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apoptotic cells, the rate of blastocyst formation, and cell numbers in the SCNT blastocysts could
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be markers of blastocyst quality.
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To further evaluate the competence of the SCNT embryos derived from the day4 group, we
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analyzed the important genes related to embryonic development. We systematically compared the
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relative expression levels of HSP70, BAX, IFNT, acrogranin, BIP and PMSB5 (Fig. 7). The
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expression levels of HSP70 [35], BAX
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reticulum stress marker) remarkably decreased.The BAX expression level was consistent with the
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result of the TMNEL assay. Apoptotic cell death in preimplantation mammalian embryos plays an
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important role in embryo development. HSP70 is a prominent cytoprotective factor that confers
(a pro-apoptosis marker), and BIP (an endoplasmic
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tolerance to cellular stress or transfection by inhibiting the induction of apoptosis [36, 37]. BAX
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and HSP70 interact under stress conditions, and BAX is suppressed in cells with high HSP70
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levels [38]. In the present study, we found no significant difference in the abundance of for HSP70
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and Bax transcripts. The apoptosis detected in our study was consistent with that of a previous
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study [39]. Previous research showed that the expression levels of BAX, HSP70, BIP and PSMB5
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are significantly high in SCNT blastocysts than in vivo, This finding suggests that SCNT embryos
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are exposed to high cellular stress in vitro than in vivo [40]. Our results showed that the exosome
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supplementation optimized the embryo culture condition as supported by the low BAX, HSP70,
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and BIP expression levels. A low BIP expression level suggested that the embryo undergoes
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minimal apoptotic damage. IFNT is a maternal recognition signal required for a successful
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pregnancy [41], Results of the present work showed high expression level of IFNT in a treated
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with exosomes. Rizol et al [42] observed a significantly high expression level of IFNT in a
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serum-free medium; this finding is consistent with the notion that the mRNA levels for this
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transcript are high in good-quality embryos. Furthermore, the amount of interferon is correlated
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with hatching rate and morphologic quality [43]. Acrogranin is positively related to blastocyst
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formation and hatching rate [7]. In the present study, the expression levels of IFNT and
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acrogranin was high in the day 4 supplementation group, This result suggested that the
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uterus-derived exosomes improved the blastocyst formation and hatching rate. Collectively, these
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results indicate a preferable embryo quality in exsome-optimized condition.
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5. Conclusions
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Bovine uterus-derived exosomes can be successfully isolated and used for in vitro SCNT
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embryo culture with a positive effect on embryo quality, The effect is reflected in the improved
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blastocyst formation, increased total cell number, increased ICM/TE ratio , decreased TUNEL
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signal, increased IFNT and acrogranin gene expression levels, and low expression levels of BAX
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and HSP70. This study was the first to report that exosomes can be successfully isolated from
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bovine early luteal phase uterus and that exosome supplementation can improve the
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develpomental capacity of SCNT embryos in vitro.
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Acknowledgement
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This research was supported by the National Major Project for Production of Transgenic Breeding (No. 2016ZX08007003) and National Natural Science Foundation of China (NO.31472094).
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[40] Cánepa MJ, Ortega NM, Monteleone MC, Mucci N, Kaiser GG, Brocco M, et al. Expression profile of genes as indicators of developmental competence and quality of in vitro fertilization and somatic cell nuclear transfer bovine embryos. Plos One. 2014;9:e108139. [41] Bazer FW, Spencer TE, Ott TL. Interferon tau: a novel pregnancy recognition signal. American Journal of Reproductive Immunology. 1997;37:412.
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[42] Rizos D, Gutiérrezadán A, Pérezgarnelo S, De LFJ, Boland MP, Lonergan P. Bovine embryo culture in the presence or absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression. Biology of Reproduction. 2003;68:236-43. [43] Hernandezledezma JJ, Mathialagan N, Villanueva C, Sikes JD, Roberts RM. Expression of bovine
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trophoblast interferons by in vitro-derived blastocysts is correlated with their morphological quality and stage of development. Molecular Reproduction & Development. 1993;36:1.
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Fig 1. Uterus-derived exosomes were identified by electron microscope analysis. (A) Transmission Electron Microscopy (TEM) images showed the presence of exosomes after gradient centrifugations and negative staining with uranyl acetate. (B) Size distribution of exosomes derived from uterine luminal fluid identified by Transmission Electron Microscopy. Bar, 100 nm.
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Fig 2. Uterus-derived exosomes were identified by immunofluorescence. The exosomes were bound to beads of a size that was in the detection range of the fluorescence microscope (4-µm diameter latex beads). The beads were then bound to fluorescence-conjugated antibody against CD9. Images were taken under epifluorescence (A, C) and DIC (B, D,). A,B: uterus-derived exosomes; C,D: IgG negative control; Bar, 10 µm.
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Fig 3. Uterus-derived exosomes proteins were identified by western blot. The NC group was treated without anti-CD9 first antibody and anti-GAPDH first antibody. Fig 4. Representative photographs of bovine SCNT blastocysts derived from NC, Day 3, Day 4, Day 5 groups.
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Fig 5. The apoptotic blastomeres in Day 4 and NC blastocysts groups. (A) Blastocysts were detected by TUNEL (green). DNA was stained by DAPI (blue) to visualize all blastomeres. Bar, 80 µm. (B) Apoptotic index of blastocysts. a.b Different superscripts indicate significant differences for each gene (p<0.05). Data presented are mean ± SE.
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Fig 6. Ratio of ICM/TE in Day 4 and NC blastocysts groups. (A) Immunostaining of CDX2. a specific marker of trophectoderm (red), as well as DAPI that stained nuclei of all blastomeres (blue). Bar, 80 µm. (B) Statistical analysis of ICM/TE in Day 4 and NC blastocysts groups. a.b Different superscripts indicate significant differences for each gene (p<0.05). Data presented are mean ± SE.
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Fig 7. Relative mRNA transcription in bovine in vitro blastocysts cultured with exsomes at the embryo cultured Day 4 of genes related with embryo quality. Data are relative to mean of internal gene H2A. Data presented are mean ± SE. a.b Different superscripts indicate significant differences for each gene (p<0.05).
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Table 1 primers sequences for real time-qPCR
Gapdh
sequence
F(3’-5’)CGACTTCAACAGCGACACTCAC R(3’-5’)CCTGTTGCTGTAGCCGAATTC
H2A.2
F(3’-5’)GAGGAGCTGAACAAGCTGTTC
F(3’-5’) CGGACACCTGACCAATGGAA
in
R(3’-5’) CTGTCACTAAGGCCACCCAG F(3’-5’)TGTTTGTTATGGAGACTGTTGGGA
Size(bp)
(°C)
158
58
NM_173979
144
58
BF 076731
155
58
XM_01081640
223
Gene
bank
access No.
5.2
58
NM_174550
114
58
NM_173894
167
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NM_00116827
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Hsp70
Tm
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R(3’-5’)TTGTGGTGGCTCTCAGTCTTC Acrogran
Product
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gene
R(3’-5’) GGTGAAAAATAGCTTGAGTTTGGC Bax
F(3’-5’) TCCCCGAGAGGTCTTTTTCCG
R(3’-5’) AGGGCCTTGAGCACCAGTTTG IFNtau
F(3’-5’) AGGTTAAGAAAGATGGGTGGAGA
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R(3’-5’) ACTGCTGACAAAGTATCGGCTAA
F(3’-5’)GATTGAAGTCACCTTTGAGATAGATGTG
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Bip
85
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NM_00107514 8.8
R(3’-5’) GATCTTATTTTTGTTGCCTGTACCTGG Psmb5
F(3’-5’) GACGTTTGCGTTTGCTTCCT
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R(3’-5’) ACCACGCCTCCATACACAAG
178
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NM_00103761 2.1
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No. embryo cultured
blastocyst(%)
hatching（%） a
Nc
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31%+0.01
Day3 Day4 Day5
189 192 186
34%+0.02a 40.3%+0.01b 34.3%+0.01a
30.3%+0.01
No. blastomeres
a
33.3%+0.01a 40.7%+0.02b 35%+0.02c
90.3a 94.7a 105.3bc 101ab
a-c
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different superscripts with same column indicate significant difference (P<0.05) day 3, day 4, day 5 suggested exosome supplementation after embryo activation for 3, 4, 5 days.
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Highlights 1. Our study firstly isolated exsomes from bovine uterine luminal fluid (ULF). 2. Uterus-derived exsomes significantly improved SCNT embryo information and hatching in vitro. 3. Uterus-derived exsomes enhanced the quality of SCNT embryos assessed by increased total cell
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number, increased ratio of ICM/TE, decreased TUNEL signal, and increased gene expression of IFNT and acrogranin as well as a low expression level with BAX，HSP70, and BIP.
4. This is the first time study the effect of uterus-derived exosomes on SCNT embryos development,
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and it may provided new methods to improve the efficiency of SCNT.
S0093-691X(18)30111-0
DOI:
10.1016/j.theriogenology.2018.03.027
Reference:
THE 14489
To appear in:
Theriogenology
Received Date: 2 January 2018 Revised Date:
13 March 2018
Accepted Date: 16 March 2018
Please cite this article as: Qiao F, Ge H, Ma X, Zhang Y, Zuo Z, Wang M, Zhang Y, Wang Y, Bovine uterus-derived exosomes improve developmental competence of somatic cell nuclear transfer embryos, Theriogenology (2018), doi: 10.1016/j.theriogenology.2018.03.027. 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.
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Revised
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Bovine uterus-derived exosomes improve developmental competence
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of somatic cell nuclear transfer embryos
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Fang Qiao a, Hui Ge a, Xiaonan Ma a, Ying Zhang a, Zhenzi Zuo a, Mengyun Wang a, Yong Zhang a,*, Yongsheng Wang a,*
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a College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, PR
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China 2 Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F
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University, Yangling, Shaanxi, 712100, PR China
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* Corresponding author: [email protected] (Yongsheng Wang); [email protected] (Yong Zhang)
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Fax: 086-029-87080092
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Abstract Exosomes widely exist in various tissues and body fluids, including blood, tissue fluid, and
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urine. In the present study, exosomes were first isolated from the early luteal phase uterus and
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confirmed through morphological examination, immunofluorescence (IF) staining of special
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membrane antigen, and Western blot. The effects of exosomes on the developmental competence
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of somatic cell nuclear transfer (SCNT) embryos were investigated. Transmission electron
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microscopy results showed that the isolated exsomes were spherical particles with a 50 nm to 150
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nm diameter. Immunostaining showed that the surface of these isolated particles were CD9
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postive, which was confirmed using Western blot. Supplementing SCNT embryos with these
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isolated exsomes on day 4 of culture significantly increased the blastocyst formation rate (31% vs.
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34%, 40.3%, and 34.3%) and hatching rate (30.3% vs. 33.3%, 40.7%, and 35%) in comparison
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with the non-supplementation (control),
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Blastocysts
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mass/trophectoderm cell ratio (48% vs 37.9%) and lower apoptosis index (2.1% vs 6.5%) than the
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control group. The gene expression analysis of the blastocysts also showed that the exsomes
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supplementation significantly enhanced the expression levels of IFNT and acrogranin and
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decreased the expression levels of HSP70, BAX and BIP. In conclusion, the present study
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indicated that the early luteal phase uterus secretes exosomes, which might play important roles in
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the development of SCNT embryos.
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Keywords：uterine exosomes, SCNT embryo, developmental potential, bovine.
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exsome
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1. Introduction In vitro culture (IVC) is a key step in producing in vitro fertilized (IVF) and somatic cell
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nuclear transfer (SCNT) embryos. However, the developmental potential of preimplantation
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embryo cultured in vitro is inferior to that cultured in vivo [1]. Moreover, SCNT embryos are
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more sensitive to culture conditions than fertilized or parthenogenetic (PA) embryos. The effect of
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the culture environment on in vitro-produced (IVP) embryos has been well documented in terms
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of aberrant gene expression profiles [2], altered inner cell mass/trophectoderm cell (ICM/TE) ratio
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[3], decreased intercellular communication [4], increased apoptosis index and cryosensitivity [5].
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Therefore, improving the IVC conditions might be a crucial factor in enhancing enhance the
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developmental efficiency of IVP embryos, especially SCNT embryos. Exosomes are a small
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membrane nanovesicles with 30-100 nm diameter and widely present in almost all tissue fluid,
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including blood, and urine, the oviduct, and the uterus [6]. Exosomes contain proteins, lipids,
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mRNAs, miRNAs, and DNA cargoes and are essential for intercellular communications [7-10].
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The Exosome-mediated transfer of these compounds into neighboring cells are indispensable for
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early embryo development [8, 11], implantation [12, 13] and pregnancy [6, 14]. The existence and
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importance of exosomes have been confirmed in regular embryo development; however, their
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effects on the development of SCNT embryos need to be elucidated.
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Initiation of mammal pregnancy requires synergistic interactions through embryonic-maternal
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cross-talk
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membrane-derived vesicles, particularly exosomes, and provided new dimensions in intercellular
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signaling [16]. Exsomes released by the endometrial epithelium into the uterine cavity are
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involved in the transfer of signaling proteins, miRNAs, and mRNAs to either the embryo or the
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adjacent endometrium. This transfer of materials affects endometrial receptivity, embryonic
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development, and implantation [17]. Our previous study showed that the addition of exsomes
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isolated from the conditioned medium of PA embryos increased the cleavage rate and blastocyst
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formation in cloned embryos [18], indicating that exogenous exosomes play crucial roles on
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embryonic development. Other studies also confirmed that bovine oviduct-derived exosomes
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improved the quality of PA embryos [6].
Studies
have
substantiated
the
essential
functions
of
cell-secreted
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[15].
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This study was conducted to investigate the effect of exosomes derived from bovine uterine
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luminal fluid (ULF) on the development of SCNT embryos. This study is the first to report the
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effects of uterus-derived components on early embryonic development. This research may
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contribute to the improvement of cloning efficiency in mammals and may provide new insights on
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assisted-reproduction technologies.
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2. Materals and methods All chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA)
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unless otherwise noted. Disposable, sterile plasticware were purchased from Nunclon (Roskilde,
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Denmark). All procedures in this experiment were approved by the Animal Care and Use
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Committee of Northwest A & F University and were performed in accordance with animal
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welfare and ethics.
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2.1 Exosomes isolation
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Ten uteri were obtained from the slaughteredhouse and were transported to the laboratory on
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ice. The uteri were in early luteal phase,which corresponds to days 4-8 of the estrous cycle (corpus
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luteum diameter: 12 mm to 18mm) [19], The uteri were then washed thrice in cold sterile PBS,
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trimmed free of tissue on a cooled surface, and gently flushed with PBS. After the first
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centrifugation at 300 × g for 10 minutes, the ULF was clarified by two sequential centrifugations
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at 10,000 × g for 60 min and 100,000 × g for 120 min. All procedures were performed at 4 ºC, and
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the exosomes were isolated and purified following a previously published protocol with minor
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modifications [20]. Briefly, after the endometrial epithelium was removed, the ULF supernatant
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was filtered through a 0.2 µm nylon filter and centrifuged again at 100,000 × g for another 60 min,
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The resulting pellet was suspended in 20 µL of PBS (pH 7.2) and stored at −80 °C.
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2.2 Identification of exosomes through transmission electron microscopy, IF,and and Western blot
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After gradient centrifugation, 7.5 µL of the pellet suspension was top loaded on 300-mesh grids
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and allowed to settle for 20 min at room temperature. The grids were then stained in 2% uranyl
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acetate, dried overnight at room temperature, and visualized with an energy-filtering transmission
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electron microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) at 120 KV.
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Immunofluorescence was conducted according to published protocols with slight modifications
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[17]. Briefly, 7.5 µL of purified pellet suspension was incubated with 5 µL of 4 µm
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aldehyde/sulfate latex beads (Life Technologies Corp., Grand Island, NY, USA) in a 30 µL final
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volume of PBS at room temperature for 15 min. PBS (170 µL) was then added, and the
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suspension was incubated in a tube rotator for 2.5 h at room temperature. Subsequently, 22 µL of
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1M glycine/PBS was added and mixed gently to block the unbound sites of the latex beads, and
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the mixture was allowed to stand on the bench for 30 min at room temperature.Afterwards, the
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beads were pelleted by centrifugation at 1,500 × g for 3 min at room temperature, and the pellets
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were washed twice with 1 mL of PBS/0.5% BSA. The exosome–bead complexes were incubated
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with anti-CD9 primary antibody (MEM-61, Thermo Fisher Scientific, Rockford, IL, USA) and
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immunoglobulin G (IgG) conjugated to fluorescein isothiocyanate isomer 1. A negative control
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antibody reaction was performed using normal mouse IgG untreated with anti-CD9 primary
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antibody. The labeled exosome–bead complexes were again pelleted, and the pellets were washed
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twice and resuspended in 20 µL of PBS/0.5% BSA. The final complexes (10 µL) were spread on a
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microscope slide with a drop of DakoCytomation fluorescent mounting medium (Dako,
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Carpinteria, CA, USA), air-dried, cover-slipped, and sealed with nail polish. Finally, the samples
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were visualized using a HAL 100 fluorescence microscope (Carl Zeiss Microscopy GmbH). The
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experiments were repeated in triplicate.
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For the Western blot assay, purified exosome precipitates were lysed in non-reducing Laemmli
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loading buffer and resolved in a 12% SDS-PAGE gel. The proteins were transferred to a PVDF
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membrane, and blocked with 10% skimmed milk for 4 h at room temperature. Primary mouse
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anti-GAPDH antibodie (1:2500 dilution, Sigma) and mouse anti-CD9 antibodies (1:1000 dilution;
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MEM-61, Thermo Fisher Scientific, Rockford, IL, USA) were mixed in TBST buffer (0.1%
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Tween 20 and 150 mM NaCl in 10 mM Tris-HCL, pH 7.4) with 5% skimmed milk and incubated
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with the membrane for 12 h at 4°C. After incubation with the primary antibodies, the blotting
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membrane was washed thrice for 15 min with TBST. The secondary anti-mouse antibody (1:1000
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dilution, Sigma) was mixed with TBST with 5% milk powder and incubated with the blotting
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membrane for 2 h at room temperature. The negative control group (NC) was not treated with
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anti-CD9 and anti-GAPDH primary antibodies. After incubation with the secondary antibodies,
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the blotting membrane was washed thrice for 15 min with TBST and visualized via
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chemiluminescence by using the ImageQuant LAS4000 biomolecular imager (GE Life Sciences).
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2.3 Oocyte collection and in vitro maturation (IVM)
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Ovaries were obtained from a local slaughterhouse and were transported to the laboratory in
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0.9% NaCl at 20–22°C. Immature cumulus oocyte complexes (COCs) were obtained by aspirating
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the follicles (2–8mm) from the ovaries. The COCs with more than three layers of compact
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cumulus cells and uniform cytoplasm were selected and used for IVM. The elected COCs were
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allowed to mature in vitro in a basic maturation medium (TCM-199, Gibco, BRL, Grand Island,
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NY, USA) supplemented with 10% (v/v) FBS, 1 µg/mL 17β-estradiol, 10 ng/ml epidermal growth
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factor, and 0.075 IU/mL human menopausal gonadotropin in humidified air with 5% CO2 at
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38.5°C for 22 h.
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2.4 Production of SCNT embryos and IVC The production of SCNT embryos was performed as previously described [21]. In brief, the in
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vitro-matured oocytes were enucleated using a 20 µm diameter aspiration pipette, microinjected
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into the perivitelline space of a female fetal fibroblast, and fused through a double electrical pulse
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stimulation. Successfully reconstructed embryos were maintained in mSOF containing 5 µg/ml
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cytochalasin B for 2 h and activated in 5 µM ionomycin for 4 min. Subsequently, the embryos
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were exposure to 1.9 mM dimethynopyridine in mSOF for 4 h. The embryos were cultured in the
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mSOF medium supplemented with or without exosomes from the luteal-phase uteri in accordance
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with the experimental design in a humidified atmosphere with 5% CO2 at 38.5°C.
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2.5 IF
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Immunofluorescence was conducted as described previously [22]. The embryos were fixed in
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4% (v/v) paraformaldehyde at 4 °C overnight, permeabilized with 0.2% (v/v) Triton X-100 in
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PBS for 20 min at room temperature, blocked with a blocking solution (Beyotime, P0102)
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overnight at 4 °C, and incubated overnight with primary anti-CDX2 mouse monoclonal antibody
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(BioGenex, Inc., San Ramon, CA) at 1:200 dilution in blocking buffer. After incubation, the
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embryos were washed in 0.1% PBS–PVA and treated with secondary Alexa Fluor 555-labeled
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goat anti-mouse IgG (1:500 dilution; Beyotime, A0459) in a dilution solution (Beyotime, P0108)
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for 2 h at room temperature. After the embryos were washed thrice in 0.1% PBS–PVA for 5 min
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per wash, nuclear labeling was performed using 4,6-diamidino-2-phenylindole hydrochloride
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(DAPI, Vysis Inc., Downers Grove, USA) for 3 min. After washing and mounting, the slides were
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examined by epifluorescence using a Nikon Eclipse Ti-S microscope (Nikon, Tokyo, Japan). All
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images were captured using the Nikon DS-Ri1 digital camera and saved in TIFF format. All the
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embryo cell nuclei were identified by their blue fluorescence, whereas the trophectoderm cell
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nuclei (CDX2 positive) exhibited red fluorescence.
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2.6 Apoptosis detection
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Apoptosis was detected using the DeadEnd Fluorometric TUNEL System (Promega, Madison,
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WI). Briefly, The blastocysts were fixed in 4% paraformaldehyde at 4 °C overnight,
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permeabilized in 0.2% Triton X-100 for 20 min, and incubated with FITC-conjugated dUTP and
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terminal deoxynucleotidyl transferase at 37 °C for 1 h in the dark. The tailing reaction was
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terminated in 2× SSC (SSC: 0.15 mol/L sodium chloride and 0.015 mol/L sodium citrate) for 15
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min. The embryos were then incubated in PBS containing 25 µg/mL RNase A for 30 min. After
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washing and DAPI staining, the embryos were mounted on slides for confocal microscopy and
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observed under Carl Zeiss LSM 510 laser confocal scanning microscope.
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2.7 Quantitative real-time PCR (q-PCR) Total RNA was extracted from day 7 blastocysts using the Cells-to-Signal™ kit (Invitrogen,
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USA) according to the manufacturer’s protocol. The cDNA was synthesized using
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PrimeScript™ RT reagent kit (TaKaRa, Japan) with a total volume of 20 µL (10 µL of 2 ×
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reaction mix, 1 µL of RT enzyme mix, 1 µL of oligo dT primer, 1 µL of random primers, 1 µL of
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DNA remover, 1 µg of RNA, and up to 20 µL of RNase-free dH2O). Reactions were performed in
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a low tube strip (Bio-Rad, GB). Each reaction mixture (20 µL) contained 2 µL (approximately
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100 ng) of cDNA template, 10 µL of SYBR® Premix Ex TII (2×), 0.8 µL of each PCR forward
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primer and reverse primer (10 µM), and 7.2 µL of dH2O. Quantitative PCR was performed using
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the CFX96 q-PCR detection system (Bio-Rad) with SYBR Premix Ex Taq™ II (TaKaRa, Japan).
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The thermal cycling conditions were as follows: 95°C for 30 s and 40 PCR cycles for 5 s at 95°C
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for DNA denaturation, 30 s at 60 °C for primer annealing, and 30 s at 72 °C for extension. The
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transcripts of the examined genes were quantified in triplicate and calculated relative to the
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housekeeping genes GAPDH and H2A.2. The specificity of the qPCR reaction was confirmed by
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the single peaks in the melt curves and by gel electrophoresis. The cDNA was replaced with water
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as negative control. The experiments were repeated at least thrice. All primers were designed
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using
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(http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The comparative cycle threshold method was
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used to quantify the expression levels. The primer sequences used for the PCR and qPCR are all
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listed in Table 1.
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2.8 Experimental design
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Primer-BLAST
software
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span
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boundaries
when
possible
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The experiments were divided into two parts. First, bovine exosomes were isolated from uteri
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in the early luteal phase, which corresponds to days 4–8 of the oestrous cycle (corpus luteum
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diameter: 12 mm to 18mm) by using differential ultracentrifugation protocol and identified using
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morphological examination, IF, and Western-blot. Second, the effects of the uterine exosomes on
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the development of SCNT embryos were assessed in terms of blastocysts formation rate, hatching
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rate,
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development-related genes. Between days3 and 4, the embryo develops into an 8-to 16-cell and
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embryo and moves from the oviduct to the uterus. To mimic the in vivo environment, we added
the
first
cellular
differentiation,
apoptosis
7
index,
and
expression
of
several
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the uterine exosomes into the culture medium on day 3 (72h), 4 (96h), and 5 (120h) of culture at
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3 × 105 exosomes/mL after the SCNT embryos activatied [6, 23].
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2.9 Statistical analysis The embryo cleavage rate, blastocyst yield, and relative mRNA abundance for the candidate
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genes were analyzed using one-way ANOVA (p<0.05). All analyses were performed using
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SigmaStat software package (Jandel Scientific, San Rafael, CA). For the total cell number and
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apoptosis index, 30 blastocysts were randomly selected from each medium group and compared
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using one-way ANOVA. Statistical significance was considered at P < 0.05 .
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3. Results
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3.1 Isolation and identification of exosomes from ULF
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As shown in Figs. 1A and 1B, exosomes were isolated from the ULF by ultracentrifugation at
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two different centrifugal forces (10,000 and 100,000 × g). Transmission electron microscopy
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showed that the isolated exosomes were spherical particles with a 50 nm to 150 nm diameter.
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These spheres were further characterized by immunostaining with CD9, a specific antibody for
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exosomal membranes. As shown in Fig. 2, CD9 was strongly laterally localized and was clearly
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visualized on the surface of the exosomes (Fig 2A and 2B). No signal was observed in the control
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group (Figs. 2C and 2D), This result indicated that our observed signal was specific. The Western
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blot also showed that the exosomal protein CD9 existed in the sample (Fig. 3). These results
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demonstrated the presence of exosomes in ULF.
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3.2 Effects of uterus-derived exosomes on the development of SCNT embryos
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The exosomes extracted from the ten uteri were diluted in 400 µL of mSOF supplemented with
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8 mg/ml BSA and added into a fresh medium at days 3(72 h), 4(96 h), and 5(120 h) after SCNT
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embryo activation. Photographs of the SCNT blastocysts are presented in Fig. 4. As shown in
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Table 2, uterine exosome supplementation on day 4 of culture significantly increased the
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blastocyst formation rate (40.3% vs. 34%, 31%, and 34.3%, respectively, P<0.05) and hatching
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rate (40.7% vs. 33.3%, 30.3%, and 35%, respectively, P<0.05) in comparison with the
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non-supplementation group (control group), day 3 and day 5 supplementation groups.
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3.3 Effects of uterus-derived exosomes on SCNT embryo quality
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Based on the findings that exosome supplementation on day 4 of culture significantly enhanced
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the blastocyst formation and hatching rate, the quality of the blastocyst from the day 4 exsome
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supplementation group were further investigated using TUNEL assay (Fig. 5), The rate of
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ICM/TE differentiation (Fig. 6), and the expression levels of several development-related genes of
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the preimplantation embryos (Fig. 7) were also examined. The exsome supplementation
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significantly reduced the apoptotic index (2.1% vs. 6.5%, P<0.05; Fig. 5), and increased the
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ICM/TE ratio (48% vs. 37.9%, P<0.05; Fig. 6) of the SCNT blastocysts compared with the NC
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group. The expression levels of IFNT and acrogranin were significantly high, and those of BAX
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(pro-apoptosis marker), HSP70 and BIP were significantly low, The expression level of PMSB5
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showed no difference in the exosome supplementation day4 blastocyst group compared with that
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in the NC group (Fig. 7).
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4. Discussion
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Uterus-derived exosomes were first found and isolated from human ULF by Ng et al. [17] and
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were later confirmed to exist in various other animals [24]. In the present study, exosomes were
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successfully isolated from fresh luteal phase uterus by ultracentrifugation and were identified
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through morphologic examination, IF, and Western blot. Previous studies suggested that bovine
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exosomes exhibit a mean size of approximately 148 nm [24]. Similarly, our results showed that
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the exosomes were mainly 100 to 150 nm in diameter. The IF and Western blot confirmed that
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CD9, an exosomal marker protein, existed in the ULF. All these parameters were consistent with
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those of previous studies, indicating the successfully isolation of the exosomes from bovine ULF
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in the present study.
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Exosomes released by the endometrial epithelium are involved in the transfer of signaling
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miRNAs and mRNAs to either the blastocyst or the adjacent endometrium. This transfer of
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materials influences endometrial receptivity and implantation success. Many components of the
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ULF are required for conceptus development [25]. The endometrium secretes substances,
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collectively termed histotroph, that govern the growth and elongation of the conceptus via its
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effects on trophectoderm proliferation, migration, attachment and adhesion to the endometrial
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luminal epithelium [26]. These uterus-derived exosomes contain various materials, such as
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proteins, miRNAs and pluripotency genes mRNAs (Oct4, Sox2, Klf4, c-Myc, and Nanog), which
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change dynamically throughout pregnancy [27]. Although we did not performend experiments to
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examine the substances in the uterus-derived exosomes, the results showing the time-dependent
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effect of the exosomes on embryonic development indicated that the exosomes might contain
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different substances at different physiological period of pregnancy.
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Despite the efforts to elucidate the function of EVs during reproductive events, the role of
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bovine uterus-derived exosomes in early embryonic development remains unknown. Our results
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were the first to show that exosomes can be successfully isolated from bovine early luteal phase
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uterus and can successfully improve the development capacity of SCNT embryos in vitro. In the
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present study, the effects of the uterus-derived exosomes on the quality of SCNT embryos were
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assessed. Our results (Table 2) showed that the group with exosome supplementation on Day 4
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exhibited a significant improvement in blastocyst formation and hatching rate compared with the
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control group. In addition, the blastocyst quality in the day4 supplementation group was
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significantly improved as assessed by the total cell number, ICM/TE ratio(Fig. 6), and apoptotic
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index (Fig. 5). The day4 nuclear transfer embryo (NTE) group showed a higher ICM/TE ratio
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(48% vs. 37.9%, P<0.05; Fig. 6) than the control group. In the literature, the ICM: TE ratio is 44%
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–50% and 17% in blastocysts derived in vivo, and in NTE, respectively [28, 29]. Placental
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abnormalities or early fetal loss might be caused by the aberrant allocation of ICM and TE cells in
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the NTE during the preimplantation stages. The structural integrity of blastocyst composed of
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ICM and TE cells, might influence postimplantation development in mammals [30, 31]. The
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exosome supplementation on the day 4 of culture improved the blastocyst structural integrity, and
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such improvement may influence the postimplantation development.
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Apoptosis is a normal physiological process in which cells die after exposure to normal or
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pathologic stimuli in vivo. Embryos produced in vitro usually exhibit a high apoptosis index,
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probably because of suboptimal culture conditions [32]. The incidence of cell death in the human
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blastocyst is correlated with the cell number and embryo quality [33, 34]. The TUNEL assay (Fig.
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5), showed a significantly lower apoptosis index in the day 4 groupthean in the control group
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(2.1% vs. 6.5%, P<0.05; Fig. 5). This result suggested that the exosome supplementation on day 4
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of culture provided a better culture condition for the bovine SCNT embryos, The incidence of
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apoptotic cells, the rate of blastocyst formation, and cell numbers in the SCNT blastocysts could
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be markers of blastocyst quality.
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To further evaluate the competence of the SCNT embryos derived from the day4 group, we
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analyzed the important genes related to embryonic development. We systematically compared the
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relative expression levels of HSP70, BAX, IFNT, acrogranin, BIP and PMSB5 (Fig. 7). The
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expression levels of HSP70 [35], BAX
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reticulum stress marker) remarkably decreased.The BAX expression level was consistent with the
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result of the TMNEL assay. Apoptotic cell death in preimplantation mammalian embryos plays an
299
important role in embryo development. HSP70 is a prominent cytoprotective factor that confers
(a pro-apoptosis marker), and BIP (an endoplasmic
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tolerance to cellular stress or transfection by inhibiting the induction of apoptosis [36, 37]. BAX
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and HSP70 interact under stress conditions, and BAX is suppressed in cells with high HSP70
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levels [38]. In the present study, we found no significant difference in the abundance of for HSP70
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and Bax transcripts. The apoptosis detected in our study was consistent with that of a previous
304
study [39]. Previous research showed that the expression levels of BAX, HSP70, BIP and PSMB5
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are significantly high in SCNT blastocysts than in vivo, This finding suggests that SCNT embryos
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are exposed to high cellular stress in vitro than in vivo [40]. Our results showed that the exosome
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supplementation optimized the embryo culture condition as supported by the low BAX, HSP70,
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and BIP expression levels. A low BIP expression level suggested that the embryo undergoes
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minimal apoptotic damage. IFNT is a maternal recognition signal required for a successful
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pregnancy [41], Results of the present work showed high expression level of IFNT in a treated
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with exosomes. Rizol et al [42] observed a significantly high expression level of IFNT in a
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serum-free medium; this finding is consistent with the notion that the mRNA levels for this
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transcript are high in good-quality embryos. Furthermore, the amount of interferon is correlated
314
with hatching rate and morphologic quality [43]. Acrogranin is positively related to blastocyst
315
formation and hatching rate [7]. In the present study, the expression levels of IFNT and
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acrogranin was high in the day 4 supplementation group, This result suggested that the
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uterus-derived exosomes improved the blastocyst formation and hatching rate. Collectively, these
318
results indicate a preferable embryo quality in exsome-optimized condition.
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5. Conclusions
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Bovine uterus-derived exosomes can be successfully isolated and used for in vitro SCNT
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embryo culture with a positive effect on embryo quality, The effect is reflected in the improved
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blastocyst formation, increased total cell number, increased ICM/TE ratio , decreased TUNEL
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signal, increased IFNT and acrogranin gene expression levels, and low expression levels of BAX
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and HSP70. This study was the first to report that exosomes can be successfully isolated from
325
bovine early luteal phase uterus and that exosome supplementation can improve the
326
develpomental capacity of SCNT embryos in vitro.
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Acknowledgement
328 329 330
This research was supported by the National Major Project for Production of Transgenic Breeding (No. 2016ZX08007003) and National Natural Science Foundation of China (NO.31472094).
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ACCEPTED MANUSCRIPT Figure legends
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Fig 1. Uterus-derived exosomes were identified by electron microscope analysis. (A) Transmission Electron Microscopy (TEM) images showed the presence of exosomes after gradient centrifugations and negative staining with uranyl acetate. (B) Size distribution of exosomes derived from uterine luminal fluid identified by Transmission Electron Microscopy. Bar, 100 nm.
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Fig 2. Uterus-derived exosomes were identified by immunofluorescence. The exosomes were bound to beads of a size that was in the detection range of the fluorescence microscope (4-µm diameter latex beads). The beads were then bound to fluorescence-conjugated antibody against CD9. Images were taken under epifluorescence (A, C) and DIC (B, D,). A,B: uterus-derived exosomes; C,D: IgG negative control; Bar, 10 µm.
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Fig 3. Uterus-derived exosomes proteins were identified by western blot. The NC group was treated without anti-CD9 first antibody and anti-GAPDH first antibody. Fig 4. Representative photographs of bovine SCNT blastocysts derived from NC, Day 3, Day 4, Day 5 groups.
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Fig 5. The apoptotic blastomeres in Day 4 and NC blastocysts groups. (A) Blastocysts were detected by TUNEL (green). DNA was stained by DAPI (blue) to visualize all blastomeres. Bar, 80 µm. (B) Apoptotic index of blastocysts. a.b Different superscripts indicate significant differences for each gene (p<0.05). Data presented are mean ± SE.
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Fig 6. Ratio of ICM/TE in Day 4 and NC blastocysts groups. (A) Immunostaining of CDX2. a specific marker of trophectoderm (red), as well as DAPI that stained nuclei of all blastomeres (blue). Bar, 80 µm. (B) Statistical analysis of ICM/TE in Day 4 and NC blastocysts groups. a.b Different superscripts indicate significant differences for each gene (p<0.05). Data presented are mean ± SE.
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Fig 7. Relative mRNA transcription in bovine in vitro blastocysts cultured with exsomes at the embryo cultured Day 4 of genes related with embryo quality. Data are relative to mean of internal gene H2A. Data presented are mean ± SE. a.b Different superscripts indicate significant differences for each gene (p<0.05).
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Table 1 primers sequences for real time-qPCR
Gapdh
sequence
F(3’-5’)CGACTTCAACAGCGACACTCAC R(3’-5’)CCTGTTGCTGTAGCCGAATTC
H2A.2
F(3’-5’)GAGGAGCTGAACAAGCTGTTC
F(3’-5’) CGGACACCTGACCAATGGAA
in
R(3’-5’) CTGTCACTAAGGCCACCCAG F(3’-5’)TGTTTGTTATGGAGACTGTTGGGA
Size(bp)
(°C)
158
58
NM_173979
144
58
BF 076731
155
58
XM_01081640
223
Gene
bank
access No.
5.2
58
NM_174550
114
58
NM_173894
167
58
NM_00116827
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Hsp70
Tm
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R(3’-5’)TTGTGGTGGCTCTCAGTCTTC Acrogran
Product
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R(3’-5’) GGTGAAAAATAGCTTGAGTTTGGC Bax
F(3’-5’) TCCCCGAGAGGTCTTTTTCCG
R(3’-5’) AGGGCCTTGAGCACCAGTTTG IFNtau
F(3’-5’) AGGTTAAGAAAGATGGGTGGAGA
9
R(3’-5’) ACTGCTGACAAAGTATCGGCTAA
F(3’-5’)GATTGAAGTCACCTTTGAGATAGATGTG
TE D
Bip
85
58
NM_00107514 8.8
R(3’-5’) GATCTTATTTTTGTTGCCTGTACCTGG Psmb5
F(3’-5’) GACGTTTGCGTTTGCTTCCT
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R(3’-5’) ACCACGCCTCCATACACAAG
178
58
NM_00103761 2.1
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ACCEPTED MANUSCRIPT Table 2 Effect of uterus-derived exosomes on in vitro development of SCNT embryos groups
No. embryo cultured
blastocyst(%)
hatching（%） a
Nc
195
31%+0.01
Day3 Day4 Day5
189 192 186
34%+0.02a 40.3%+0.01b 34.3%+0.01a
30.3%+0.01
No. blastomeres
a
33.3%+0.01a 40.7%+0.02b 35%+0.02c
90.3a 94.7a 105.3bc 101ab
a-c
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different superscripts with same column indicate significant difference (P<0.05) day 3, day 4, day 5 suggested exosome supplementation after embryo activation for 3, 4, 5 days.
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Highlights 1. Our study firstly isolated exsomes from bovine uterine luminal fluid (ULF). 2. Uterus-derived exsomes significantly improved SCNT embryo information and hatching in vitro. 3. Uterus-derived exsomes enhanced the quality of SCNT embryos assessed by increased total cell
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number, increased ratio of ICM/TE, decreased TUNEL signal, and increased gene expression of IFNT and acrogranin as well as a low expression level with BAX，HSP70, and BIP.
4. This is the first time study the effect of uterus-derived exosomes on SCNT embryos development,
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and it may provided new methods to improve the efficiency of SCNT.