BMP-1 participates in the selection and dominance of buffalo follicles by regulating the proliferation and apoptosis of granulosa cells

Accepted Manuscript BMP-1 Participates in the Selection and Dominance of Buffalo Follicles by Regulating the Proliferation and Apoptosis of Granulosa Cells Xiaocan Lei, Kuiqing Cui, Zhipeng Li, Jie Su, Jianrong Jiang, Haihang Zhang, Qingyou Liu, Deshun Shi PII:

S0093-691X(15)00631-7

DOI:

10.1016/j.theriogenology.2015.11.011

Reference:

THE 13417

To appear in:

Theriogenology

Received Date: 25 June 2015 Revised Date:

10 November 2015

Accepted Date: 14 November 2015

Please cite this article as: Lei X, Cui K, Li Z, Su J, Jiang J, Zhang H, Liu Q, Shi D, BMP-1 Participates in the Selection and Dominance of Buffalo Follicles by Regulating the Proliferation and Apoptosis of Granulosa Cells, Theriogenology (2015), doi: 10.1016/j.theriogenology.2015.11.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Revised

BMP-1 Participates in the Selection and Dominance of Buffalo Follicles by

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Regulating the Proliferation and Apoptosis of Granulosa Cells

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Xiaocan Lei, Kuiqing Cui, Zhipeng Li, Jie Su, Jianrong Jiang, Haihang Zhang,

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Qingyou Liu*, Deshun Shi*

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Animal Reproduction Institute, State Key Laboratory for Conservation and Utilization

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of Subtropical Agro-bioresources, Guangxi University, Nanning, China

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X. Lei and K. Cui contributed equally to this work.

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*Corresponding author. Tel.: +86 771 3239202; fax: +86 771 3239202.E-mail

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addresses: [email protected] (D. Shi) and [email protected] (Q. Liu).

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ACCEPTED MANUSCRIPT ABSTRACT

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BMP-1/TLD-related metalloproteinases play a key role in morphogenesis via the

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proteolytic maturation of a number of ECM proteins and the activation of a subset of

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growth factors of the TGF-β superfamily. Recent data indicated that BMP-1 is

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expressed in sheep ovarian follicles and showed a protease activity. The aim of the

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current study was to characterize the function of the buffalo BMP-1 gene in

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folliculogenesis. A 3195 bp buffalo BMP-1 mRNA fragment was firstly cloned and

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sequenced, which contained a whole 2967 bp CDS. The multialigned results

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suggested that BMP-1 is highly conserved among different species both at the nucleic

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acid and the amino acid level. BMP-1 is located in the oogonium of the fetal buffalo

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ovary, as well as in the granulosa cells (GCs) and the oocytes of adult ovary from the

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primordial to the large antral follicles. Further study showed that BMP-1 promoted

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cell cycle and proliferation and inhibited apoptosis in in vitro cultured GCs. Adding

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BMP-1 recombinant protein to the culture medium of the GCs increased the

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expression of the key cell cycle regulators such as Cyclin D1 and Cyclin D2. And

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down-regulated the expression of cell apoptosis pathway genes such as Cytochrome C,

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Fas, FasL and Chop .etc, both at the mRNA and the protein levels. It also up-regulated

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the expression of PAPP-A, IGF system and VEGF etc., which play important roles in

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the selection and dominance of growth follicles. The opposite results were observed

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by adding BMP-1 antibody to the investigation groups. This study suggests that

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BMP-1 regulates the proliferation and apoptosis of in vitro cultured GCs by changing

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the expression pattern of related genes, and may potentially promote the selection and

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ACCEPTED MANUSCRIPT 45

dominance of the buffalo follicles.

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Key words: Buffalo; BMP-1; Folliculogenesis; Proliferation; Apoptosis

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1. Introduction Bone Morphogenetic Proteins (BMPs) comprise an extensive group of

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phylogenetically conserved growth factors of which over 20 members have been

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identified [1]. Bone morphogenetic protein-1 (BMP-1) was originally identified in

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bone extracts capable of inducing bone formation at ectopic sites [2]. Unlike the other

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BMPs such as BMP-2 and BMP-4, which belong to the transforming growth factor

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beta (TGF-β) super family, BMP-1 is a zinc-dependent metalloproteinase that belongs

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to the astacin family [3]. BMP-1 has been reported in different species, including

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human [4], mouse [5], xenopus [6], drosophila [7], sea urchin [8] and chick [9] , as

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having a similar structure. It contains a NH2-terminal activation region and an

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astacin-like protease domain and is followed by different numbers of EGF-like motifs

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and a CUB protein-protein interaction domains [10], and a mammalian homolog of

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tolloid was identified (mTLD), which turned out to be a splice-variant encoded by the

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same gene as BMP-1 [11].

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BMP-1 is expressed and has distinct temporal functions in different isoforms

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during the morphogenesis [12, 13]. Three XBMP-1 transcripts (2.9, 5.2, and 6.6 kb)

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were found in the blastula and gastrula stages of xenopus by Northern Blot [6], which

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increased gradually from the morula to the swimming tadpole stages [14]. The over

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expression of BMP-1 in early xenopus embryos inhibited the development of

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dorso-anterior structures [15]. In sea urchin, the highest level of suBMP-1 mRNA was

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in late gastrula stage of the embryos, which suggested that suBMP-1 is a secreted

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protein that subsequently associates with a cell surface component [8]. Furthermore,

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BMP-1-null mice generated a syndrome of a persistent herniation of the gut in the

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umbilical region and were embryonic lethal [16, 17].

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BMP-1 is the prototype of a family of putative proteases that is implicated in

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pattern formation during development in diverse organisms [18]. BMP1 cleaves

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human and mouse IGFBP3 at a single conserved site, resulting in a markedly reduced

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ability of cleaved IGFBP3 to bind IGF-I or to block IGF-I-induced cell signaling. In

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contrast, such cleavage is shown to result in the enhanced IGF-I independent ability of

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cleaved IGFBP3 to block FGF-induced proliferation and to induce Smad

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phosphorylation [19]. BMP-1 plays a major role in the cleavage of latent TGF-β

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binding proteins (LTBPs), which releases the complex formed by TGF-β1 and LAP

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(propeptide) from the ECM [20]. It was also shown that BMP-1 contributes to

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maintaining high levels of active TGF-β1 in tissues by promoting the degradation of

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two TGF-β antagonists (soluble betaglycan and CD109 [21]). BMP-1 activates

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several other members of the TGF-β or IGF superfamilies, such as GDF-8/11 [22, 23],

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BMP-2/4 (chordin; [24, 25]) and IGF-1/2 (IGFBP3, [26]). Similarly, BMP-1 cleaves

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several proteins, including endorepellin, which is endowed with strong angiogenic

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properties [27]. In addition, BMP-1 can turn prolactin and growth hormone into

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potent anti-angiogenic molecules [28, 29] and abolish the pro-metastatic potential of

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angiopoietin-like protein 2 [30]. Recently, it was reported that BMP-1 was present in

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GCs at all stages of sheep follicular development both at the mRNA and the protein

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level [31], which suggested a new physiological role for BMP-1 metalloproteinases in

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mammalian folliculogenesis, but the underlying mechanisms need to be explored. Buffalo is one of the most important domestic animals distributed in tropical and

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subtropical regions and provides better milk, meat and draft for agriculture [32]. The

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Chinese swamp buffalo has a lower reproductive ability, and this limits the production

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of this species. Therefore, there is an urgent need to improve their production traits by

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genetic manipulation technology, and to determinate the function of more genes

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involved in the folliculogenesis of buffalo. To our knowledge, expression pattern and

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function of BMP-1 in the folliculogenesis of swamp buffalo were seldomly reported.

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The current study was performed to investigate the expression pattern of BMP-1, as

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well as its function during the folliculogenesis of the swamp buffalo.

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2. Materials and methods

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2.1. Cloning and analysis of buffalo BMP-1

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Three adult swamp buffalo ovaries were collected from the local slaughterhouse

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and the total RNA was extracted using the Trizol reagent (Ambion, Life Technologies,

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USA) according to the manufacturer's instruction. Three independent preparations

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were used. The first-stranded cDNA was synthesized from 2 µg of total RNA for

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RT-PCR by using the Prime ScriptTM 1st strand cDNA synthesis kit (Takara, Japan).

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A

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R:5′-GTCTCCCATCCCTGCC-3′) were designed based on the sequence of bovine

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BMP-1 (XP_002689817.1). Then, a touchdown PCR (TD PCR) was performed with

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All assessments were conducted in three biological replicates. The PCR products were

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purified using a TIAN Gen Mini Purification Kit (TIANGEN BIOTECH, Beijing CO.,

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Ltd), inserted into the pMD18-T vector (Takara, Japan) and transformed into DH 5α

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Escherichia coli (Stored in the laboratory). The positive clones were sequenced by the

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automated sequencing method (BGI-Guangzhou, China).

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The alignment of the nucleotide sequences was established with the NCBI Blast

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program [http://www.ncbi.nlm.nih.gov/BLAST]. The open reading frame and protein

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prediction

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[http://www.ncbi.nlm.nih.gov/gorf/gorf.html]. The protein domain architecture was

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predicted on [http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1]. The

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alignment of the highly conserved sequence, the Zn2+-binding sequence in the signal

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peptide and metalloendopeptidase were performed onsite [http://www.uniprot.org/].

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The multi-alignments were carried out on Clustalx 1.83 and GeneDoc software, and a

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phylogenetic tree was constructed using the Neighbor-Joining method with the

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MEGA 5 program and visualized by TreeView software.

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2.2. Immunohistochemistry

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Fetal and adult swamp buffalo ovaries were obtained from the local slaughter

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house. They were fixed in 4% paraformaldehyde (PFA) (P-6148, sigma), dehydrated

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and paraffin embedded. Serial sections of 5-µm-thick were cut using a Leica RM

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2235 rotary microtome. The sections were processed in 1:49 APES: acetone,

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de-parafinized and rehydrated. Then, the sections were incubated with 3% hydrogen

ACCEPTED MANUSCRIPT peroxide in methanol, boiled in 10 mM sodium citrate buffer, and permeabilized in

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1% Triton X-100. After blocking with 5% BSA, the sections were incubated with the

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goat polyclonal BMP-1 (sc-27324, SANTA) antibody (diluted 1:100 in 1% Tween-20

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in PBS (PBS-T)) at 4℃ overnight. After three 5-min washes with PBS-T, the sections

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were incubated with rabbit anti-goat Biotin-SP-conjugated antibody (1:100,

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SA00004-4, Protein Tech Group, Inc.) and Peroxidase-conjugated Streptavidin (1:100,

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SA00001-0, Protein Tech Group, Inc.) separately and followed another 5-min washes

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in PBS-T. The sections were incubated with a DAB color development kit for 2 min

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and then counterstained with hematoxylin at RT (room temperature). Finally, the

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sections were dehydrated in a graded series of ethanol to 100% and mounted with

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Neutral Balsam. The negative controls were generated in which the primary antibody

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was replaced with rabbit IgG. The sections were observed with an Olympus DP70

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digital camera mounted on a Leica DMR microscope with Nomarski optics.

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2.3. Immunofluorenscence

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The in vitro matured (IVM) of cumulus-oocytes complex (COCs) and the

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denuded oocytes were performed using a method from our laboratory [33]. All of the

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samples were collected and fixed with 4% PFA in PBS for 1 h at room temperature

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(RT) respectively. They were washed with PBS containing 0.1% TritonX-100 and

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0.3% BSA (TBP) and permeabilized with 1% Triton X-100 in PBS, and blocked with

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PBS supplemented with 1% BSA at RT for 1 h. Afterwards, they were incubated with

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the primary goat polyclonal anti-BMP-1 (sc-27324, SANTA, 1:200) antibody at 4℃

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overnight. After washing for three times within TBP, they were incubated with

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ACCEPTED MANUSCRIPT Fluorescein conjugated rabbit anti-goat IgG (H+L) (1:100, Protein Tech Group, Inc.,

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Chicago, SA00003-2) for 1 h at RT (in the dark) and then washed another three times.

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The samples were counterstained with 10 µg/mL PI (propidium iodide) (Sigma) for

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10 min at RT (in the dark), washed in TBP and mounted on the slides with fight

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quenches agent. The samples were observed under a laser scanning confocal

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microscope (Zeiss LSM510 Meta).

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2.4. In vitro culture of GCs

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The GCs were collected from 3 to 6 mm buffalo follicles with a 10 mL injection

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syringe by differential centrifugation according the protocol reported by Lucie [34].

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The contamination of theca cells was judged to be < 1% based on the comparison of

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the relative expression of CYP17A1 and LHCGR in freshly isolated granulosa cells

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and theca cells by qPCR (data not shown). After 3 washes with DMEM, 106/mL GCs

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were cultured in 6-cm dishes with 3 mL DMEM containing 10% FBS. The different

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doses of the BMP-1 recombinant protein (0, 5, 15, 25 and 50 ng/mL [35]) and BMP-1

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antibody (0, 50, 100, 200 and 400 ng/mL) were respectively added into the culture

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medium. After 72 h, the GCs were collected for total RNA and protein extraction (Fig.

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S3). The GCs were also cultured in a 96-well plate for a cell proliferation assay and

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6-well plates for separate cell cycle and apoptosis analyses.

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2.5. RNA analysis (real-time PCR)

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Quantitative real-time PCR (qPCR) was performed to determine the gene

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expression pattern of BMP-1 and other genes. Buffalo GCs were cultured in vitro for

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72 h, and when the cells number was reached approximately 5×106 total RNA was

ACCEPTED MANUSCRIPT extracted with the Trizol reagent (Ambion, Life Technologies, USA) according to the

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manufacturer's instructions on three independent preparations. Then, the first-strand

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cDNA from the GCs were synthesized from 2 µg of total RNA for RT-PCR by using

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the Prime ScriptTM 1st strand cDNA synthesis kit (Takara, Japan). The first-strand

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cDNA from oocyte and COCs (cumulus and oocyte complex) was prepared with the

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Cells-to-cDNA™ II Kit (Applied Biosystems) according to the manufacture

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instructions. The assays of the candidate genes, including Cyclin D1, Cyclin D2,

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Cytochrome C, Fas, FasL, Chop, etc., as well as PAPP-A, the IGF system and VEGF

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were carried out using specific primers, as shown in table 1, on the ABI PRISM 7500

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Real Time System (Applied Biosystems). The reaction mixture (20 µL) contained 10

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µL SYBR qPCR Super Mix-UDG with Rox (Takara, Japan) and was run at 95℃ for

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10 min, followed by 40 amplification cycles of 95℃ for 30 s and 60℃ or 55℃ for 1

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min. The relative expression level of the assayed genes was calculated by the

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comparative Ct method using 18S rRNA as a reference gene.

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2.6. Western Blot Analysis

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Buffalo GCs were cultured in vitro for 72 h, and when the cells number was

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reached approximately 5×106 the GCs were collected and lysated with RIPA buffer.

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Briefly, the separated proteins were separated by in SDS PAGE and electrically

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transferred to a nitrocellulose filter membrane, and then, the membrane was blocked

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in 50 mM Tris, 150 mM NaCl, and 0.1% Tween (TBST) containing 5% skimmed

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milk for 45 min at RT, followed by incubation overnight at 4℃ with rabbit polyclonal

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anti-Fas, Bax and Caspase-9 antibodies (Proteintech Group, Inc, 1:1000, 1:800 and

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ACCEPTED MANUSCRIPT 1:500 respectively) and the mouse monoclonal anti-β-actin antibody (1:2000). After

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three 10 min washes in TBST, the membranes were incubated for 1 h at RT with

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peroxidase-conjugated goat anti-rabbit IgG (Proteintech Group, Inc, 1:3000) and

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peroxidase-conjugated goat anti-mouse IgG (Proteintech Group, Inc, 1:3000)

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respectively. Finally, the membranes were washed three times in TBST and processed

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for detection by ECL with the Bio-Rad ChemiDoc system.

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2.7. Cell proliferation assay

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Cell proliferation was analyzed using the CCK-8 assay as reported by Yong

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Zhang [36]. Briefly, the buffalo GCs were cultured in 96-well plates at a density of

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1×104 per well with different concentrations of BMP-1 recombinant protein or

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antibody in growth medium for 72 h and then incubated with cck-8 for 1.5 h. After the

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incubation, the color reaction was measured at 450 nm by using an Enzyme

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Immunoassay Analyzer (Bio-Rad, Hercules, CA, USA). The proliferation activity was

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calculated for five times for each group with the different concentrations of BMP-1

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recombinant protein and antibody.

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2.8. Cell cycle analysis

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GCs were treated with different concentrations of BMP-1 recombinant protein or

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antibody and cultured in 6-well plates for 72 hours. After harvesting, the cells were

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washed and fixed with 70% ethanol overnight at -20℃. The fixed cells were pelleted,

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washed with ice-cold PBS, and resuspended in staining solution with 50

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µg/mL propidium iodide, 0.1% Triton-X-100, 0.1% sodium citrate, and 100 µg/mL

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RNase. After a 30 min incubation, the cells were acquired and analyzed by the Merck

ACCEPTED MANUSCRIPT Millipore guava® easyCyte HT System. Twenty thousand events were analyzed for

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each sample, and the appropriate gating was used to select the standardized cell

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population.

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2.9. Cells apoptosis assay

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Apoptosis detection was performed using the FITC Annexin V Apoptosis

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Detection Kit (BD Biosciences, USA). The buffalo GCs were treated with different

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concentrations of BMP-1 antibody and cultured in 6-well plates for 72 h. The cells

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were harvested, washed with PBS, resuspended in 1× Annexin V binding buffer, and

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stained with annexin V and propidium iodide for 15 min at RT in the dark. Apoptosis

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was determined using the Merck Millipore guava® easyCyte HT System or observed

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under a laser scanning confocal microscope (Zeiss LSM510 Meta) (Fig. S4). The

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distribution of the cell population in the different quadrants was analyzed with

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quadrant statistics. The lower left quadrants consisted of viable cells, lower right

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quadrants the early apoptotic cells, and the upper right quadrants the late-apoptotic or

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necrotic cells.

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2.10. Statistical analysis

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The differences between groups were analyzed by one-way ANOVA and the

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least significant difference (LSD) post hoc test. The results are summarized as the

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means ± SEM from three independent observations. Each in vitro experiment was

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performed at a different time. The statistical analyses were performed by using SPSS

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17.0 program for Windows. P < 0.05 was considered statistically significant.

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3. Results

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3.1. Molecular cloning and bioinformatics analysis of buffalo BMP-1 gene A 3195 bp BMP-1 cDNA fragment was amplified and sequenced from the RNA

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samples of the swamp buffalo ovary, which contained a 2967 bp open reading frame

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extending from 145 bp to 3111 bp, encoding a 988 aa protein (KC007442.1, Fig. S1).

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The alignment with the BLAST software revealed that swamp buffalo BMP-1 is a

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highly conserved gene, which shared 98%, 96%, 95%, 95% and 95% similarity at the

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amino acid level with that of Cricetulus Griseus (EGV91962.1), Sus Scrofa

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(XP_001927808.2), Human (AAI42954.1), Bovine (XP_002689817.1) and Mouse

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(NP_033885.2). The N-terminal signal sequence of buffalo BMP-1 is followed by the

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21 amino acid sequence YKDPCKAAAFLGDIALDEEDL (Fig. 1), and contains an

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active sites consensus sequence that is typical of metalloendoproteases, namely

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HEXXHXXGFXHE (Fig. 1). All of the indicated proteases in the different species

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exhibit extremely high sequence similarity around this active site, which was

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highly conserved during evolution, as shown in the phylogenetic tree (Fig. S2). The

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predicted domain of buffalo BMP-1 contained a signal peptide, a preregion, a Zinc

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binding domain, followed by five CUB domains and two EGF-like domains and was

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homologous among the different species (Fig. S1 and Fig. 2).

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3.2. Expression pattern of BMP-1 during the follicular genesis of the swamp buffalo

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Buffalo BMP-1 was observed predominantly in the oogonium of the fetal ovary

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by immunohistochemistry (Fig. 3). BMP-1 is especially localized in the GCs of all

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categories of follicles, from the primordial to the large antral follicle stages in

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adulthood ovary (Fig. 3). Occasionally, in the large antral follicles and the mature

ACCEPTED MANUSCRIPT follicles, the staining intensity in the membrane of the oocyte was stronger, and a faint

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staining was observed in theca cells. In the buffalo COCs and the denuded oocytes, as

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shown in Fig. 4A, positive BMP-1 immunostaining was detected on the membrane of

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both the GCs and the oocytes. BMP-1 was more highly expressed in the COCs than

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that of the denuded oocytes, and showed higher expression in the mature COCs and

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the denuded oocytes than the immature ones (Fig. 4B).

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3.3. The effect of BMP-1 on in vitro cultured GCs

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To determine the effect of BMP-1 on in vitro cultured GCs, different

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concentrations of BMP-1 recombinant protein or antibody were added to the culture

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medium. The 25 ng/mL BMP-1 recombinant protein group significantly promoted the

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proliferation of the in vitro cultured GCs (p<0.05) (Fig. 5A). On the contrary, the

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BMP-1 antibody groups generated the opposite results and significantly inhibited the

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proliferation of the GCs in a dose-dependent manner (p<0.05, Fig. 5B). The effect of

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BMP-1 on the cell cycle progression of the GCs was further determined by a flow

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cytometer. Treatment with the BMP-1 recombinant protein significantly reduced the

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cell percentage in the G0/G1 phase and increased the amount of cells in the S phase of

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the cell cycle (Fig. 6A, 6C). A significant accumulation in the S phase cell population

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(38.28% ± 1.23%) was observed when the cells were treated for 72 h with 25 ng/mL

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BMP-1 recombinant protein. The percentage of cells in S phase decreased from

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31.68% ± 1.56% (control) to 28.57% ± 1.38%, 27.56% ± 1.47%, 24.78% ± 1.96% and

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21.10% ± 2.07%, when treated with increasing concentrations of the BMP-1 antibody

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(50-400 ng/mL) (Fig. 6B, Fig. 6D).

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ACCEPTED MANUSCRIPT The effect of BMP-1 on apoptosis in the GCs was determined by flow cytometric

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analysis using Annexin V/PI (Fig. 7). When the concentration of the BMP-1 antibody

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was increased from 50 ng/mL to 400 ng/mL, the population of early apoptotic cells

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increased from 12.93% (control) to 18.22%, 19.16%, 23.38% and 42.86%, while the

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Annexin V/PI double positive cells increased from 5.01% (control) to 5.20%, 6.07%

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and 6.69%.

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3.4. BMP-1 affects the expression pattern of cell cycle and apoptosis-related genes

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The qPCR results showed that the cell cycle related gene expression levels of

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Cyclin D1 and Cyclin D2 were up-regulated more than 5-fold in the BMP-1

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recombinant protein groups (Fig. 8 A, E), and in contrast they were down-regulated in

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a dose dependent manner in the BMP-1 antibody groups, which showed that 400

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ng/mL of BMP-1 antibody significantly suppressed the expression of Cyclin D1 and

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Cyclin D2 (p<0.05, Fig. 8 B, F). The apoptosis-related genes, including Fas, Fas L,

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TNFα, TNFR1, cytochrome C, Apaf1, Chop, Caspase-3, Caspase-8, Caspase-9 and

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Bax were down-regulated in different degrees in the BMP-1 recombinant protein

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groups (Fig. 8 C, E). In contrast, these genes were significantly (p<0.05) up-regulated

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by adding 400 ng/mL BMP-1 antibody (Fig.8 D, F). The western blot analysis proved

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that BMP-1 recombinant protein reduced the expression of Fas, Bax, and Caspase-9

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(Fig. 9), while the BMP-1 antibody increased the expression of these genes at the

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protein level.

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3.5. BMP-1 affects the genes expression in the dominance and selection of the follicles

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As BMP-1 promotes the proliferation and inhibits the apoptosis of GCs, we

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ACCEPTED MANUSCRIPT hypothesized that BMP-1 may play an important role in follicle selection and

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dominance. The results showed that the BMP-1 recombinant protein significantly

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increased the members of the IGF and VEGF family, as well as PAPP-A and AMH

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(Fig. 10). In contrast, the BMP-1 antibody significantly decreased the expression of

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these genes in a dose-dependent manner.

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4. Discussion

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Recently, researchers have focused on the matrix metalloproteinase, which takes

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on an important role during folliculogenesis and embryo development in different

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species [37, 38]. In the present study, the isolation and characterization of swamp

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buffalo BMP-1 cDNA was determined, which is closely related to Human BMP-1, the

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original members belongs to astacin family [2]. The alignment data and the

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phylogenetic tree from amino acid sequences showed that BMP-1 is highly conserved

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among buffalo and other species. The 21 amino acid sequence of buffalo BMP-1

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appears to contain an unpaired cysteine, which acted as a 'cysteine switch' and

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functions in the proregion of matrix metalloproteinases [39]. In addition, the majority

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of zinc-dependent metallopeptidases containing the sequence HEXXHXXGFXH in

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their primary structure, which is involved in the binding of zinc and can be grouped

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together as a super-family called metzincins [12].

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Additionally, the predicted protein domain architecture of buffalo BMP-1 has

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high similarity with the other species. Among these domains, the CUB domain is a

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structural motif of approximately 110 residues found almost exclusively in

330

extracellular and plasma membrane-associated proteins. While the CUB1 is important

ACCEPTED MANUSCRIPT for the secretion of the molecule, when CUB1 is interchanged with other CUB

332

domains this may result in the retention of the proteins by the cells [40], Mutants

333

lacking the CUB2 domain are poor C-proteinases. In contrast, mutants lacking the

334

EGF-like and CUB3 domains exhibit full C-proteinases activity. The EGF-like repeat

335

is a six-cysteine conserved motif found in a number of proteins such as secreted

336

growth factors, adhesion molecules, signaling proteins, transmembrane receptors, and

337

components of the ECM [41]. In summary, our results showed that the cDNA of

338

buffalo BMP-1 is a new metalloproteinase that belongs to the astacin family.

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331

Reports have also confirmed the expression of matrix metalloproteinase-14

340

(MMP-14) and MMP-2 during human folliculogenesis [42]. The present data showed

341

that BMP-1 was expressed throughout buffalo folliculogenesis, which is consistent

342

with the observations in ovine [43]. In addition, the identification of the BMP-1

343

expression pattern in COCs and the oocyte in this study may have direct implications

344

for the regulation and modulation of the buffalo folliculogenesis, as well the

345

maturation of the oocyte in vitro. Previous studies showed that suBMP-1 mRNA was

346

expressed at a low level in the unfertilized egg and was maximum at the hatched

347

blastula stage, and showed a modest decrease at later stages of in the sea urchin [8].

348

The stage-dependent expression of Xenopus BMP-1 suggests that it participates in

349

gastrulation and the differentiation of the developing organs. The overexpression of

350

BMP-1 and Xld increases the activity of Xenopus TGF-β-related BMPs during the

351

blastula and gastrula stages and these may include BMP-2, BMP-4, BMP-7 [44, 45].

352

However, BMPs signaling is important for folliculogenesis in many species [1, 46-48].

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ACCEPTED MANUSCRIPT BMPs, their receptors and Smads have variable levels of mRNA in the follicles of

354

goat [49], and play important role in mediating cumulus cell expansion in porcine and

355

murine animals [50]. In addition, BMP-1 is known to be responsible for cleavage of

356

Chordin in mammals [17, 31, 40], the antagonist of BMPs, including BMP-2/4 [24,

357

25], GDF-8/11 [22, 23], which indicates that BMP-1 possesses important function

358

during folliculogenesis that involves interacting with BMPs. MMP-1, MMP-3 and

359

MMP-9 are synthesized and secreted by GCs in different follicles in the chicken ovary

360

[51]. This study proved buffalo BMP-1 is located on the layers of the GCs and the

361

oocyte, which coincided with the report showing that BMP-1 is a membrane protein

362

that is secreted out and subsequently associates with a cell surface component [8]. So

363

above all, we conclude that BMP-1 may take on an important role during

364

folliculogenesis in the swamp buffalo.

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353

In addition, the present study proved that BMP-1 promotes proliferation and cell

366

cycle progression, and inhibits the apoptosis of GCs in vitro. In fact, MMP-1

367

induces lung alveolar epithelial cell migration and proliferation, and protects cells

368

from apoptosis [52]. MMP-8 also promotes vascular smooth muscle cell proliferation

369

as well [53]. BMP-1 is a cell surface component that may interact with

370

apoptosis-related factors on the cell membrane. This study showed the first evidence

371

that BMP-1 suppresses the genes expression of Fas, FasL, TNF-α and TNFR1, etc., in

372

the GCs. For example, Fas and FasL are expressed at higher levels in bovine GCs in

373

the atretic subordinate follicles relative to healthy dominant follicles on day 5 of the

374

estrous cycle [54]. Treatment with TNF-α and IFN-γ may induce apoptosis in bovine

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365

ACCEPTED MANUSCRIPT GCs [55], while TNF-α-deficient mice exhibit increased GC proliferation[56]. In

376

addition, Apaf1 and Caspase-9 are expressed in GCs and are demonstrated to cause

377

follicular atresia in mice and pigs [57, 58]. Therefore, our results suggested that once

378

with BMP-1 expression declines the apoptosis of cells become irreversible.

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375

In the present study, BMP-1 up-regulated the expression of PAPP-A，which acts

380

on the ovary as a growth-promoting metalloproteinase and could release and increase

381

the bioactivity of IGF in close proximity to the IGF receptor [59]. In a similar manner

382

as the IGF system, VEGF is involved in the proliferation of the capillaries that

383

accompany the selection of the preovulatory follicle and support the growth of the

384

dominant follicle [60, 61]. The direct injection of VEGF into the ovary increases the

385

vasculature [62], and the number of antral follicles and inhibits apoptosis [63].

386

Simultaneously, MMP-1 (both the mRNA and protein) remained low in the

387

pre-hierarchical and pre-ovulatory follicles but increased in post-ovulatory follicles of

388

the chicken ovary, while both MMP-3 and MMP-9 expression levels increased during

389

follicular maturation [51]. BMP-1/Tld metalloprotease could weaken the follicular

390

wall via proteolysis of the extracellular matrix (ECM) and the connective tissues in

391

the follicles at the ovulation stage [64]. Deleting the DNA sequences encoding the

392

active site of the astacin-like protease domain of BMP-1 in mouse resulted in a

393

persistent herniation of the gut in the umbilical region, and the mice did not survive

394

beyond birth [16]. Meanwhile, the results of this study have proved that BMP-1

395

functions during the folliculogenesis of buffalo and mainly regulates the follicle

396

selection and dominance.

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ACCEPTED MANUSCRIPT 397

4.1. Conclusion In summary, our results suggest that BMP-1 has the potential to promote the

399

selection and dominance of buffalo follicles by regulating the proliferation and

400

apoptosis of the GCs.

401

Acknowledgments

402

This work was supported by China high-tech 863 Project (2013AA102504) and

403

National Natural Science Fund of China (NSFC 31260552).

404

Competing interests

405

The authors state that there is no conflict of interest to declare.

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406 407 REFERENCES

409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428

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members in folliculogenesis. Wiley Interdiscip Rev Syst Biol Med. 2010;2:117-25. [49] Costa J, Passos M, Leitão C, Vasconcelos G, Saraiva M, Figueiredo J, et al. Levels of mRNA for bone morphogenetic proteins, their receptors and SMADs in goat ovarian follicles grown in vivo and in vitro. Reproduction, Fertility and Development. 2012;24:723-32. [50] Gilchrist RB, Ritter LJ. Differences in the participation of TGFB superfamily signalling pathways mediating porcine and murine cumulus cell expansion. Reproduction. 2011;142:647-57. [51] Zhu G, Kang L, Wei Q, Cui X, Wang S, Chen Y, et al. Expression and regulation of MMP1, MMP3, and MMP9 in the chicken ovary in response to gonadotropins, sex hormones, and TGFB1. Biology of reproduction. 2014:biolreprod. 113.114249. [52] Herrera I, Cisneros J, Maldonado M, Ramírez R, Ortiz-Quintero B, Anso E, et al. Matrix metalloproteinase (MMP)-1 induces lung alveolar epithelial cell migration and proliferation, protects from apoptosis, and represses mitochondrial oxygen consumption. Journal of Biological Chemistry. 2013;288:25964-75. [53] Xiao Q, Zhang F, Grassia G, Hu Y, Zhang Z, Xing Q, et al. Matrix metalloproteinase-8 promotes vascular smooth muscle cell proliferation and neointima formation. Arteriosclerosis, thrombosis, and vascular biology. 2014;34:90-8. [54] Porter D, Harman R, Cowan R, Quirk S. Relationship of Fas ligand expression and atresia during bovine follicle development. Reproduction. 2001;121:561-6. [55] Vickers SL, Cowan RG, Harman RM, Porter DA, Quirk SM. Expression and activity of the Fas antigen in bovine ovarian follicle cells. Biology of reproduction. 2000;62:54-61. [56] Cui L-l, Yang G, Pan J, Zhang C. Tumor necrosis factor α knockout increases fertility of mice. Theriogenology. 2011;75:867-76. [57] Robles R, Tao X-J, Trbovich AM, Maravei DV, Nahum R, Perez GI, et al. Localization, regulation and possible consequences of apoptotic protease-activating factor-1 (Apaf-1) expression in granulosa cells of the mouse ovary. Endocrinology. 1999;140:2641-4. [58] Matsui T, Manabe N, Goto Y, Inoue N, Nishihara S, Miyamoto H. Expression and activity of Apaf1 and caspase-9 in granulosa cells during follicular atresia in pig ovaries. Reproduction. 2003;126:113-20. [59] Oxvig C. The role of PAPP-A in the IGF system: location, location, location. Journal of Cell Communication and Signaling. 2015:1-11. [60] Berisha B, Schams D, Kosmann M, Amselgruber W, Einspanier R. Expression and localisation of vascular endothelial growth factor and basic fibroblast growth factor during the final growth of bovine ovarian follicles. Journal of Endocrinology. 2000;167:371-82. [61] Bruno J, Matos M, Chaves R, Celestino J, Saraiva M, Lima-Verde I, et al. Angiogenic factors and ovarian follicle development. Anim Reprod. 2009;6:371-9. [62] SHIMIZU T. Promotion of ovarian follicular development by injecting vascular endothelial growth factor (VEGF) and growth differentiation factor 9 (GDF-9) genes. Journal of Reproduction and Development. 2006;52:23-32.

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[63] Quintana R, Kopcow L, Sueldo C, Marconi G, Rueda NG, Barañao RI. Direct injection of vascular endothelial growth factor into the ovary of mice promotes follicular development. Fertility and Sterility. 2004;82:1101-5. [64] Ohnishi J, Ohnishi E, Shibuya H, Takahashi T. Functions for proteinases in the ovulatory process. Biochimica et Biophysica Acta (BBA)-Proteins & Proteomics. 2005;1751:95-109.

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Sequence(5’-3’)

1

BMP1

2

Bcl-2

3

Bax

4

Cytochrome C

5

Apaf-1

6

Caspase-9

7

Caspase-3

8

Fas

9

Fas L

10

TNFR1

11

TNF-α

12

Caspase-8

13

Chop

14

Cyclin D1

15

Cyclin D2

16

PAPP-A

17

AMH

18

VEGFR1

19

VEGFR2

20

IGFII

F:ATGAGGTGAATGGGGTGA R:AGGCTGGGCACTTGTAGA F:GTGGATGACCGAGTACCTGAAC R:AGACAGCCAGGAGAAATCAAAC F:GACACAGACTCTCCCCGA R:GAAGGAAGTCCAATGTCCA

Product （bp） ）

GeneBank No.

60℃

96

KC007442.1

55℃

124

U92434.1

55℃ 55℃ 55℃

TE D

EP

F:GTCCTGGTGAACAAACT

R:CGGGTTGGAAATGAACTT F:GCTGGAGGTCTGTGAGGAG

R:CTTGATGGAGTTGTCGGTG F:TGACCTTTGTCTCCACTGACT R:TACACCTCCTCTCCCACATC F:GTGGTGCTGCTGCTAAAGATG

R:TCGGACAGGCTGATGAGGAG F:GTCTCGTCTCCCAGCATC R:TGTCTCGCAGGTCAAAAG F:CCTTTTGTTGCTTTCGGT R:TGGCTCTGTTTCTCCTTTG F:GCGGTTGTACCAGTCCAC R:TCGCGTGATGACTCAGAG

184

NM_173894.1

147

NM_001046061.2

170

NM_001191507.1

60℃

240

NM_001205504.1

55

264

NM_001077840.1

55

225

NM_174662.2

55

140

NM_001098859.2

55

227

U90937.1

55

224

EU276079.1

55

229

NM_001045970.2

55

88

NM_001078163.1

55

158

BC112798.1

55

186

NM_001076372.1

55

154

AF421141.1

55

104

NM_173890.1

60

150

JN175354.1

60

251

JN175355.1

55

205

NM_174087.3

M AN U

F:TGAGAAGGGCAAGAAGATT R:TTGGCATCTGTGTAAGAGAA F:GGAGGAAAAAGTGAAAAATG R:AAGAGATAACAGGAATGCCA F:CTCGCTTTGGGACGCTCTG R:TTTCATGGGTCATCCTGTTTTGC F:CAGAACTGGACTGTGGTATTG R:AATCGGTAGAAAAGGACTCAT F:CAGTCGGGGCTCACTACTCA R:GGCAGGAGTTCGCTTCAGTAA F:AGGCATACAGCATCATCTTT R:CCATAGGTGTCTTCCCATT F:GGGAATACTGGGGTGAAAC R:GCCTGGGTCCTGAGAGTC F:CCTCTTCTCCTTCCTCCTG R:CATACGAGTCCCACCACC F:AAGAGAATGTTGGAGGAAAA R:CCTGGCTCAAAAAAAACTTA F:ACCTGTGTTTCACCCCCT R:CCTCAGTAAGCCAAGCCA

Annealing Temperature

RI PT

Gene Name

SC

NO.

AC C

Table 1 Primers for QRT-PCR and RT-PCR

ACCEPTED MANUSCRIPT

22

IGF-IIR

23

18S

24

BMP1 for RT-PCR

F:GATCCCGTGTTCTTCTACGTTC

R:AAGCCTCCCACTATCAACAGAA F:GCAGATTTATTTCTTCTCCCAC R:CACTCAAACTCGTAGAAGCA F:GAAGGAAGTCCAATGTCCA R:GATGGGCGGCGGAAAATTG F:CAGTCCTCCGCTTCCC R:GTCTCCCATCCCTGCC

55

101

NM_001244612.1

55

187

NM_174352.2

55℃

79

NR_036642.1

2967

RI PT

IGF-IR

AC C

EP

TE D

M AN U

SC

21

ACCEPTED MANUSCRIPT FIGURE LEGENDS

RI PT

Fig. 1. Alignment of the highly conserved 21-amino acid sequence in the proregion of the BMP1-related metalloproteinases in most animals. The boxed 12-amino acid consensus sequence encompasses the Zn2+-binding site of the astacin endopeptidases and is essential for protease function in most animals whose sequence can be found in the NCBI. The signature sequence is part of an approximately 143-amino acid sequence, which is the entire mature crayfish astacin and the catalytic or protease domain of all of the members of the family. Fig. 2. Comparison of the similar protein domain architecture of the swamp buffalo BMP1 and other human bone morphogenetic protein 1 (huBMP1) related proteins, with number of amino acids in each domain. The domains are aligned relative to the astacin protease domain. All of the sequences were deduced from the cDNA sequences.

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Fig. 3. Immunohistochemistry analysis of BMP-1 localiztion in the swamp buffalo genital ridge and the fetal and adult ovaries. The BMP-1 protein staining is in brown by an immunostain using the BMP1 antibody (A, B, C, D, E and F) or the preimmuno serum with the diluent (negative control; G, H). PROFs, primordial follicles; PRIFs, primary follicles; ANFs, antral follicles; MATFs, mature follicles; OO, oocyte; GC, granulosa cells; TC, theca cells; FF, follicular fluid.

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Fig. 4. The expression pattern of BMP-1 in the swamp buffalo denuded oocyte and cumulus–oocyte complexes (COCs) before and after in vitro maturation (IVM). (A) Confocal microscope images of BMP-1 (FITC) in the immature oocyte, immature COCs, mature oocyte, and mature COCs with an anti-BMP1 antibody. The yellow color corresponds to the overlap of the fluorescein (green) and propidium iodide (red) staining. Scale bars represent 100 µm. (B) Real-time RT-PCR analysis of the relative expression level of BMP1 in the swamp buffalo denuded oocyte and cumulus–oocyte complexes (COCs) before and after in vitro maturation (IVM) at the mRNA level.

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Fig. 5. BMP-1 regulates the proliferation of the granular cells of buffalo in vitro. (A) A CCK8 assay showing that the BMP-1 recombinant protein enhanced the granular cell proliferation. (B) A CCK8 assay showing that the BMP-1 antibody inhibited the granular cells proliferation. The growth index as assessed at 72 h. Fig. 6. BMP-1 regulates the cell cycle of the granular cells from the buffalo in vitro. (A and C) The granular cells were treated with the BMP-1 recombinant protein at different concentrations. (B and D) The granular cells were treated with the BMP-1 antibody at different concentrations. After 72 hours of treatment, the results showed the cell cycle distributions which have been summarized and presented as the figure shown. Fig. 7. BMP-1 regulates the apoptosis of granular cells from buffalo in vitro. BMP-1 increases the percentage of apoptotic cells after treatment with different concentrations of the BMP-1 antibody in a dose-dependent manner. After 72 hours of treatment, the results are presented as the mean±S.E., and values of p<0.05 were regarded as statistically significant compared to the untreated control. The lower left quadrants show the viable cells, the lower right quadrants show

ACCEPTED MANUSCRIPT the early apoptotic cells, and the upper right quadrants show the late apoptotic/necrotic cells.

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Fig. 8. BMP-1 affects the expression of genes associated with the cell cycle and apoptosis at the mRNA level. Granular cells were treated with 5-50 ng/mL BMP-1 recombinant protein and 50-400 ng/mL BMP-1 antibody (dose-dependent study) for 72 h respectively to determine its effect on cell cycle and apoptosis genes. The cells were harvested and cell lysates RNA was extracted from the cell lysates and transcribed into cDNA. The assay was confirmed by using 18S as a reference gene. The results shown here are from a representative experiment that was repeated three times with similar results. A, C, and E show the results of the BMP-1 recombinant protein separately, and B, D and F show the results of the BMP-1 antibody separately.

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Fig. 9. BMP-1 affects the expression of genes associated with the cell cycle and apoptosis at the protein level. Western blot analysis of Fas, Bax and caspase-9 from granular cells on treated with the BMP-1 recombinant protein (A) and BMP-1 antibody (B). β-actin was used as the internal control.

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Fig. 10. BMP-1 affects the expression of genes associated with the follicle selection and dominance at the mRNA level. The granular cells were treated with 5-50 ng/mL BMP-1 recombinant protein and 50-400 ng/mL BMP-1 antibody (dose-dependent study) for 72 h to determine its effect on cell cycle and apoptosis genes. The cells were harvested and RNA was extracted from cell lysates and transcribed into cDNA. The assay was confirmed by using 18S as a reference gene. The results shown here are from a representative experiment that was repeated three times with similar results. A and C showed the results of the BMP-1 recombinant protein separately, B and D showed the results of the BMP-1 antibody separately.

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Fig. 11. The mechanism of how BMP-1 participates in the follicular genesis in the swamp buffalo.

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Figure 1

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Figure 2

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Figure 3

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A

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Figure 5

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12.93% 2

3

10

4

10

R2

69.92% 0

23.36% 1

2

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10 10 10 Green Fluorescence (GRN-HLog)

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400 ng/mL

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10 4 Red Fluorescence (RED-HLog) 10 1 10 2 10 3

18.22% 1

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10 10 10 Green Fluorescence (GRN-HLog)

10 4

6.69%

Red Fluorescence (RED-HLog) 101 10 2 103

0.03%

10 0

10 0

10

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50 ng/mL

Plot P02, gated on P01.R1

Red Fluorescence (RED-HLog) 10 1 10 2 10 3

10 4

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76.54%

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74.74% 0

19.15%

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10 10 10 Green Fluorescence (GRN-HLog)

100 ng/mL

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10 10 10 Green Fluorescence (GRN-HLog)

R2

6.07%

0.04%

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5.20%

0.04%

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10

Plot P02, gated on P01.R1

Red Fluorescence (RED-HLog) 10 1 10 2 10 3

5.01%

0.05%

10 0

10 0

10 4

Plot P02, gated on P01.R1

Red Fluorescence (RED-HLog) 10 1 10 2 10 3

10 4

Figure 7

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Figure 8

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Figure 9

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Figure 10

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Figure 11

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BMP-1 promotes cell cycle and proliferation, inhibits apoptosis of buffalo granular cells.




BMP-1 up-regulates expression of cell cycle regulators such as Cyclin D1 and

Cytochrome C, Fas, FasL and Chop.

BMP-1 accelerates expression of PAPP-A, IGF system, VEGF .etc, which play

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an important role in selection and dominance of the follicles.

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Cyclin D2, as well as down regulates expression of cell apoptosis related genes as