Expression of adiponectin and its receptors (AdipoR1 and AdipoR2) in goat ovary and its effect on oocyte nuclear maturation in vitro

Accepted Manuscript Expression of adiponectin and its receptors (AdipoR1 and AdipoR2) in goat ovary and its effect on oocyte nuclear maturation in vitro Bruna S.P. Oliveira, Joana A.S. Costa, Elizabete T. Gomes, Diogo M.F. Silva, Sandra M. Torres, Pedro L.J. Monteiro, Jr., Antônio S. Santos Filho, Maria Madalena P. Guerra, Gustavo F. Carneiro, Aurea Wischral, André M. Batista PII:

S0093-691X(17)30398-9

DOI:

10.1016/j.theriogenology.2017.08.013

Reference:

THE 14233

To appear in:

Theriogenology

Received Date: 5 May 2017 Revised Date:

30 July 2017

Accepted Date: 10 August 2017

Please cite this article as: Oliveira BSP, Costa JAS, Gomes ET, Silva DMF, Torres SM, Monteiro Jr. PLJ, Santos Filho AntôS, Guerra MMP, Carneiro GF, Wischral A, Batista AndréM, Expression of adiponectin and its receptors (AdipoR1 and AdipoR2) in goat ovary and its effect on oocyte nuclear maturation in vitro, Theriogenology (2017), doi: 10.1016/j.theriogenology.2017.08.013. 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.

Revised

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Expression of adiponectin and its receptors (AdipoR1 and AdipoR2) in goat ovary

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and its effect on oocyte nuclear maturation in vitro

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Bruna S. P. Oliveiraa, Joana A. S. Costaa, Elizabete T. Gomesa, Diogo M. F. Silvaa,

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Sandra M. Torresa, Pedro L. J. Monteiro Jrb, Antônio S. Santos Filhoc, Maria Madalena

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P.Guerraa, Gustavo F. Carneirod, Aurea Wischrala, André M. Batistaa,*

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a

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Pernambuco, Brazil.

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Department of Animal Science, University of São Paulo, Piracicaba, SP, Brazil.

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Agronomic Institute of Pernambuco, Arcoverde, PE, Brazil.

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UAG/University Federal Rural of Pernambuco, Garanhuns, PE, Brazil.

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Department of Veterinary Medicine, Federal Rural University of Pernambuco, Recife,

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* Corresponding author: Department of Veterinary Medicine, Rural Federal

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University of Pernambuco, Dom Manoel de Medeiros street, s/n, Dois Irmãos, CEP

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52171-900, Recife, Pernambuco, Brazil. Tel.: +55 81 3320 6414; fax: +55 81 3320

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6057. E-mail address: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT

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Adiponectin is an adipokine secreted primarily by adipocytes and is involved in the

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control of male and female reproductive functions. Circulating levels of adiponectin are

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inversely correlated with body fat mass, and its biological effects are predominantly

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mediated through two receptors, AdipoR1 and AdipoR2. The aim of the present study

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was to verify the expression of the adiponectin system (adiponectin and its receptors,

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AdipoR1 and AdipoR2) in goat ovary using qPCR and immunohistochemistry analyses

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and further investigate the in vitro effects of recombinant adiponectin (5 µg/mL and 10

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µg/mL) on goat oocyte nuclear maturation. We demonstrated that the mRNA and

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proteins of the adiponectin system are present in goat ovary. Gene and protein

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expression of AdipoR1 and AdipoR2 was detected in follicular cells (oocyte, cumulus,

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granulosa and theca) of small and large antral follicles, while adiponectin mRNA was

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not detected in oocytes from small and large follicles or in large follicle cumulus cells.

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Finally, addition of various concentrations of adiponectin in maturation medium

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affected the number of oocytes that reached metaphase II. In conclusion, in the present

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study, we detected expression of adiponectin and its receptors AdipoR1 and AdipoR2 in

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goat ovarian follicles. Furthermore, we demonstrated that recombinant adiponectin

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increases nuclear maturation of goat oocytes in vitro.

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Keywords: follicle, ovary, goat, adipose tissue, gene expression

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1. Introduction

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Increased livestock productivity has become a major research topic in response

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to increases in the population and consumption [1]. In this context, understanding the

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reproductive physiology of these animals is key to improving reproductive techniques

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and, consequently, increasing productivity. In ruminants, reproductive function can be 2

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influenced at different levels of the hypothalamic-pituitary-gonadal axis by energetic

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alterations related to diet [2], with a well-known relationship between energetic balance

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and reproduction [3]. Adipose tissue, once recognized only for its role as an energy reservoir, has been

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identified as an important modulator of this relationship [4,5]. Now recognized as an

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endocrine organ, adipose tissue releases a wide variety of protein factors called

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adipokines [6]. These adipokines have important effects on various physiological

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processes, including reproduction [7]. Adiponectin, a 30 kDa, 244 amino acid protein,

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has an N-terminal signal sequence, a species-dependent variable sequence, a collagen

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domain, and a C-terminal globular domain [8,9]. Its secretion is inversely related to

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adipose tissue levels [10], and its circulating levels are two to three times higher in

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females than those in males [11].

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Adiponectin acts through two specific membrane receptors, AdipoR1 and

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AdipoR2 [12]. These receptors are formed by seven transmembrane domains, with an

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intracellular N-terminus and an extracellular C-terminus, and have an opposite

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orientation to that of classical G-proteins-coupled receptors [13,14]. The adiponectin

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system was identified in different organs of the female reproductive system in various

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species. Adiponectin, AdipoR1 and AdipoR2 were observed in ovarian cells of cattle

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[15], chickens [16], mice [17] and swine [18]. AdipoR1 and AdipoR2 were also

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identified in ovarian fish cells [19] and humans [20,21]. Adiponectin protein was also

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visualized in ovine follicular cells [22].

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The literature suggests that adiponectin has a steroidogenic effect on ovarian

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function. In pigs, in vitro studies have shown that adiponectin reduced basal

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testosterone secretion in internal theca cells; in granulosa cells, it increased secretion of

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estradiol and, in combination with insulin, increased secretion of progesterone [23]. In

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progesterone and estradiol [20]. In chickens, adiponectin was also shown to modify the

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progesterone secretion pattern induced by LH, FSH or IGF-1 in granulosa cells [16].

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This adipokine effect was also verified in oocyte maturation in vitro. In pigs,

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adiponectin promoted nuclear maturation of treated oocytes [18]; however this effect

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was not observed in cattle [15,24].

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Regarding caprine species, no reports were found on the adiponectin system and

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its influence on female reproduction. Thus, the objective of this work was first to

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investigate mRNA and protein expression of the adiponectin system (adiponectin,

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AdipoR1 and AdipoR2) in goat ovary and second to study the effects of recombinant

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adiponectin on goat oocyte maturation in vitro.

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2. Materials and methods

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2.1.Tissue collection

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Antral follicles were dissected from the ovaries of adult goats (Capra hircus),

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collected independently of the stage of estrous cycle, obtained from a commercial

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slaughterhouse and transported to the laboratory in saline solution on ice. Isolated

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follicles were classified based on the diameter as small (<3 mm) and large (≥ 3 mm)

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antral follicles.

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Follicles were pooled (10-15 follicles/category) separately into petri dishes in

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phosphate-buffered saline (PBS) and then sectioned under a stereomicroscope (Nikon

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Corporation, Tokyo, Japan). Granulosa cells (GCs) adhering to follicle wall were

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removed by gentle scraping with a scalpel blade. After removal of all cumulus-oocyte

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complexes (COCs) from the petri dish, GCs were recovered by centrifugation at 1200 g

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for 1 min. Then, the theca cell layers (TC) were vortexed for 1 min in 1 ml PBS, 4

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transferred to 1 ml of fresh buffer and centrifuged for 1 min. The GC and TC pellets

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were homogenized in 0.5 ml of TRI® Reagent (Invitrogen, Thermo Fisher Scientific,

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Inc., Carlsbad, CA, USA) and stored at - 80°C until RNA extraction. Cumulus-oocyte complexes were aspirated from small and large antral follicles.

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Cumulus cells were mechanically separated by careful and repeated pipetting until no

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adherent cumulus cells could be observed under the stereomicroscope. Pools of 10-15

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denuded oocytes and cumulus cells of 10-15 COCs, of each category of follicle, were

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collected and immediately subjected to total RNA extraction using TRI® reagent. Six

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samples of each tissue pool were analyzed.

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Cross-contamination of granulosa and theca cells was tested by detection of

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mRNA encoding the cytochrome P450 aromatase (CYP19A1) and 17α-hydroxylase

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(CYP17A1) in each sample by PCR as described by Batista et al. [25]. The presence of

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CYP19A1 amplicons in theca samples or CYP17A1 in granulosa samples indicated

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cross-contamination, and such samples were discarded.

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2.2.Total RNA extraction and cDNA synthesis

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Total RNA was extracted from each cell pool (oocytes, cumulus cells, GCs and

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TCs) using TRI® reagent. Samples were homogenized in 500 µl of TRI® reagent and

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extracted with 100 µl chloroform; after separation of the aqueous phase, RNA was

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precipitated with isopropanol and washed in 70% (v/v) ethanol, and the RNA pellet was

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eluted in MilliQ water. Then, to eliminate possible DNA contamination, all total RNA

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samples were treated with RNase-free DNase (RQ1, Promega, Madison, USA)

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according to the manufacturer's recommended protocol. Total RNA was quantified by

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measuring the absorbance at 260 nm, and RNA purity was determined by the 260/280

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nm absorbance ratio using a NanoVue spectrophotometer (GE Healthcare Life

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Sciences Chicago, Illinois, United States). For complementary DNA (cDNA), 1 µg of total RNA was mixed with 0.5 µg

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random hexamers (Promega, Madison, WI, USA) and 0.5 µg oligo-d(T) primer

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(Promega, Madison, WI, USA). The mixture was then subjected to denaturation at 70°C

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for 5 min and immediately chilled in ice water for 5 min. Subsequently, 13 µL of cDNA

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master mix consisting of 1 µL reverse transcriptase (GoScript™; Promega), 4 µL 5×

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reaction buffer (GoScript™; Promega), 4 µL MgCl2 (25 mM; Promega), 1 µL dNTP

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working stock (0.5mM; Promega), and 3 µL of nuclease-free water was added. The

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samples underwent annealing at 25°C for 5 min, extension at 42°C for 60 min and

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inactivation at 70°C for 15 min.

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2.3.Real-time PCR

Quantitative real-time polymerase chain reaction (qPCR) was performed using a

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Rotor-Gene Q Real-Time PCR System (Qiagen, Hilden, Germany). Primers used were

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designed based on the published sequences for goat adiponectin (GenBank:

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KF452236.1),

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JX573540.1).

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dehydrogenase (GAPDH) and ubiquitin (UBQ) have been previously described [26].

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Primers sequences are shown in Table 1.

(GenBank:

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AdipoR1

Primers

for

the

HQ846828.1)

reference

genes

and

AdipoR2

(GenBank:

glyceraldehyde-3-phosphate

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The qPCR assay was performed in a final reaction volume of 15 µL containing 2

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µL of the standardized sample at a 500 µg/mL concentration, 7.5 µL of qPCR Master

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Mix (GoTaq® qPCR Master Mix; Promega, Madison, USA), 0.5 µL of each forward

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and reverse primer and 4.5 µl nuclease free water. The protocol consisted of one

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activation phase (95°C for 2 min) and one unfolding phase (95°C for 15 sec) followed

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by 40 annealing/extension cycles (60°C for 60 sec). Samples were measured in duplicate with a negative control for each primer

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evaluated. Calculation of the relative expression level of adiponectin, AdipoR1 and

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AdipoR2 genes was conducted based on the comparative cycle threshold method

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(∆∆CT) [27] and normalized using the geometrical means of reference gene expression

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levels: GAPDH and UBQ. Expression of adiponectin, AdipoR1 and AdipoR2 genes was

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calculated by the equation 2−∆∆CT, where ∆CT value was determined by subtracting the

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corresponding geometrical means of reference genes (GAPDH and UBQ) CT value

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from the specific CT of the target. Calculation of ∆∆CT involved using the highest

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sample ∆CT value (i.e., the sample with the lowest target expression) as an arbitrary

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constant to subtract from all other ∆CT sample values.

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2.4.Immunohistochemistry

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Goat ovaries were collected from a commercial abattoir, bisected, and fixed in

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4% paraformaldehyde in PBS for 24 hours. Fixed tissues were subsequently dehydrated,

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diaphanized and included in paraffin. Serial sections (4 µm) were mounted on silanized

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slides (S4651; Sigma, St. Louis, USA), dried overnight at 37°C, deparaffinized in

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xylene and hydrated in decreasing concentrations of ethanol.

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Endogenous peroxidase activity was blocked by tissue incubation in a

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peroxidase blocking reagent (Dako, Carpinteria, USA) for 30 min. Antigen retrieval

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was performed by immersing the sections in citrate buffer (pH 6.0) in a microwave

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oven. Then, non-specific reactions were blocked with normal goat serum (1:10; Dako,

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Glostrup, Denmark) in PBS for 30 min. The rabbit polyclonal antibodies anti-

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adiponectin (N-20-R, sc-17044, Santa Cruz Biotechnology, Santa Cruz, CA, USA), 7

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99184; Santa Cruz Biotechnology) diluted 1:50 in blocking buffer were incubated with

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tissues overnight at 4°C. Antibodies used in this study are recommended for the

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detection of adiponectin and its receptors in most animal species, including rat, human,

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equine, swine, canine and bovine. Sections were then washed in PBS and incubated

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with a peroxidase-conjugated polymer (EnVision ™ + Dual Link System-HRP; Dako,

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Carpinteria, USA) in a humidified chamber at room temperature for 30 min.

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Immunostaining was revealed by incubation at room temperature with 3-3'-

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diaminobenzidine

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counterstained with hematoxylin, photographed and examined by a Leica DM 500 light

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microscope coupled with a Leica ICC50 HD camera and EZ LAS 4.3 software (Leica

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Microsystems Nussloch GmbH, Nussloch, Germany). In the negative controls, the

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primary antibody was omitted from the procedure or primary antibodies were

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preincubated with blocking peptide (sc-17044P, Santa Cruz Biotechnology, Santa Cruz,

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CA, USA) or adiponectin at a ratio of 1:100. No immunoreaction was observed in the

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control preparations.

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(DAB,

2.5.In vitro maturation (IVM)

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Adult goat ovaries were collected from commercial abattoir and transported to

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the laboratory at 38°C in PBS containing antibiotic/antimycotic solution (Ab/Am, 100

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U/mL penicillin, 100 µg/mL streptomycin and 0.25 µg/mL amphotericin B; Gibco, Life

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Technologies, Grand Island, NY, USA) within two hours of slaughter. COCs were

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obtained by aspiration of antral follicles between 2 and 8 mm in diameter with an 18G

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needle connected to a 10 mL syringe, which was previously filled with 3 mL of

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HEPES-buffered TCM199 and supplemented with Ab/Am solution. The samples were 8

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selected under a stereomicroscope (Nikon Corporation, Tokyo, Japan), and only those

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with at least one layer of compact cumulus cells and homogeneous cytoplasm were

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selected. COCs were washed three times in serum-free maturation medium (TCM-199-

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Bicarbonate, containing 2 mM L-glutamine, 0.3 mM sodium pyruvate and 50 µg/mL

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gentamycin) and randomly distributed on 35 mm petri dishes (Nunc, Roskilde,

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Denmark) containing 25-35 oocytes in 80 µL drops of serum-free maturation medium

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with or without adiponectin (0, 5 or 10 µg/mL) or 10% (v/v) fetal bovine serum (FBS)

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as a positive control, under mineral oil. These adiponectin concentrations were chosen

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based on plasma and follicular fluid levels reported in earlier studies in goats [28, 29],

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and bovine [24]. COCs were cultured for 27 h at 38.5° C in humidified atmosphere of

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5% CO2. Recombinant human adiponectin (rh) (full-length human adiponectin, RD

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172023100) derived from mammalian cells (HEK-293 cells) was purchased from

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BioVendor Laboratory Medicine (Heidelberg, Germany). This experiment was repeated

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seven times (n = 660 oocytes).

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After the maturation period, some oocytes were denuded of cumulus cells by

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careful and repeated pipetting in PBS-PVP. The denuded oocytes were incubated with

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10 µg/mL Hoechst 33342 in PBS-PVP for 30 min at room temperature, washed three

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times in PBS-PVP and mounted on slides with the mounting medium ProLong® Gold

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(Molecular Probes, Life Technologies, Eugene, OR, USA), covered by coverslips

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supported by paraffin columns and sealed with nail polish. Samples were observed

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using a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Inc., Göttingen, Germany).

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Images were acquired using AxioVision software and an AxioCam MRm digital camera

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(Carl Zeiss, Inc., Göttingen, Germany). The oocytes were classified according to the

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nuclear configuration as germinal vesicle (GV; Fig. 1A), germinal vesicle breakdown

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(GVBD; Fig. 1B), metaphase I (MI; Fig. 1C) and metaphase II (MII; Fig. 1D).

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2.6.Statistical analyses

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Categorical data were analyzed using the GLIMMIX Procedure of SAS fitted to

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a binary distribution. However, due the outcome 0 or 100% some analyses were

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performed using Fisher’s exact test using the FREQ procedure of SAS. Data were tested

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for normality of residuals, and data with residuals not normally distributed were

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transformed before analysis. Quantitative PCR data were analyzed using the GLIMMIX

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procedure of SAS fitted to a Gaussian distribution with the fixed effects of cell type and

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follicle size and the random effect of the sample.

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The GLIMMIX procedure (ANOVA) of SAS was used with generalized linear

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model methodology. For the data analyses, a Gaussian distribution was used. The

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variables were analyzed using a mathematical model that included the effects of the

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treatment. The residual method was used to calculate the denominator degrees of

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freedom to approximate the F tests in the mixed models. The relative proportion of the

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oocytes in each nuclear stage of maturation was calculated with the following equation:

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Relative proportion = number of the oocytes in each stage/total of oocytes. The

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statistical comparisons were performed using means adjusted by the least squares mean

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method.

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Differences with P ≤ 0.05 were considered significant, and those with 0.05 < P ≤

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0.10 were considered tendencies. Results are presented as the mean ± standard error of

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the mean (SEM).

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3. Results

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3.1.Analysis of gene expression qPCR revealed adiponectin mRNA in cumulus cells from small follicles and in

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GCs and TCs of both follicular sizes but not in oocytes, regardless of size, or in the

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cumulus cells from large follicles (Figure 2A).

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Adiponectin mRNA levels in small antral follicles were significantly higher (P

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<0.05) in the cumulus cells compared to those in GCs and TCs, and mRNA levels did

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not differ between GCs and TCs. In large antral follicles, adiponectin mRNA levels

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were significantly higher (P <0.05) in granulosa than those in TCs (Figure 2A).

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AdipoR1 and AdipoR2 expression was detected in all evaluated cell types

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(oocytes, cumulus cells, GCs and TCs) of large and small antral follicles. AdipoR1 and

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AdipoR2 showed significant differences (P <0.05) between the cell types derived only

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from large antral follicles. AdipoR1 expression levels were significantly (P < 0.05)

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lower in TCs compared with those in cumulus and GCs but did not differ (P > 0.05)

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compared to oocyte levels (Figure 2B). AdipoR2 mRNA levels in TCs were

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significantly (P < 0.05) lower than those of other cell types (Figure 2C).

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3.2. Protein expression analysis

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Positive immunolocalisation for adiponectin, AdipoR1 and AdipoR2 were found

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in oocyte, cumulus cells and granulosa cells of the follicles at all stages of development

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(Fig. 3). Oocytes from preantral and antral follicles showed intense and uniform

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cytoplasmic staining for the adiponectin (Fig. 3A-C), AdipoR1 (Fig. 3E-G) and

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AdipoR2 (Fig. 3I, J and L) proteins. In addition, theca cells showed moderate

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immunostaining for all proteins (Fig. 3D, H and K). Control tissue samples showed no

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positive staining (Fig. 3X, Y and Z).

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3.3. Adiponectin effects on meiotic progression

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Figure 4 shows the effects of adiponectin on goat oocyte meiotic maturation.

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The proportion of GV or GVBD stage-blocked oocytes (Figure 4A-B) was significantly

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reduced (P <0.05) in the 5 or 10 µg/mL adiponectin-treated groups compared to that of

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the control group. Maturation in the presence of 5 µg/mL adiponectin resulted in higher

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rates of MI compared to those of the group without adiponectin (P <0.05; Figure 4C).

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Additionally, the proportion of oocytes that progressed to MII was significantly higher

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(P <0.05) in the 5 and 10 µg/mL adiponectin-treated groups (Figure 4D) than that in the

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control groups. Groups treated with adiponectin showed similar percentages of MII

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compared to the group cultured with FBS.

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4. Discussion

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This study demonstrated the presence of the adiponectin system in goat follicular

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cells. Although expression and immunolocalization of the adiponectin system has

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already been studied in other species, as well as in different cell types, the gene and

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protein expression of adiponectin and its receptors had not been studied in goats.

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Our results demonstrated that adiponectin was not expressed at detectable levels

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in oocytes of small and large goat antral follicles or cumulus cells from large antral

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follicles. These results differ from those reported by Maillard et al. [15] and Tabandeh

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et al. [30], who found adiponectin expression in oocytes and cumulus cells of bovine

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antral follicles, indicating that there may be species-specific differences in ovarian

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expression of the adiponectin gene. The lack of adiponectin mRNA in oocytes and cumulus cells from large antral

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follicles is intriguing. However, mRNA samples from several pools of denuded oocytes

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and cumulus cells from large antral follicles were analyzed, and none generated

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amplicons after qPCR. Therefore, it is unlikely to be a methodological problem because

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the reference genes GAPDH and UBQ were easily amplified in these samples, and we

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detected AdipoR1 and AdipoR2 mRNAs in oocytes and cumulus cells.

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Quantitatively, relative adiponectin expression in cumulus cells was significantly

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higher than that in GCs and TCs from small follicles. In large follicles, relative

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expression in GCs was significantly higher compared with that in TCs. These data differ

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from what has been observed in chicken ovaries, in which the expression of adiponectin

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in TCs was 10-30-fold higher than that in GCs [16]. In addition, adiponectin expression

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was also low in GCs of mice [17]. However, Maillard et al. [15] observed good

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adiponectin expression in bovine GCs.

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Receptor expression analysis showed decreased expression of AdipoR1 and

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Adipo2 in TCs of large follicles compared with expression of receptors in GCs. These

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results differ from those found by Tabandeh et al. [30] in cattle, where AdipoR1 and

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AdipoR2 expression was higher in large follicle TCs than that in GCs. Our findings also

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differ from results observed in mice, in which receptor expression in GCs was lower

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than in TCs [17]. However, similar results were observed in chickens, where AdipoR1

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expression was twice as high in GCs as that in TCs, and AdipoR2 expression was

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similar in both cell types [16]. This discrepancy suggests a species-specific mode of

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action of the adiponectin system on follicular development.

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immunolocalized in oocytes, cumulus cells, GCs and TCs in ovarian follicles at all

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stages of development. These findings corroborate those obtained in bovines [15], mice

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[17] and humans [21] and further support the physiological relevance of this hormone

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on ovarian function.

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Adiponectin protein was detected in the oocytes by immunohistochemistry,

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although no mRNA was observed, indicating that adiponectin must be produced

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elsewhere and then transported to the oocyte. This is likely because adiponectin is a

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secreted protein, is found in follicular fluid [29], and binds to its receptors on granulosa,

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cumulus and oocyte cells; receptor-bound adiponectin has been shown to be internalized

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in several cell lines [31,32]. The detection of adiponectin appears to be specific because

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preincubation of the antibody with blocking peptide abolished staining.

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Oocyte meiotic maturation and acquisition of developmental competence is an

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essential physiological process for survival of the species. The presence of AdipoR1 and

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AdipoR2 mRNA and protein in both oocytes and cumulus cells suggests that both cell

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types are sensitive to adiponectin and that adiponectin may also affect oocyte

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maturation.

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In the present work, we demonstrated that adiponectin addition (5 or 10 µg/mL)

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during IVM affected goat oocyte meiotic maturation. However, conflicting results have

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been reported in the literature. In cattle, supplementation of IVM medium with

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adiponectin (5 or 10 µg/mL) did not affect bovine oocyte meiotic maturation [15,24]. In

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pigs, however, Chappaz et al. [18] reported that recombinant porcine adiponectin (30

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µg/mL) significantly decreased the proportion of immature oocytes, suggesting that

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adiponectin accelerated porcine oocyte meiotic maturation.

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ACCEPTED MANUSCRIPT These apparently controversial IVM results reported in the literature are

342

probably dependent on the species studied or on the culture medium used. The

343

experiment described in the present study was performed with serum-free medium and

344

in the absence of any growth factor or hormone other than adiponectin. However, other

345

studies have usually added gonadotropins or growth factors to the culture media; these

346

additives could have influenced the action of adiponectin or affected its pathways,

347

resulting in different responses. Thus, possible interactions between these additives and

348

the possible pathways by which adiponectin affects oocyte maturation remain unclear

349

and require further investigation.

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In conclusion, this study detected the expression of adiponectin and its receptors,

351

AdipoR1 and AdipoR2, in goat ovarian follicles. In addition, adiponectin was shown to

352

enhance the progression of goat oocyte nuclear maturation in vitro. The present findings

353

provide evidence for paracrine/autocrine effects of the analyzed hormone. However,

354

future studies are needed to elucidate the mechanisms underlying the effect of

355

adiponectin on meiotic maturation and to understand the role of the adiponectin system

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in goat fertility.

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Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as

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prejudicing the impartiality of the research reported.

Acknowledgements The authors thank FACEPE (Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco) for financial support (APQ-1115-5.05/14).

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[11] Combs TP, Berg AH, Rajala MW, Klebanov S, Iyengar P, Jimenez-Chillaron JC, et al. Sexual differentiation, pregnancy, calorie restriction, and aging affect the

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[12] Yamauchi T, Iwabu M, Okada-Iwabu M, Kadowaki T. Adiponectin receptors: A

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2005;26:439–51. doi:10.1210/er.2005-0005. [14] Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yokomizo T, Kita S, et al. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature

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[15] Maillard V, Uzbekova S, Guignot F, Perreau C, Ramé C, Coyral-Castel S, et al.

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Effect of adiponectin on bovine granulosa cell steroidogenesis, oocyte maturation and embryo development. Reprod Biol Endocrinol 2010;8:23. doi:10.1186/14777827-8-23.

[16] Chabrolle C, Tosca L, Crochet S, Tesseraud S, Dupont J. Expression of

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adiponectin and its receptors (AdipoR1 and AdipoR2) in chicken ovary: Potential

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role in ovarian steroidogenesis. Domest Anim Endocrinol 2007;33:480–7.

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doi:10.1016/j.domaniend.2006.08.002. [17] Chabrolle C, Tosca L, Dupont J. Regulation of adiponectin and its receptors in rat ovary by human chorionic gonadotrophin treatment and potential involvement

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of adiponectin in granulosa cell steroidogenesis. Reproduction 2007;133:719–31.

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doi:10.1530/REP-06-0244.

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[18] Chappaz E, Albornoz MS, Campos D, Che L, Palin MF, Murphy BD, et al.

Adiponectin enhances in vitro development of swine embryos. Domest Anim

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Endocrinol 2008;35:198–207. doi:10.1016/j.domaniend.2008.05.007.

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[19] Nishio SI, Gibert Y, Bernard L, Brunet F, Triqueneaux G, Laudet V. Adiponectin and adiponectin receptor genes are coexpressed during zebrafish embryogenesis

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and regulated by food deprivation. Dev Dyn 2008;237:1682–90.

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[20] Chabrolle C, Tosca L, Ramé C, Lecomte P, Royère D, Dupont J. Adiponectin increases insulin-like growth factor I-induced progesterone and estradiol

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secretion in human granulosa cells. Fertil Steril 2009;92:1988–96.

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[21] Comim F V., Hardy K, Franks S. Adiponectin and its receptors in the ovary:

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Further evidence for a link between obesity and hyperandrogenism in polycystic

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ovary syndrome. PLoS One 2013;8:1–9. doi:10.1371/journal.pone.0080416.

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[22] Ortega HH, Rey F, Velazquez MML, Padmanabhan V. Developmental programming: effect of prenatal steroid excess on intraovarian components of

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insulin signaling pathway and related proteins in sheep. Biol Reprod

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2010;82:1065–75. doi:10.1095/biolreprod.109.082719.

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[23] Kiezun M, Smolinska N, Maleszka A, Dobrzyn K, Szeszko K, Kaminski T. Adiponectin expression in the porcine pituitary during the estrous cycle and its

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effect on LH and FSH secretion. Am J Physiol Endocrinol Metab

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2014;307:E1038-46. doi:10.1152/ajpendo.00299.2014.

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[24] Divar MR, Kafi M, Mohammadi A, Azari M. The In Vitro Effect of Adiponectin on Early Bovine Embryonic Development and Transcriptomic Markers of Oocyte

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Competence. J Fertil Vitr - IVF-Worldwide, Reprod Med Genet Stem Cell Biol

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2016;4:1–7. doi:10.4172/2375-4508.1000172.

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[25] Batista AM, Silva DMF, Rêgo MJBM, Silva FLM, Silva ECB, Beltrão EIC, et al.

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The expression and localization of leptin and its receptor in goat ovarian follicles.

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Anim Reprod Sci 2013;141:142–7. doi:10.1016/j.anireprosci.2013.08.007.

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[26] Frota IM a, Leitão CCF, Costa JJN, Brito IR, van den Hurk R, Silva JR V.

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Stability of housekeeping genes and expression of locally produced growth

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factors and hormone receptors in goat preantral follicles. Zygote 2011;19:71–83.

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doi:10.1017/S0967199410000080.

[27] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-

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time quantitative PCR and. Methods 2001;25:402–8.

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doi:10.1006/meth.2001.1262.

[28] Guzel S, Yibar A, Belenli D, Cetin I, Tanriverdi M. The concentrations of

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adipokines in goat milk: relation to plasma levels, inflammatory status, milk

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quality and composition. J Vet Med Sci 2017;79:602-607. doi:10.1292/jvms.16-

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0061.

[29] Wang Z, Meng C, Ding X, Zhang G, Wang F. Effect of body condition on

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follicle transcriptome in estrous. Peerj Prepr 2016;4:1–34.

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doi:https://doi.org/10.7287/peerj.preprints.2177v1.

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[30] Tabandeh MR, Hosseini A, Saeb M, Kafi M, Saeb S. Changes in the gene expression of adiponectin and adiponectin receptors (AdipoR1 and AdipoR2) in

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ovarian follicular cells of dairy cow at different stages of development.

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Theriogenology 2010;73:659–69. doi:10.1016/j.theriogenology.2009.11.006.

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[31] Ding Q, Wang Z, Chen Y. Endocytosis of adiponectin receptor 1 through a clathrin- and Rab5-dependent pathway 2009;19:317–27.

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doi:10.1038/cr.2008.299.

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[32] Almabouada F, Diaz-ruiz A, Rabanal-ruiz Y, Peinado JR, Vazquez-martinez R,

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Malagon MM. Adiponectin Receptors Form Homomers and Heteromers

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Exhibiting Distinct Ligand Binding and Intracellular Signaling 2013;288:3112–

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25. doi:10.1074/jbc.M112.404624.

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Table Legend

492

Table 1. Oligonucleotide primer sequences used to perform qPCR.

493

Figure Legends

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Figure 1. Representative images of goat oocytes stained with Hoechst 33342 and

496

evaluated under fluorescence microscopy. Oocytes in germinal vesicle (GV, A),

497

germinal vesicle breakdown (GVBD, B), metaphase I (MI, C), and metaphase II (MII,

498

D) stages are shown.

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Figure 2. Adiponectin (A); AdipoR1 (B) and AdipoR2 (C) mRNA expression in oocyte

501

– O, cumulus cells – C, granulosa cells – G and theca cells – T isolated from small (< 3

502

mm) and large (≥ 3 mm) goat antral follicles. Different superscripts denote the statistical

503

significance between different cells types isolated from follicles in the same size

504

category (P < 0.05).

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Figure 3. Immunolocalization of adiponectin, AdipoR1 and AdipoR2 in goat ovaries.

507

Positive staining of adiponectin, AdipoR1 and AdipoR2 was observed in oocytes and

508

granulosa cells of primordial (A, E and I), primary (B and I) and secondary follicles (C,

509

F and J). Antral follicles with positive immunostaining of adiponectin, AdipoR1 and

510

AdipoR2 in oocytes, cumulus, theca and granulosa cells (D, G, H, K and L). No staining

511

was observed in the negative control (X, Y and Z). g, granulosa; t, theca, c, cumulus; o,

512

oocytes.

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Figure 4. Effects of recombinant adiponectin (0, 5 or 10 µg/mL) on the progression of

515

meiotic maturation of goat oocytes in vitro matured for 27 h. Germinal vesicle (GV, A),

21

ACCEPTED MANUSCRIPT 516

germinal vesicle breakdown (GVBD, B), metaphase I (MI, C), and metaphase II (MII,

517

D). The data are expressed as the mean ± SEM. Different superscripts denote the

518

statistical significance among experimental groups within the same stage of meiotic

519

maturation (P < 0.05).

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532 533 534 535 536

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Revised Highlighted

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Expression of adiponectin and its receptors (AdipoR1 and AdipoR2) in goat ovary

542

and its effect on oocyte nuclear maturation in vitro

543

Bruna S. P. Oliveiraa, Joana A. S. Costaa, Elizabete T. Gomesa, Diogo M. F. Silvaa,

544

Sandra M. Torresa, Pedro L. J. Monteiro Jrb, Antônio S. Santos Filhoc, Maria Madalena

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P.Guerraa, Gustavo F. Carneirod, Aurea Wischrala, André M. Batistaa,*

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546 547

a

548

Pernambuco, Brazil.

549

b

Department of Animal Science, University of São Paulo, Piracicaba, SP, Brazil.

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C

Agronomic Institute of Pernambuco, Arcoverde, PE, Brazil.

551

d

UAG/University Federal Rural of Pernambuco, Garanhuns, PE, Brazil.

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Department of Veterinary Medicine, Federal Rural University of Pernambuco, Recife,

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* Corresponding author: Department of Veterinary Medicine, Rural Federal

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University of Pernambuco, Dom Manoel de Medeiros street, s/n, Dois Irmãos, CEP

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52171-900, Recife, Pernambuco, Brazil. Tel.: +55 81 3320 6414; fax: +55 81 3320

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6057. E-mail address: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT

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Adiponectin is an adipokine secreted primarily by adipocytes and is involved in the

568

control of male and female reproductive functions. Circulating levels of adiponectin are

569

inversely correlated with body fat mass, and its biological effects are predominantly

570

mediated through two receptors, AdipoR1 and AdipoR2. The aim of the present study

571

was to verify the expression of the adiponectin system (adiponectin and its receptors,

572

AdipoR1 and AdipoR2) in goat ovary using qPCR and immunohistochemistry analyses

573

and further investigate the in vitro effects of recombinant adiponectin (5 µg/mL and 10

574

µg/mL) on goat oocyte nuclear maturation. We demonstrated that the mRNA and

575

proteins of the adiponectin system are present in goat ovary. Gene and protein

576

expression of AdipoR1 and AdipoR2 was detected in follicular cells (oocyte, cumulus,

577

granulosa and theca) of small and large antral follicles, while adiponectin mRNA was

578

not detected in oocytes from small and large follicles or in large follicle cumulus cells.

579

Finally, addition of various concentrations of adiponectin in maturation medium

580

affected the number of oocytes that reached metaphase II. In conclusion, in the present

581

study, we detected expression of adiponectin and its receptors AdipoR1 and AdipoR2 in

582

goat ovarian follicles. Furthermore, we demonstrated that recombinant adiponectin

583

increases nuclear maturation of goat oocytes in vitro.

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Keywords: follicle, ovary, goat, adipose tissue, gene expression

5. Introduction

588

Increased livestock productivity has become a major research topic in response

589

to increases in the population and consumption [1]. In this context, understanding the

590

reproductive physiology of these animals is key to improving reproductive techniques 24

ACCEPTED MANUSCRIPT 591

and, consequently, increasing productivity. In ruminants, reproductive function can be

592

influenced at different levels of the hypothalamic-pituitary-gonadal axis by energetic

593

alterations related to diet [2], with a well-known relationship between energetic balance

594

and reproduction [3]. Adipose tissue, once recognized only for its role as an energy reservoir, has been

596

identified as an important modulator of this relationship [4,5]. Now recognized as an

597

endocrine organ, adipose tissue releases a wide variety of protein factors called

598

adipokines [6]. These adipokines have important effects on various physiological

599

processes, including reproduction [7]. Adiponectin, a 30 kDa, 244 amino acid protein,

600

has an N-terminal signal sequence, a species-dependent variable sequence, a collagen

601

domain, and a C-terminal globular domain [8,9]. Its secretion is inversely related to

602

adipose tissue levels [10], and its circulating levels are two to three times higher in

603

females than those in males [11].

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Adiponectin acts through two specific membrane receptors, AdipoR1 and

605

AdipoR2 [12]. These receptors are formed by seven transmembrane domains, with an

606

intracellular N-terminus and an extracellular C-terminus, and have an opposite

607

orientation to that of classical G-proteins-coupled receptors [13,14]. The adiponectin

608

system was identified in different organs of the female reproductive system in various

609

species. Adiponectin, AdipoR1 and AdipoR2 were observed in ovarian cells of cattle

610

[15], chickens [16], mice [17] and swine [18]. AdipoR1 and AdipoR2 were also

611

identified in ovarian fish cells [19] and humans [20,21]. Adiponectin protein was also

612

visualized in ovine follicular cells [22].

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The literature suggests that adiponectin has a steroidogenic effect on ovarian

614

function. In pigs, in vitro studies have shown that adiponectin reduced basal

615

testosterone secretion in internal theca cells; in granulosa cells, it increased secretion of

25

ACCEPTED MANUSCRIPT estradiol and, in combination with insulin, increased secretion of progesterone [23]. In

617

human primary granulosa cells, adiponectin increased IGF-1-induced secretion of

618

progesterone and estradiol [20]. In chickens, adiponectin was also shown to modify the

619

progesterone secretion pattern induced by LH, FSH or IGF-1 in granulosa cells [16].

620

This adipokine effect was also verified in oocyte maturation in vitro. In pigs,

621

adiponectin promoted nuclear maturation of treated oocytes [18]; however this effect

622

was not observed in cattle [15,24].

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Regarding caprine species, no reports were found on the adiponectin system and

624

its influence on female reproduction. Thus, the objective of this work was first to

625

investigate mRNA and protein expression of the adiponectin system (adiponectin,

626

AdipoR1 and AdipoR2) in goat ovary and second to study the effects of recombinant

627

adiponectin on goat oocyte maturation in vitro.

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6. Materials and methods

630

6.1.Tissue collection

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Antral follicles were dissected from the ovaries of adult goats (Capra hircus),

632

collected independently of the stage of estrous cycle, obtained from a commercial

633

slaughterhouse and transported to the laboratory in saline solution on ice. Isolated

634

follicles were classified based on the diameter as small (<3 mm) and large (≥ 3 mm)

635

antral follicles.

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Follicles were pooled (10-15 follicles/category) separately into petri dishes in

637

phosphate-buffered saline (PBS) and then sectioned under a stereomicroscope (Nikon

638

Corporation, Tokyo, Japan). Granulosa cells (GCs) adhering to follicle wall were

639

removed by gentle scraping with a scalpel blade. After removal of all cumulus-oocyte

640

complexes (COCs) from the petri dish, GCs were recovered by centrifugation at 1200 g 26

ACCEPTED MANUSCRIPT 641

for 1 min. Then, the theca cell layers (TC) were vortexed for 1 min in 1 ml PBS,

642

transferred to 1 ml of fresh buffer and centrifuged for 1 min. The TC and GC pellets

643

were homogenized in 0.5 ml of TRI® Reagent (Invitrogen, Thermo Fisher Scientific,

644

Inc., Carlsbad, CA, USA) and stored at - 80°C until RNA extraction. Cumulus-oocyte complexes were aspirated from small and large antral follicles.

646

Cumulus cells were mechanically separated by careful and repeated pipetting until no

647

adherent cumulus cells could be observed under the stereomicroscope. Pools of 10-15

648

denuded oocytes and cumulus cells of 10-15 COCs, of each category of follicle, were

649

collected and immediately subjected to total RNA extraction using TRI® reagent. Six

650

samples of each tissue pool were analyzed.

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Cross-contamination of granulosa and theca cells was tested by detection of

652

mRNA encoding the cytochrome P450 aromatase (CYP19A1) and 17α-hydroxylase

653

(CYP17A1) in each sample by PCR as described by Batista et al. [25]. The presence of

654

CYP19A1 amplicons in theca samples or CYP17A1 in granulosa samples indicated

655

cross-contamination, and such samples were discarded.

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6.2.Total RNA extraction and cDNA synthesis Total RNA was extracted from each cell pool (oocytes, cumulus cells, TCs and

659

GCs) using TRI® reagent. Samples were homogenized in 500 µl of TRI® reagent and

660

extracted with 100 µl chloroform; after separation of the aqueous phase, RNA was

661

precipitated with isopropanol and washed in 70% (v/v) ethanol, and the RNA pellet was

662

eluted in MilliQ water. Then, to eliminate possible DNA contamination, all total RNA

663

samples were treated with RNase-free DNase (RQ1, Promega, Madison, USA)

664

according to the manufacturer's recommended protocol. Total RNA was quantified by

665

measuring the absorbance at 260 nm, and RNA purity was determined by the 260/280

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nm absorbance ratio using a NanoVue spectrophotometer (GE Healthcare Life

667

Sciences Chicago, Illinois, United States). For complementary DNA (cDNA), 1 µg of total RNA was mixed with 0.5 µg

669

random hexamers (Promega, Madison, WI, USA) and 0.5 µg oligo-d(T) primer

670

(Promega, Madison, WI, USA). The mixture was then subjected to denaturation at 70°C

671

for 5 min and immediately chilled in ice water for 5 min. Subsequently, 13 µL of cDNA

672

master mix consisting of 1 µL reverse transcriptase (GoScript™; Promega), 4 µL 5×

673

reaction buffer (GoScript™; Promega), 4 µL MgCl2 (25 mM; Promega), 1 µL dNTP

674

working stock (0.5mM; Promega), and 3 µL of nuclease-free water was added. The

675

samples underwent annealing at 25°C for 5 min, extension at 42°C for 60 min and

676

inactivation at 70°C for 15 min.

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6.3.Real-time PCR

Quantitative real-time polymerase chain reaction (qPCR) was performed using a

680

Rotor-Gene Q Real-Time PCR System (Qiagen, Hilden, Germany). Primers used were

681

designed based on the published sequences for goat adiponectin (GenBank:

682

KF452236.1),

683

JX573540.1).

684

dehydrogenase (GAPDH) and ubiquitin (UBQ) have been previously described [26].

685

Primers sequences are shown in Table 1.

(GenBank:

EP

AdipoR1

Primers

for

the

HQ846828.1)

reference

genes

and

AdipoR2

(GenBank:

glyceraldehyde-3-phosphate

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The qPCR assay was performed in a final reaction volume of 15 µL containing 2

687

µL of the standardized sample at a 500 µg/mL concentration, 7.5 µL of qPCR Master

688

Mix (GoTaq® qPCR Master Mix; Promega, Madison, USA), 0.5 µL of each forward

689

and reverse primer and 4.5 µl nuclease free water. The protocol consisted of one

28

ACCEPTED MANUSCRIPT 690

activation phase (95°C for 2 min) and one unfolding phase (95°C for 15 sec) followed

691

by 40 annealing/extension cycles (60°C for 60 sec). Samples were measured in duplicate with a negative control for each primer

693

evaluated. Calculation of the relative expression level of adiponectin, AdipoR1 and

694

AdipoR2 genes was conducted based on the comparative cycle threshold method

695

(∆∆CT) [27] and normalized using the geometrical means of reference gene expression

696

levels: GAPDH and UBQ. Expression of adiponectin, AdipoR1 and AdipoR2 genes was

697

calculated by the equation 2−∆∆CT, where ∆CT value was determined by subtracting the

698

corresponding geometrical means of reference genes (GAPDH and UBQ) CT value

699

from the specific CT of the target. Calculation of ∆∆CT involved using the highest

700

sample ∆CT value (i.e., the sample with the lowest target expression) as an arbitrary

701

constant to subtract from all other ∆CT sample values.

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6.4. Immunohistochemistry

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Goat ovaries were collected from a commercial abattoir, bisected, and fixed in

705

4% paraformaldehyde in PBS for 24 hours. Fixed tissues were subsequently dehydrated,

706

diaphanized and included in paraffin. Serial sections (4 µm) were mounted on silanized

707

slides (S4651; Sigma, St. Louis, USA), dried overnight at 37°C, deparaffinized in

708

xylene and hydrated in decreasing concentrations of ethanol.

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Endogenous peroxidase activity was blocked by tissue incubation in a

710

peroxidase blocking reagent (Dako, Carpinteria, USA) for 30 min. Antigen retrieval

711

was performed by immersing the sections in citrate buffer (pH 6.0) in a microwave

712

oven. Then, non-specific reactions were blocked with normal goat serum (1:10; Dako,

713

Glostrup, Denmark) in PBS for 30 min. The rabbit polyclonal antibodies anti-

714

adiponectin (N-20-R, sc-17044, Santa Cruz Biotechnology, Santa Cruz, CA, USA), 29

ACCEPTED MANUSCRIPT anti-AdipoR1 (H-40, sc-99183, Santa Cruz Biotechnology) or anti-AdipoR2 (M -44, sc-

716

99184; Santa Cruz Biotechnology) diluted 1:50 in blocking buffer were incubated with

717

tissues overnight at 4°C. Antibodies used in this study are recommended for the

718

detection of adiponectin and its receptors in most animal species, including rat, human,

719

equine, swine, canine and bovine. Sections were then washed in PBS and incubated

720

with a peroxidase-conjugated polymer (EnVision ™ + Dual Link System-HRP; Dako,

721

Carpinteria, USA) in a humidified chamber at room temperature for 30 min.

722

Immunostaining was revealed by incubation at room temperature with 3-3'-

723

diaminobenzidine

724

counterstained with hematoxylin, photographed and examined by a Leica DM 500 light

725

microscope coupled with a Leica ICC50 HD camera and EZ LAS 4.3 software (Leica

726

Microsystems Nussloch GmbH, Nussloch, Germany). In the negative controls, the

727

primary antibody was omitted from the procedure or primary antibodies were

728

preincubated with blocking peptide (sc-17044P, Santa Cruz Biotechnology, Santa Cruz,

729

CA, USA) or adiponectin at a ratio of 1:100. No immunoreaction was observed in the

730

control preparations.

733

SC

Carpinteria,

USA).

Finally,

sections

were

TE D

M AN U

Dako,

EP

732

(DAB,

6.5.In vitro maturation (IVM)

AC C

731

RI PT

715

Adult goat ovaries were collected from commercial abattoir and transported to

734

the laboratory at 38°C in PBS containing antibiotic/antimycotic solution (Ab/Am, 100

735

U/mL penicillin, 100 µg/mL streptomycin and 0.25 µg/mL amphotericin B; Gibco, Life

736

Technologies, Grand Island, NY, USA) within two hours of slaughter. COCs were

737

obtained by aspiration of antral follicles between 2 and 8 mm in diameter with an 18G

738

needle connected to a 10 mL syringe, which was previously filled with 3 mL of

739

HEPES-buffered TCM199 and supplemented with Ab/Am solution. The samples were 30

ACCEPTED MANUSCRIPT 740

selected under a stereomicroscope (Nikon Corporation, Tokyo, Japan), and only those

741

with at least one layer of compact cumulus cells and homogeneous cytoplasm were

742

selected. COCs were washed three times in serum-free maturation medium (TCM-199-

744

Bicarbonate, containing 2 mM L-glutamine, 0.3 mM sodium pyruvate and 50 µg/mL

745

gentamycin) and randomly distributed on 35 mm petri dishes (Nunc, Roskilde,

746

Denmark) containing 25-35 oocytes in 80 µL drops of serum-free maturation medium

747

with or without adiponectin (0, 5 or 10 µg/mL) or 10% (v/v) fetal bovine serum (FBS)

748

as a positive control, under mineral oil. These concentrations were chosen based on

749

plasma and follicular fluid levels reported in earlier studies in goats [28, 29], and bovine

750

[24]. COCs were cultured for 27 h at 38.5° C in humidified atmosphere of 5% CO2.

751

Recombinant human adiponectin (rh) (full-length human adiponectin, RD 172023100)

752

derived from mammalian cells (HEK-293 cells) was purchased from BioVendor

753

Laboratory Medicine (Heidelberg, Germany). This experiment was repeated seven

754

times (n = 660 oocytes).

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M AN U

SC

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743

After the maturation period, some oocytes were denuded of cumulus cells by

756

careful and repeated pipetting in PBS-PVP. The denuded oocytes were incubated with

757

10 µg/mL Hoechst 33342 in PBS-PVP for 30 min at room temperature, washed three

758

times in PBS-PVP and mounted on slides with the mounting medium ProLong® Gold

759

(Molecular Probes, Life Technologies, Eugene, OR, USA), covered by coverslips

760

supported by paraffin columns and sealed with nail polish. Samples were observed

761

using a Zeiss Axioplan fluorescence microscope (Carl Zeiss, Inc., Göttingen, Germany).

762

Images were acquired using AxioVision software and an AxioCam MRm digital camera

763

(Carl Zeiss, Inc., Göttingen, Germany). The oocytes were classified according to the

AC C

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755

31

ACCEPTED MANUSCRIPT 764

nuclear configuration as germinal vesicle (GV; Fig. 1A), germinal vesicle breakdown

765

(GVBD; Fig. 1B), metaphase I (MI; Fig. 1C) and metaphase II (MII; Fig. 1D).

766

6.6.Statistical analyses

RI PT

767

Categorical data were analyzed using the GLIMMIX Procedure of SAS fitted to

769

a binary distribution. However, due the outcome 0 or 100% some analyses were

770

performed using Fisher’s exact test using the FREQ procedure of SAS. Data were tested

771

for normality of residuals, and data with residuals not normally distributed were

772

transformed before analysis. Quantitative PCR data were analyzed using the GLIMMIX

773

procedure of SAS fitted to a Gaussian distribution with the fixed effects of cell type and

774

follicle size and the random effect of the sample.

M AN U

SC

768

The GLIMMIX procedure (ANOVA) of SAS was used with generalized linear

776

model methodology. For the data analyses, a Gaussian distribution was used. The

777

variables were analyzed using a mathematical model that included the effects of the

778

treatment. The residual method was used to calculate the denominator degrees of

779

freedom to approximate the F tests in the mixed models. The relative proportion of the

780

oocytes in each nuclear stage of maturation was calculated with the following equation:

781

Relative proportion = number of the oocytes in each stage/total of oocytes. The

782

statistical comparisons were performed using means adjusted by the least squares mean

783

method.

EP

AC C

784

TE D

775

Differences with P ≤ 0.05 were considered significant, and those with 0.05 < P ≤

785

0.10 were considered tendencies. Results are presented as the mean ± standard error of

786

the mean (SEM).

787

32

ACCEPTED MANUSCRIPT 788

7. Results

789

7.1.Analysis of gene expression qPCR revealed adiponectin mRNA in cumulus cells from small follicles and in

791

GCs and TCs of both follicular sizes but not in oocytes, regardless of size, or in the

792

cumulus cells from large follicles (Figure 2A).

RI PT

790

Adiponectin mRNA levels in small antral follicles were significantly higher (P

794

<0.05) in the cumulus cells compared to those in GCs and TCs, and mRNA levels did

795

not differ between GCs and TCs. In large antral follicles, adiponectin mRNA levels

796

were significantly higher (P <0.05) in granulosa than those in TCs (Figure 2A).

M AN U

SC

793

AdipoR1 and AdipoR2 expression was detected in all evaluated cell types

798

(oocytes, cumulus cells, GCs and TCs) of large and small antral follicles. AdipoR1 and

799

AdipoR2 showed significant differences (P <0.05) between the cell types derived only

800

from large antral follicles. AdipoR1 expression levels were significantly (P < 0.05)

801

lower in TCs compared with those in cumulus and GCs but did not differ (P > 0.05)

802

compared to oocyte levels (Figure 2B). AdipoR2 mRNA levels in TCs were

803

significantly (P < 0.05) lower than those of other cell types (Figure 2C).

806

EP

805

7.2. Protein expression analysis

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804

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797

Positive immunolocalisation for adiponectin, AdipoR1 and AdipoR2 were found

807

in oocyte, cumulus cells and granulosa cells of the follicles at all stages of development

808

(Fig. 3). Oocytes from preantral and antral follicles showed intense and uniform

809

cytoplasmic staining for the adiponectin (Fig. 3A-C), AdipoR1 (Fig. 3E-G) and

810

AdipoR2 (Fig. 3I, J and L) proteins. In addition, theca cells showed moderate

33

ACCEPTED MANUSCRIPT 811

immunostaining for all proteins (Fig. 3D, H and K). Control tissue samples showed no

812

positive staining (Fig. 3X, Y and Z).

813

7.3.Adiponectin effects on meiotic progression

RI PT

814

Figure 4 shows the effects of adiponectin on goat oocyte meiotic maturation.

816

The proportion of GV or GVBD stage-blocked oocytes (Figure 4A-B) was significantly

817

reduced (P <0.05) in the 5 or 10 µg/mL adiponectin-treated groups compared to that of

818

the control group. Maturation in the presence of 5 µg/mL adiponectin resulted in higher

819

rates of MI compared to those of the group without adiponectin (P <0.05; Figure 4C).

820

Additionally, the proportion of oocytes that progressed to MII was significantly higher

821

(P <0.05) in the 5 and 10 µg/mL adiponectin-treated groups (Figure 4D) than that in the

822

control groups. Groups treated with adiponectin showed similar percentages of MII

823

compared to the group cultured with FBS.

824

825

8. Discussion

TE D

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815

This study demonstrated the presence of the adiponectin system in goat follicular

827

cells. Although expression and immunolocalization of the adiponectin system has

828

already been studied in other species, as well as in different cell types, the gene and

829

protein expression of adiponectin and its receptors had not been studied in goats.

AC C

830

EP

826

Our results demonstrated that adiponectin was not expressed at detectable levels

831

in oocytes of small and large goat antral follicles or cumulus cells from large antral

832

follicles. These results differ from those reported by Maillard et al. [15] and Tabandeh

833

et al. [30], who found adiponectin expression in oocytes and cumulus cells of bovine

34

ACCEPTED MANUSCRIPT 834

antral follicles, indicating that there may be species-specific differences in ovarian

835

expression of the adiponectin gene. The lack of adiponectin mRNA in oocytes and cumulus cells from large antral

837

follicles is intriguing. However, mRNA samples from several pools of denuded oocytes

838

and cumulus cells from large antral follicles were analyzed, and none generated

839

amplicons after qPCR. Therefore, it is unlikely to be a methodological problem because

840

the reference genes GAPDH and UBQ were easily amplified in these samples, and we

841

detected AdipoR1 and AdipoR2 mRNAs in oocytes and cumulus cells.

SC

RI PT

836

Quantitatively, relative adiponectin expression in cumulus cells was significantly

843

higher than that in GCs and TCs from small follicles. In large follicles, relative

844

expression in GCs was significantly higher compared with that in TCs. These data differ

845

from what has been observed in chicken ovaries, in which the expression of adiponectin

846

in TCs was 10-30-fold higher than that in GCs [16]. In addition, adiponectin expression

847

was also low in GCs of mice [17]. However, Maillard et al. [15] observed good

848

adiponectin expression in bovine GCs.

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842

Receptor expression analysis showed decreased expression of AdipoR1 and

850

Adipo2 in TCs of large follicles compared with expression of receptors in GCs. These

851

results differ from those found by Tabandeh et al. [30] in cattle, where AdipoR1 and

852

AdipoR2 expression was higher in large follicle TCs than that in GCs. Our findings also

853

differ from results observed in mice, in which receptor expression in GCs was lower

854

than in TCs [17]. However, similar results were observed in chickens, where AdipoR1

855

expression was twice as high in GCs as that in TCs, and AdipoR2 expression was

856

similar in both cell types [16]. This discrepancy suggests a species-specific mode of

857

action of the adiponectin system on follicular development.

AC C

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849

35

ACCEPTED MANUSCRIPT In the present study, adiponectin and its receptors AdipoR1 and AdipoR2 were

859

immunolocalized in oocytes, cumulus cells, GCs and TCs in ovarian follicles at all

860

stages of development. These findings corroborate those obtained in bovines [15], mice

861

[17] and humans [21] and further support the physiological relevance of this hormone

862

on ovarian function.

RI PT

858

Adiponectin protein was detected in the oocytes by immunohistochemistry,

864

although no mRNA was observed, indicating that adiponectin must be produced

865

elsewhere and then transported to the oocyte. This is likely because adiponectin is a

866

secreted protein, is found in follicular fluid [29], and binds to its receptors on granulosa,

867

cumulus and oocyte cells; receptor-bound adiponectin has been shown to be internalized

868

in several cell lines [31,32]. The detection of adiponectin appears to be specific because

869

preincubation of the antibody with blocking peptide abolished staining.

M AN U

SC

863

Oocyte meiotic maturation and acquisition of developmental competence is an

871

essential physiological process for survival of the species. The presence of AdipoR1 and

872

AdipoR2 mRNA and protein in both oocytes and cumulus cells suggests that both cell

873

types are sensitive to adiponectin and that adiponectin may also affect oocyte

874

maturation.

EP

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870

In the present work, we demonstrated that adiponectin addition (5 or 10 µg/mL)

876

during IVM affected goat oocyte meiotic maturation. However, conflicting results have

877

been reported in the literature. In cattle, supplementation of IVM medium with

878

adiponectin (5 or 10 µg/mL) did not affect bovine oocyte meiotic maturation [15,24]. In

879

pigs, however, Chappaz et al. [18] reported that recombinant porcine adiponectin (30

880

µg/mL) significantly decreased the proportion of immature oocytes, suggesting that

881

adiponectin accelerated porcine oocyte meiotic maturation.

AC C

875

36

ACCEPTED MANUSCRIPT These apparently controversial IVM results reported in the literature are

883

probably dependent on the species studied or on the culture medium used. The

884

experiment described in the present study was performed with serum-free medium and

885

in the absence of any growth factor or hormone other than adiponectin. However, other

886

studies have usually added gonadotropins or growth factors to the culture media; these

887

additives could have influenced the action of adiponectin or affected its pathways,

888

resulting in different responses. Thus, possible interactions between these additives and

889

the possible pathways by which adiponectin affects oocyte maturation remain unclear

890

and require further investigation.

SC

RI PT

882

In conclusion, this study detected the expression of adiponectin and its receptors,

892

AdipoR1 and AdipoR2, in goat ovarian follicles. In addition, adiponectin was shown to

893

enhance the progression of goat oocyte nuclear maturation in vitro. The present findings

894

provide evidence for paracrine/autocrine effects of the analyzed hormone. However,

895

future studies are needed to elucidate the mechanisms underlying the effect of

896

adiponectin on meiotic maturation and to understand the role of the adiponectin system

897

in goat fertility.

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891

900 901 902 903 904 905

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as

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899

EP

898

prejudicing the impartiality of the research reported.

Acknowledgements The authors thank FACEPE (Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco) for financial support (APQ-1115-5.05/14).

906

37

ACCEPTED MANUSCRIPT 907

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[10] Campos DB, Palin M-F, Bordignon V, Murphy BD. The “beneficial” adipokines in reproduction and fertility. Int J Obes 2008;32:223–31.

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[11] Combs TP, Berg AH, Rajala MW, Klebanov S, Iyengar P, Jimenez-Chillaron JC, et al. Sexual differentiation, pregnancy, calorie restriction, and aging affect the

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adipocyte-specific secretory protein adiponectin. Diabetes 2003;52:268–76.

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review of their structure, function and how they work. Best Pract Res Clin

944

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[13] Kadowaki T, Yamauchi T. Adiponectin and adiponectin receptors. Endocr Rev

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2005;26:439–51. doi:10.1210/er.2005-0005. [14] Yamauchi T, Kamon J, Ito Y, Tsuchida A, Yokomizo T, Kita S, et al. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature

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[15] Maillard V, Uzbekova S, Guignot F, Perreau C, Ramé C, Coyral-Castel S, et al.

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Effect of adiponectin on bovine granulosa cell steroidogenesis, oocyte maturation and embryo development. Reprod Biol Endocrinol 2010;8:23. doi:10.1186/14777827-8-23.

[16] Chabrolle C, Tosca L, Crochet S, Tesseraud S, Dupont J. Expression of

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adiponectin and its receptors (AdipoR1 and AdipoR2) in chicken ovary: Potential

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doi:10.1016/j.domaniend.2006.08.002. [17] Chabrolle C, Tosca L, Dupont J. Regulation of adiponectin and its receptors in rat ovary by human chorionic gonadotrophin treatment and potential involvement

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of adiponectin in granulosa cell steroidogenesis. Reproduction 2007;133:719–31.

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Adiponectin enhances in vitro development of swine embryos. Domest Anim

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[19] Nishio SI, Gibert Y, Bernard L, Brunet F, Triqueneaux G, Laudet V. Adiponectin and adiponectin receptor genes are coexpressed during zebrafish embryogenesis

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and regulated by food deprivation. Dev Dyn 2008;237:1682–90.

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[20] Chabrolle C, Tosca L, Ramé C, Lecomte P, Royère D, Dupont J. Adiponectin increases insulin-like growth factor I-induced progesterone and estradiol

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secretion in human granulosa cells. Fertil Steril 2009;92:1988–96.

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Further evidence for a link between obesity and hyperandrogenism in polycystic

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ovary syndrome. PLoS One 2013;8:1–9. doi:10.1371/journal.pone.0080416.

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insulin signaling pathway and related proteins in sheep. Biol Reprod

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[23] Kiezun M, Smolinska N, Maleszka A, Dobrzyn K, Szeszko K, Kaminski T. Adiponectin expression in the porcine pituitary during the estrous cycle and its

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effect on LH and FSH secretion. Am J Physiol Endocrinol Metab

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2014;307:E1038-46. doi:10.1152/ajpendo.00299.2014.

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[24] Divar MR, Kafi M, Mohammadi A, Azari M. The In Vitro Effect of Adiponectin on Early Bovine Embryonic Development and Transcriptomic Markers of Oocyte

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Competence. J Fertil Vitr - IVF-Worldwide, Reprod Med Genet Stem Cell Biol

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2016;4:1–7. doi:10.4172/2375-4508.1000172.

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The expression and localization of leptin and its receptor in goat ovarian follicles.

990

Anim Reprod Sci 2013;141:142–7. doi:10.1016/j.anireprosci.2013.08.007.

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[26] Frota IM a, Leitão CCF, Costa JJN, Brito IR, van den Hurk R, Silva JR V.

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Stability of housekeeping genes and expression of locally produced growth

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factors and hormone receptors in goat preantral follicles. Zygote 2011;19:71–83.

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doi:10.1017/S0967199410000080.

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time quantitative PCR and. Methods 2001;25:402–8.

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1000 1001 1002

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[28] Guzel S, Yibar A, Belenli D, Cetin I, Tanriverdi M. The concentrations of adipokines in goat milk: relation to plasma levels, inflammatory status, milk quality and composition. J Vet Med Sci 2017;79:602-607. doi:10.1292/jvms.16-

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[29] Wang Z, Meng C, Ding X, Zhang G, Wang F. Effect of body condition on

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1005 1006

[30] Tabandeh MR, Hosseini A, Saeb M, Kafi M, Saeb S. Changes in the gene expression of adiponectin and adiponectin receptors (AdipoR1 and AdipoR2) in

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ovarian follicular cells of dairy cow at different stages of development.

1008

Theriogenology 2010;73:659–69. doi:10.1016/j.theriogenology.2009.11.006.

1009

[31] Ding Q, Wang Z, Chen Y. Endocytosis of adiponectin receptor 1 through a clathrin- and Rab5-dependent pathway 2009;19:317–27.

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doi:10.1038/cr.2008.299.

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1010

[32] Almabouada F, Diaz-ruiz A, Rabanal-ruiz Y, Peinado JR, Vazquez-martinez R,

1013

Malagon MM. Adiponectin Receptors Form Homomers and Heteromers

1014

Exhibiting Distinct Ligand Binding and Intracellular Signaling 2013;288:3112–

1015

25. doi:10.1074/jbc.M112.404624.

M AN U

1016 1017 1018 1019

1024 1025 1026 1027

EP

1023

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1022

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1020 1021

SC

1012

1028 1029 1030 1031

42

ACCEPTED MANUSCRIPT 1032

Table Legend

1033

Table 1. Oligonucleotide primer sequences used to perform qPCR.

1034

Figure Legends

1036

Figure 1. Representative images of goat oocytes stained with Hoechst 33342 and

1037

evaluated under fluorescence microscopy. Oocytes in germinal vesicle (GV, A),

1038

germinal vesicle breakdown (GVBD, B), metaphase I (MI, C), and metaphase II (MII,

1039

D) stages are shown.

SC

RI PT

1035

1040

Figure 2. Adiponectin (A); AdipoR1 (B) and AdipoR2 (C) mRNA expression in oocyte

1042

– O, cumulus cells – C, granulosa cells – G and theca cells – T isolated from small (< 3

1043

mm) and large (≥ 3 mm) goat antral follicles. Different superscripts denote the statistical

1044

significance between different cells types isolated from follicles in the same size

1045

category (P < 0.05).

TE D

M AN U

1041

1046

Figure 3. Immunolocalization of adiponectin, AdipoR1 and AdipoR2 in goat ovaries.

1048

Positive staining of adiponectin, AdipoR1 and AdipoR2 was observed in oocytes and

1049

granulosa cells of primordial (A, E and I), primary (B and I) and secondary follicles (C,

1050

F and J). Antral follicles with positive immunostaining of adiponectin, AdipoR1 and

1051

AdipoR2 in oocytes, cumulus, theca and granulosa cells (D, G, H, K and L). No staining

1052

was observed in the negative control (X, Y and Z). g, granulosa; t, theca, c, cumulus; o,

1053

oocytes.

AC C

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1047

1054 1055

Figure 4. Effects of recombinant adiponectin (0, 5 or 10 µg/mL) on the progression of

1056

meiotic maturation of goat oocytes in vitro matured for 27 h. Germinal vesicle (GV, A),

43

ACCEPTED MANUSCRIPT germinal vesicle breakdown (GVBD, B), metaphase I (MI, C), and metaphase II (MII,

1058

D). The data are expressed as the mean ± SEM. Different superscripts denote the

1059

statistical significance among experimental groups within the same stage of meiotic

1060

maturation (P < 0.05).

AC C

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M AN U

SC

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1057

44

21

ACCEPTED MANUSCRIPT 1

Table 1. Oligonucleotide primer sequences used to perform qPCR.

2 Gene

Primer sequences (5'-3')

GAPDH

Ubiquitin

CYP17A1

Forward

5'-GAAGGTGGTGTTCGGGATGT-3'

Reverse

5'-CAGTGTGGAAGAGCCAGGAG-3'

Forward

5'-GCTCTCAGGCTCAGGAAAGG-3'

Reverse

5'-AAGCCAGACGCCTCTACAAG-3'

Forward

5'- TGTTTGTGATGGGCGTGAACCA-3'

Reverse

5'-ATGGCGTGGACAGTGGTCATAA-3'

Forward

5'-GAAGATGGCCGCACTCTTCTGAT-3'

Reverse

5’-ATCCTGGATCTTGGCCTTCACGTT-3'

RI PT

5'-CCGCATAGACCCCATTGTGA-3'

Forward

5'-ACTGAATGCCTTTGCCCTGT-3'

Reverse

5'-CTGATTATGTTGGTGACCGCC-3'

Forward

5'-CATCCTCAATACCAGGTCCCA-3'

Reverse

5'-GGTTTCCTCTCCACATACCCA-3'

3

AC C

EP

CYP19A1

Reverse

KF452236.1

HQ846828.1

SC

AdipoR2

5'-GCTCCGTGCTCCTCTATCTG-3'

M AN U

AdipoR1

Forward

TE D

Adiponectin

Accession no.

JX573540.1

AJ431207.1

GI:57163956

AF251387

AY148883

22

ACCEPTED MANUSCRIPT 1

Figure 1

4 5 6 7 8 9 10

AC C

3

EP

TE D

M AN U

SC

RI PT

2

23

ACCEPTED MANUSCRIPT 11

Figure 2

AC C

EP

TE D

M AN U

SC

RI PT

12

13 14

24

ACCEPTED MANUSCRIPT 15

Figure 3

SC

RI PT

16

17

M AN U

18 19 20 21

25 26 27 28 29 30 31 32 33

EP

24

AC C

23

TE D

22

25

ACCEPTED MANUSCRIPT 34

Figure 4

M AN U

SC

RI PT

35

36

40 41 42 43 44 45 46 47 48

EP

39

AC C

38

TE D

37

ACCEPTED MANUSCRIPT Highlights •

Expression of the members of the adiponectin system was determined in the ovary of goats. Adiponectin mRNA was not expressed at detectable levels in oocytes.

•

AdipoR1 and AdipoR2 expression was detected in all evaluated follicle cells.

•

Adiponectin was shown to enhance the progression of goat oocyte nuclear

RI PT

•

AC C

EP

TE D

M AN U

SC

maturation in vitro.