Apelin and apelin receptor at different stages of corpus luteum development and effect of apelin on progesterone secretion and 3β-hydroxysteroid dehydrogenase (3β-HSD) in pigs

Accepted Manuscript Title: Apelin and apelin receptor at different stages of corpus luteum development and effect of apelin on progesterone secretion and 3␤-hydroxysteroid dehydrogenase (3␤-HSD) in pigs Authors: Marta R´oz˙ ycka, Patrycja Kurowska, Małgorzata Grzesiak, Małgorzata Kotula-Balak, Wacław Tworzydło, Christelle Rame, Ewa Gregoraszczuk, Joelle Dupont, Agnieszka Rak PII: DOI: Reference:

S0378-4320(17)30728-5 https://doi.org/10.1016/j.anireprosci.2018.03.021 ANIREP 5799

To appear in:

Animal Reproduction Science

Received date: Revised date: Accepted date:

12-9-2017 6-3-2018 19-3-2018

Please cite this article as: R´oz˙ ycka M, Kurowska P, Grzesiak M, Kotula-Balak M, Tworzydło W, Rame C, Gregoraszczuk E, Dupont J, Rak A, Apelin and apelin receptor at different stages of corpus luteum development and effect of apelin on progesterone secretion and 3␤-hydroxysteroid dehydrogenase (3␤-HSD) in pigs, Animal Reproduction Science (2010), https://doi.org/10.1016/j.anireprosci.2018.03.021 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.

Apelin and apelin receptor at different stages of corpus luteum development and effect of apelin on progesterone secretion and 3β-hydroxysteroid dehydrogenase (3β-HSD) in pigs

Short title: Apelin in porcine corpus luteum

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Marta Różyckaa, Patrycja Kurowskaa, Małgorzata Grzesiakb, Małgorzata Kotula – Balakc, Wacław Tworzydłod, Christelle Ramee, Ewa Gregoraszczuka, Joelle Duponte, Agnieszka Raka*

a

Department of Physiology and Toxicology of Reproduction, Institute of Zoology and Biomedical Research, Jagiellonian University in Krakow, 30-387 Krakow, Poland b

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Department of Animal Physiology and Endocrinology, University of Agriculture in Krakow, 30-059 Krakow, Poland c

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Department of Endocrinology, Institute of Zoology and Biomedical Research, Jagiellonian University in Krakow, 30-387 Krakow, Poland d

INRA, Unité Physiologie de la Reproduction et des Comportements, 37-380 Nouzilly, France

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e

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Department of Developmental Biology and Invertebrate Morphology, Institute of Zoology and Biomedical Research, Jagiellonian University in Krakow, 30-387 Krakow, Poland

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Corresponding author: dr hab. Agnieszka Rak, Department of Physiology and Toxicology

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of Reproduction, Institute of Zoology and Biomedical Research, Jagiellonian University

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in Krakow, Gronostajowa 9, 30-387 Krakow, Poland; e-mail: [email protected]

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ABSTRACT Recent studies have suggested that apelin has a role in controlling female reproduction. The aims of the present study were, firstly, to investigate the gene expression (mRNA and protein) and immunolocalization of apelin and its receptor APJ in corpora lutea (CL) of pigs

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collected during the early (CL1), middle (CL2) and late (CL3) luteal phase. Using real time PCR and immunoblotting techniques, it was observed that apelin gene expression was similar in CL1 and CL2, and less in CL3, while relative abundance APJ mRNA and abundance of the

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protein were similar in CL1 and CL3 and greater in CL2. There was apelin staining in the

cytoplasm of both small (SC) and large (LC) luteal cells with the greatest intensity in CL2. In

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the cytoplasm of CL1, only a few SC cells stained for APJ; in CL2, APJ was located in the cell

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membrane of LC and in the cytoplasm of SC; and in CL3 was located in the membrane with

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moderate cytoplasmic APJ staining. Intense APJ staining was noted in epithelium of blood

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vessels of CL2-3. Secondly, there was an effect of apelin on progesterone (P4) secretion in CL2 and on the molecular mechanisms of these cells. Stimulatory effects of apelin on P4 secretion,

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3β–hydroxysteroid dehydrogenase (HSD) activity and protein abundance were observed and this was inhibited in response to APJ and adenosine 5’-monophosphate-activated protein kinase

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(AMPKα) kinase blockers. In conclusion, the presence of apelin/APJ in the CL of pigs and

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stimulatory effects of apelin on P4 secretion and 3β-HSD levels suggest potential auto/paracrine regulation by apelin in the luteal phase of the estrous cycle. Moreover, the involvement of APJ

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and AMPKα kinase in apelin activity in CL was confirmed.

Keywords: Apelin; Apelin receptor; Progesterone; Corpus luteum; Pig

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1. Introduction The corpus luteum (CL) is a transient endocrine gland that differentiates from the thecal and granulosal cells of the ovarian follicle after ovulation. Its formation and limited lifespan in the mammalian ovary is important for fertility, as the CL produces progesterone (P4), the

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essential steroid hormone required for embryo implantation and maintenance of pregnancies until placental development (Stouffer, 2003; Stouffer et al., 2013). Recent studies, particularly those involving genome and cellular analyses, have increased the understanding of local factors

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associated with the development, functional lifespan and regression of the CL.

There is compelling evidence for interactive functions among metabolic hormones, such

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as ghrelin, leptin, or resistin, and female reproduction (Rak-Mardyła, 2013; Rak-Mardyła et al.,

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2013, 2014). Apelin is a regulator of ovarian physiology (Roche et al., 2016, 2017; Rak et al.,

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2017). Apelin was originally identified in stomach extracts of cattle as the endogenous ligand

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of the orphan G protein-coupled receptor APJ (Tatemoto et al., 1998). It is derived from a 77-

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amino-acid preproapelin that is cleaved into a 55-amino-acid fragment and then into shorter forms. This adipokine is involved in a broad range of physiological functions such as fluid

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homeostasis, regulation of food intake and energy metabolism (Taheri et al., 2002, Bertrand et al., 2015). Moreover, the apelin peptide is a potent angiogenic factor inducing endothelial cell

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(EC) proliferation, migration, and development of blood vessels in vivo (Kasai et al. 2004, Cox et al. 2006). Apelin was described as a biomarker of several pathologies including diabetes, obesity, cardiovascular disease, endometriosis, cancer and polycystic ovarian syndrome

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(Rayalam et al., 2011; Perjes et al., 2014; Narayanan et al., 2015; Roche et al., 2016). Amounts of apelin are increased in obese humans compared with lean control individuals (Boucher et al., 2005). The expression of the apelin and its receptor APJ genes has been detected in human, cattle, rhesus monkey and pig ovaries (Shirasuna et al., 2008; Schilffarth et al., 2009; Shimizu et al., 2009; Fuhua and Stouffer, 2012; Roche et al., 2016, 2017; Rak et al., 2017). In humans, 3

the apelin/APJ genes are expressed in different ovarian cells such as granulosal, thecal cells and oocytes (Roche et al., 2016). Schilffarth et al. (2009) demonstrated that in ovaries of cattle apelin/APJ decreased at the end of the luteal phase and decreased during CL regression, suggesting the role of apelin in CL formation and the luteolytic endocrine cascade pathway. These findings were consistent with data of Shirasuna et al., (2008) where it was demonstrated

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that the expression of both the apelin/APJ genes is isolated to the smooth muscle cells of luteal arterioles in the CL of cattle, suggesting that the apelin/APJ system may be associated with the

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vascular function in the CL.

Data concerning the role of apelin in the physiology of pigs are limited. In a study of Del

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Ry et al., (2009), the apelin gene was initially sequenced for Sus scrofa for future applications

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to molecular biology studies. In a previous study, it was documented that there was gene

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expression and a direct role of apelin on ovarian follicular cells steroidogenesis and

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proliferation in pigs (Rak et al., 2017). There are no reports describing the expression of apelin/APJ genes in corpus luteum, particularly as related to actions on progesterone secretion.

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The aims of the present study, therefore, were to evaluate a) the relative abundances of mRNA and abundance of protein of apelin/APJ during different stages of CL development; b)

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level.

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immunolocalization in CLs; and c) direct in vitro effects of apelin on P4 secretion and 3βHSD

2. Materials and methods

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2.1. Reagents and antibodies Fetal bovine serum (FBS, heat-inactivated) was purchased from Biowest (Nuaile, France)

and phosphate-buffered saline (PBS) from Gibco Life Technologies (Paisley, UK). The M199 medium, antibiotic-antimycotic solution (10,000 units/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin B), Tris, Na-deoxycholate, Nonidet NP-40, sodium dodecyl sulfate (SDS), protease inhibitors (EDTA-free), dithiothreitol (DTT), Tween 20, bromophenol blue, 1 4

bromo-3-chloro-propane, 3,3′-diaminobenzidine, pregnenolone (P5, cat. # P9129) and human recombinant apelin - 13 (cat. # A6469) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Human recombinant apelin-13 was utilized in this experiment as porcine apelin was not readily available at the onset of the experiment. The homology of human (GeneBank accession number NP_059109.3) and Sus scrofa (GeneBank accession number XP_003360494.1) apelin

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is 83%. Apelin-13 is the main circulatory form and its biological activity is greater than apelin17 or -36 (Tatemoto et al., 1998, Habata et al., 1999). Consumables for reverse transcription

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(RT) were obtained from Sigma (l’Isle d’Abeau Chesnes, France), and Moloney Murine Leukemia Virus reverse transcriptase and the RNase inhibitor were obtained from Promega (Madison, WI, USA). The APJ antagonist, ML221, was obtained from TOCRIS Bioscience

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(Bristol, UK). A Bradford protein assay kit was purchased from Bio-Rad Laboratories

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(Hercules, CA, USA). Compound C and polyvinylidene difluoride (PVDF) membrane were

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obtained from Merck Millipore (Darmstadt, Germany).

Antibodies against apelin (sc-293441), APJ (sc-33823), 3βHSD (sc-30820), horseradish

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peroxidase-conjugated antibody (sc-2020) and Western blotting luminol reagent (cat. # sc2048) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin

goat

anti–rabbit

IgG

(BA-1000),

avidin-biotin-peroxidase

complex

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biotinylated

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(A5316) antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA). Secondary

(StreptABComplex-HRP, PK-4000) were obtained from Vector Laboratories.

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2.2. Animals and sample collection Pig ovaries were collected from mature (7-8 mo of age), crossbred gilts (Large White and

Polish Landrace) at a local abattoir. Average weight of these animals was 120 to 140 kg. The veterinarian determined the age, weight and sex of all animals before slaughter. Approximately 15 min elapsed between slaughter and ovary collection. Ovaries were collected in a bottle filled with sterilized saline and transported to the laboratory. The CLs were classified, based on 5

morphological criteria, as early (CL1: 1-2 d after ovulation; n = 6), middle (CL2: 7-10 d after ovulation; n = 6) and late luteal phase (CL3: 13-15 d after ovulation; n = 6) using criteria reported in a previously published study (Rak-Mardyła et al., 2012). Assays for apelin/APJ gene expression involved CLs being isolated from three different stages (CL1-CL3) of the luteal phase, followed by immediate freezing in liquid nitrogen and storage at −70 °C until RNA

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extraction. For determination of protein amount of apelin/APJ, CLs were homogenized twice

in ice-cold lysis buffer, which contained 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5% Na-

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deoxycholate, 0.5% NP-40, 0.5% SDS and protease inhibitor (EDTA-free). The lysates were

clarified by centrifugation at 15.000 × g at 4 C for 30 min, and protein content in the lysates

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was determined with the Bradford reagent using BSA as the standard. After homogenization,

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the supernatants were collected and stored at -20 °C. For apelin/APJ immunohistochemical determination, CLs were fixed in 10% buffered formalin, embedded in paraplast, cut into 5-μm

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thick sections, and mounted onto 3′3′-aminopropyl-triethoxysaline–coated slides (SigmaAldrich).

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2.3. Corpus luteum cell culture

In vitro effects of apelin on P4 secretion and 3β-HSD level (activity and protein

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expression) were evaluated using luteal cells obtained from the middle luteal phase (CL2), as

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results of the first experiment indicated the greatest APJ gene expression in this phase. For luteal tissue assessments, pools of freshly excised CLs were obtained from three animals in the same phase of the estrous cycle, was minced mechanically and then underwent trypsinization

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using 0.25% trypsin in PBS for 10 min at 37 °C, according to the technique described by Gregoraszczuk (1983). Subsequently, the cells were separated by decanting and the procedure was repeated three times. Cells were subsequently centrifuged and re-suspended in M199 medium supplemented with 10% FBS to yield a suspension of 3.5 × 105 cells/mL medium. Cell viability was measured using the Trypan blue exclusion test and was determined as 80% to 6

85%. After incubation for 24 h, media were changed to fresh M199 (supplemented with 5% FBS) and apelin at concentrations of 0.02, 0.2, 2 and 20 ng/mL was added for the next 24 h. Selection of apelin concentrations was based on results of a previously published study (Rak et al., 2017). For assessment of 3β-HSD activity, CL2 cells were incubated for 24 h in M199 (supplemented with 5% FBS) containing apelin at concentrations of 0.02, 0.2, 2 and 20 ng/mL

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and P5 at 5 µg/mL. At the end of the incubation period, the medium was collected for analysis of P4 content. Cells were transferred into ice-cold lysis buffer and total cell lysates were

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prepared and stored at -20 °C for subsequent determination of abundance of the 3β-HSD

protein. Apelin-induced activation of AMPKα was studied by pre-treating cells for 1 h with the AMPKα antagonist, Compound C (10 µM), after which apelin (2 ng/mL) was added. Results

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of previous studies indicate the AMPKα gene is expressed in CL and is involved in the

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regulation of P4 secretion in various species, including pigs (Tosca et al., 2006, Santiquet et al.,

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2014, Bertoldo et al., 2015). In addition, it was clearly documented in a previous study that

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apelin induced an increase in abundance of AMPKα protein in ovarian follicular cells of pigs

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and regulated P4 synthesis (Rak et al., 2017). Activation of APJ was evaluated by pre-treating cells for 1 h with the APJ antagonist, ML221 (10 µM), after which apelin (2 ng/mL) was added.

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Selection of the concentrations of these compounds was based on preliminary research and results of a previous study (Rak et al., 2017). After incubation for 48 h, culture medium was

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collected and stored at −20 °C for quantitation of P4 concentrations.

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2.4. RNA isolation, cDNA synthesis and real time polymerase chain reaction (PCR) Total RNA was extracted using Trizol reagent according to the manufacturer’s procedure

(Sigma Aldrich, Saint Quentin Fallavier, France), as described previously (Reverchon et al. 2014). The RT procedures were subsequently conducted. Briefly, 1 µg of total RNA was reverse transcribed for 1 h at 37 °C in a final reaction volume of 20 µL, containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 200 µM of each deoxynucleotide triphosphate 7

(Amersham, Piscataway, NJ), 50 pmol of oligo(dT) 15, 5 U of ribonuclease inhibitor and 15 U of MMLV reverse transcriptase. After RT, porcine cDNA from CL was diluted 1:5. Real time PCR was performed in a 20-µL final volume containing 10 μL iQ SYBR Green supermix (BioRad), 0.25 μL of each primer (10 µM), 4.5 μL of water and 5 µL of template. The cDNA templates were amplified and detected with the MYIQ Cycler real time PCR system (Bio-Rad)

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using the protocol previously described by us (Rak et al., 2017). The abundance of two housekeeping genes – PPIA (cyclophilin A) and RPL19 were examined and normalized

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according by Vandesompele et al., (2002). For each gene, relative abundance of mRNA was

calculated according to primer efficiency and Cq expression = E-Cq. These two housekeeping genes had changes in expression between the three stages of CL development. The data,

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therefore, were normalized to the geometric mean of PPIA and RPL19 (this combination was

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stable) using the processes that were previously reported that the geometric mean of multiple

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housekeeping genes can be used as an accurate normalization factor (Vandesompele et al.

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2002). Normalized values of relative abundance (R) were calculated according to the following

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equation:

 CtGene ( EGene ) ,  CtPpia  CtRpl19 ( geometric mean( EPpia ; ERpl )) 19

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R

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where Ct is the cycle threshold and E is the PCR efficiency for each primer pair. The description of the different primers is: RPL19 (forward “5’- AACTCCCGTCAGCAGATCC -3’ and reverse

5’-

AGTACCCTTCCGCTTACCG

-3’)

and

PPIA

(forward

“5’-

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CACAAACGGTTCCCAGTTTT -3’ and reverse 5’- TGTCCACAGTCAGCAATGGT -3’). The

specific

primers

for

apelin

and

APJ

were:

apelin

(forward

5’-

AAGGCAACGTCCGCTATTTG -3’ and reverse “5’- ATGGGGCCCTTGTGGGAGA -3’), and

APJ

(forward

“5’-

ACCTTGGTGCCGTTCTCGG

-3’

and

reverse

5’-

CTCAGCTTCGACCGCTACCT-3’). Normalized values of relative abundance (R) were 8

calculated according to the following equation: where Ct is the cycle threshold and E is the PCR efficiency for each primer pair. The specificity of the amplified fragment sequence was assessed by Beckman Coulter Genomics (Essex, United Kingdom). The efficiency was between 1.8 and 2. 2.5. Western blot

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Protein (40 μg) was reconstituted directly in the appropriate amount of sample buffer: 125 nM TRIS (pH 6.8), 4% SDS, 25% glycerol, 4 mM EDTA, 20 mM DTT and 0.01%

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bromophenol blue. Samples were separated by 12% SDS-polyacrylamide gel electrophoresis

(SDS-PAGE) and proteins were transferred to nitrocellulose membranes. Membranes were then

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washed and non-specific binding sites were blocked with 5% w/v BSA and 0.1% v/v Tween 20

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in 0.02 M Tris-buffered saline (TBS) for 1 h. Membranes were then incubated overnight at 4

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ºC with anti-apelin, -APJ and -3β-HSD antibody diluted at 1: 200 in TBS/Tween. After

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incubation with the primary antibody, the membranes were washed with TBS and 0.02% Tween 20 (TBST) and incubated for 1 h with horseradish peroxidase-conjugated antibody diluted at

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1:5000 at room temperature in TBST. Signals were detected by chemiluminescence using Western blotting luminol reagent. Blots were visualised using the ChemiDocTM and all of the

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bands were quantified using Image LabTM 2.0 Software (BioRad Laboratories). Blots were

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stripped and probed for β-actin. 2.6. Immunohistochemistry Slide-mounted sections of CL were deparaffinized in xylene, rehydrated gradually

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through a series of ethanol dilutions and rinsed in water. The slides were treated using an unmasking procedure with microwave heating (2 × 600 W) in 0.01 M citrate buffer (pH 6.0), followed by 30 min in 0.3% (v/v) H2O2 in TBS (pH 7.4) to quench endogenous peroxidase activity. Blocking of non-specific binding sites was performed with 5% (v/v) normal goat serum prior to incubation with primary antibodies anti-apelin (dilution 1:50) or -APJ (dilution 1:100). 9

After overnight incubation at 4 °C in a humidified chamber, sections were washed with TBST. The antigens were visualized using secondary biotinylated goat anti–rabbit IgG (dilution 1:300; 1.5 h at room temperature), StreptABComplex-HRP (40 min at room temperature) and 3,3′diaminobenzidine as a chromogen. Sections were then counterstained with hematoxylin QS (Vector Laboratories), dehydrated and mounted using DPX (Sigma-Aldrich). For a negative

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control reaction, sections were incubated with non-immune rabbit IgG (cat. No. NI01,

Calbiochem, Darmstadt, Germany) instead of primary antibodies and processed as above. All

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slides were processed immunohistochemically at the same time, so that staining intensity among serial sections of CL could be compared. Selected sections were photographed using a Nikon Eclipse Ni-U microscope and DMRLeica microscope equipped with Nomarski

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interference contrast.

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2.7. ELISA

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The concentrations of P4 in the media were determined by enzyme immunoassays (EIA) using commercial ELISA kits (DRG Diagnostic, Germany). All samples were evaluated in

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duplicate in the same assay. The limit of the P4 assay sensitivity was 0.045 ng/mL, and the inter- and intra-procedure precision had coefficients of variation of 4.34% and 6.99%,

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respectively. The range of the assay was 0 to 40 ng/mL. Each treatment was conducted in

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quadruplicate (four wells each) for three separate experiments. 2.8. Statistical analysis Each experiment was repeated three times (n = 3). Statistical analysis was performed

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using Statistical 6.0. Data were analyzed using a one-way or two-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) multiple range test. All date are expressed as the mean  SEM. Groups that were significantly different (P < 0.05) are indicated in the figures with different letters or by *(P < 0.05), **(P < 0.01) and ***(P < 0.001). Data points with the same letters were not significantly different. 10

3. Results 3.1. Relative abundance of mRNA and abundance of protein for apelin and APJ in CL at different stages of development Using real-time PCR in porcine CL at different stages of development, relative abundances of apelin mRNA were similar in CL1 and CL2, and less in CL3, while relative

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abundance of APJ mRNA was similar in CL1 and CL3, and greater in CL2 (Fig. 1A; P < 0.05). These results were confirmed by Western blot and densitometry analysis that indicated

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abundance of apelin/APJ proteins was also dependent on CL development: the least apelin

abundance was in CL3 and CL1 and the greatest abundance of APJ in CL2 (Fig. 1B; P < 0.05).

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3.2. Immunolocalization of apelin and APJ in CL at different stages of development

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Luteal phase stage – dependent apelin immunostaining intensity and APJ

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immunolocalization was observed. Apelin staining was exclusively in the cytoplasm of both

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small and large luteal cells with the greatest intensity of staining in CL2 (Fig. 2A-C). In CL3,

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apelin was present in a few of the luteal cells of both types (Fig. 2C). The APJ was localized in both the cytoplasm and membrane of small and large luteal

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cells. In CL1, only a few small luteal cells had weak, cytoplasmic APJ immunostaining. In CL2, the APJ was located in the cell membrane of large luteal cells while it was located in cytoplasm

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of small luteal cells. (Fig. 2E). In the small luteal cells, the staining was of moderate intensity. In luteal cells of CL3, there was membrane and moderate cytoplasmic APJ immunostaining (Fig. 2F). Additionally, intense APJ staining was noted in epithelium of blood vessels of CL2

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and CL3 (Fig. 2E, F). There was no positive staining in controls, when primary antibodies were omitted (Fig. 2C, F, insets). 3.3. Effect of apelin on luteal progesterone secretion

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Apelin, at concentrations of 0.2, 2.0 and 20 ng/mL, increased P4 secretion by CL2 cells (49.557, 48.179 and 40.51 ng/mL, respectively, compared with 28.02 ng/mL in control; Fig. 3A; P < 0.05). The role of AMPKα in the regulation of apelin-induced effects through APJ on luteal P4 secretion was studied using the potent antagonists of APJ (ML221) and AMPKα (Compound

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C). The APJ and AMPKα antagonists inhibited the stimulatory activity of apelin (2 ng/mL) on P4 secretion compared with the values of the control group, suggesting that APJ through

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AMPKα activation is involved in the action of apelin on luteal P4 secretion (Fig. 3B; P < 0.05). 3.4. Effect of apelin on 3βHSD protein and activity

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Western blot analysis revealed that treatment of CL2 cells with 2 and 20 ng/mL apelin

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increased protein of 3β-HSD (Fig. 4A; P < 0.01, P < 0.001, respectively). The enzymatic

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activity of 3β-HSD was measured by the conversion of P5 into P4. The P4 synthesis

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(representing 3β-HSD activity), which was determined following the addition of P5 over a 4-

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h incubation period, increased from 28.06 to 43.21 ng/mL. Apelin, at concentrations of 2 and 20 ng/mL, increased P5-stimulated progesterone synthesis to a greater extent than observed in

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cells treated with P5 alone. Values were 64.38 and 61.52 ng/mL, respectively, compared to 43.21 ng/mL with P5 alone (P < 0.05; Fig. 4B).

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Apelin at 0.2, 2 and 20 ng/mL concentrations also increased P5-stimulated 3β-HSD

protein to a greater extent than that of cells treated with P5 alone (P < 0.05).

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4. Discussion

The results of this study demonstrate, for the first time, relative abundance of mRNA and abundance of protein of both apelin and its receptor APJ at different stages of CL development in pigs. Relative abundance of apelin mRNA and abundance of protein were similar in CL from early and middle luteal phases of the estrous cycle, but were less in the late luteal phase. Relative 12

abundance of APJ mRNA and abundance of protein were similar in CL from early and late luteal phases and was similar in CL during the mid-luteal phase. The differences in the abundance of proteins between individual CL are probably hormonally controlled by steroid hormones secreted by luteal cells, the concentrations of which change during the luteal phases. Immediately after ovulation, P4 secretion is relatively less and is greater in CL2 compared to

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secretion during CL regression. There are similar fluctuations in testosterone (T) concentrations

as those of P4 during the luteal phases. The aromatase inhibitory actions of P4 prevent the

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conversion of T to estradiol (E2), therefore, the greatest amounts of E2 occur when P4 concentrations are least (Gregoraszczuk, 1992). The greatest secretion of P4 and expression of the APJ gene during the mid-luteal phase of the estrous cycle indicates P4 can affect APJ

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regulation via an autocrine pathway. This action of P4 is further confirmed by the results of

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Shimizu et al. (2009) where it was observed that there was an increase in relative abundance of

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APJ mRNA in ovarian granulosal cells of cattle in vitro after P4 administration. Published data

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also indicate that relative abundance of apelin mRNA is regulated by other factors, such as

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luteinizing hormone (LH), insulin-like growth factor 1 (IGF1) or insulin (Boucher et al., 2005; Wei et al., 2005; Shimizu et al., 2009; Roche et al., 2016). Moreover, in a previous study

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gonadotropins and steroids hormones increased the relative abundance of resistin in ovarian follicles of pigs (Rak et al. 2015). Results from this previous study are consistent with those of

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other studies where it was reported that, during the luteal phase in cattle, relative abundance of mRNA for apelin/APJ increased from the early to late luteal phase of the estrous cycle, followed

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by a decrease in regressing CL (Shirasuna et al., 2008; Schilffarth et al., 2009). In addition, relative abundance of apelin/APJ mRNA changed during pregnancy in the CL of cattle, gradually increasing between 1 and 7 months of pregnancy and then there was a marked decrease during the eighth month. In contrast, the relative abundance of APJ mRNA in CL during pregnancy is relatively constant and similar to that during the mid-leuteal phase of the

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estrous cycle (Schiffarth et al. 2009). Immunohistochemical analysis resulted in the finding that apelin staining was exclusively present in the cytoplasm of both small and large luteal cells with the greatest intensity staining in CL2. In CL3, apelin was present in a few luteal cells of both types. The APJ was localized in both the cytoplasm and membranes of small and large luteal cells. In CL1, there was faint staining of only a few small luteal cells and for cytoplasmic

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APJ immunostaining. In CL2,the APJ was located in the cell membrane of large luteal cells

while it was located in the cytoplasm of small luteal cells. In the large luteal cells, the staining

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was of moderate intensity. In luteal cells of CL3, the membrane and cytoplasm had moderate

APJ immunostaining. Additionally, there was intense APJ staining in the epithelium of blood vessels of CL2 and CL3. Previously published data by Shirasuna et al. (2008) provided evidence

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that apelin and APJ was present in luteal arteries, indicating the involvement of apelin in CL

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angiogenesis. In the present study, there were greater amounts of APJ in mid-luteal phase CL,

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when blood vessel growth is occurring, and this indirectly confirms that apelin is involved in

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angiogenesis of CL.

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Overall, the expression of both components (ligand and receptor) of the apelin signaling system allows for analysis of the direct role of apelin in CL function, and specifically P4

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production. Although the CL secretes many different hormones, P4 is of predominant

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importance because it is necessary for transforming the endometrium to a state receptivity for blastocyst implantation and for maintaining an early pregnancy. After ovulation, differentiation of follicular cells into luteal cells capable of producing P4, is accomplished by the increased

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activity of enzymes necessary for the conversion of cholesterol to P4. 3β-HSD converts 5-ene3β–hydroxysteroids to the 4-ene-3-oxo configuration and, therefore, has an essential role in the biosynthesis of hormonally active steroids, such as P4 (Strauss and Miller, 1991). Any dysfunction of the CL can result in failure of embryo implantation or early abortion. In the present study, the first in vitro evidence was obtained for the direct stimulatory effect of apelin 14

on luteal P4 synthesis in pigs, involving an increase in 3β-HSD activity. Previously published data demonstrated effects of apelin in male (Sandal et al., 2015) and female (Roche et al., 2016; Rak et al., 2017) steroid production. For example, chronic central infusion of apelin-13 resulted in a decreased concentration of testosterone in male rats (Sandal et al., 2015). The findings of the present study are, however, consistent with those from other research where it was

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documented in in vitro experiments with cattle (Roche et al., 2016) and human (Roche et al.,

2017) granulosal cells, as well as pig ovarian follicles cells (Rak et al., 2017) that apelin

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stimulated P4 secretion. The mechanism of apelin action on P4 secretion in the present experiments was demonstrated by using selective blockers of the APJ receptor (ML221) and AMPKα kinase (Compound C). With these approaches, the stimulatory effect of apelin on P4

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secretion was inhibited in response to the both APJ receptor and AMPKα kinase blockers,

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confirming that APJ and AMPKα kinase are involved in the apelin action in CL. The results

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obtained are consistent with results of studies conducted on molecular mechanism of apelin

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action on P4 secretion in human (Roche et al., 2016), pig (Rak et al., 2017) and cattle (Roche

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et al., 2017) ovarian follicular cells. Moreover, the positive effect of apelin on P4 production and the abundance of the 3β-HSD protein in human ovarian cells was dependent on activation

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of the MAPK3/1 kinase pathway as well as Akt (Roche et al., 2016). Based on results of the present experiments which indicated a stimulatory effect of apelin on luteal P4 secretion, it is

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suggested that apelin is another factor which can regulate CL development. Apelin as a hormone functioning through paracrine or autocrine pathways may affect blood vessel

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development and P4 secretion and thus support the maturation and function of the CL of pigs. In conclusion, the present study provides evidence, for the first time, that apelin and APJ

are dynamically expressed during the estrous cycle in the CL of pigs with relative extent of apelin gene expression being similar in CL1 and CL2, then decreasing in CL3, while APJ gene expression was similar in CL1 and CL3 and greater in CL2. There were stimulatory effects of 15

apelin on P4 secretion, 3β-HSD activity and abundance of protein involved APJ and AMPKα activation. In summary, the presence of apelin/APJ in the CL of pigs and the direct stimulatory effects of apelin on luteal P4 secretion and abundance of 3β-HSD suggest potential auto/paracrine regulation by apelin in the luteal phase of the estrous cycle.

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Declaration of interest

Acknowledgements This

research

was

supported

by

Jagiellonian

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The authors declare no conflict of interest regarding the publication of this article.

University

in

Krakow:

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DSC/MND/WBiNoZ/IZ/5/2015, K/ZDS/006310 and by Ministry of Science and Higher

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Education for the PHC project under the bilateral Polish-France Agreement "POLONIUM"

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(2016–2017) between Agnieszka Rak and Joelle Dupont.

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Figure legends Fig. 1. Relative abundances of apelin (A) mRNA, and abundance of apelin receptor (APJ) (B) and protein (46 kDa) (C) and APJ (42 kDa) (D) in different stages of corpus luteum (CL) development. CL were collected during early (CL1: 1–2 d after ovulation, n=6), middle (CL2:

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7–10 d after ovulation, n=6) and late luteal phase (CL3: 13–15 d after ovulation, n=6). The data are plotted as the mean ± SEM. Different letters indicate differences between the groups (P < 0.05).

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Fig. 2. Representative micrographs of apelin (A-C) and apelin receptor (APJ; D-F) immunostaining in the corpora lutea (CL) of pigs. CL were obtained from the early (CL1: 1–2

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d after ovulation; A and D), middle (CL2: 7–10 d after ovulation; B and E) and late luteal phase

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(CL3: 13–15 d after ovulation; C and F) of the estrous cycle. Immunostaining with DAB and

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counterstaining with hematoxylin. Bar = 50 µm, n = 5/each group. Apelin (A-C) is present

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exclusively in cytoplasm while APJ (D-F) in cytoplasm and membrane of large (LC) and small (SC) luteal cells of CL1-CL3. (E, F) APJ staining in epithelium of blood vessels (arrowheads).

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No positive stating is visible when primary antibodies are omitted (C, F, insets). Fig. 3. Effect of apelin on luteal cell progesterone secretion. (A) Mature corpus luteum (CL)

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cells were cultured with apelin (0.02, 0.2, 2 or 20 ng/mL). After incubation for 24 h, the medium

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was collected and hormone concentration was determined. (B) Mature CL cells were pretreated for 1 h with the AMPKα antagonist, Compound C (10 µM) or APJ antagonist, ML221 (10 µM), after which apelin at 2 ng/mL (AP2) was added. After incubation for 48 h, the medium was

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collected and analyzed for progesterone content. At least three different experiments (n = 3), each in quadruplicate, were performed. All data are expressed as the mean ± SEM. Significance between control and apelin treatments is indicated by *P < 0.05. Different letters indicate differences between groups (P < 0.05).

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Fig. 4. Effect of apelin on abundance of luteal 3β-HSD (42 kDa) protein and activity. (A) Mature corpus luteum cells were cultured with apelin (0.02, 0.2, 2 or 20 ng/mL). After incubation for 24 h, the cells were collected and abundance of 3β-HSD protein was determined. (B, C) To assess 3β-HSD activity, mature luteal cells in M199 containing 5% FBS were incubated for 24 h with apelin at 0.02, 0.2, 2 or 20 ng/mL, with or without 5 µg/mL

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pregnenolone (P5). After incubation for 24 h, the medium was collected for measurement of P4

concentration, while cells were analyzed for abundance of 3β-HSD protein. At least three

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different experiments (n = 3), each in quadruplicate, were performed. All data are expressed as

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the mean ± SEM. Different letters indicate differences between groups (P < 0.05).

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