Theriogenology 81 (2014) 1139–1147

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Immunolocalization of GnRHRI, gonadotropin receptors, PGR, and PGRMCI during follicular development in the rabbit ovary R.X. Lan a, F. Liu a, Z.B. He a, C. Chen a, S.J. Liu a, Y. Shi a, Y.L. Liu b, Y. Yoshimura c, M. Zhang a, *,1 a

College of Animal Science & Technology, Sichuan Agricultural University, Ya’an, Sichuan 625014, China Chengdu Research Base of Giant Panda Breeding, Chengdu 610081, China c Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 October 2013 Received in revised form 10 January 2014 Accepted 31 January 2014

The aim of this study was to investigate the presence and localization of gonadotropinreleasing hormone receptor-I (GnRHRI), gonadotropin receptors (FSHR, LHR), progesterone receptor (PGR), and progesterone receptor membrane-binding component-I (PGRMCI) in the different developmental stages of the rabbit follicle. The ovaries were collected from four healthy New Zealand white rabbits, and the mRNA expression and protein levels of GnRHRI, FSHR, LHR, PGR, and PGRMCI were examined with real-time PCR and immunohistochemistry. The results showed that GnRHRI, FSHR, LHR, PGR, and PGRMCI mRNA was expressed in the ovary; furthermore, we show cell-type specific and follicular development stage-specific expression of these receptors at the protein level. Specifically, all of the receptors were detected in the oocytes from the primordial to the tertiary follicles and in the granulosa and theca cells from the secondary and tertiary follicles. In the mature follicles, all receptors were primarily localized in the granulosa and theca cells. In addition, LHR was also localized in the granulosa cells from the primordial and primary follicles. With follicular development, the expression level of all of the receptors, except GnRHRI, in the follicles showed a tendency to decrease because the area of the follicle increased sharply. The expression level of GnRHRI, FSHR, and PGR in the granulosa and theca cells showed an increasing trend with ongoing follicular development. Interestingly, the expression level of FSHR in the oocytes obviously decreased from the primary to the tertiary follicles, whereas LHR in the oocytes increased from the secondary to tertiary follicles. In conclusion, the expression of GnRHRI, the gonadotropin receptors, PGR, and PGRMCI decreased from the preantral follicles (primordial, primary, and secondary follicles) to the tertiary follicles. The expression of GnRHRI and LHR in the oocytes increased from the secondary to the tertiary follicles, whereas FSHR decreased from the primary to the tertiary follicles. The expression of GnRHRI and PGR in the granulosa and theca cells increased from the secondary to the mature follicles. These observations suggest that these receptors play roles in follicular development and participate in the regulation of follicular development. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: GnRHRI Gonadotropin receptors PGR PGRMCI Rabbit Follicular development

* Corresponding author. Tel.: 0086-835-2885345; fax: 0086-8352886080. E-mail address: [email protected] (M. Zhang). 1 Present address: College of Animal Science & Technology, Sichuan Agricultural University, No 46, Xinkang St., Ya’an, Sichuan Province, 625014, China. 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.01.043

1. Introduction Follicular development, ovulation, and luteinization are complex processes that are regulated by endocrine hormones

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such as the GnRH from the hypothalamus and the pituitary gonadotropins (FSH and LH) [1]. It is well known that GnRH is involved in the regulation of ovarian development through paracrine/autocrine pathways [2]. GnRH is released in a pulsatile manner and regulates the biosynthesis and secretion of gonadotropins from the pituitary [3]. In addition to the hypothalamus and pituitary gland, GnRH and its receptor (gonadotropin-releasing hormone receptor-I (GnRHRI)) have also been shown to be expressed in extra-pituitary tissues including the ovary [2]. Indeed, in the rabbit ovary, evidence has indicated that there are specific GnRHRI binding sites in oocytes, granulosa cells, thecal cells, ovarian surface epithelial cells, and in the CLs [4]. However, nothing is known about its expression in specific follicular cell types during development. The gonadotropins FSH and LH play essential roles in follicular growth and steroid function [5]. The development and maturation of the follicles are dependent on the successive actions of FSH and LH, which are mediated by their receptors FSHR and LHR, respectively [6]. The expression of the FSHR and LHR proteins in the rabbit CLs has been reported [4]. Similarly, the expression of FSHR was detected in granulosa cells [7,8], theca cells [7] and oocytes [9–11] in other species, and LHR mRNA was localized in the theca cells, large follicles, CLs, and the surface epithelial cells of the mouse ovary [12]. Moreover, the FSHR and LHR transcripts have been detected in neonatal mouse ovary from D3 to D15 [13] and in preimplantation embryos [11]. These observations suggest that gonadotropin receptors are expressed in the ovary very early during the formation of the gonads and in early folliculogenesis during the neonatal period. However, in the rabbit, the expression and location of FSHR and LHR in the ovarian follicles has not yet been described. The interaction of FSH and LH stimulates the production of sexual steroid hormones by their receptors, such as estradiol and progesterone (P4) [14]. Progesterone is a steroid hormone that is known to influence follicular growth, ovulation, and luteinization [15]. Progesterone also acts directly on granulosa cells to inhibit apoptosis [16]. It is generally accepted that the effects of P4 are mediated by its interaction with the genomic progesterone receptor (PGR). Progesterone receptor is a member of the nuclear receptor superfamily of transcription factors and consists mainly of two different isoforms, PGR-A and PGR-B, both encoded by a single gene by transcription from alternative promoters [17]. A study in the monkey ovary has indicated that the PGR was detected in the granulosa cells of the primordial and primary follicles [18], and a study in the human ovary has shown that the PGR was detected in the granulosa cells of the primordial, preantral, and antral follicles; in the theca cells of the preantral and antral follicles; and in the CLs [19]. However, studies in the rat [20,21] have conclusively demonstrated that the PGR is not expressed in the granulosa cells of the developing follicles and luteal cells. A recent study in the mouse has shown PGR localization in the theca and in the granulosa cells of the mature follicles as well as the CLs. There are conflicting results in different species on the expression of the PGR in the ovary; therefore, it is essential to examine the expression of the PGR in the rabbit ovary. Another protein that could mediate the action of P4 is progesterone receptor membrane component I (PGRMCI) [22]. PGRMCI was first isolated from the porcine liver [23]

and is a relatively small protein (28 kDa) that possesses a short N-terminal extracellular domain, a single transmembrane domain, and a cytoplasmic domain [24]. The finding that PGRMC1 participates in the action of P4 in the ovary is supported by the observations that PGRMCI has been detected in the mouse granulosa [25] and luteal cells [26,27] and in bovine [28], rat [22], and human oocytes [29]. Furthermore, an antibody to PGRMCI completely attenuates the anti-apoptotic action of P4 [22]. Although these studies provide strong evidence that PGRMCI mediates the action of P4 in the ovary, nothing is known about its location and expression in the specific follicular cell types during follicular development. However, to the best of our knowledge, information on the expression pattern of the reproductive hormone receptors in the developing follicles of the rabbit is still limited. Notably, reports that have examined whether PGRMCI is expressed in the rabbit follicle and changes in its expression profile during follicular development are scarce. The aim of this study was to investigate the spatiotemporal expression of GnRHRI, FSHR, LHR, PGR, and PGRMCI at the mRNA and protein levels during rabbit follicular development. 2. Materials and methods 2.1. Animals and tissue collection Four mature female New Zealand white rabbits (3.18  0.40 kg body weight and 5–6 months of age) were housed individually in cages under controlled light (12 hour light:12 hour darkness) and temperature (18  C–24  C) conditions and provided with free access to water and food. The protocols for animal use for these experiments were approved by the Institutional Animal Care and Use Committee of the Sichuan Agricultural University. After being treated with a suitable dose of an anesthetic (containing sodium pentobarbital and procaine), the left-side ovaries were carefully collected and rinsed with RNase-free PBS and then frozen at 80  C for total mRNA extraction; the right-side ovary was fixed in 4% formaldehyde (vol/vol) for 72 hours at room temperature and subsequently dehydrated through a graded ethanol series and then cleared with xylene and finally embedded in paraffin for histological and immunohistochemical examination. 2.2. Antibodies The primary antibodies were a goat polyclonal antihuman GnRHR (sc-8681), goat polyclonal anti-human FSHR (sc-7798), goat polyclonal anti-human LHR (sc26341), mouse monoclonal anti-human PR (sc-810), and mouse monoclonal anti-human PGRMCI (sc-271275), all of which were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A biotin-conjugated universal goat/ mouse-IgG secondary antibody kit (SA2003) containing normal rabbit serum, a rabbit anti-goat/mouse secondary antibody, and a streptavidin–biotin complex (SABC) was used for the goat primary antibodies. A biotin-conjugated universal mouse-IgG secondary antibody kit (SA2001) containing normal goat serum, a goat anti-mouse secondary antibody, and the SABC was used for the primary

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from 65  C to 95  C with 0.5  C increments, during which the fluorescence data were collected to evaluate the specific amplification. The relative differences for a gene in the different groups were determined using the comparative OO cycle threshold ( CT) method [30]. The resulting values were converted to fold changes compared with the GAPDH OO OOCT signal by raising two to the CT power (2). To confirm the product’s specificity, each PCR product was analyzed by 1.5% agarose gel electrophoresis followed by sequencing to confirm that these were the correct DNA fragments.

antibodies from mouse. Both kits were purchased from Wuhan Boster (Wuhan, China). 2.3. Real-time PCR Total RNA was extracted from the ovaries with Trizol according to the manufacturer’s instructions (TaKaRa, Dalian, China). The collected supernatant (400 mL) was precipitated with 400 mL of isopropanol, followed by washing with precooled 75% ethanol. The precipitated total RNA samples were dried and then dissolved in 1 mL RNase-free dH2O. The amount and purity of the total RNA were quantified by measuring the optical density at 260 and 280 nm; the RNA integrity was checked by 1.5% agarose gel electrophoresis. The cDNA synthesis was performed using a PrimerScript RT reagent kit with gDNA Eraser (TaKaRa) according to the manufacturer’s instructions. First, the genomic DNA elimination reaction was performed. The 10-mL reaction solution contained 2 mL 5 gDNA Eraser Buffer, 1 mL gDNA Eraser,1 mg total RNA, and 4 mL RNase-free dH2O. The reaction proceeded at room temperature for 5 minutes. Then, 4 mL 5 PrimerScript buffer, 1 mL 5 PrimerScript RT enzyme mix, 1 mL RT primer mix, and 4 mL RNase-free dH2O were added to the solution. The reaction was performed in a programmable thermal controller PTC-100 (MJ Research Inc.) and incubated at 37  C for 15 minutes, followed by heat inactivation at 85  C for 5 seconds. Real-time PCR analysis for GnRHRI, FSHR, LHR, PGR, and PGRMCI expression in the ovary was performed using a CFX-96 (Bio-Rad). The 25-mL real-time PCR solution mixture contained 12.5 mL SYBR premix, 1 mL sense and antisense primers (stock concentration 10 mmol/L), 8.5 mL water, and 2 mL cDNA. The primers used are listed in Table 1. The rabbit GAPDH and PGR and the Homo sapiens PGRMCI mRNA sequences were obtained from the GenBank of the National Center for Biotechnology Information of the National Institutes of Health (http://www.ncbi.nlm.nih.gov/ cgi-bin/genbank). The specific primers were designed using Primer Premier 5.0 software, and the GnRHRI primer was as previously described [4]. The annealing temperature for each primer set is given in Table 1. The real-time PCR was performed with an initial incubation at 95  C for 30 seconds, followed by 40 cycles at 95  C for 5 seconds, and then annealing for 30 seconds; during this period, the real-time fluorescence data were collected. A melting-curve protocol was performed by repeating the 95  C heating for 10 seconds,

2.4. Histology Serial tissue sections were cut at 4 mm thickness and mounted on Fisherbrand Superfrost Plus Microscope Slides (Fisher Scientific, USA). The sections were deparaffinized, rehydrated, and rinsed in distilled water. Finally, they were stained with hematoxylin for 5 minutes and then eosin for 40 seconds, dehydrated, and mounted. For each ovary sample, we made five slides, and each slide had six tissue sections mounted on it. The diameter of the follicles and oocytes and the thickness of the zona pellucida (ZP) from the different developmental stages of the follicles were measured using a Nikon-90i microscope (Nikon, Japan) linked to a computerbased image analyzer (ACT-2U software; Nikon, Japan). The primordial, primary, secondary, tertiary, and mature follicles were defined according to a previous reference [31]. 2.5. Immunohistochemistry These sections were subjected to antigen retrieval at 120  C for 5 minutes in a citrate buffer solution (10 mmol/L, pH 6) after being deparaffinized and rehydrated. To block the endogenous peroxidase activity, the sections were dipped in 3% H2O2 (vol/vol) in methanol for 10 minutes and then incubated with the corresponding serum for 20 minutes. Subsequently, the sections were incubated with either a goat polyclonal anti-GNRHRI primary antibody, goat polyclonal anti-FSHR primary antibody, goat polyclonal anti-LHR primary antibody, mouse monoclonal antiPR primary antibody, or a mouse monoclonal anti-PGRMCI antibody at 4  C overnight. The primary antibodies were diluted 1:200 in PBS. These sections were then incubated with the corresponding secondary antibodies (diluted

Table 1 Primers used for real-time PCR. Gene name

Sequence (50 –30 )

Product length (bp)

Annealing temperature ( C)

Accession number

GAPDH

F: TGTTTGTGATGGGCGTGAA R: CCTCCACAATGCCGAAGT F: TGATCCACCTCA CAA ATG GA R: ATGAAGGACCCGTGTCAGAG F: GAGGAATGCCATTGA ACTGAGG R: AAGGTTGGAGAACACATCTG F: CTG GAG AAG ATG CAC AAT GG R: AATTAGCCTCTGAATGGACTC F: GCTGCCCCCGCTACCAAAGG R: TCCCCTGGGCAGCACCTTGT F: GCCCAACCTTTACTCCA R: TCGTCGTCGTCGCTGTC

150

57.4

NC-013676.1

191

61.3

AY781779

150

57

AY429104.1

118

57

S57793

108

61

NM-001082267

205

57

NC-013676.1

GnRHRI FSHR LHR PGR PGRMCI

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1:100 in PBS) and the SABC for 1 hour each at room temperature. The immunoreaction products were visualized using a 3,30 -diaminobenzidine (DAB) kit (Wuhan Boster, China). The sections were counterstained with hematoxylin, sequentially dehydrated and mounted. To confirm the specificity of the immunostaining, the primary antibody was replaced with PBS (Fig. 2A to E). The sections were examined under a light microscope (Olympus BX-51, Japan) with image analysis software (NIS Element, Nikon, Tokyo, Japan). The integrated optical density (IOD) of the positive immunostaining was analyzed in the area of interest (AOI), such as the follicle, oocyte, granulose, and theca cell layer, using Image-Pro plus software, and then the optical density (OD) was calculated as follows: OD ¼ IOD/AOI. Six tissue sections for each ovary from each animal were mounted on a slide, six different eyefields from each section were analyzed for the OD, and the ODs were averaged to obtain a mean optical density (mOD). The mOD value represents the expression density of the specific receptors. 2.6. Statistical analysis The mODs from the different developmental stages of the follicles were compared. The data were analyzed by one-way ANOVA. Duncan’s multiple comparison was used for the detection of significant differences using SAS 9.1 software. Pvalue

Immunolocalization of GnRHRI, gonadotropin receptors, PGR, and PGRMCI during follicular development in the rabbit ovary.

The aim of this study was to investigate the presence and localization of gonadotropin-releasing hormone receptor-I (GnRHRI), gonadotropin receptors (...
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