- Research
- Open Access
The effect of progesterone replacement on gene expression in the corpus luteum during induced regression and late luteal phase in the bonnet monkey (Macaca radiata)
- Padmanaban S Suresh1,
- Kadthur C Jayachandra1 and
- Rudraiah Medhamurthy1Email author
https://doi.org/10.1186/1477-7827-9-20
© Suresh et al; licensee BioMed Central Ltd. 2011
- Received: 21 September 2010
- Accepted: 3 February 2011
- Published: 3 February 2011
Abstract
Background
In higher primates, although LH/CG play a critical role in the control of corpus luteum (CL) function, the direct effects of progesterone (P4) in the maintenance of CL structure and function are unclear. Several experiments were conducted in the bonnet monkey to examine direct effects of P4 on gene expression changes in the CL, during induced luteolysis and the late luteal phase of natural cycles.
Methods
To identify differentially expressed genes encoding PR, PR binding factors, cofactors and PR downstream signaling target genes, the genome-wide analysis data generated in CL of monkeys after LH/P4 depletion and LH replacement were mined and validated by real-time RT-PCR analysis. Initially, expression of these P4 related genes were determined in CL during different stages of luteal phase. The recently reported model system of induced luteolysis, yet capable of responsive to tropic support, afforded an ideal situation to examine direct effects of P4 on structure and function of CL. For this purpose, P4 was infused via ALZET pumps into monkeys 24 h after LH/P4 depletion to maintain mid luteal phase circulating P4 concentration (P4 replacement). In another experiment, exogenous P4 was supplemented during late luteal phase to mimic early pregnancy.
Results
Based on the published microarray data, 45 genes were identified to be commonly regulated by LH and P4. From these 19 genes belonging to PR signaling were selected to determine their expression in LH/P4 depletion and P4 replacement experiments. These 19 genes when analyzed revealed 8 genes to be directly responsive to P4, whereas the other genes to be regulated by both LH and P4. Progesterone supplementation for 24 h during the late luteal phase also showed changes in expression of 17 out of 19 genes examined.
Conclusion
These results taken together suggest that P4 regulates, directly or indirectly, expression of a number of genes involved in the CL structure and function.
Keywords
- Progesterone Receptor
- Luteal Phase
- Corpus Luteum
- Late Luteal Phase
- Bonnet Monkey
Background
In mammals, the secretion of progesterone (P4) by corpus luteum (CL) is absolutely essential for establishment and, in some species, maintenance of pregnancy. In higher primates, LH and chorionic gonadotropin (CG) have been suggested to be the principal trophic factors responsible for P4 secretion in the CL [1]. Whether P4 plays a role in the maintenance of structure and function of CL has not been fully elucidated in higher primates. Rothchild postulated that P4 is the primary stimulus of its own secretion and that intraluteal P4, among other effects such as control of structural integrity and steroidogenic capacity, is responsible for regulation of production of luteolysin, the prostaglandin (PG) F2α, within the CL[2, 3]. More recent studies have provided several lines of evidence, some of them with mechanistic insights, in support of the direct effects of P4 on CL. Expression of progesterone receptor (PR) isoforms in CL have been reported in several mammalian species [4–6]. Several studies have suggested that by way of its proliferative and anti-apoptotic actions, P4 functions as survival factor of CL in human [7], rat [8–10], and cattle [11, 12]. Like other steroid nuclear receptors, PR utilizes a plethora of cofactors termed coactivators or corepressors to regulate gene expression [13]. The PR cofactors identified to date include coactivators like (SRC1-3, CBP/p300 and NCOA1-3) [13] and corepressors like NCOR1-2 involved in modulation of PR activity in vivo [14, 15].
We have recently standardized a GnRH R antagonist-induced luteolysis model system in the monkey in which induced luteolysis could be reversed by exogenous LH administration [16]. Employing this model system, microarray analysis of differentially expressed genes in CL tissue during induced luteolysis and LH replacement following induced luteolysis has been determined [16]. The GnRH R antagonist-induced regressed CL with ablated LH action, yet capable of responding to LH replacement, affords an ideal situation to examine direct effects of P4 on CL structure and function. Extensive tissue remodeling with breakdown and renewal of extracellular matrix (ECM) that occurs during spontaneous luteolysis requires participation of matrix metalloproteinases (MMPs) and their tissue inhibitors, TIMPs [17–19]. It has been reported that decrease in P4 levels during late luteal phase of the non fertile cycle is associated with changes in expression of ECM regulators [20]. It remains to be determined whether P4 regulates expression of tissue proteinases, especially following conception. The purpose of this study was to examine effects of P4 action on gene expression changes and function of CL. Experiments were carried out to determine expression of genes in CL tissue that encode different elements of PR complex and few downstream targets of PR activation throughout the luteal phase, after LH/P4 depletion, after P4 replacement and after P4 supplementation during the late luteal phase.
Methods
Reagents
Oligonucleotide primers were synthesized by Sigma-Genosys, Bangalore, India. DyNAzyme™ II DNA polymerase (F-501L) was purchased from Finnzymes, Espoo, Finland. Moloney murine leukemia virus (MMuLV) reverse transcriptase (RT) and 100 bp DNA ladder were obtained from MBI Fermentas GmbH (St. Leon-Rot, Germany). Power SYBR® Green PCR master mix was obtained from Applied Biosystems, Foster City, CA, USA. GnRH R antagonist [Cetrorelix®; (CET)] was a kind gift from Asta Medica, Frankfurt, Germany. ALZET® Osmotic pump Model 2ML1 (infusion rate 10 μl/h) was obtained from Alza Corporation, Palo Alto, CA. Antibodies specific to phospho-p38 (9211), phospho-p42/44 MAPK (9101), p38 MAPK (9212), p42/44 MAPK (9102), pMKK3/6 (9231), p38 MAPK assay kit (9820), MMP-9 (G657) and Phototype-HRP Western detection system with horseradish peroxidase-linked anti-rabbit IgG (7071) were purchased from Cell Signaling Technology, Inc. Danvers, MA. Antibodies specific to PR (sc-538), NCOA1 (sc-8995), NCOA2 (also designated as GRIP1; sc-8996), NCOA3 (sc-25742) were procured from Santa Cruz Biotechnology, Santa Cruz, CA. Crystalline P4 (P0130) and all other reagents were purchased from Sigma Aldrich Corp. St. Louis, MO or sourced locally.
Animal protocols, blood samples and CL collection
Experimental protocols involving monkeys in this study were approved by the Institutional Animal Ethics Committee of the Indian Institute of Science, Bangalore. Adult female bonnet monkeys (Macaca radiata) weighing 3.3-5.1 kg were utilized for the study. The general care and housing of monkeys have been described elsewhere [21]. In this study, one day after occurrence of peak E2 surge was designated as day 1 of the luteal phase, and CL was collected on designated days of the luteal phase and/or after administration of different treatments (see below). To retrieve CL from experimental monkeys, ovaries were accessed by performing laparotomy under aseptic conditions on ketamine hydrochloride (15 mg/kg BW) and/or pentobarbital sodium (8-12 mg/kg BW) anesthetized monkeys. Under sterile conditions, the excised CL was transferred to a petri dish containing filter paper, wiped dry, weighed, cut into 4-5 pieces, placed in individual sterile cryovials, snap-frozen in liquid nitrogen and stored at -70°C until analysis.
Experiment 1: Examination of differentially expressed genes during induced luteolysis and LH replacement experiments
List of expanded forms of gene symbols used
Sl. No. | Gene symbol | Gene name |
---|---|---|
1 | PR | Progesterone receptor |
2 | PGRMC1 | Progesterone receptor membrane component 1 |
3 | FKBP4 | FK506 binding protein 4 |
4 | FKBP5 | FK506 binding protein 5/peptidyl-prolyl cis-trans isomerase FKBP5-like (M. mulatta) |
5 | NCOA1 | Nuclear receptor coactivator 1 |
6 | NCOA2 | Nuclear receptor coactivator 2 |
7 | NCOA3 | Nuclear receptor coactivator 3 |
8 | NCOR1 | Nuclear receptor corepressor 1 |
9 | NCOR2 | Nuclear receptor corepressor 2 |
10 | WNT7A | Wingless-type MMTV integration site family, member 7A |
11 | AREG | Amphiregulin |
12 | BMP5 | Bone morphogenetic protein 5 |
13 | CDH2 | Cadherin 2, type 1, N-cadherin (neuronal)/cadherin-2-like (M. mulatta) |
14 | HOXA1 | Homeobox A1 |
15 | IHH | Indian hedgehog |
16 | ADAMTS1 | A disintegrin-like and metallopeptidase with thrombospondin type 1 motif, 1/ADAM metallopeptidase with thrombospondin type 1 motif, 1 |
17 | MMP9 | Matrix metallopeptidase 9 |
18 | MMP2 | Matrix metallopeptidase 2 |
19 | TIMP3 | Tissue inhibitor of metalloproteinase 3/TIMP metallopeptidase inhibitor 3 (M. mulatta) |
Experiment 2: Expression of various components of PR signaling complex and PR target genes throughout the luteal phase
To study expression of PR, various components of PR signaling complex and some of the downstream target genes of PR signaling, corpora lutea (n = 3 animals/stage of luteal phase) were collected from monkeys at early (day 5), mid (day 8), and late (day 14) stage of the luteal phase of the menstrual cycle as reported previously [27]. Also, CL (n = 3 animals) was collected from monkeys on day 1 of menstrual cycle (d1M), a time point when luteolytic events are manifested. Blood samples were collected from monkeys on the day of CL collection for determination of P4 concentration.
Experiment 3: Effects of P4 replacement on expression of PR, coactivators, corepressors and P4target genes during induced luteolysis
We have recently reported that replacement of LH post LH/P4 depletion leads to brisk and sustained increase in P4 concentration suggesting rescue of CL function following reestablishment of LH levels [16]. In the present experiments, we determined the direct effects of P4 on CL during absence of luteotrophic stimulus. The different components of the PR signaling complex and expression of few genes considered as target of PR activation were examined. In experiment 3.1, with a view to mimic mid luteal phase circulating P4 concentration, exogenous P4 was administered through implantation of P4 filled ALZET pumps to monkeys depleted of LH/P4 (P4 replacement model). For this purpose, pilot experiments were carried out in adult female monkeys to examine the feasibility of providing P4 as continuous infusion by way of implantation of P4 filled ALZET pump. Keeping in mind the limitation on use of organic solvents compatible with the pump, initially 16 mg of P4 was dissolved in 300 μl of ethanol and the solution was made up to 2 ml by propylene glycol and the entire solution was transferred to 2 ML1 ALZET pump. It was determined that three, 32.5 mg of P4 filled pumps that provided infusion of 487.5 μg/30 μl/h, were required to be implanted in anesthetized monkeys to establish circulating P4 concentration higher or in the range of mid luteal phase concentration. In order to determine the time course of P4 secretion immediately before and at different time intervals after injection of CET (LH/P4 depletion), monkeys on day 7 of the luteal phase (n = 3 animals) were administered CET (150 μg/kg BW, s.c.) and blood samples were collected twice daily until onset of menses. In experiment 3.2, three groups of monkeys (n = 3-4 animals/group) were administered 5.25% glucose (vehicle for CET treatment; VEH; group 1), CET [150 μg/kg BW; (LH/P4 depletion); CET; group 2] and CET (150 μg/kg BW) followed 24 h later with implantation of P4 filled three ALZET pumps designed to infuse 487.5 μg of P4/30 μl/h for 24 h [CET+P4; (P4 replacement); group 3]. Blood samples were collected immediately before and at different time intervals throughout the experiment. In group 3, ALZET pumps were removed 24 h later, and further blood samples were collected to monitor circulating P4 levels. Corpora lutea were retrieved from monkeys of all three groups (VEH, CET and CET+P4).
Experiment 4: Effects of P4supplementation on CL function during late luteal phase
An experiment was conducted to gain insight into direct effects of increased P4 during rescue of CL function that occurs during late luteal phase of the fertile menstrual cycle. On day 13 of the luteal phase of non-mated females, three P4 filled ALZET pumps (97.5 mg of P4) were implanted for 24 h, CL (n = 3 animals) was harvested and the pumps were removed 24 h later. Blood samples were collected immediately before and at different time intervals during implantation and after retrieval of CL as well as removal of implants for determining the P4 secretion pattern. For purposes of comparison, CL (n = 3 animals) was harvested from untreated monkeys (untreated control) on day 14 of the luteal phase of the menstrual cycle.
RNA isolation
Total RNA was isolated from CL tissues obtained from different experiments using TRI reagent according to manufacturer's instructions. RNA samples were analyzed using NanoDrop ND-1000 UV-VIS spectrophotometer and samples with A260/A280 values ~1.8 -1.9 were selected for further analyses.
Real-time RT-PCR analysis
List of Primers used for real time RT-PCR analysis
S. No. | Gene name | Primer sequence (5'to 3') | Annealing temp (OC) | Product size (bp) |
---|---|---|---|---|
1 | PR F | GCCACATTCAACACCCACTT | 57.4 | 134 |
PR R | CCTTCAGCTCAGTCATGACG | |||
2 | NCOA1 F | CTGCACGTGGGGGATCAT | 60.4 | 146 |
NCOA1 R | CCTGGCTCATCTGGAGGGT | |||
3 | NCOA2 F | CATGACCTCAGTGACCTCCGT | 60.4 | 116 |
NCOA2 R | CCATTCCAGGCAGCTGGTTT | |||
4 | NCOA3 F | CACCACAGGGCAGATGAGTG | 60.4 | 135 |
NCOA3 R | TGTGGGGGGCTACTCATG | |||
5 | NCOR1 F | GCATCGAGCTGCTGTTATCCC | 62.9 | 145 |
NCOR1 R | TCTCTGCCGCTGCTCCTCC | |||
6 | NCOR2 F | AAGCAGCGAGCGGCTGCCAT | 72.6 | 143 |
NCOR2 R | TGCTGAGGGGCGTCGCTCTC | |||
7 | FKBP4 F | GGGGACCGAGTCTTTGTCCACT | 69.9 | 124 |
FKBP4 R | CCCAAGCCTTGATGACCTCCC | |||
8 | MMP-2 F | GCCACATTCTGGCCTGAGCT | 62.4 | 151 |
MMP-2 R | CCAGGCTGGTCAGTGGCTTG | |||
9 | MMP-9 F | CTGGAGGTTCGACGTGAAG | 55 | 155 |
MMP-9 R | AACTCACGCGCCAGTAGAAG | |||
10 | TIMP3 F | CAAGTACCAGTACCTGCTGACA | 55.4 | 122 |
TIMP3 R | GATAGTTCAGCCCCTTGC | |||
11 | BMP5 F | CCTGAAGAGTCGGAGTACTCAG | 59.2 | 139 |
BMP5 R | GACTCTGGGTGGTCAGAGGA | |||
12 | CDH2 F | CATCCCTCCAATCAACTTGCC | 59.2 | 131 |
CDH2 R | GAGGCTGGTCAGCTCCTG | |||
13 | HOXA1 F | CTTCGCAGGACCAGGTCACTC | 65.1 | 124 |
HOXA1 R | GGTCCGAGGGGTAGGCTCG | |||
14 | IHH F | GCCGCGCGGTGGACATCA | 71.9 | 126 |
IHH R | CGGAGCAATGCACGTGGGCC | |||
15 | FKBP5 F | GGTGAAGCCCAGCTGCTC | 66.7 | 114 |
FKBP5 R | CTGGCACATGGAGATCTGC | |||
16 | PRMC1F | CGCCGCTCAACCTGCTGCT | 69.7 | 139 |
PRMC1 R | GTGAAGTCGCGCCGCTTGAG | |||
17 | ADAMTS 1 F | GCCGACTGGGAAAGCGGA | 66.6 | 120 |
ADAMTS 1 R | CCAGTTCCTGTGGGCTGTCC | |||
18 | WNT7A F | AGCTGGGCTACGTGCTCAAGG | 68.2 | 138 |
WNT7A R | CCAGGTCCGTGTCCATGGG | |||
19 | AREG F | GTTGCCCCAGAGACCGAGACG | 72.6 | 113 |
AREG R | CAGCATAATGGCCTGAGCCGA | |||
20 | L19 F | GCCAACTCCCGTCAGCAGA | 60 | 154 |
L19 R | TGGCTGTACCCTTCCGCTT |
Hormone assays
E2 and P4 concentrations in serum were determined by specific RIA as reported previously [21].
Immunoblotting analysis
Immunoblot analyses and in vitro P38MAPK (P38) assays of CL tissue lysates were carried out as per published procedures. Assays were carried out with equal amount of protein lysates from the luteal tissue of various treatments as reported previously from the laboratory [27].
Statistical analysis
Data were expressed as mean ± SEM. Statistical evaluation of mean differences of serum P4 concentrations and real-time RT-PCR expression among different experimental groups were analyzed by one-way ANOVA, followed by the Newman-Keuls multiple comparison tests (PRISM GraphPad, version 4.0; GraphPad Software Inc., San Diego, CA) and Student's t- test to compare between two groups. A P value of <0.05 was considered statistically significant. The linear regression analysis on microarray and real time RT-PCR data was done as reported earlier [16].
Results
Identification and validation of differentially expressed genes in LH/P4depletion and LH replacement experiments
The experimental details of CL collection from LH/P4 depletion (CET-induced luteolysis) and LH replacement (CET+LH) models and the microarray data comparing these models deposited in NCBI's Gene Expression Ominibus (#GSE7827 and #GSE8371) have been described previously [16]. The published microarray data was mined for P4← responsive genes, and 45 differentially expressed genes were identified based on criteria of >1.5 fold change in both LH/P4 depletion and LH replacement experiments. We narrowed down to the 45 genes by comparing our previous data on LH replacement studies with P4 -responsive genes reported in literature for other tissues [28, 29]. From the 45 genes identified, 19 genes belonging to steroidogenesis and P4 target/regulation related genes (PR, PR binding proteins, cofactors, corepressors and some of the genes considered as downstream targets of PR activation) considered necessary for CL structure and function were selected for further studies. It should be noted that of the 19 genes, expression of only 12 genes was examined during different stages of the luteal phase in experiment 2 and expression of all 19 genes was determined in experiments 3 and 4.
Microarray and real time RT-PCR expression analyses of genes associated with PR signaling/regulation in CL tissue from monkeys of LH/P 4← depletion and LH replacement models. (A) Effects of LH/P4 depletion (CET treatment; n = 5 animals each for VEH and CET treatments) and (B) LH replacement (CET+LH treatments; n = 3 animals each for CET+PBS and CET+LH treatments) on gene expression changes in the CL tissue. Microarray data deposited at GEO with accession #s GSE7827 and GSE8371 were analyzed. Expression of P4 responsive genes identified from the microarray data (i.e. having >1.5 fold cut off over VEH treatment) is expressed as fold expression change (mean ± SEM) and further validated by real time RT-PCR analyses. The real time RT-PCR analysis was determined using ΔΔCt method (see materials and methods). RT-PCR and microarray data are presented mean ± SEM of fold change above control.
Expression of PR cofactors and its downstream target genes throughout the luteal phase
Analysis of expression of genes encoding PR, its cofactors and P 4 target genes in CL during different stages of the luteal phase in monkeys. A) Real time RT-PCR fold change in expression (represented as mean ± SEM; n = 3 animals/stage of the luteal phase) of various genes associated with PR signaling and its target genes. The fold expression change at early stage was set as 1 and fold changes in other stages were expressed in relation to the early stage. Bars with different letters indicate significant (P < 0.05) change in the expression of the individual gene examined. B) Immunoblot analysis of PR, NCOA1, NCOA2 and NCOA3 expression in CL collected during various stages of the luteal phase. Immunoblots were analyzed by densitometry, and the densitometric value of early stage CL for each protein was set as 1 and values at other stages of CL were expressed in relation to the early stage. For NCOA1 protein, the upper band indicated by asterisk was considered for densitometric analysis. The statistical analysis details for densitometric values for each protein are provided in the results section.
Effects of P4replacement on expression of PR, coactivators and corepressors during induced luteolysis
Determination of luteal function after LH/P 4 depletion and P 4 replacement treatments in the monkey. A) Circulating P4 levels before and after LH/P4 depletion [post CET administration (150 μg/kg BW) indicated by arrow; n = 3 animals] on day 7 of the luteal phase. P4 levels are represented only up to 72 h for purposes of comparing the levels with P4 replacement experiment [see panel C below]. Panel B depicts experimental protocol for VEH (5.25% glucose), LH/P4 depletion [CET (150 μg/kg BW)] and P4 replacement (CET+P4). The duration of P4 treatment initiated 24 h post LH/P4 depletion (n = 3 animals) in the form of P4 filled ALZET pumps is indicated by 'in' and 'out' words and arrows. The retrieval of CL after treatments is indicated by asterisk. C) Effects of VEH treatment (n = 3 animals), LH/P4 depletion (CET) (n = 3 animals) and [CET+P4 (n = 3 animals)] on circulating serum P4 levels. Data are presented as mean ± SEM. Before initiation of treatments, P4 values for each time point were pooled and represented as shaded bars (bars with dotted lines represent P4 concentrations after vehicle treatment). Following LH/P4 depletion and P4 replacement, P4 values are shown as open bars and solid bars, respectively. Serum P4 levels at 12 and 24 h after P4 implant withdrawal and CL removal are shown as open bars. Bars with different letters are significantly (P < 0.05) different.
Analysis of expression of genes associated with PR, its cofactors and P 4 target genes in CL of monkeys treated with VEH (5.25% glucose injection at mid luteal phase), CET (LH/P 4 depletion) and CET+P 4 (P 4 replacement). A) Real time RT-PCR expression analysis of various genes associated with PR signaling and its target genes. The fold expression change for VEH group was set as 1 and values for other groups were expressed in relation to the VEH group. For details on fold change calculation for real time RT-PCR analysis, see legend to Figure 1. Bars with different letters are significantly (P < 0.05) different for the individual genes examined. B) Immunoblot analysis of PR, NCOA1, NCOA2 and NCOA3 proteins in CL after LH/P4 depletion and P4 replacement. Immunoblots are analyzed by densitometry and densitometric value for VEH group is set as 1 fold and values for other groups were expressed in relation to the VEH group. For NCOA1 protein, the upper band indicated by asterisk was considered for densitometric analysis.
The fold expression change of ADAMTS-1 mRNA after LH/P4 depletion was lower compared to VEH treatment (P < 0.05) and the expression increased significantly (P < 0.05) following P4 replacement (Figure 4A). Fold change in expression of MMP-9 mRNA increased post LH/P4 depletion, and decreased significantly post P4 replacement (P < 0.05; Figure 4A) indicating indirect/direct regulation of both ADAMTS-1 and MMP-9 mRNAs by P4 at the transcription level. Further, the fold change in expression of other genes like MMP-2 and TIMP3 increased significantly following LH/P4 depletion, but P4 replacement had no effect (P < 0.05; Figure 4A).
Changes in activation levels of MAPKs during LH/P 4 depletion and P 4 replacement treatments. Tissue lysates were prepared from CL of monkeys receiving VEH, CET (LH/P4 depletion) and CET+P4 (P4 replacement) treatments. The immunoblots shown are from one of three independent experiments (CL from one monkey at each time point was used per experiment). Immunoblots for phospho MAPKs and total MAPKs were analyzed by densitometry and the fold change in the protein levels of phospho/total MAPKs compared between treatments are represented numerically. β-actin was used as loading control.
Effects of P4supplementation on gene expression changes during the late luteal phase
Effects of P 4 supplementation on luteal function during the late luteal phase. Panel A, circulating P4 levels during P4 supplementation (n = 3 animals) with exogenous treatment by way of ALZET pumps for 24 h beginning on day 13 of the luteal phase to mimic high P4 levels similar to mid luteal phase. Panel B, real time RT-PCR expression analysis (mean ± SEM) of various genes associated with PR signaling and PR activated target genes are represented. Panel C, immunoblot analysis for PR, NCOA1-3 and MMP-9 proteins late luteal phase CL after P4 supplementation. Monkey term placenta tissue lysate was used as positive control for immunoblot analysis. The blots are analyzed by densitometry and densitometric values for untreated control group were set as 1 and values in P4 supplemented group were expressed in relation to the control group. The immunoblots shown are from one of three independent experiments (CL from one monkey at each time point was used per experiment). β-actin was used as loading control.
Discussion
Together with earlier reports for presence of PR in CL of many other species, the results of expression of PR, various binding proteins, cofactors and P4 regulation of expression of few genes in the present study further confirm P4 actions within the CL. The importance of P4 in the regulation of CL structure and function has been demonstrated in rats. It was shown that P4 administration during postpartum period had an anti apoptotic action in the CL [9]. Although the rat CL does not express nuclear PR, it was suggested that P4 mediates its action by binding to its membrane receptors [30]. In recent years, the membrane receptor-mediated actions of P4 appear to have emerged as an important mechanism for activation of P4 signal transduction pathway. It was reported that rat luteal cells express membrane and progestin binding proteins, progestin membrane receptor (PMR) α, PMR β, PMR γ, membrane component 1 (PRMC1/PGMRC1) and Rda 288 [30]. In a recent study, it was demonstrated that PGMRC1-dependent mechanism appears to promote human granulose/luteal cell survival [31] confirming the novel membrane-bound progestin receptors with defined actions. In the present study, expression of PGRMC1 was demonstrated in the CL and its expression became lower after LH/P4 depletion. This finding was similar to the earlier observation reported previously in the rhesus macaque [6]. Further restoration of PGRMC1 expression was observed after LH replacement in LH/P4 ablated rhesus macaque [6]. In contrast, in the present study PGRMC1 expression was not restored following LH replacement in LH/P4 depleted monkeys. The difference in the findings between the two studies could be attributed to differences in the treatment protocols employed and/or differences in the response to LH replacement observed between the two macaques.
Rothchild [2] aptly described CL as the most unique endocrine gland in the body, since there appears to be large interspecies variations in the mechanisms of regulation of its function. In higher primates, it is well established that the sole trophic stimulus for P4 secretion is the pulsatile secretion of pituitary LH. It is possible that P4 may not have an important role in its own secretion, but it might have an important role in maintaining structural integrity of the CL capable of responding to the luteotrophic stimulus. Hutchison and Zeleznik [32] demonstrated the rescue of CL function in the hypothalamus lesioned monkeys after reestablishment of pulsatile GnRH treatment indicating the resilience of CL tissue to the deprivation of luteotrophic support for long periods. In our studies, it was confirmed that CL was responsive to luteotrophic support for up to 48 h following inhibition of pituitary LH secretion [16]. We utilized LH/P4 depleted and LH replacement model systems that did not require extreme surgical procedures, yet allow examining the CL function during LH/P4 depletion period as well as hormone replacement period. During induced luteolysis, even though LH replacement had profound effects on expression of several genes [16], but this could be the result of both direct as well as indirect effects of LH. In the present study, examination of direct effects of P4 on CL tissue revealed changes in expression of PR and NCOR1, but expression of many other genes involved in PR activation were not significantly affected. Since, the duration of P4 replacement lasted only for 24 h, for the effect to be apparent a longer duration of P4 replacement might be required. Alternatively, perhaps the effects of P4 on expression of genes may be observed only when high intraluteal P4 levels as seen in functional CL is achieved. In the present study, only circulating P4 levels were mimicked but more studies are required to achieve the high intra luteal P4 levels and determine its effect on the luteal expression of genes.
Tissue proteinases and genes associated with tissue remodelling namely, TIMPs, MMPs and ADAMTS-1 have been examined in the CL tissues of higher primates. In the present study, the findings of higher expression of TIMP3 and MMP-9 was observed during latter part of the luteal phase which is in accordance with similar findings reported for CL of the rhesus macaque [18]. It was previously reported that ADAMTS-1 expression was high in early CL but declined thereafter at other stages of the luteal phase in the rhesus macaque [22]. In the present study, however, no discernible pattern of expression of ADAMTS-1 was observed during the luteal phase. It should be pointed out that in the rhesus macaque the highest expression was seen only at day 2 of the luteal phase, while in the present study the day 2 CL was not examined. Treatment with GnRH R antagonists [i.e. LH/P4 depletion models] in both the macaques resulted in decreased expression of ADAMTS-1 at mid luteal phase. Interestingly, LH replacement, but not steroid replacement, prevented the decreased ADAMTS-1 expression seen after GnRH R antagonist treatment in the rhesus macaque, but in the present study both LH (observed only in real time RT-PCR analysis) and P4 replacement treatments restored or increased its expression. The reason for different findings on ADAMTS-1 expression after P4 (in the present study) or progestin (rhesus monkey; [22]) treatments is difficult to explain and perhaps, related to treatment protocol employed in both these studies. Also, the unexpected decrease in ADAMTS-1 expression seen after P4 supplementation at late luteal phase observed in the present study is difficult to explain. Additional experiments are necessary to address the regulation of ADAMTS-1 expression in the CL by LH and P4.
Stouffer and Young, 2004 [19] reported a differential expression of MMP-9 with regulation of its expression to be at different levels involving both transcriptional and translational mechanisms in the CL of the rhesus macaque. In the present study, expression of MMP-9 in LH/P4 depletion and LH replacement studies remained high, but P4 replacement decreased its expression, suggesting MMP-9 expression appear to be regulated by P4. In accordance with our data, regulation of MMP-9 expression by P4 had been reported by other investigations in studies involving human endometrial explants and rabbit cervix [33, 34]. It should be pointed out that MMP-9 expression during spontaneous luteolysis [data not shown] and P4 supplementation during late luteal phases did not show significant change indicating that regulation of MMP-9 expression may be dependent on circulating LH as well as P4 concentrations. The decrease in expression of MMP-2 mRNA post P4 replacement during late luteal phase was similar to the reported decrease in MMP-2 expression in the human CL after treatment with hCG to mimic early pregnancy [20]. It is possible that LH and P4 may synergistically act to regulate MMP-2 mRNA expression during the late luteal phase following P4 replacement as both are present in this model system, however, the molecular mechanisms for this synergistic action needs to be clarified. Decreased expression of pro-MMP-9, pro-MMP-2 and MMP-2 in endometrial cancer cell lines after in vitro administration of medroxy progesterone acetate, an synthetic progesterone preparation, [35] suggests that ECM remodelling could well be controlled by P4 as well as by LH in the CL tissue.
List of genes whose expression identified to be regulated by P4 treatment
Sl. No. | Gene | Changes in levels after | Regulation by P4 | ||
---|---|---|---|---|---|
LH/P4 depletion | P4 replacement | ||||
1 | PR | mRNA | up | down | Y |
Protein (PR B) | up | down | Y | ||
Protein (PR A) | - | - | N | ||
2 | NCOA1 | mRNA | - | up | N |
Protein | up | - | N | ||
3 | NCOA2 | mRNA | - | - | N |
Protein | up | down | Y | ||
4 | NCOA3 | mRNA | - | - | N |
Protein | up | down | Y | ||
5 | PGRMC1 | down | - | N | |
6 | FKBP4 | down | - | N | |
7 | FKBP5 | - | - | N | |
8 | NCOR1 | up | down | Y | |
9 | NCOR2 | up | - | N | |
10 | WNT7A | up | down | Y | |
11 | AREG | down | down | N | |
12 | BMP5 | up | down | Y | |
13 | CDH2 | up | - | N | |
14 | HOXA1 | up | down | Y | |
15 | IHH | up | - | N | |
16 | ADAMTS1 | down | up | Y | |
17 | MMP9 | up | down | Y | |
18 | MMP2 | up | - | N | |
19 | TIMP3 | up | - | N |
In summary, experiments were performed to examine direct effects of P4← on the regulation of expression of genes in the monkey CL. The first experiment comprised of analysis of previously published microarray data to identify differentially expressed genes considered as target of P4 action and validation of some of these genes by real time RT-PCR analysis. In the second experiment, expression of many of the genes identified in the first experiment was examined throughout the luteal phase. In the third experiment, the direct effects of P4← on expression of many of these genes was examined following P4← replacement in monkeys depleted for circulating endogenous LH and P4← by way of inhibition of pituitary gonadotropin secretion. In the fourth experiment, the effects of P4← supplementation on expression of genes during the late luteal phase with declining endogenous P4← levels were examined. This experiment was done to determine whether increased P4← seen during early pregnancy regulate expression of these genes. The results from these studies indicated that P4← appears to regulate expression of many of the genes in the CL.
Conclusions
Experiments were carried out to assess the direct effects of P4 on CL function in monkeys. Expression of some of the genes involved in PR signaling and genes considered as targets of LH and P4 was analyzed in LH/P4 depletion and P4 replacement model as well as P4 supplementation model during the late luteal phase, and the results indicated that expression of number of genes appeared to be regulated directly or indirectly by P4. These results suggest that replacement of P4 during LH/P4 depletion (induced luteolysis model) is suitable for assessing the effects of P4 on CL function. Further, these studies suggest that CL could serve as a target tissue for examining genomic and non genomic actions of P4.
Declarations
Acknowledgements
We are grateful to Dr. Basavanagouda and staff of Primate Research Laboratory for assistance with surgeries and Mr. Kunal BS for help rendered in the preparation of MS. Financial support from Department of Biotechnology, Government of India, to conduct these studies is gratefully acknowledged.
Authors’ Affiliations
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