The online version of this article (doi:10.1186/1477-7827-10-25) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
KBS & RM participated in designing, conducting experiments, analysis of results and preparation of manuscript. AK participated in analysis of data and preparation of manuscript. All authors read and approved the final manuscript.
In higher primates, during non-pregnant cycles, it is indisputable that circulating LH is essential for maintenance of corpus luteum (CL) function. On the other hand, during pregnancy, CL function gets rescued by the LH analogue, chorionic gonadotropin (CG). The molecular mechanisms involved in the control of luteal function during spontaneous luteolysis and rescue processes are not completely understood. Emerging evidence suggests that LH/CGR activation triggers proliferation and transformation of target cells by various signaling molecules as evident from studies demonstrating participation of Src family of tyrosine kinases (SFKs) and MAP kinases in hCG-mediated actions in Leydig cells. Since circulating LH concentration does not vary during luteal regression, it was hypothesized that decreased responsiveness of luteal cells to LH might occur due to changes in LH/CGR expression dynamics, modulation of SFKs or interference with steroid biosynthesis.
Since, maintenance of structure and function of CL is dependent on the presence of functional LH/CGR its expression dynamics as well as mRNA and protein expressions of SFKs were determined throughout the luteal phase. Employing well characterized luteolysis and CL rescue animal models, activities of SFKs, cAMP phosphodiesterase (cAMP-PDE) and expression of SR-B1 (a membrane receptor associated with trafficking of cholesterol ester) were examined. Also, studies were carried out to investigate the mechanisms responsible for decline in progesterone biosynthesis in CL during the latter part of the non-pregnant cycle.
The decreased responsiveness of CL to LH during late luteal phase could not be accounted for by changes in LH/CGR mRNA levels, its transcript variants or protein. Results obtained employing model systems depicting different functional states of CL revealed increased activity of SFKs [pSrc (Y-416)] and PDE as well as decreased expression of SR-B1correlating with initiation of spontaneous luteolysis. However, CG, by virtue of its heroic efforts, perhaps by inhibition of SFKs and PDE activation, prevents CL from undergoing regression during pregnancy.
The results indicated participation of activated Src and increased activity of cAMP-PDE in the control of luteal function in vivo. That the exogenous hCG treatment caused decreased activation of Src and cAMP-PDE activity with increased circulating progesterone might explain the transient CL rescue that occurs during early pregnancy.
Additional file 1: Table S1: List of primers employed for semi-quantitative RT-PCR analysis. (PDF 19 KB)12958_2011_964_MOESM1_ESM.PDF
Additional file 2: Table S2: List of primers employed for qPCR analysis. (PDF 10 KB)12958_2011_964_MOESM2_ESM.PDF
Additional file 3: Figure S1: Schematic representation of LH/CGR gene depicting exons, number of nucleotides in each exon region and various structural domains formed by each group of exons. The arrows indicate the multiple primer sets designed around the alternatively spliced regions to detect various splice variants of LH/CGR by RT-PCR analysis. The positions of forward primers, F1 and F2 on exon 7 and 5, while position of their respective reverse primers, R1 and R2 on exon 11 are represented. The positions of two other primer sets (F3-R3 and F4-R4) spanning the extreme 3' end of exon 11 region and within the exon 9 region are also represented. The details of splice variants of LH/CGR reported in literature and the calculated PCR product size for each of the possible splice variants detected employing multiple primer sets F1-R1 and F2-R2 in the present study are shown. (PDF 32 KB)12958_2011_964_MOESM3_ESM.PDF
Additional file 4: Figure S2: The blast analysis of nucleotide sequences obtained after sequencing the upper and lower bands obtained using multiple primer pair set F2-R2 (exon5-11). Shown here is the multiple sequence alignment of the PCR product sequence [(A) LH/CGR upper band (718 bp) and (B) lower band (532 bp)] compared with Gen Bank database sequence of human and monkey species depicting the sequence identity (*). (PDF 1 MB)12958_2011_964_MOESM4_ESM.PDF
Additional file 5: Figure S3: (A) Schematic representation of FSHR gene depicting exons, various structural domains formed by exons and number of nucleotides in each exon region. The arrows indicate the primer set representing position of forward primers (F1&F2) and reverse primers (R1&R2) designed around the extracellular domain and hinge regions to detect FSHR mRNA transcripts (sizes 680 and 151 bp). (B) Semi-quantitative RT-PCR analysis to determine FSHR mRNA expression in the monkey CL during different stages of the luteal phase (E: early, M: mid, L: late and D1M: day 1 of menses). L-19 mRNA was used as internal control and the relative expression was calculated following densitometric analysis. Each bar represents mean ± SEM values (n = 3 CL/stage). No significant differences (p > 0.05; denoted by the same letter on individual bars) in expression was seen throughout the luteal phase. (C) The qPCR analysis for FSHR mRNA expression in the monkey CL during different stages of the luteal phase. The fold change in mRNA expression at each stage CL compared to early stage is represented in each bar as mean ± SEM values (n = 3 CL/stage). FSHR expression was high at mid luteal phase, but the expressions at late and D1M were low. Individual bars with different letters are significantly different (p < 0.05). (PDF 29 KB)12958_2011_964_MOESM5_ESM.PDF
Additional file 6: Figure S4: Semi-quantitative RT-PCR analysis of PDE4D isoforms [PDE4D3 (A), PDE4D5 (B) and PDE4D6 (C)] was performed in CL tissue collected from monkeys during different stages of the luteal phase. The expression of housekeeping gene, L19, was used as the internal control. Shown here is a representative gel picture of PDE4D isoforms and L-19 PCR amplification products. (PDF 34 KB)12958_2011_964_MOESM6_ESM.PDF
Additional file 7: Figure S5: (A) Tissue cAMP levels in the monkey CL collected during different stages of the luteal phase. Individual bars with different letters indicate statistical significance (p < 0.05). (B) Tissue cAMP levels from animals treated with Vehicle (VEH), CET and CET + LH (LH replacement). Individual bars with different letters indicate statistical significance (p < 0.05). (PDF 15 KB)12958_2011_964_MOESM7_ESM.PDF
Authors’ original file for figure 112958_2011_964_MOESM8_ESM.pdf
Authors’ original file for figure 212958_2011_964_MOESM9_ESM.pdf
Authors’ original file for figure 312958_2011_964_MOESM10_ESM.pdf
Authors’ original file for figure 412958_2011_964_MOESM11_ESM.pdf
Authors’ original file for figure 512958_2011_964_MOESM12_ESM.pdf
Authors’ original file for figure 612958_2011_964_MOESM13_ESM.pdf
Authors’ original file for figure 712958_2011_964_MOESM14_ESM.pdf
Authors’ original file for figure 812958_2011_964_MOESM15_ESM.pdf
Richards JS: New signaling pathways for hormones and cyclic adenosine 3',5'-monophosphate action in endocrine cells. Mol Endocrinol. 2001, 15 (2): 209-218. 10.1210/me.15.2.209. PubMed
Yadav VK, Medhamurthy R: Dynamic changes in mitogen-activated protein kinase (MAPK) activities in the corpus luteum of the bonnet monkey (Macaca radiata) during development, induced luteolysis, and simulated early pregnancy: a role for p38 MAPK in the regulation of luteal function. Endocrinology. 2006, 147 (4): 2018-2027. CrossRefPubMed
Taylor CC, Limback D, Terranova PF: Src tyrosine kinase activity is related to luteinizing hormone responsiveness: genetic manipulations using mouse MA10 Leydig cells. Endocrinology. 1996, 137 (12): 5735-5738. 10.1210/en.137.12.5735. PubMed
Andersen JM, Dietschy JM: Relative importance of high and low density lipoproteins in the regulation of cholesterol synthesis in the adrenal gland, ovary, and testis of the rat. J Biol Chem. 1978, 253 (24): 9024-9032. PubMed
Spady DK, Dietschy JM: Sterol synthesis in vivo in 18 tissues of the squirrel monkey, guinea pig, rabbit, hamster, and rat. J Lipid Res. 1983, 24 (3): 303-315. PubMed
Azhar S, Nomoto A, Leers-Sucheta S, Reaven E: Simultaneous induction of an HDL receptor protein (SR-BI) and the selective uptake of HDL-cholesteryl esters in a physiologically relevant steroidogenic cell model. J Lipid Res. 1998, 39 (8): 1616-1628. PubMed
Connelly MA, Klein SM, Azhar S, Abumrad NA, Williams DL: Comparison of class B scavenger receptors, CD36 and scavenger receptor BI (SR-BI), shows that both receptors mediate high density lipoprotein-cholesteryl ester selective uptake but SR-BI exhibits a unique enhancement of cholesteryl ester uptake. J Biol Chem. 1999, 274 (1): 41-47. 10.1074/jbc.274.1.41. CrossRefPubMed
Priyanka S, Jayaram P, Sridaran R, Medhamurthy R: Genome-wide gene expression analysis reveals a dynamic interplay between luteotropic and luteolytic factors in the regulation of corpus luteum function in the bonnet monkey (Macaca radiata). Endocrinology. 2009, 150 (3): 1473-1484. PubMedCentralCrossRefPubMed
Selvaraj N, Medhamurthy R, Ramachandra SG, Sairam MR, Moudgal NR: Assessment of luteal rescue and desensitization of macaque corpus luteum brought about by human chorionic gonadotrophin and deglycosylated human chorionic gonadotrophin treatment. J Biosci. 1996, 21 (4): 497-510. 10.1007/BF02703214. CrossRef
DNA Data Bank of Japan. [ http://blast.ddbj.nig.ac.jp/]
National center for Biological information. [ http://blast.ncbi.nlm.nih.gov/Blast.cgi]
GenomeNet. [ http://www.genome.jp/tools/clustalw/]
Yamashita S, Nakamura K, Omori Y, Tsunekawa K, Murakami M, Minegishi T: Association of human follitropin (FSH) receptor with splicing variant of human lutropin/choriogonadotropin receptor negatively controls the expression of human FSH receptor. Mol Endocrinol. 2005, 19 (8): 2099-2111. 10.1210/me.2005-0049. CrossRefPubMed
Nishimori K, Dunkel L, Hsueh AJ, Yamoto M, Nakano R: Expression of luteinizing hormone and chorionic gonadotropin receptor messenger ribonucleic acid in human corpora lutea during menstrual cycle and pregnancy. J Clin Endocrinol Metab. 1995, 80 (4): 1444-1448. 10.1210/jc.80.4.1444. PubMed
Cameron JL, Stouffer RL: Gonadotropin receptors of the primate corpus luteum. II. Changes in available luteinizing hormone- and chorionic gonadotropin-binding sites in macaque luteal membranes during the nonfertile menstrual cycle. Endocrinology. 1982, 110 (6): 2068-2073. 10.1210/endo-110-6-2068. CrossRefPubMed
Smith GD, Sawyer HR, Mirando MA, Griswold MD, Sadhu A, Reeves JJ: Steady-state luteinizing hormone receptor messenger ribonucleic acid levels and endothelial cell composition in bovine normal- and short-lived corpora lutea. Biol Reprod. 1996, 55 (4): 902-909. 10.1095/biolreprod55.4.902. CrossRefPubMed
Gromoll J, Schulz A, Borta H, Gudermann T, Teerds KJ, Greschniok A, Nieschlag E, Seif FJ: Homozygous mutation within the conserved Ala-Phe-Asn-Glu-Thr motif of exon 7 of the LH receptor causes male pseudohermaphroditism. Eur J Endocrinol. 2002, 147 (5): 597-608. 10.1530/eje.0.1470597. CrossRefPubMed
Carvalho CR, Carvalheira JB, Lima MH, Zimmerman SF, Caperuto LC, Amanso A, Gasparetti AL, Meneghetti V, Zimmerman LF, Velloso LA, et al: Novel signal transduction pathway for luteinizing hormone and its interaction with insulin: activation of Janus kinase/signal transducer and activator of transcription and phosphoinositol 3-kinase/Akt pathways. Endocrinology. 2003, 144 (2): 638-647. 10.1210/en.2002-220706. CrossRefPubMed
Shiraishi K, Ascoli M: Lutropin/choriogonadotropin stimulate the proliferation of primary cultures of rat Leydig cells through a pathway that involves activation of the extracellularly regulated kinase 1/2 cascade. Endocrinology. 2007, 148 (7): 3214-3225. 10.1210/en.2007-0160. PubMedCentralCrossRefPubMed
Taylor CC, Terranova PF: Lipopolysaccharide inhibits rat ovarian thecal-interstitial cell steroid secretion in vitro. Endocrinology. 1995, 136 (12): 5527-5532. 10.1210/en.136.12.5527. PubMed
Stouffer RL, Ottobre JS, Molskness TA, Zelinski-Wooten MB: The function and regulation of the primate corpus luteum during the fertile menstrual cycle. Prog Clin Biol Res. 1989, 294: 129-142. PubMed
Chandrasekaran A, Toh KY, Low SH, Tay SK, Brenner S, Goh DL: Identification and characterization of novel mouse PDE4D isoforms: molecular cloning, subcellular distribution and detection of isoform-specific intracellular localization signals. Cell Signal. 2008, 20 (1): 139-153. 10.1016/j.cellsig.2007.10.003. CrossRefPubMed
Bolger GB, Erdogan S, Jones RE, Loughney K, Scotland G, Hoffmann R, Wilkinson I, Farrell C, Houslay MD: Characterization of five different proteins produced by alternatively spliced mRNAs from the human cAMP-specific phosphodiesterase PDE4D gene. Biochem J. 1997, 328 (Pt 2): 539-548. PubMedCentralCrossRefPubMed
O'Connell JC, McCallum JF, McPhee I, Wakefield J, Houslay ES, Wishart W, Bolger G, Frame M, Houslay MD: The SH3 domain of Src tyrosyl protein kinase interacts with the N-terminal splice region of the PDE4A cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5). Biochem J. 1996, 318 (Pt 1): 255-261. PubMedCentralCrossRefPubMed
Dodge-Kafka KL, Bauman A, Mayer N, Henson E, Heredia L, Ahn J, McAvoy T, Nairn AC, Kapiloff MS: cAMP-stimulated protein phosphatase 2A activity associated with muscle A kinase-anchoring protein (mAKAP) signaling complexes inhibits the phosphorylation and activity of the cAMP-specific phosphodiesterase PDE4D3. J Biol Chem. 2010, 285 (15): 11078-11086. 10.1074/jbc.M109.034868. PubMedCentralCrossRefPubMed
Reaven E, Nomoto A, Leers-Sucheta S, Temel R, Williams DL, Azhar S: Expression and microvillar localization of scavenger receptor, class B, type I (a high density lipoprotein receptor) in luteinized and hormone-desensitized rat ovarian models. Endocrinology. 1998, 139 (6): 2847-2856. 10.1210/en.139.6.2847. PubMed
Li X, Peegel H, Menon KM: In situ hybridization of high density lipoprotein (scavenger, type 1) receptor messenger ribonucleic acid (mRNA) during folliculogenesis and luteinization: evidence for mRNA expression and induction by human chorionic gonadotropin specifically in cell types that use cholesterol for steroidogenesis. Endocrinology. 1998, 139 (7): 3043-3049. 10.1210/en.139.7.3043. PubMed
Connelly MA: SR-BI-mediated HDL cholesteryl ester delivery in the adrenal gland. Mol Cell Endocrinol. 2009, 300: (1-2):83-88. 10.1016/j.mce.2008.11.023. CrossRef
Reaven E, Zhan L, Nomoto A, Leers-Sucheta S, Azhar S: Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis. J Lipid Res. 2000, 41 (3): 343-356. PubMed
- Involvement of Src family of kinases and cAMP phosphodiesterase in the luteinizing hormone/chorionic gonadotropin receptor-mediated signaling in the corpus luteum of monkey
Shah B Kunal
- BioMed Central
Neu im Fachgebiet Gynäkologie und Geburtshilfe
Meistgelesene Bücher aus dem Fachgebiet
e.Med Kampagnen-Visual, Mail Icon II