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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Reproductive Biology and Endocrinology 1/2012

Involvement of Src family of kinases and cAMP phosphodiesterase in the luteinizing hormone/chorionic gonadotropin receptor-mediated signaling in the corpus luteum of monkey

Zeitschrift:
Reproductive Biology and Endocrinology > Ausgabe 1/2012
Autoren:
Shah B Kunal, Asaithambi Killivalavan, Rudraiah Medhamurthy
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1477-7827-10-25) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

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.

Abstract

Background

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.

Methods

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.

Results and discussion

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.

Conclusions

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.
Zusatzmaterial
Additional file 1: Table S1: List of primers employed for semi-quantitative RT-PCR analysis. (PDF 19 KB)
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Additional file 2: Table S2: List of primers employed for qPCR analysis. (PDF 10 KB)
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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)
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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)
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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)
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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)
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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)
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Authors’ original file for figure 1
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Authors’ original file for figure 5
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Authors’ original file for figure 6
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Authors’ original file for figure 7
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Authors’ original file for figure 8
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