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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

Leptin (LEP) and leptin receptor (LEP-R) expression was shown to change throughout the luteal phase in several species and may be involved in steroid hormone production. In the bitch, leptin but not LEP-R protein was detected in the non-pregnant corpus luteum (CL). Until now, no further information has been available on their expression levels and role in CL function. Our objective was to compare time-related changes in luteal LEP and LEP-R mRNA levels during the non-pregnant luteal phase, pregnancy and after aglepristone treatment in mid-gestation. CLs were collected by ovariohysterectomy at different time points: day (d) 5, 15, 25, 35, 45, 65 after ovulation (p.o.) in non-pregnant bitches; pre-implantation, post-implantation, mid-gestation, during prepartum luteolysis; 24 and 72 h after aglepristone injection. Non-pregnant LEP expression was lowest on d5 p.o., increased thereafter and fell again on d45 (P ≤ 0.04). LEP-R expression was not altered (P = 0.07). In pregnant bitches, neither LEP nor LEP-R mRNA levels varied over time (P = 0.201 and P = 0.150, respectively). Aglepristone treatment caused substantial downregulation of luteal LEP expression by 72h post-treatment (P ≤ 0.01). However, LEP-R expression did not follow the same course (P = 0.193). Our results indicate that both LEP and LEP-R mRNA are present in the canine CL during the non-pregnant luteal phase and pregnancy. LEP expression changes significantly over time in non-pregnant dogs and after aglepristone administration and thus, it may play a role in luteal steroidogenesis and regression.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

Leptin has been shown to control reproductive processes at the hypothalamic-pituitary level and peripherally in reproductive tissues, for example the ovary, where it is expressed together with its receptors suggesting an autocrine/paracrine effect. In the cyclic bovine corpus luteum, LEP and LEP-R increased significantly by the mid-luteal phase and were low again in the regressing CL. During pregnancy, expression level was comparable to that of mid- and late-luteal phase and did not change afterwards (Sarkar et al. 2010). Leptin may influence steroidogenesis either directly or indirectly by modulating the actions of metabolic hormones, for example GH, insulin, IGF-I. These effects may differ between species, stage of the CL, in vitro culture conditions and hormone dosages used. In a study on dispersed bovine luteal cells, leptin (10 ng/ml) alone had no effect but acted synergistically with IGF-I increasing progesterone (P4) production (Nicklin et al. 2007). There are ample data on the relationship of leptin and adiposity in the dog; however, insufficient information is available on its involvement in reproduction. Saleri et al. (2003) found higher serum levels in female than in male dogs, which also varied by cycle stage showing a significant increase during estrus compared to pro-estrus and diestrus. Leptin was detected by immunohistochemistry in the non-pregnant canine ovary with abundance in luteinized granulosa cells and in the CL, but the phase of the CL was not specified. Luteal LEP-R was not found (Sorace et al. 2006). The reproductive physiology of the dog differs from that of many other mammals as the length of the non-pregnant luteal phase exceeds the gestational luteal lifespan, and the CL is the sole source of progesterone. Therefore, regulatory processes and the role of players involved in the formation, maintenance and regression of the CL may be different from other species. Our goal was to characterize changes in luteal LEP and LEP-R gene expressions during the course of the luteal phase and pregnancy and to see whether aglepristone, a progesterone-receptor blocker, administered to mid-gestation bitches induces alterations in the expression levels of LEP and LEP-R.

Materials and Methods

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

Groups of 4-5 healthy bitches (2-8 years, different breeds) were ovariohysterectomized on d5, 15, 25, 35, 45, 65 p.o. (ovulation: P4 = 5 ng/ml) during the non-pregnant diestrus and on pregnancy d8-12 (pre-implantation, n = 5; embryos detected in uterine flushes), d18-25 (post-implantation; n = 5), d35-45 (mid-gestation; n = 5) and during prepartum luteolysis (n = 3; P4 < 3 ng/ml in two consecutive samples). Additionally, mid-gestation dogs were treated with aglepristone (Alizin®; Virbac, Carros Cedex, France; 10 mg/kg BW s.c. twice, 24h apart) and ovariohysterectomized 24 (n = 5) and 72h (n = 5) after the second treatment.

CLs were collected, preserved for RNA extraction, and total RNA was isolated as described previously (Kowalewski et al. 2006). Samples were DNase-treated (RQ1 RNase-free DNase; Promega, Dübendorf, Switzerland) and reverse-transcribed using 100 ng and 200 ng RNA from each CL sample for LEP and LEP-R, respectively. Primers and probes for the semi-quantitative real-time (TaqMan) PCR were designed based on known canine leptin (GenBank accession number NM_001003070) and leptin receptor (long form, GenBank accession number NM_001024634) sequences; LEP (forward): 5′-GGG TCG CTG GTC TGG ACT T-3′, LEP (reverse): 5′-CTG TTG GTA GAT GGC CAA CGT-3′, LEP TaqMan Probe: 5′-TCC TGG GCT CCA ACC AGT CCT GAG T-3′, LEP-R (forward): 5′-CAT TTG CGG AGG GAT GGT T-3′, LEP-R (reverse): 5′-AGC GGT TTC ACC ACG GAA T-3′, LEP-R TaqMan Probe: 5′-TTG ACT CTT CAC CAA CGT GTG TGG TTC C-3′. Efficiency of PCR reactions was close to 100%. Samples were run in duplicates; autoclaved water served as negative control. Canine GAPDH and cyclophyllin A were used as reference genes; details on both TaqMan systems have already been provided by Kowalewski et al. (2011). Reverse transcription and semi-quantitative real-time PCR were performed according to our protocols (Kowalewski et al. 2011).

Only valid data (relative amounts of reference genes in accordance with each other per sample) were considered, and their average amount was used for the calculation of relative gene expression (RGE) based on the ΔΔCt method (Kowalewski et al. 2006). Data were logarithmically transformed to approximate normal distribution (Kolmogorov–Smirnov test, p > 0.05), and parametric one-way anova followed by Bonferroni multiple comparisons were performed. Results are presented as mean ± SE. Aglepristone-treated dogs were evaluated together with mid-gestation bitches who served as non-treated controls. Level of significance was set at p < 0.05. Statistical calculations were carried out with IBM SPSS Statistics for Windows, Version 19.0 (Armonk, NY, USA)

Results

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

Changes in luteal LEP expression during the non-pregnant luteal phase were significant. Lowest mRNA levels were detected on d5 p.o. and were significantly decreased by the end of diestrus compared to the mid-luteal phase (p ≤ 0.04; Fig. 1). LEP-R mRNA did not change through the course of the luteal phase (p = 0.07; Fig. 1). In the pregnant group, neither LEP nor LEP-R RGE showed significant changes over time (p = 0.201 and p = 0.15, respectively; Fig. 2). LEP expression was markedly downregulated by 72h after aglepristone treatment (p ≤ 0.01) without concomitant changes in LEP-R expression (p = 0.193; Fig. 3).

image

Figure 1. Relative leptin and leptin receptor gene expression in corpora lutea obtained from non-pregnant bitches in the luteal phase. Different superscripts within leptin or leptin receptor denote significant differences between days. Bars represent the mean and whiskers the SE. ln RGE: relative gene expression after log-transformation

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image

Figure 2. Relative leptin and leptin receptor gene expression in corpora lutea obtained from pregnant bitches before (d8-12 of pregnancy) and after implantation. Different superscripts within leptin or leptin receptor denote significant differences between days. Bars represent the mean and whiskers the SE. ln RGE: relative gene expression after log-transformation

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image

Figure 3. Relative leptin and leptin receptor gene expression in corpora lutea obtained from pregnant bitches (d35-45 of pregnancy) before (non-treated control) and after aglepristone treatment. Different superscripts within leptin or leptin receptor denote significant differences between days. Bars represent the mean and whiskers the SE. ln RGE: relative gene expression after log-transformation

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Discussion

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

Our study is the first to describe the presence and changes of LEP and LEP-R mRNA during the lifespan of the canine CL. LEP expression was substantially upregulated between d15-35 of the non-pregnant luteal phase. This coincides with the period when generally high serum P4 levels are detected in the bitch. Steroidogenic acute regulatory protein (StAR) mRNA concentration started to increase shortly after ovulation until d25, fell abruptly on d35 and remained low thereafter in non-pregnant dogs (Kowalewski and Hoffmann 2008). We found high LEP mRNA levels when those of StAR, a key regulator of luteal steroidogenesis, in the dog were also increased. This implies that LEP may be involved in the autocrine/paracrine regulation of steroid hormone production within the canine CL. We were not able to show the same trend during pregnancy. This may be due to differences in the physiological role of leptin in pregnancy or to different time points of tissue collection. The abundance of LEP-R may alter local bioavailability of leptin. However, at least at the mRNA level observed in the present study, LEP-R expression did not change significantly during either diestrus or pregnancy despite numerically higher values on d45 and 65 p.o. or at prepartum luteolysis, respectively, compared to the earlier phases (see Figs 1 and 2). Because of the small sample size and large variation between individual animals, we might not have been able to show clear differences, provided they indeed exist, over the course of time. This may warrant further investigation on a larger number of animals. We demonstrated that aglepristone significantly downregulated LEP mRNA levels shortly after its administration. In a previous study (Kowalewski et al. 2009), aglepristone inhibited StAR and 3β-hydroxysteroid dehydrogenase gene expressions and induced luteolysis. Even though the underlying cellular mechanism is not known, this progesterone-receptor blocker may cause luteal regression by suppressing gene expressions of key luteotropic factors. Further studies are needed to elucidate the role of LEP and LEP-R in CL function in the bitch.

Author contributions

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References

O. Balogh contributed to the conception and design of the experiment, data collection and analysis, interpretation of results, manuscript writing and final revision for submission. M.P. Kowalewski and I.M. Reichler contributed equally to the design of the experiment, data analysis and interpretation, manuscript revision and final approval.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Conflict of interest
  9. Author contributions
  10. References
  • Kowalewski M, Hoffmann B, 2008: Molecular cloning and expression of StAR protein in the canine corpus luteum during dioestrus. Exp Clin Endocrinol Diabetes 116, 158161.
  • Kowalewski MP, Schuler G, Taubert A, Engel E, Hoffmann B, 2006: Expression of cyclooxygenase 1 and 2 in the canine corpus luteum during diestrus. Theriogenology 66, 14231430.
  • Kowalewski MP, Beceriklisoy HB, Aslan S, Agaoglu AR, Hoffmann B, 2009: Time related changes in luteal prostaglandin synthesis and steroidogenic capacity during pregnancy, normal and antiprogestin induced luteolysis in the bitch. Anim Reprod Sci 116, 129138.
  • Kowalewski MP, Meyer A, Hoffmann B, Aslan S, Boos A, 2011: Expression and functional implications of Peroxisome Proliferator—Activated Receptor Gamma (PPARγ) in canine reproductive tissues during normal pregnancy and parturition and at antiprogestin induced abortion. Theriogenology 75, 877886.
  • Nicklin LT, Robinson RS, Marsters P, Campbell BK, Mann GE, Hunter MG, 2007: Leptin in the bovine corpus luteum: receptor expression and effects on progesterone production. Mol Reprod Dev 74, 724729.
  • Saleri R, Tirelli M, Grasselli F, Dondi M, Arisi M, 2003: Sexual dimorphism in leptin blood levels in the dog (in Italian). Veterinaria 17, 4751.
  • Sarkar M, Schilffarth S, Schams D, Meyer HHD, Berisha B, 2010: The expression of leptin and its receptor during different physiological stages in the bovine ovary. Mol Reprod Dev 77, 174181.
  • Sorace M, Tripodi L, Tripodi A, Groppetti D, Cremonesi F, 2006: Leptin: pharmacological aspects in gynecology. Clin Exp Obstet Gynecol 33, 113116.