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Contents

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

Matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), vascular endothelial growth factor (VEGF)-A, VEGF-A receptor (Flt-1) and KiSS-1 receptor (KiSS-1R) all play a role in trophoblast invasion in a number of mammalian species. However, mRNA expression of factors regulating trophoblast invasion has not been studied in dogs. Abnormal expression of these factors at the end of canine gestation may contribute to placental retention and/or subinvolution of placental sites. Therefore, we sought to determine the relative mRNA expression of these factors in canine chorioallantois tissue at 61 ± 1 day past the LH surge (pre-term; n = 4), following elective c-section at 64 ± 1 day past the LH surge prior to first-stage labour (pre-labour; n = 4) and following natural delivery (parturient; n = 3). Total RNA was isolated, real-time RT-PCR was performed, and relative expression was calculated using the relative quantitation (inline image) method. MMP-9 mRNA expression was significantly higher in pre-term samples compared to pre-labour and parturient samples. The results showed no significant difference between MMP-2, TIMP-2, VEGF-A and Flt-1 mRNA expression among the three groups. KiSS-1R mRNA was not expressed in any tissues studied. Gene expression of MMP-9 may be related to the onset of labour, whereas MMP-2, VEGF-A, Flt-1, TIMP-2 and KiSS-1R mRNA do not appear to play a role at the end of gestation in the dog.


Introduction

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

The completion of parturition involves the delivery of the placenta. Complications arise when the placenta is not properly delivered within a defined time following delivery of the foetus. Two common placental abnormalities in the dog are placental retention and subinvolution of placental sites (SIPs), which involve pathologic trophoblast invasion. Placental retention increases the risk of the bitch developing post-partum uterine infections and metritis (Wallace 1994). In addition, placental retention can be life-threatening if the bitch becomes very ill (e.g. fever, anorexia, depression). The mechanisms behind placental retention remain unknown.

Subinvolution of placental sites (SIPS) is characterized by abnormally deep invasion of the trophoblasts into the myometrium following delivery of the placenta (Al-Bassam et al. 1981), resulting in a failure or delay in uterine involution (Beck and McEntee 1966). Instead of degenerating after parturition, trophoblasts persist within the endometrium, preventing endometrial blood vessels from developing thromboses, resulting in persistent bleeding (Al-Bassam et al. 1981). In addition, trophoblasts may on rare occasions erode through the serosa resulting in peritonitis (Beck and McEntee 1966). The factors involved with SIPs are unknown.

Many molecular factors are involved in trophoblast invasion as well as placental release during parturition. Some of these factors include matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs), vascular endothelial growth factor (VEGF)-A, VEGF-A receptor (Flt-1) and the KiSS-1 receptor (KiSS-1R) (Lash et al. 1999; Zhang et al. 2011; Maj and Kankofer 1997; Xu et al. 2002). MMPs and TIMPs regulate normal trophoblast invasion and migration in humans (Librach et al. 1991). MMP-2 and MMP-9 are likely responsible for conditions of abnormal placentation in humans (Zhang et al. 2011). KiSS-1 and KiSS-1R may also play a role in the regulation of human trophoblast invasion (Zhang et al. 2011), while VEGF-A may be important in the initiation of trophoblast invasion (Lash et al. 1999).

Detachment and release of the placenta at parturition in the human (Xu et al. 2002), cow (Walter and Boos 2001) and rat (Lei et al. 1996) relies on enzymatic degradation of the extracellular matrix likely by MMP activity. Disorders affecting the regulation of MMPs have been proposed to contribute to placental retention in the cow (Maj and Kankofer 1997; Walter and Boos 2001). Knowledge of the factors involved in normal placental release during parturition and pathologic placental conditions during the post-partum period in the dog is lacking. To the authors' knowledge, gene expression of VEGF-A, Flt-1, KiSS-1R, MMP-2, MMP-9 and TIMP-2 in the canine placenta has not been previously published. The aim of this study was to determine whether MMP-2, MMP-9, TIMP-2, VEGF-A, Flt-1 and KiSS-1R are expressed in the canine placenta. Once established, the objective was to compare expression patterns during late gestation in the bitch, around the initiation of labour and after delivery.

Materials and Methods

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

Tissue collection

Canine chorioallantoic tissue was collected from various breeds following elective pregnancy termination by ovariohysterectomy at 61 ± 1 day past the LH surge (late gestation; n = 4), following elective C-section at 64 ± 1 day past the LH surge prior to first-stage labour (pre-labour; n = 4), and following natural delivery (parturient; n = 3). Prior to being flash frozen in liquid nitrogen, the marginal haematoma was dissected away and any excess tissue and blood were removed. Tissue was then stored at −80°C until analysed.

RNA isolation

Total RNA was isolated using the TRIzol Plus RNA Purification kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. RNA concentration and purity was determined using a spectrophotometer (NanoPhotometer; IMPLEN, Munich, Germany). Total RNA samples were submitted to the Center for Genome Research and Biocomputing (CGRB) at Oregon State University to measure RNA quality. RNA integrity numbers (RIN) were obtained for each sample using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

TaqMan real-time RT-PCR

RNA was reverse transcribed to cDNA using oligo dT as a primer with the SuperScript III First-Strand Synthesis System according to the manufacturer's instructions (Invitrogen). Concentrations of the cDNA samples were measured using the DNA setting on a spectrophotometer (NanoPhotometer, IMPLEN) and then diluted 10-fold for RT-PCR. TaqMan primers and probes of proprietary sequences developed by the company for canine MMP-2, MMP-9, TIMP-2, VEGF-A, Flt-1 and KiSS-1R (Table 1) were used for real-time RT-PCR on the ABI 7300 RT-PCR machine (Applied Biosystems, Carlsbad, CA, USA). Default cycling conditions were used (Table 2). Gene expression was normalized to mRNA expression of HPRT-1 based on its stable expression in all tissues studied (Ct mean ± SEM; 32.7 ± 0.52). The first parturient sample collected was used as a calibrator sample. All RT-PCRs were performed in triplicate with a final reaction volume of 20 μl each. Relative expression was calculated using the inline image method.

Table 1. TaqMan primers and probes used for real-time RT-PCR
Gene nameAssay IDRefSeqExon boundaryAssay locationAmplicon length
MMP-2Cf02623423_m1 XM_535300.2 3–452058
MMP-9Cf02621845_m1NM_001003219.112–13201559
TIMP-2Cf02623335_m1NM_001003082.11–223481
VEGF-ACf02623453_m1NM_001003175.17–890662
Flt-1Cf02696454_g1 XM_534520.2 26–273489116
KiSS-1RCf02715933_u1 XM_850105.1 3–3635141
HPRT-1Cf026262558_m1NM_001003357.18–9605129
Table 2. Real-time RT-PCR cycling conditions
StageTemperature (°C) and time (mins)Replications
Stage 150.0°C for 2 : 001
Stage 295.0°C for 10 : 001
Stage 395.0°C for 0 : 15 then 60.0°C for 1 : 0040

Statistical analysis

Statistical analysis was performed using a one-way anova with Bonferroni corrections to make comparisons between the three groups. Statistical significance was calculated using the Stata 12.0 software (StataCorp LP, College Station, TX, USA). Data were expressed as mean ± standard deviation (SD). Significance was defined as p < 0.05.

Results

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

All RNA samples used in the synthesis of cDNA for real-time RT-PCR studies were determined to be of good quality based on a RIN ≥ 7.0. MMP-9 mRNA expression was significantly higher in pre-term samples compared with pre-labour and parturient samples (Table 3). Although not statistically significant (p = 0.21), there is a similar apparent decrease in the expression of VEGF-A prior to parturition (Table 3). There was no significant difference in mRNA expression between the samples for MMP-2, TIMP-2 or Flt-1 (Table 3). KiSS-1R was not expressed in any of the placentas studied as the Ct values for KiSS-1R were greater than 38 cycles (data not shown).

Table 3. Relative quantitation (mean ± SD) of placental gene expression at three time points prior to parturition
 Pre-term (n = 4)Pre-labour (n = 4)Parturient (n = 3)
  1. a

    p < 0.05 compared to Pre-labour and Parturient.

MMP-915.2 ± 4.30a2.16 ± 1.234.27 ± 2.32
MMP-20.62 ± 0.191.57 ± 0.891.34 ± 1.0
TIMP-20.08 ± 0.040.37 ± 0.269.31 ± 14.4
VEGF-A0.98 ± 0.540.40 ± 0.300.71 ± 0.26
Flt-10.99 ± 0.250.64 ± 0.300.77 ± 0.26

Discussion

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

Molecular changes in the canine placenta in the periparturient period have not been previously described in the literature. Therefore, the aim of this study was to characterize changes in the expression of genes known in other species to regulate trophoblast invasion from late gestation to immediately following parturition. In the current study, MMP-9 mRNA expression was significantly higher in pre-term tissues compared with pre-labour and parturient tissues. MMPs have been shown to play an essential role in the regulation of trophoblast invasion in humans (Librach et al. 1991), so these factors may also be important in the abnormal regulation of trophoblast invasion in SIPs.

Previous research has also described the involvement of MMPs in placental release at parturition in cattle and humans. Walter and Boos (2001) suggested that MMP-2 and MMP-9 may be important in the detachment of placental membranes at parturition in the cow. A similar role at the end of pregnancy for MMP-9 has been proposed in the human placenta by Xu and coworkers, who demonstrated an increase in MMP-9 at labour, but no change in MMP-2 (Xu et al. 2002). MMP-9 has been proposed to be crucial in the degradation of the extracellular matrix at the end of gestation for successful detachment of placental membranes in the human (Xu et al. 2002). In the present study, MMP-9 mRNA expression was significantly increased during late gestation and then decreased around the initiation of labour suggesting that while it may play a role during this period in the dog, this role may be different than that reported in humans and cows.

The relationship of these factors in canine placental retention remains unknown. Maj and Kankofer (1997) suggested that a lack of active MMP-2 in maternal and foetal parts of retained placenta could contribute to the improper release of foetal membranes at the time of parturition.

Lack of MMP inhibition has been demonstrated to be important in placental release. Walter and Boos (2001) reported a decrease in TIMP-2 expression at parturition in cows and suggested this decrease was required for normal placental release. The decrease in TIMP-2 in their study differs from what was found in the current study as TIMP-2 mRNA expression was not significantly different between the three study groups. This suggests the role of TIMP-2 in the dog may differ slightly from that in the cow. However, the relationship of all these factors to canine placental retention will require further study.

VEGF-A and associated receptor, Flt-1, are important in initiating trophoblast invasion in humans (Lash et al. 1999). In addition, Flt-1 placental mRNA increased in women who experienced spontaneous term delivery compared those who underwent an elective term C-section (Lee et al. 2010). Based on these studies, we expected to see a change in VEGF-A and Flt-1 mRNA expression at the end of gestation in dogs, but this was not the case. In the current study, VEGF-A and Flt-1 were not significantly different between the periparturient groups. One possibility for the different expression patterns in the dog compared with the human at the end of gestation may be the difference in placental types. The human placenta is more invasive than the canine placenta, which may necessitate a lesser role of VEGF-A and Flt-1 in the dog at the time of parturition.

Previous studies in humans suggest the primary role of KiSS-1 and its receptor KiSS-1R occurs during the first trimester of pregnancy where it acts to suppress trophoblast invasion by inhibiting protease activity of factors such as MMP-2 (Bilban et al. 2004). In addition, KiSS-1 placental mRNA increased in women who experienced spontaneous term delivery compared those who underwent an elective term C-section (Torricelli et al. 2008). Studies in vitro have shown that KiSS-1 transfection of human trophoblast cells decreases cell invasion (Zhang et al. 2011) and an upregulation has been reported in women with abnormal placentation such as pre-eclampsia (Zhang et al. 2011). There was no expression of KiSS-1R mRNA in any of the canine tissues in the current study based upon a cut-off for relevant expression that was chosen according to the upper limit of the possible detection range (40 cycles) following a starting cDNA amount of 10 ng (Goni et al. 2009). The receptor does not appear to play a role or be present in these tissues at this time in the dog. Further studies are needed to investigate the KiSS-1 gene itself as well as the expression of KiSS-1R earlier in canine gestation.

In summary, MMP-9 mRNA expression was significantly different in the pre-term groups compared with pre-labour and parturient groups. While the increase in MMP-2 mRNA expression prior to labour was not statistically significant, it warrants recognition because of the relevance to previous studies in other species. The results of this study suggest that parturition influences the expression of MMP-9 in the dog, which is similar to what has been reported in humans and cattle. However, further studies are needed to determine whether changes in mRNA expression of MMP-2, MMP-9, TIMP-2, VEGF-A, Flt-1 and KiSS-1R play a role in canine placental abnormalities such as retained placental membranes and excessive trophoblast invasion (SIPs) in dogs.

Acknowledgements

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

The authors are grateful for the technical support of Anne-Marie Girard in the Center for Genome Research and Biocomputing (CGRB) at Oregon State University.

Author contributions

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

Fellows performed all experiments and wrote manuscript. Hazzard assisted with tissue collection and edited manuscript. Kutzler provided oversight and funding for all experiments and edited manuscript.

Funding

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

This research was funded by the Department of Animal and Rangeland Sciences, College of Agricultural Sciences, Oregon State University, Corvallis, OR, USA.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. Author contributions
  10. Funding
  11. References
  • Al-Bassam MA, Thomson RG, O'Donnell L, 1981: Involution abnormalities in the postpartum uterus of the bitch. Vet Pathol 18, 201218.
  • Beck AM, McEntee K, 1966: Subinvolution of placental sites in a post partum bitch. A case report. Cornell Vet 56, 269277.
  • Bilban M, Ghaffari-Tabrizi N, Hintermann E, Bauer S, Molzer S, Zoratti C, Malli R, Sharabi A, Hiden U, Graier W, Knöfler M, Andeae F, Wagner O, Quaranta V, Desoye G, 2004: Kisspeptin-10, a KiSS-1/metastatin-derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts. J Cell Sci 117, 13191328.
  • Goni R, García P, Foissac S, 2009: The qPCR data statistical analysis. Integromics White Paper, 1-9.
  • Lash GE, Cartwright JE, Whitley GS, Trew AJ, Baker PN, 1999: The effects of angiogenic growth factors on extravillous trophoblast invasion and motility. Placenta 20, 661667.
  • Lee KJ, Shim SH, Kang KM, Kang JH, Park DY, Kim SH, Farina A, Shim SS, Cha DH, 2010: Global gene expression changes induced in the human placenta during labor. Placenta 31, 698704.
  • Lei H, Furth EE, Kalluri R, Chiou T, Tilly KI, Tilly JL, Elkon KB, Jeffrey JJ, Strauss JF III, 1996: A program of cell death and extracellular matrix degradation is activated in the amnion before the onset of labor. J Clin Invest 98, 19711978.
  • Librach CL, Web Z, Fitzgerald ML, Chiu K, Corwin NM, Esteves RA, Grobelny D, Galardy R, Damsky CH, Fisher SJ, 1991: 92-kD type IV collagenase mediates invasion of human cytotrophoblasts. J Cell Biol 113, 437449.
  • Maj JG, Kankofer M, 1997: Activity of 72-kDa and 92-kDa matrix metalloproteinases in placental tissues of cows with and without retained fetal membranes. Placenta 18, 683687.
  • Torricelli M, Galleri L, Voltolini C, Biliotti G, Florio P, De Bonis M, Petraglia F, 2008: Changes of placental Kiss-1 mRNA expression and maternal/cord kisspeptin levels at preterm delivery. Reprod Sci 15, 779784.
  • Wallace MS, 1994: Management of parturition and problems of the periparturient period of dogs and cats. Semin in Vet Med Surg (Small Anim) 9, 2837.
  • Walter I, Boos A, 2001: Matrix metalloproteinases (MMP-2 and MMP-9) and tissue inhibitor-2 of matrix metalloproteinases (TIMP-2) in the placenta and interplacental uterine wall in normal cows and in cattle with retention of fetal membranes. Placenta 22, 473483.
  • Xu P, Alfaidy N, Challis JRG, 2002: Expression of matrix metalloproteinase (MMP)-2 and MMP-9 in human placenta and fetal membranes in relation to preterm and term labor. J Clin Endocrinol Metabol 87, 13531361.
  • Zhang H, Long Q, Ling L, Gao A, Li H, Lin Q, 2011: Elevated expression of KiSS-1 in placenta of preeclampsia and its effect on trophoblast. Reprod Biol 11, 99115.