| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Pregnancy |
Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada J2S 7C6
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
placenta, pregnancy, implantation, seasonal reproduction, uterus
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The peri-implantation period in mammals is characterized by morphological and functional changes in the uterine cells accompanied by vascular remodelling. Angiogenesis is a key event for the proliferative processes in the uterus and is required for both placental and embryonic development [11–14]. Vascular endothelial growth factor (VEGF) is the principal factor responsible for regulation of vascular changes [15]. VEGF is a homodimeric glycoprotein of 40–45 kDa and is best known for its potent endothelial cell-specific mitogenic activity, but it also plays a role in increasing vascular permeability [16–19]. Several isoforms have been identified to date, and these differ in the number of amino acids in the final protein. The VEGF gene has eight exons, seven introns, and a coding region of around 14 kilobases [20]. The isoforms are the result of alternative exon splicing from a single gene [20]. The isoforms share the same function, and the main difference among them lies in their ability to bind to heparin [15, 21]. Such differential heparin-binding properties are related to the bioavailability of the several isoforms [22]. The major isoforms identified in humans are comprised of 121, 165, and 189 amino acids [23]. One of these, VEGF 121, is a soluble protein in its free form and has no heparin-binding properties. VEGF 165 is secreted and bound to the cell surface and extracellular matrix (ECM), and VEGF 189 is almost completely bound to the ECM [23]. VEGF protein appears to become available to endothelial cells in at least two ways: as freely diffusible proteins (VEGF 121 and 165 in humans) or after protease activation and cleavage of the longer isoforms bound to the ECM [15]. Among the factors that upregulate VEGF are the ovarian steroid hormones (see [24] for review) and prostaglandins (see [25] for review). VEGF is expressed in the endometrium [26] and is an important factor in regulation of the events of early implantation and establishment of the placenta [27].
VEGF effects on angiogenesis are dependent upon its binding to tyrosine kinase receptors, Flt-1 (fms-like tyrosine kinase, also known as VEGFR-1) and KDR (kinase domain region, also known as VEGFR-2) (see [28] for review). These receptors play an important role in transduction of the VEGF signal during implantation [27].
Gestation in mink displays unique characteristics, including obligate diapause, implantation through the zona pellucida, and formation of an endotheliochorial placenta. Mitogenic activity of the endothelial cells of the maternal placental microvascular is accelerated during the early part of gestation [29]. Mink placenta differs from that of other carnivores in retention of the fetal chorionic villi, allowing the maternal blood vessels to maintain their architecture [30]. Given the peculiarities in this species, it was of considerable interest to explore the expression of the angiogenic factors involved in the implantation process, including VEGF isoforms and the VEGF receptors KDR and Flt-1. We also evaluated the involvement of the embryo in the angiogenic process.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
All animal treatment protocols were approved by the Comité de déontologie, Faculté de médecine vétérinaire, Université de Montréal in accordance with regulations of the Canadian Council of Animal Care.
Mink of the Dark and Pastel varieties were purchased and maintained on a commercial farm (A. Richard, St. Damase, PQ, Canada). Females were mated to fertile males twice, 7 days apart, throughout the first 2 wk of March, according to standard husbandry procedures. Prolactin injection induces embryo activation [4] and implantation [5, 6], and a standard protocol consisting of daily i.m. injections of 1 mg/kg prolactin (Sigma, St. Louis, MO) was employed beginning approximately 1 wk following last mating and continuing for 12 days. Implantation, as indicated by the presence of uterine swelling(s), occurred on the 13th day after initiation of prolactin injections.
Uterine tissues were collected from three animals selected randomly every second day starting on the day of the first prolactin injection (Day 0) and continuing for 19 days. For nonimplanted females, uteri were flushed for embryo recovery and frozen in liquid nitrogen immediately following flushing. For implanted females, implantation chambers were frozen individually. Samples were kept at -70°C until analyzed.
The assignment of samples into categories was based upon gross and microscopic inspection of the uterus and embryos. Diapause samples were collected prior to prolactin injection, the uterus had no implantation chambers, and only embryos in diapause (approximately 200 µm) were found during flushing. The activation period was divided into early and late activation, according to the number of prolactin injections received and the size of flushed embryos [31], i.e., late activated embryos were near 2 mm in diameter. Implantation was confirmed by microscopic evidence of embryo attachment and trophoblast invasion. Pseudopregnant animals were not mated but received two injections of GnRH (10 µg/kg Factrel; Ayerst, Guelph, ON, Canada) 7 days apart during the mating period to induce ovulation, and samples were obtained 30 days later, allowing natural increases in prolactin and progesterone levels to take place. To establish whether there is basal expression of VEGF, uteri from anestrous females were obtained prior to the beginning of the mink breeding season, when ovarian steroids are not present in significant amounts and therefore are not expected to regulate VEGF.
RNA Extraction and Purification and Reverse Transcription
Tissues were homogenized in buffer RLT (Qiagen, Mississauga, ON, Canada) with 0.12 M ß-mercaptoethanol (Sigma), and RNA was purified using an RNeasy Protect Mini kit (Qiagen) as recommended by the manufacturer. Total RNA was measured by spectrophotometry at 260 nm, and 1.5 µg/sample of total RNA was used for reverse transcription (RT) with the Omniscript RT kit (Qiagen) according to the instructions from the manufacturer.
Mink-Specific cDNA Cloning
VEGF primers were designed based on homologous sequences (GenBank nos.) of human (AF022375), mouse (NM_009505), and cow (M32976) (Table 1). Homologous sequences of human (KDR: AF035121; Flt-1: AF063657), rat (KDR: U93306; Flt-1: D28498), and mouse (KDR: X70842; Flt-1: L07297) were also used for designing primers for the VEGF receptors (Table 1). PCR products of the expected size obtained with these primers were excised and purified using a gel extraction kit (Qiagen). Purified cDNA was then ligated into a pGEM-T Easy Vector System I (Promega Corp., Nepean, ON, Canada) according to the instructions of the manufacturer and further transformed into competent Escherichia coli strain XL-1 blue. Plasmids were isolated with a QIAprep Spin Miniprep kit (Qiagen) and sequenced using an ABI PRISM 310 sequencer (Applied Biosystems, Foster City, CA). At least three independent samples were sequenced for verification of the transcripts. The primers for KDR and Flt-1 based on these homologous sequences were used for our studies.
|
Mink-specific primers (AY158156), except primer ex6 which was based on the homologous sequences, were chosen for all three VEGF isoforms (Fig. 1). The forward primer (ex3) utilized for all three VEGF isoforms was the same and is located at the third exon, and the reverse primers differed to allow for amplification of the individual isoforms (Table 1). Mink-specific primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used (AF076283) as a control (Table 1).
|
Semi-quantitative RT Polymerase Chain Reaction
Uterine tissues were examined from three animals in each of the following reproductive states: anestrous, diapause, early embryo activation, late embryo activation, implanted (implantation sites and interimplantation sites), and pseudopregnant. All results are presented as the mean ± SEM of the three individual samples per group.
Relative abundance of the VEGF isoforms 120, 164, and 188 and the receptors KDR and Flt-1 was determined by semiquantitative RT polymerase chain reaction (PCR) using GAPDH as a control for RNA quantity and RT efficiency. For each individual product analyzed, the number of cycles was chosen by subjecting the RT products to PCRs of 17–35 cycles. The quantification procedure was performed by choosing the number of cycles retaining amplification in the exponential phase. The number of cycles chosen for GAPDH, VEGF 120, and VEGF 164 were 23, 32, and 28, respectively. For VEGF 188 and the receptors KDR and Flt-1, 31 cycles were employed.
The semiquantitative reactions were carried out for the chosen number of cycles in a final volume of 50 µl and using Taq DNA polymerase (Amersham Biosciences Corp., Baie d'Urfe, PQ, Canada). Amplifications were carried out with annealing conditions of 58°C for 40 sec for VEGF 120 and 59°C for 40 sec for the remaining products. PCR products were separated in a 1.8% agarose gel and stained in ethidium bromide. Densities of the amplified fragments were analyzed using the Collage software (Photodyne, New Berlin, WI). Results were expressed as a density ratio of the target gene to the control (GAPDH).
Immunohistochemical Analysis of VEGF
Tissues fixed in Zamboni solution were used to demonstrate expression of VEGF throughout the peri-implantation period. A Vectastain ABC kit (Vector Laboratories, Burlington, ON, Canada) was used according to the manufacturer's protocol. Deparaffinized and hydrated sections were immersed in methanol containing 0.75% hydrogen peroxide for 20 min for quenching of any endogenous peroxidase activity. Sections were then washed in Tris-buffered saline (TBS), and retrieval of antigens was performed by microwave treatment twice for 5 min in TBS. Sections were then incubated with normal goat serum at room temperature for 30 min. A rabbit polyclonal antibody (VEGF: A-20; Santa Cruz Biotechnology, Santa Cruz, CA) raised against residues 1–20 at the amino terminus of human VEGF, thus recognizing all isoforms used during this study, was added at a concentration of 0.6 µg/ml diluted in normal serum, and tissues were incubated overnight at 4°C. The same antibody was previously used to immunolocalize VEGF in mink [32]. After washes in PBS, incubation with biotinylated second antibody took place for 45 min. Following PBS washes, a complex of avidin-biotin-peroxidase was applied for 45 min. Positive reactions were identified by the use of the peroxide substrate 3,3' diaminobenzidine (Sigma) at a concentration of 0.6 mg/ml.
Statistical Analysis
The ratio of target gene:GAPDH was used as a value for each sample, and data were analyzed using the least square ANOVA and the general linear model procedures of SAS (Cary, NC). When significant differences in treatments were found, comparisons of means were further performed by the methods of orthogonal contrasts and the Duncan multiple range test.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
All three VEGF isoforms were present in anestrous, diapause, activated (early and late), implanted (implantation site and interimplantation site), and pseudopregnant uteri of mink. The relative abundance for all three isoforms of VEGF increased throughout implantation (Fig. 2). Expression of VEGF 120 was significantly different among groups (P < 0.01) (Fig. 2). Individual mean comparisons revealed that anestrous uteri had the lowest level of VEGF 120 mRNA, which was significantly lower than that of implanted and pseudopregnant samples (P < 0.05). Diapause and early activated uteri displayed significantly lower VEGF 120 levels relative to implanted uteri (P < 0.05) (Fig. 2).
|
Expression of VEGF 164 also differed significantly among groups (P < 0.01). Anestrous uteri had the lowest level of VEGF 164 mRNA, which was significantly lower than that of early and late activated, implanted, and pseudopregnant uteri (P < 0.05). Samples from diapause and early activation had lower VEGF 164 levels than did late activated, implanted, and pseudopregnant uteri (P < 0.05); diapause VEGF 164 levels were not significantly different from anestrous levels (Fig. 2).
Expression of VEGF 188 differed significantly among groups (P < 0.01). Anestrous uteri had the lowest level of VEGF 188 mRNA, which was significantly lower relative to late activated, implanted, and pseudopregnant uteri (P < 0.05). Late activated and pseudopregnant samples had the highest levels mRNA levels, which differed significantly from anestrous, diapause and early activated samples (P < 0.05), and implanted samples had levels between those of the early activated and the late activated and pseudopregnant samples (Fig. 2).
Further analyses were performed using orthogonal contrasts, in which the different stages were grouped and contrasted to allow comparisons of biological interest. The contrasts chosen were as follows: anestrous vs. pregnant (including all the other groups), diapause vs. activated (early and late), early activated vs. late activated, late activated and implanted vs. pseudopregnant, implanted vs. nonimplanted (diapause, early and late activated), and implantation site vs. interimplantation site. Results for VEGF isoforms are depicted in Table 2.
|
VEGF Localization During the Peri-Implantation Period
Mink uterine tissues from the peri-implantation period were stained for VEGF protein. In uteri samples taken from diapause (obligate delay), VEGF staining was observed in the glandular epithelium, very little or no staining was present on the luminal epithelium, and no staining was present in the subepithelial stroma (Fig. 3, A and B). Following activation of the embryo, VEGF staining was observed in the luminal and glandular epithelium, but the stromal bed consistently lacked expression of VEGF (Fig. 3, E and F). In implanted uteri, VEGF was strongly expressed in the luminal and glandular epithelium and in the subepithelial stroma (Fig. 3, G and H). At the site of embryo attachment and implantation, the first layer of trophoblast cells leading the invasion into the uterus displayed a strong VEGF signal (Fig. 3, G and H). Samples taken from pseudopregnant animals presented a pattern of VEGF localization similar to that in activated samples, with positive staining in the luminal and glandular epithelium and lack of signal in the stromal bed (Fig. 3, I and J). In uterine samples collected during anestrous, no significant localization of VEGF protein was found (Fig. 3, L and M).
|
Semiquantification of VEGF Receptors KDR and Flt-1
We also investigated expression of the VEGF receptors KDR and Flt-1 throughout the process of implantation. Expression of KDR mRNA differed among groups (P < 0.05). Anestrous and pseudopregnant uteri had lower levels of this receptor than did implanted uteri (P < 0.05) (Fig. 4).
|
Expression of Flt-1 mRNA also differed among groups (P < 0.01). Anestrous and pseudopregnant uteri had lower levels of this receptor than did activated and implanted uteri (P < 0.05). Diapause levels were significantly lower than those at implantation sites (P < 0.05) but did not differ from levels in any other group (Fig. 4). Orthogonal contrast analysis was performed for the receptors using the same comparisons as for the VEGF isoforms (Table 2).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A second candidate for the regulation of angiogenic factors during implantation is prolactin, because this hormone is an important effector of the termination of diapause [5]. Prolactin receptors are present in the mink uterus during early gestation, and their abundance is influenced by ovarian steroids [35]. No evidence for or against direct regulation of VEGF expression in the mink uterus by prolactin is yet available.
A third possibility is that the activated or implanting embryo plays a role in regulating VEGF either locally or globally in the uterus. This possibility has been previously suggested for the hamster [36] and is supported in the present study by the distinct differences that were noted in VEGF expression between mink uteri containing blastocysts in diapause and those with activated embryos. VEGF staining was limited to the glandular epithelium during diapause, whereas in activated uteri VEGF was localized in the glandular epithelium and the luminal epithelium. Nonetheless, similar increases in the quantity and extent of VEGF expression, both in terms of mRNA abundance and protein localized to glandular and endometrial epithelium during the implantation process, were also observed in uteri of pseudopregnant animals. The pattern in the uterus of the pseudopregnant animals resembles that seen during late embryo activation and early implantation. The mRNA results revealed that the increase in VEGF did not differ in samples taken from the implantation site relative to those from interimplantation areas, indicating that the changes are not specific local effects of embryo invasion. Thus, we concluded that VEGF expression is independent of embryonic influence in the mink. This independence distinguishes mink from other mammals studied to date.
Very strong VEGF signal was localized in invasive hypertrophied trophoblast cells. In contrast, the outermost layer of trophoblast cells, which are not in direct contact with uterine tissue, showed no positive staining for VEGF. Similar VEGF localization within the invasive trophoblast cells has been observed in murine implantation [37]. At later times in gestation, VEGF-positive staining is present in the cytotrophoblast and syncytiotrophoblast layers of the mink placenta [32], which indicates evolution of VEGF expression in the two trophoblast cell types as gestation progresses.
Matsumoto et al. [27] suggested that although preimplantation expression of VEGF is steroid regulated, the onset of implantation and decidualization shifts this regulation to cyclooxygenase 2 (COX-2)-derived prostaglandins. Uterine expression of COX-2 is associated with implantation in mice, and COX-2-deficient mice display defective implantation and decidualization [38, 39]. Studies by Lim et al. [40] revealed that COX-2-derived prostaglandin (PG) I2 is involved in implantation and decidualization, and its action is mediated by the peroxisome proliferator-activated receptor delta (PPAR
). In the mink, COX-2 expression is a transient event that occurs at the time of trophoblast attachment and invasion [41]. This finding concurs with elevated expression of VEGF in the present study. Further, endometrial expression of PPAR has been observed in mink following implantation [42]. This finding reinforces the view that PGs contribute to upregulation of VEGF during this time in early gestation. PGE2 effectively induces VEGF expression in other tissues [43], and its expression was shown in the mouse uteri during implantation [40]. Preliminary data indicate that mink activated embryos produce PGE2 [42]. Another candidate for upregulation of VEGF during early gestation is PGJ, which stimulates VEGF expression in human macrophages, activating gene expression through PPAR
-mediated processes [43]. PPAR and its heterodimerization partner RXR
are strongly expressed in human trophoblasts, and RXR is present in decidual cells [44]. Preliminary information indicates that PPAR is expressed in the trophoblast at the time of implantation [42], placing PPAR as a proximal candidate for VEGF regulation following implantation. The occurrence of the ligands for PPAR at implantation remains to be investigated.
VEGF acts through the tyrosine kinase receptors KDR and Flt-1 [45]. In this study, expression of KDR in the uteri of pseudopregnant mink was low in comparison to samples from implanted animals. In addition, uterine samples derived from implantation sites displayed greater expression of Flt-1 relative to uteri from diapause or from pseudopregnancy. Studies in the mouse have shown very low accumulation of KDR mRNA in the uterus on the first 2 days of pregnancy; however, on Days 3 and 4, these genes were distinctly expressed in the stromal bed [37]. On Days 5–8 of mouse gestation, the decidual beds accumulated KDR and Flt-1 mRNAs [37]. In the rabbit, mRNA for both receptors was present in the uterus at several stages, with high levels at estrus and just prior to implantation [46]. In the hamster, expression of the receptors was also correlated with the progression of embryo implantation [36]. In the current studies, elevation of VEGF receptor expression likewise appeared to be associated with implantation in mink. Unlike the VEGF isoforms, both receptors were at their lowest levels in pseudopregnant uteri, which indicates that the implanting embryo plays a role in regulating expression of VEGF receptors. The factors produced by embryos or by the uterus in the presence of the embryo during implantation remain obscure, but the eicosanoids and their receptors are excellent candidates.
We used a unique animal model, the mink, that shows obligate developmental arrest in the embryo, progesterone-dependent embryo activation, a lack of decidual response, and an endotheliochorial placenta to study the factors involved in angiogenesis during the peri-implantation period. Three VEGF isoforms are upregulated during the peri-implantation period, as are the VEGF receptors KDR and Flt-1. Upregulation of VEGF during the implantation process is dependent on maternal factors, presumably gonadal steroids, whereas the presence of the embryo appears to regulate the VEGF receptors.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Bruce D. Murphy, Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, 3200 rue Sicotte, St-Hyacinthe, PQ, Canada J2S 7C6. FAX: 450 778 8103; murphyb{at}medvet.umontreal.ca
Received: 15 November 2002.
First decision: 26 November 2002.
Accepted: 18 December 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  J. A Desmarais, M. Cao, A. Bateman, and B. D Murphy Spatiotemporal expression pattern of progranulin in embryo implantation and placenta formation suggests a role in cell proliferation, remodeling, and angiogenesis Reproduction, August 1, 2008; 136(2): 247 - 257. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A Desmarais, F. L Lopes, H. Zhang, S. K Das, and B. D Murphy The Peroxisome Proliferator-Activated Receptor Gamma Regulates Trophoblast Cell Differentiation in Mink (Mustela vison) Biol Reprod, November 1, 2007; 77(5): 829 - 839. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. L. Lopes, J. Desmarais, S. Ledoux, N. Y. Gevry, P. Lefevre, and B. D. Murphy Transcriptional Regulation of Uterine Vascular Endothelial Growth Factor during Early Gestation in a Carnivore Model, Mustela vison J. Biol. Chem., August 25, 2006; 281(34): 24602 - 24611. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
