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BOR - Papers in Press, published online ahead of print May 18, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.040808
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BIOLOGY OF REPRODUCTION 73, 663–671 (2005)
DOI: 10.1095/biolreprod.105.040808
© 2005 by the Society for the Study of Reproduction, Inc.

Effects of Embryo Culture on Angiogenesis and Morphometry of Bovine Placentas During Early Gestation1

Jeremy R. Miles 3, Charlotte E. Farin 4, Karina F. Rodriguez 4, Joseph E. Alexander 4, and Peter W. Farin 2, 3

Departments of Population Health and Pathobiology3 Animal Science,4 North Carolina State University, Raleigh, North Carolina 27606


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of this study was to determine the effects of undefined and semidefined culture systems for in vitro embryo production on angiogenesis and morphometry of bovine placentas during early gestation. Blastocysts produced in vivo were recovered from superovulated Holstein cows and served as controls. Blastocysts produced in vitro were exposed to either serum-supplemented medium with cumulus cell coculture (in vitro-produced with serum; IVPS) or modified synthetic oviductal fluid medium without serum or coculture (mSOF). Single blastocysts from each production system were transferred into heifers. Fetuses and placentas were recovered on Day 70 of gestation. Cotyledonary tissues were obtained for quantification of vascular endothelial growth factor (VEGF) and peroxisome proliferator-activated receptor-gamma (PPARG) mRNA and protein. Samples of placentomes were prepared for immunocytochemistry and histological analysis. Placentas from the mSOF group were heavier and had the fewest placentomes, least placental fluid, and lowest placental efficiency (fetal weight/placental weight) compared with the in vivo and IVPS groups. There was no effect of embryo culture system on volume densities of fetal villi or maternal endometrium within placentomes. The volume density of fetal pyknotic cells was increased in placentomes in the mSOF group compared with the in vivo and IVPS groups. Placentomes in the mSOF group had decreased densities of blood vessels and decreased levels of VEGF mRNA in cotyledonary tissue. In conclusion, compared with placentas from embryos produced in vivo or in vitro using an undefined culture system, placentas from embryos produced in vitro using a semidefined culture system exhibited a greater degree of aberrant development of the placenta during early gestation.

developmental biology, embryo, gene regulation, in vitro fertilization, placenta


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abnormal placentation has been observed following transfer of in vitro-produced embryos in cattle [1–4] and sheep [5]. Placental abnormalities associated with in vitro production of embryos have included an increased incidence of hydrallantois [1, 4–6] and decreased number of placentomes [7], as well as variations in feto-maternal contact [3, 4], and placental vascular development [4]. Furthermore, the production of embryos by nuclear transfer (NT; cloning) has also been associated with severe abnormalities of the placenta and pregnancy loss in cattle [8–11], sheep [12, 13], and mice [14–16]. Recently, we have shown that during late gestation, bovine placentas from embryos produced in vitro had decreased feto-maternal contact as measured by surface area of placentomes and volume density of fetal villi compared with their in vivo counterparts [4]. However, placentas from embryos produced in vitro had an increased density of maternal blood vessels and an increased ratio of blood vessels-to-placentome surface area compared with controls [4]. These findings support the hypothesis that during late gestation in the cow, placentas from embryos produced in vitro have enhanced vascular development, which may be compensating for decreased feto-maternal contact.

Placental angiogenesis is critical for increasing placental blood flow throughout gestation [17, 18], thereby ensuring proper availability of nutrients to the developing fetus, regulation of gas exchange, and elimination of waste products. Molecular regulation of angiogenesis in the placenta is driven by angiogenic factors including vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGFB), and angiopoietins [17, 19, 20]. In sheep, VEGF and FGFB act on endothelial cells to stimulate placental angiogenesis [18]. In our previous study on late-gestation bovine placentas, we found, interestingly, no differences between placentas from in vivo- or in vitro-produced embryos in the levels of expression of mRNA for VEGF or the levels of protein for VEGF in either caruncular or cotyledonary tissues [4]. However, caruncular tissues from in vitro-produced embryos had increased levels of protein for peroxisome proliferator-activated receptor-gamma (PPARG) compared with controls, suggesting that enhanced vascular development in bovine placentas may be regulated by PPARG [4]. PPARG has also been associated with vascular development of the placenta in mice [21] and tissue remodeling of the placenta in humans [22]. Alterations in expression of angiogenic factors, such as VEGF and, potentially, PPARG, may initiate abnormalities in vascular development with placentas from embryos produced in vitro.

The establishment of adequate embryo culture systems is essential for the production of a live, healthy offspring following embryo transfer. Successful in vitro systems have included the use of undefined (i.e., coculture, serum supplementation, or both), semidefined (i.e., BSA supplementation), and defined (i.e., amino acids and polyvinyl alcohol supplementation) media [23–25]. In cattle and sheep, extensive comparisons of culture systems have been made using end points that include rate of blastocyst development [24, 26–30], embryo morphometry [31, 32], pregnancy rate [2, 33–35], fetal and placental weights [2, 5], and birth weight [34–36]. Developmental abnormalities of the fetus, placenta, and offspring have been predominantly attributed to the presence of serum in the culture medium [5, 28, 34, 37]. In sheep, placental weight was greater for placentas from embryos produced using serum-supplemented coculture medium compared with placentas from embryos produced in vivo or in vitro using BSA-supplemented medium [5]. In addition, a higher incidence of polyhydramnios (≥23%) was observed in sheep placentas derived from cocultured embryos [5]. Similarly, a higher incidence of hydrallantois was observed in bovine pregnancies resulting from embryos produced using serum-supplemented coculture medium [1].

The overall objective of this study was to determine the effects of in vitro production of embryos using either undefined or semidefined culture systems on the morphometry and angiogenesis of placentas during early gestation in cattle. Specifically, we directly compared placentas at Day 70 of gestation from bovine embryos produced in vivo (control group) or in vitro using either a coculture medium supplemented with serum (undefined) or modified synthetic oviductal fluid medium supplemented with BSA (semidefined) for 1) gross and histological morphometry, 2) mRNA and protein expression for VEGF and PPARG in cotyledonary tissue, and 3) morphometry of blood vessels within the cotyledonary (fetal) and caruncular (maternal) components of placentomes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Hormones

Tissue culture medium (M-199 with Earle salts) was purchased from Gibco BRL (Grand Island, NY). Equine pituitary LH (11.5 NIH LH-S1 units/mg) and porcine pituitary FSH (50 mg/vial Armour FSH Standard) preparations were obtained from Sigma Chemical Co. (St. Louis, MO). Fatty acid-free BSA was purchased from Roche Applied Sciences (Indianapolis, IN). All other culture reagents were tissue culture grade and were purchased from Sigma Chemical Co.

TRI-Reagent was purchased from Molecular Research Center, Inc. (Cincinnati, OH). DNase and random hexamers were purchased from Promega (Madison, WI). SuperScript II reverse transcriptase and dNTPs were purchased from Invitrogen Co. (Carlsbad, CA). Polymerase chain reaction (PCR) purification kits were purchased from Qiagen (Valencia, CA). Taq polymerase was purchased from Roche. SYBR Green dye was purchased from Molecular Probes, Inc. (Eugene, OR). All primers for PCR and real-time PCR were custom synthesized by either Sigma-Genosys (Woodlands, TX) or Qiagen Operon (Alameda, CA). For detection of VEGF protein by Western blot and immunocytochemistry, an anti-VEGF polyclonal rabbit antibody (sc-152) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). For detection of PPARG protein by Western blot, an anti-PPARG polyclonal rabbit antibody (107100) was purchased from Cayman Chemical (Ann Arbor, MI).

Production of Embryos

All procedures and protocols involving the use of cattle were approved by the Institutional Animal Care and Use Committee at North Carolina State University. For in vivo embryo production, donor cows were nonlactating Holstein cows, 3 y of age or older from the North Carolina State University Dairy Farm. Donor cows were synchronized using two i.m. injections of 25 mg prostaglandin F2{alpha} (PGF2{alpha}; Lutalyse; Pharmacia & Upjohn Co., Kalamazoo, MI) 14 days apart. Donor cows were superovulated with 400 mg FSH (Folltropin, Vetrapharm Canada, London, ON) administered in decreasing doses over a 4-day period beginning on Days 10, 11, 12, or 13 of the estrous cycle (Day 0 = estrus). On the morning and evening of the third day of FSH treatment, estrus was induced using two i.m. injections of 25 mg of PGF2{alpha}. Donors were artificially inseminated at 12 and 24 h after detection of first standing heat with thawed frozen semen from the same proven Holstein bull. Embryos were collected by nonsurgical uterine flushing on Day 7 (Day 0 = first detected estrus).

For in vitro embryo production, ovaries of Holstein cows were obtained from a local abattoir and held in saline with 0.75 µg/ml penicillin for 4–6 h during transport to the laboratory. Cumulus-oocyte complexes (COCs) were aspirated, matured, and fertilized in vitro as previously described [7]. Briefly, COCs were aspirated from 2- to 7-mm follicles and washed five times in modified Tyrode medium (TL-Hepes). Groups of 20 to 30 COCs were matured for approximately 22 h in M-199 supplemented with 10% heat-inactivated estrus cow serum (ECS), 10 µg/ml LH, 5 µg/ ml FSH, 1 µg/ml estradiol, 200 µM sodium pyruvate, and 50 µg/ml gentamicin. All cultures were incubated at 5% CO2 in air with 100% humidity. Following the maturation period, COCs were washed once and placed in fertilization medium that consisted of heparin-supplemented Tyrode albumin lactate pyruvate medium with 6 mg/ml fatty acid-free BSA [38]. Thawed frozen semen from the same Holstein bull used to inseminate the donor cows for in vivo embryo production was used for in vitro fertilization. Motile spermatozoa were collected using the swim-up procedure [38], and a final concentration of 1 x 106 spermatozoa per ml was used for fertilization in 0.75 ml of fertilization medium. Spermatozoa and COCs were coincubated for 18–20 h. Following incubation, presumptive zygotes were washed six times with TL-Hepes and placed into one of two culture systems for the 168-h embryo culture period. The two culture systems used were in vitro-produced with serum (IVPS; M-199 supplemented with 10% ECS [7]) and modified synthetic oviductal fluid (mSOF; mSOF containing 0.6% BSA [32]). Embryos for the IVPS group were cultured with their cumulus investments in 1 ml of medium in an atmosphere of 5% CO2 in air with 100% humidity. Culture medium for the IVPS group was changed at 48-h intervals throughout the 168-h culture period [7]. Embryos for the mSOF group had their cumulus investments removed after fertilization by moderate vortexing, and embryos were cultured undisturbed in 1 ml of medium in an atmosphere of 90% N2:5%O2:5% CO2 throughout the 168-h culture period [32].

Transfer of Embryos

Angus heifers 1 y of age or older served as embryo recipients. Heifers were given two i.m. injections of 25 mg of PGF2{alpha} 10 to 12 days apart to synchronize estrus. Grade 1 blastocysts [39] from in vivo or in vitro production systems were transferred in TL-Hepes medium singly into the uterine horn ipsilateral to the ovary bearing the corpus luteum of recipient heifers at Day 7 of the estrous cycle.

Recovery of Fetuses and Placental Tissue

At Day 70 of gestation (63 days after transfer), a total of 18 pregnant recipients (n = 6 each for in vivo, IVPS, and mSOF groups) were slaughtered. Fetuses and their placentas were removed from the reproductive tracts and physical measurements were taken, including fetal body weight, wet placental weight, number of placentomes, and placental fluid (amniotic plus chorioallantoic fluid) volume. Samples of cotyledonary tissue were obtained by careful manual separation of cotyledonary tissue from the placentome. These tissues were immediately snap-frozen in liquid nitrogen and stored at –80°C for whole cell RNA (wcRNA) and protein extraction. Center segments cut from whole placentomes of individual placentas were stored in 10% neutral buffered formalin for histology and immunocytochemistry.

Reverse Transcription and Verification of PCR Products

Whole cell RNA from cotyledonary tissue was extracted using a previously reported protocol [4]. Briefly, wcRNA was extracted using TRI-Reagent and dissolved in diethyl pyrocarbonate-treated water. Concentrations of wcRNA were determined by absorbance at 260 nm. The quality and integrity of the wcRNA was assessed based on the ratio of absorbance at 260 and 280 nm and visualization of 28S and 18S rRNA bands in ethidium bromide-stained agarose gels (data not shown). Each sample of wcRNA was treated with DNase (1.5 units) for 20 min at 37°C. Following DNase inactivation, wcRNA was then reverse transcribed using random hexamers and SuperScript II reverse transcriptase under conditions recommended by the manufacturer. Following cDNA synthesis, samples were purified using the Qiagen PCR purification kit as recommended by the manufacturer and stored at 4°C.

Forward and reverse primer sequences used to amplify mRNA for VEGF-1, PPARG, and H2A histone family, member Z (H2AZ) are found in Table 1. The VEGF-2 forward and reverse primer pair, specifically designed to detect all five VEGF isoforms [40], was also used (Table 1). Each PCR reaction consisted of 50 ng equivalents of cotyledonary cDNA from a control (in vivo) placenta, 1.6 µM of the appropriate forward and reverse primers, 16 µM dNTPs, 2 µl of 10x PCR buffer (Roche), and 2.5 U of Taq polymerase in a 20-µl reaction. A negative control lacking cDNA was included in each PCR assay. All PCR reactions were run in 96-well PCR plates. Plates were placed into an iCycler thermocycler (Bio-Rad, Richmond, CA). Each PCR program consisted of 90 sec at 95°C, followed by 35 cycles of 30 sec denaturation at 94°C and 45 sec annealing at the appropriate temperature. Following amplification cycles, a primer extension for 5 min at 72°C was used. The primer sequences used for PCR as well as the specific isoforms detected, annealing temperatures, and expected product lengths are shown in Table 1. The specificity of the PCR products using the VEGF-1, VEGF-2, PPARG, and H2AZ primer pairs were each verified by sequence analysis.


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  		TABLE 1. Primer sequences used for PCR analysis of cDNA from bovine cotyledonary tissues

Real-Time Quantitative PCR Analysis

VEGF-1, PPARG, and H2AZ primers were used for quantification of VEGF and PPARG mRNA levels by real-time reverse transcription-PCR as previously reported [4]. Briefly, each PCR reaction consisted of 50 ng equivalents of cDNA, 1.6 µM of the appropriate forward and reverse primers, 16 µM dNTPs, 2 µl of 10x PCR buffer, 2 µl of 2x SYBR Green I dye, 1 µl of 200 nM Fluorescein dye (Bio-Rad), and 2.5 U of Taq polymerase in a 20-µl reaction. Melt-curve analysis and gel electrophoresis were used to confirm product length after amplifications were complete (data not included). Before quantification of VEGF and PPARG, raw cycle threshold (CT) values were converted to the linear term using the formula [41]. Expression of VEGF and PPARG mRNA in cotyledonary tissues was expressed relative to the expression of the endogenous control mRNA, H2AZ. Levels of VEGF and PPARG mRNAs are expressed as a ratio of linearized CT values for the gene of interest to the linearized CT values for H2AZ in individual samples.

Expression of mRNA for either VEGF or PPARG in cotyledonary tissue was analyzed in one assay (in vivo, n = 6; IVPS, n = 6; mSOF, n = 6). Each assay contained reference samples of cotyledonary tissue from a random control bovine placenta at approximately Day 70 of gestation (fetal crown-rump length = 10.0 cm). The reference sample was used to determine the intraassay coefficient of variation (CV) for the linearized CT values. The intraassay CV of the VEGF assay was 9.8% and the intraassay CV of the PPARG assay was 4.9%.

Western Blot

Protein was extracted from cotyledonary tissue according to the method described by Miles et al. [4]. Briefly, protein was resuspended in a cold buffer (13.3 µl buffer per milligram of tissue) consisting of 1% (v/ v) Triton X-100, 2 mM EDTA, aprotinin (20 µg/ml), leupeptin (20 µg/ ml), 1 mM PMSF, and 20 mM Hepes. Total protein was quantified using bicinchoninic acid protein assay (Pierce, Rockford, IL) according to the manufacturer's recommended protocol.

Expression of VEGF and PPARG protein was evaluated using a modified Western blot protocol [42]. Briefly, a total of 20 mg of protein from each sample of cotyledonary tissues was separated on 12% (w/v) SDS-PAGE gels under nonreducing conditions. Following electrophoresis, the polyacrylamide gels were transferred to nitrocellulose membranes (Bio-Rad) using a semidry transfer system (Bio-Rad). Following transfer, polyacrylamide gels were stained with Coomassie Brilliant blue protein dye to ensure consistent loading of protein for each sample (data not shown). VEGF and PPARG antibody binding was detected using the BM Chemiluminescence Western blotting kit (Mouse/Rabbit; Roche). Blots were incubated overnight at 4°C with a 1:500 dilution of VEGF polyclonal antibody or 1:750 dilution of PPARG polyclonal antibody. Specificity of the VEGF and PPARG antibodies was verified using blocking peptides obtained from Santa Cruz Biotechnology, Inc. and Cayman Chemical, respectively. Blots were exposed to Kodak X-OMAT-AR film (Eastman Kodak, Rochester, NY) and binding was quantified using computer-assisted video image analysis (Optimas Visual Imaging System 6.1; Optimas Corporation, Bothell, WA).

Morphometry of Placentomes

Samples of intact placentomes were embedded in paraffin and 5-µm sections of placentome tissues were prepared. Sections were deparaffinized, dehydrated, and stained with hematoxylin-eosin. Stereological end points, including the volume densities of fetal villi, caruncular endometrium, fetal binucleate cells, fetal pyknotic cells, and maternal pyknotic cells, were evaluated by point count methods [43, 44] using computer-assisted image analysis (Optimas Visual Imaging System 6.1). For analysis of fetal villi, caruncular endometrium, and fetal binucleate cells, 10 fields of view representing a total of 8.12 x 106 µm2 of each placentome tissue was examined using a 100-point grid system [44]. For analysis of fetal and maternal pyknotic cells, 10 fields of view representing a total of 6.01 x 104 µm2 of each placentome tissue was examined using a 256-point grid system [44].

Immunocytochemical localization of VEGF protein was used to identify vascular beds for morphometric analysis [4]. Briefly, antigen availability within tissue sections was enhanced using Target Unmasking Fluid (BD Pharmingen, San Jose, CA) as recommended by the manufacturer. Immunoreactivity for VEGF was detected using a 1:50 dilution of the VEGF polyclonal antibody and an incubation period of 1 h at room temperature. Specificity of the VEGF antibody was verified using blocking peptide obtained from Santa Cruz Biotechnology, Inc. Binding of VEGF antibody within placentome sections was visualized with diaminobenzidine tetrahydrochloride containing nickel (Vector Laboratories, Burlingame, CA).

For analysis of maternal and fetal blood vessels, 20 fields of view representing a total of 4.8 x 105 µm2 of tissue was examined from each placentome section. Point count methodology with a 256-point grid system [44] was used to determine the volume densities of maternal and fetal blood vessels. For determination of total blood vessel volume density, the volume densities of maternal and fetal blood vessels were added.

Statistical Analysis

Proportional data for fetal and placental weights were analyzed using the Chi-square test [45, 46]. All other data were analyzed using the general linear model procedure for analysis of variance [45, 46]. When a significant F statistic was determined, means were separated using the Duncan multiple-range test [45, 46]. Means were considered statistically different at P ≤ 0.05, and tendencies between P = 0.06 and P = 0.10. Results are reported as least-squares means ± SEM.

The model for analysis of fetal body weight, placental weight, and placental efficiency (fetal weight/placental weight, [47]) included only the main effect of treatment (in vivo, IVPS, and mSOF) because the main effects of sex of fetus and the interaction of treatment by sex of fetus were nonsignificant, and these effects did not increase the R2 value. The model for analysis of placental fluid volume, number of placentomes, histological morphometry of placentas, and mRNA for VEGF and PPARG included the main effects of treatment, sex of fetus, interaction of treatment by sex of fetus, and the covariate of placental weight. For the analysis of protein for VEGF and PPARG, the model included the main effects of treatment, sex of fetus, protein gel, all two-way interactions between main effects, and the covariate of placental weight.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Morphometry of Fetuses and Placentas

Morphometry of fetuses and placentas resulting from the transfer of in vivo-, IVPS-, and mSOF-produced embryos is summarized in Table 2. The mean body weights of fetuses derived from these three sources of embryos were not different. However, the range of fetal body weight was more extreme for fetuses in the IVPS (12.9 g) and mSOF (28.2 g) groups compared with in vivo controls (5.6 g). In addition, the proportion of fetuses heavier than 41 g (+2 SD above the mean fetal body weight for the in vivo group) was greater (P = 0.05) in the mSOF group (3 of 6; 50%) compared with the in vivo group (0 of 6; 0%). There was also an increased proportion of heavier fetuses in the IVPS group (2 of 6; 33%) compared with controls (0%); however, this difference in proportions was not significant (P = 0.12).


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  		TABLE 2. Morphometry of bovine fetuses and placentas recovered on Day 70 of gestation for blastocysts produced in vivo or in vitro using either an undefined (IVPS) or semi-defined (mSOF) medium.a

Placentas were heavier (P = 0.02) from the mSOF group compared with those from the in vivo and IVPS groups (Table 2). The range of placental weight was also more extreme for placentas in the mSOF group (173.4 g) compared with those from the IVPS (76.0 g) and in vivo (79.5 g) groups. In addition, the proportion of placentas heavier than 177 g (+2 SD above the mean placental weight for the in vivo group) was greater (P = 0.05) in the mSOF group (3 of 6; 50%) compared with that of the control group (0 of 6; 0%). The mean placental fluid volume tended to be less (P = 0.07) in the mSOF group compared with fluid volumes in the in vivo and IVPS groups. Placentome number was also less (P = 0.02) in the mSOF group compared with the in vivo and IVPS groups. Of interest, placental efficiency (fetal weight/placental weight) was less (P = 0.01) for the mSOF group compared with the IVPS group. Placental efficiency was similar for the control group compared with both the IVPS and mSOF groups.

Figure 1 shows the maternal and fetal components of a placentome at Day 70 of gestation. Based on morphometric analysis of placentomes, the volume densities of fetal villi as well as maternal endometrium were not different between the three groups (Table 2). However, the volume density of fetal binucleate cells was increased (P = 0.04) in the IVPS group compared with in vivo controls. Placentomes from the mSOF group displayed an intermediate volume density of fetal binucleate cells between the in vivo and IVPS groups. There was no effect of treatment on the pyknotic cell volume density within the maternal endometrium. However, the volume density of pyknotic cells within the fetal villi was increased (P = 0.01) in placentas from the mSOF group compared with either the in vivo or IVPS groups.



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  		FIG. 1. A section of bovine placentome at Day 70 of gestation from the in vivo group stained with hematoxylin-eosin that was used for assessment of volume densities of caruncular endometrium (CE), fetal villi (FV), fetal binucleate cells (arrow), maternal pyknotic cells, and fetal pyknotic cells (circle). Bar = 10 µm

Expression of VEGF and PPARG mRNA

Figure 2 displays the amplification products following reverse transcription-PCR using the VEGF-2 primer pair and cDNA from cotyledonary tissue of placentas from the in vivo, IVPS, and mSOF groups. Three bands were visualized at expected lengths corresponding to the VEGF120, VEGF164, and VEGF188 isoforms [40] for cotyledonary tissue from the in vivo, IVPS, and mSOF groups. The predominant isoform in cotyledonary tissue from all three groups appeared to be VEGF164.



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  		FIG. 2. Ethidium bromide-stained agarose gel of VEGF amplification products from bovine cotyledonary tissues. The gel depicts PCR products from a reaction using the VEGF-2 primer pair. Lane 1, base pair (bp) marker (100 bp ladder); lane 2, negative control sample; lane 3, random cotyledon sample; lane 4, in vivo cotyledon sample; lane 5, IVPS cotyledon sample; lane 6, mSOF cotyledon sample. The amplification products represent expected product lengths for VEGF120 (186 bp), VEGF164 (318 bp), and VEGF188 (390 bp) isoforms, respectively [40]

The expression of VEGF mRNA was less (P = 0.02) in cotyledonary tissue from placentas in the mSOF group (0.17 ± 0.07) compared with the in vivo (0.46 ± 0.06) and IVPS (0.50 ± 0.06) groups (Fig. 3A). In contrast, the expression of PPARG mRNA was not different in cotyledonary tissue from placentas in the in vivo (1.1 ± 0.3), IVPS (1.1 ± 0.4), and mSOF (0.5 ± 0.4) groups (Fig. 3B).



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  		FIG. 3. A) Expression of VEGF mRNA (least-squares means ± SEM) in bovine cotyledonary tissues of placentas from embryos produced in vivo (n = 6), IVPS (n = 6), or mSOF (n = 6). B) Expression of PPARG mRNA (least-squares means ± SEM) in bovine cotyledonary tissues of placentas from embryos produced in vivo (n = 6), IVPS (n = 6), or mSOF (n = 6). a,bP = 0.02

VEGF and PPARG Protein

Binding of VEGF and PPARG antibodies to proteins in cotyledonary tissue from embryos produced either in vivo or in vitro resulted in bands at approximately 20 kDa and 50 kDa, corresponding to VEGF164 and PPARG1, respectively (data not shown). Preincubation of each antibody with their respective blocking peptide eliminated the antibody signal. The antibody used to quantify VEGF protein has been shown to bind VEGF121, VEGF165, and VEGF189 isoforms in the human uterus [48]. For Western blot analysis of VEGF protein in the current study, multiple bands were detected in some samples. We chose to quantify the band at approximately 20 kDa corresponding to VEGF164 because 1) this band was consistently expressed in all samples, and 2) the assessment of VEGF mRNA levels was based on quantification of the VEGF164 isoform amplification product. Thus, there was consistency between assessment of both VEGF protein and mRNA levels in this study. The expression of VEGF protein in cotyledons was not different between the three treatment groups (Table 3). Similarly, the expression of PPARG protein did not differ in cotyledons from the in vivo, IVPS, or mSOF groups (Table 3).


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  		TABLE 3. Expression of VEGF and PPARG protein in cotyledonary tissues at Day 70 of gestation from embryos produced in vivo or in vitro by either an undefined (IVPS) or semi-defined (mSOF) medium.a

Placental Vascular Morphometry

Blood vessels within the maternal endometrium and fetal villi were visualized using immunohistochemical staining for VEGF protein (Fig. 4). The volume density of fetal blood vessels was decreased (P = 0.03) in placentomes from the mSOF group (4.2% ± 0.4%) compared with the in vivo (5.7% ± 0.3%) and IVPS (6.1% ± 0.4%) groups (Fig. 5). The volume density of maternal blood vessels within placentomes tended to be decreased (P = 0.08) in placentomes from the mSOF group (3.5% ± 0.4%) compared with placentomes from the in vivo group (4.9% ± 0.3%) with the density of maternal blood vessels intermediate in the IVPS group (4.3% ± 0.3%; Fig. 5). Similarly, the density of blood vessels within the total placentome (maternal plus fetal components) was decreased (P = 0.03) in the mSOF group (7.7% ± 0.7%) compared with the in vivo (10.5% ± 0.6%) and IVPS (10.4% ± 0.6%) groups (Fig. 5).



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  		FIG. 4. A representative transverse section of a bovine placentome at Day 70 of gestation resulting from the transfer of a blastocyst produced in vivo. The section was incubated with a primary antibody to VEGF165 with binding visualized by staining tissue sections with diaminobenzidine tetrahydrochloride containing nickel. This staining procedure was used to distinguish blood vessels within both the caruncular endometrium (CE) (large arrow) and the fetal villi (FV) (small arrow). Bar = 10 µm



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  		FIG. 5. Volume densities of blood vessels (least-squares means ± SEM) within the fetal villi, caruncular endometrium, and total placentome at Day 70 of gestation resulting from bovine embryos produced in vivo (n = 6), IVPS (n = 6), or mSOF (n = 5) culture media. a,bP = 0.03; c,dP = 0.08


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study directly compared morphometry and angiogenesis of placentas during early gestation from bovine blastocysts produced in vivo or in vitro using either an undefined medium system (IVPS) or a semidefined medium system (mSOF). The mean values for fetal body weight were not statistically different between the three groups. However, the range in fetal weights in both the IVPS and mSOF groups was consistent with previous reports on fetal and birth weights in cattle [2, 4, 7, 49–51] and sheep [5, 34] following transfer of embryos produced in vitro. The observation that fetal body weights from the mSOF group had the greatest range and the highest percentage of heavier fetuses compared with the in vivo group does not support the premise that problems associated with aberrant growth of fetuses from embryos produced in vitro is primarily due to the presence of serum in culture medium [2, 3, 5, 34, 37]. Several reports have indicated that bovine embryos [31, 32] and calves [51] resulting from embryos cultured in serum-free, semidefined systems had developed abnormally compared with in vivo controls. Variation observed in the body weights of fetuses from embryos produced using mSOF medium in the current study could potentially be due to build up of ammonia in undisturbed culture medium. Gardner et al. [52] suggested that excessive ammonia generated from the breakdown of amino acids may accumulate during culture. In addition, McEvoy et al. [53] has linked excessive ammonia in maternal rations to fetal oversize in sheep. Alternatively, embryos cultured in mSOF medium may have abnormal expression of imprinted genes compared with embryos in the in vivo and IVPS groups, resulting in a greater deviation in fetal body weight. A widely recognized hypothesis to explain aberrant fetal and placental development from in vitro-produced and cloned embryos is abnormal epigenetic reprogramming of the maternal and paternal genome (i.e., imprinting) during embryonic development [54, 55]. Furthermore, Doherty et al. [56] has demonstrated differential imprinted gene expression of H19 in mouse embryos cultured in two different culture systems (Whitten medium and KSOM containing amino acids).

The current study demonstrated that at Day 70 of gestation bovine placentas from embryos produced using mSOF medium were heavier and had a more extreme range in weight than placentas from embryos produced in vivo and in vitro using a serum-supplemented medium (IVPS). Lee et al. [57] found no difference in the weights of placentas from embryos produced using SOF medium compared with artificial insemination at Day 50 of gestation; however, placental weight was more variable in the SOF group. In the present study, no difference was found in placental weights between the in vivo and IVPS groups. We have previously found that placentas resulting from IVPS embryos were heavier during late gestation than in vivo controls [4]. This discrepancy in placental weight between early (Day 70) and late (Day 222) gestation suggests that deviations in growth of placentas from IVPS-derived embryos occur at different stages of gestation. The increased weight of placentas from embryos produced using serum-supplemented medium appears to occur after Day 70 of gestation.

Fetal weight is highly correlated with placental weight throughout gestation in the cow [58]. In several mammalian species, including cattle, the capacity of the placenta has been measured by placental efficiency (fetal weight/placental weight) [47]. In the current study, placental efficiency was lowest for the mSOF group, suggesting reduced capacity of placentas from mSOF embryos to support fetal growth during gestation. Placental efficiency of the in vivo control group was intermediate between the mSOF and IVPS groups.

Placentas from the mSOF group had less placental fluid volume and fewer placentomes compared with the in vivo and IVPS groups. The decreased placental fluid in the mSOF group was unexpected because placental weight was greater than the in vivo and IVPS groups, and placental weight has previously been shown to be highly correlated with placental fluid volume [58]. Traditionally, chorioallantoic fluid was considered to serve as a reservoir for fetal waste because of the direct relationship between the allantois and the fetal urogenital tract [59, 60]. Accordingly, decreased placental fluid volume observed in the mSOF group may indicate abnormalities with the fetal renal system resulting in inadequate maintenance of fetal fluid volumes [61]. Alternatively, recent evidence in sheep regarding amino acid composition of the chorioallantoic fluid suggests that this fluid is both fetal and maternal in origin, and its volume is maintained via a placental transport mechanism [62]. Therefore, the decrease in placental fluid observed in the mSOF group could be explained by decrease solute transfer for the maternal compartment, which may be reflective of the decreased number of placentomes observed in these placentas.

No differences were observed between the in vivo, IVPS, or mSOF groups in the volume densities of maternal endometrium or fetal villi at Day 70 of gestation. In contrast, during late gestation (Day 222), we found that the density of fetal villi was decreased in placentomes derived from IVPS embryos compared with controls, suggesting decreased feto-maternal contact area [4]. Taken together, these findings indicate that fetal villous development at Day 70 of gestation is similar for placentas from embryos produced either in vivo or in vitro, and that alterations in villous formation of placentas from in vitro-produced embryos is not evident until later in gestation when more extensive branching of villi occurs [63].

The volume density of fetal binucleate cells was greater in placentomes in the IVPS group compared with the in vivo group. These findings are consistent with a previous study in which we found increased fetal binucleate cells in placentas from embryos produced using serum-supplemented medium (IVPS), as well as serum-restricted medium, at Day 70 of gestation compared with in vivo controls (unpublished results). In placentas from NT-derived embryos, Ravelich et al. [64] reported that binucleate cells were increased at Days 50, 100, and 150 of gestation compared with placentas from either artificially inseminated or in vitro-produced embryos. Of interest, these authors found no difference in binucleate cells of placentas from in vitro-produced embryos compared with those artificially inseminated at Days 50, 100, and 150 of gestation [64]. The increased volume density of binucleate cells in placentas from the IVPS group at Day 70 of gestation imply that these placentomes may have an increased availability of placental lactogens and pregnancy-associated glycoproteins, suggesting enhanced placental development [65, 66].

Placentomes from the mSOF group had a greater volume density of fetal pyknotic cells compared with those from the in vivo or IVPS groups. This finding implies that fetal villi of placentomes from mSOF embryos had increased cell death, either by apoptosis or necrosis [67, 68]. Excessive apoptosis or necrosis within the placental villi of humans at term has been associated with preeclampsia, a pathological condition characterized by abnormally shallow invasion of extravillous cytotrophoblast in the decidua [69]. The increased incidence of cell death observed in the fetal villi of the mSOF group may indicate compromised placental development. Alternatively, it has been suggested that apoptosis may contribute to placental remodeling in the sheep, resulting in expansion of the villous architecture [70]. Therefore, the increase in cell death in fetal villi of the mSOF group may indicate enhanced remodeling of the placentome, thereby resulting in more complex villous structure.

Proper development of the placental vasculature is critical for determining the rate of placental blood flow, and thereby providing adequate exchange of respiratory gases, nutrients, and waste between the mother and fetus [18]. Vascular development of the placenta is initiated by vasculogenesis and subsequently controlled by branching and nonbranching angiogenesis [19]. Molecular regulation of angiogenesis in the placenta is driven by angiogenic factors, including VEGF, FGFB, and angiopoietins [17, 19, 20]. In the present study, the levels of VEGF mRNA were lower in cotyledonary tissue in the mSOF group. This finding implies that the fetal component of the placentome had decreased angiogenesis as assessed by levels of VEGF mRNA. Of interest, no differences due to treatment were found for VEGF protein in cotyledonary tissue. This discrepancy may be due to a lack of sensitivity of the Western blot technique to detect relatively small differences in VEGF protein levels between treatments.

The five isoforms of VEGF mRNA identified in mammalian tissues include VEGF120, VEGF144, VEGF164, VEGF188, and VEGF205 [40]. In most tissues, the most abundant isoform expressed is VEGF164, which has been shown to possess the greatest mitogenic and vascular permeability properties [40, 71]. In bovine cotyledonary tissue at Day 70 of gestation, VEGF120, VEGF164, and VEGF188 were detected regardless of treatment group. Furthermore, the predominant VEGF isoform detected in cotyledons at Day 70 of gestation was VEGF164. These findings are consistent with our previous report [4] in which VEGF120, VEGF164, and VEGF188 were detected in bovine cotyledons in late gestation (Day 222 of gestation), and the predominant isoform in these tissues was VEGF164. In ovine placentas from Days 60 to 140 of gestation, VEGF120, VEGF144, VEGF164, and VEGF188 were present, and the predominant isoform was VEGF164 [40].

PPARG is a transcription factor that has been shown to up-regulate VEGF mRNA expression in vascular smooth muscle cells [72] and macrophages [73]. Similarly, PPARG null mice have been shown to be embryonically lethal due to an interference of terminal differentiation of the trophoblast and placental vascularization [21]. In the human trophoblast, PPARG has been shown to regulate differentiation of extravillous cytotrophoblast [22]. Furthermore, increased levels of protein for PPARG in bovine caruncular tissues from embryos produced in vitro were associated with enhanced vascular development in these placentomes [4]. Although PPARG mRNA and protein were expressed in cotyledonary tissues of all three treatment groups at Day 70 of gestation, no differences due to treatment were found for either mRNA or protein levels. This finding suggests that PPARG may not be associated with decreased expression of VEGF mRNA in cotyledons from the mSOF group. It is possible that PPARG expression may be altered before Day 70 of gestation, which then resulted in the observed decrease in expression of VEGF mRNA in cotyledons from the mSOF group at Day 70 of gestation. Alternatively, PPARG may not be the primary regulator of VEGF in the bovine placenta, but rather VEGF in the bovine placenta may be regulated by other factors such as hypoxia or estradiol [18, 71].

The volume densities of fetal and total blood vessels in placentomes were decreased in the mSOF group compared with the in vivo and IVPS groups. In addition, the density of maternal blood vessels in placentomes tended to be decreased in the mSOF group. These findings suggest that vascular development was deficient in both the maternal and fetal components of placentomes resulting from embryos produced using mSOF medium. This deficiency in vasculature of placentomes may have been due to decreased expression of mRNA for VEGF or perhaps other angiogenic factors such as FGFB or angiopoietins [17, 18].

In summary, placentas from embryos produced using the semidefined mSOF medium had a greater degree of aberrant development during early gestation than placentas from embryos produced in vivo or in vitro using an undefined serum-supplemented medium (IVPS). Placentas from embryos produced using mSOF were heavier and had decreased placental efficiency, fewer placentomes, and decreased placental fluid. In addition, placentomes in the mSOF group had an increased volume density of fetal pyknotic cells. Placentomes in the mSOF group had decreased densities of blood vessels and decreased levels of VEGF mRNA in cotyledonary tissue, suggesting insufficient vascular development. Taken together, these observations suggest that the use of the semidefined mSOF medium does not appear to alleviate developmental abnormalities of the placenta associated with in vitro production of embryos, but rather may intensify these problems during early gestation.


   ACKNOWLEDGMENTS
 
We thank Drs. John Gadsby, Jorge Piedrahita, and Robert Petters for their critical review of the manuscript. Special thanks to Martin's Abattoir & Wholesale Meats, Inc., Godwin, NC, for providing assistance with collection of tissues for this study. We also thank KAMAR Inc., Steamboat Springs, CO, for donating KAMAR Heatmount detection patches.


   FOOTNOTES
 
1 Supported by the U.S. Department of Agriculture Animal Health Formula Funds and the State of North Carolina. Back

2 Correspondence: Peter W. Farin, Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606-1499. FAX: 919 513 6464; peter_farin{at}ncsu.edu Back

Received: 9 February 2005.

First decision: 8 March 2005.

Accepted: 13 May 2005.


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TOP
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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