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BOR - Papers in Press, published online ahead of print December 17, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.020198
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BIOLOGY OF REPRODUCTION 70, 1114–1120 (2004)
DOI: 10.1095/biolreprod.103.020198
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Expression of Trophoblast Cell-Specific Pregnancy-Related Genes in SomaticCell-Cloned Bovine Pregnancies1

Osman V. Patel4, Osamu Yamada4, Keiichiro Kizaki3,4, Toru Takahashi4, Kei Imai4, Seiya Takahashi5, Yoshiaki Izaike4, Linda A. Schuler6, Toshiaki Takezawa4, and Kazuyoshi Hashizume2,4

National Institute of Agrobiological Sciences,4 Tsukuba, Ibaraki 305-8602 Japan National Institute of Livestock and Grassland Sciences,5 Tsukuba, Ibaraki 305-0901 Japan School of Veterinary Medicine,6 University of Wisconsin, Madison, Wisconsin 53706


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We compared the expression of bovine prolactin-related protein-1 (bPRP-1), placental lactogen (bPL), and pregnancy-associated glycoproteins-1 (bPAG-1) and -9 (bPAG-9) genes in artificially inseminated (AI) and nuclear transferred (NT) cows during the first trimester of gestation using real-time reverse transcription-polymerase chain reaction and in situ hybridization. Placentomal (cotyledonary, caruncular) and interplacentomal (intercotyledonary, intercaruncular) tissues of AI and NT cows carrying either motile (M) or immotile (IM) fetuses were examined. Transcripts for bPL and bPAG-9 were lower (P < 0.01) in the fetal membranes of NT (n = 4) cows at Day 30 of gestation, compared with AI (n = 4) cows. There was no difference in the mean (± SEM) levels of expressions of bPRP-1, bPL, and PAG-1 in the placentomal and interplacentomal tissues of AI (n = 5) and NT (M, n = 4) cows at Day 60 of gestation. The mRNAs for bPRP-1, bPL, bPAG-1, and bPAG-9 genes were higher (P < 0.01) in the caruncular tissue of AI cows, compared with NT (IM, n = 4) cows at Day 60 of gestation. Expression of bPRP-1, bPL, bPAG-1, and bPAG-9 in the placentomal and interplacentomal tissues of the NT (n = 3) group varied considerably more, compared with the AI (n = 4) group at Day 100 of gestation. These findings suggest defective binucleate cell-specific gene transcriptional commands in NT cows.

conceptus, placenta, pregnancy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One of the significant advances of the past decade was the production of viable offspring by somatic cell cloning [1, 2]. Subsequently, live births using adult somatic cells have been reported in the major commercially farmed cloven-hoofed species [1, 3–5]. However, a major impediment to the widespread application of the technique has been the high proportion of embryonic/fetal losses, especially during the first trimester of gestation, and increased perinatal/neonatal mortality [1, 6–9]. A plethora of abnormalities affecting different systems has been described in this group of animals [4, 7, 9–12], but a common finding in most clone-associated pathologies is atypical placentation [4, 7, 9–13]. In sheep, cotyledonary agenesis to hypoplasia has been described [9, 14]. Fetomegaly to placentomegaly, including aberrations of the cytoskeletal architecture of the placenta, especially of the invading trophoblast cells, is reported in mice [2, 12, 15, 16]. In cloned cattle, placentomal agenesis, rudimentary cotyledons, decreased numbers of cotyledons, aberrations in the allantoic epithelium and vascularization, and hydrops of fetal membranes are common [7, 8, 10, 13, 17]. The trophoblast cells induce the repertoire of genes involved in implantation and placentogenesis in all mammals [18]. Faulty gene expressions during implantation and placentogenesis have been suggested to contribute to the pathologies observed in cloned offspring [12, 19–21]. Several studies have reported deviations from normal expression patterns of a number of imprinted genes in different species [12, 15, 20, 22–25].

A unique feature of bovine placentation is the migration of trophoblast-derived binucleate cells (BNCs) at implantation and continuing across gestation through chorionic tight junctions to fuse with the uterine epithelial cells [26, 27]. BNCs are directly involved in modification of the uterine epithelium beginning at implantation and continuing until term [26, 27]. Hence, the BNC plays a pivotal role in fetomaternal communication in the bovine. The said cells produce an array of proteinaceous compounds including bovine placental lactogens (bPL), prolactin-related protein-1 (bPRP-1), and pregnancy-associated glycoproteins (bPAG) [28–30]. These proteins are detectable in the peripheral circulation following definitive attachment of the trophoblast to the endometrium [31–33]. Heyman et al. [34] reported significantly higher peripheral levels of pregnancy-associated protein in cows carrying cloned pregnancies, especially after Day 50 of gestation. Incidentally, all the previous studies looking at abnormal gene expression patterns in cloned animals focused primarily on imprinted genes using the semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) method for their investigations [22–24]. However, the effect of cloning on the in vivo expression of trophoblast cell-specific genes, particularly in cattle, has not been investigated. Therefore, in this study, changes in the expression and localization of bPRP-1, bPL, bPAG-1, and bPAG-9 in the placentomal (cotyledonary, caruncular) and interplacentomal (intercotyledonary, intercaruncular) tissues during the first trimester of pregnancies derived from artificially insemination (AI) and nuclear transfer (NT) cattle were examined using the real-time RT-PCR method and in situ hybridization.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Source for Nuclear Transferred Somatic Cells

The somatic cell nuclear embryos used in this experiment were derived from bovine ovarian cumulus cells. Ovaries were obtained from a local abattoir and transported at 25–30°C to the laboratory. Cumulus oocyte complexes (COCs) were aspirated from follicles 2–8 mm in diameter and placed into sterile plastic tubes. Cumulus cells were harvested from the COCs, and cultured in Dulbecco minimal essential medium plus 10% fetal bovine serum (FBS), 100 IU/ml penicillin G potassium, and 100 µg/L streptomycin at 37.5°C in a humidified atmosphere of 5% CO2 and 95% air. For nuclear transfer, frozen-thawed cells were subcultured up to confluence and utilized following culture in serum-deficient medium for 5 days.

Nuclear Transfer

The details of producing nuclear embryos are well described by Kato et al. [3]. Briefly, COCs with at least two to three compact layers of cumulus cells were selected and cultured for 20 h in TCM 199 (GIBCO BRL, Tokyo, Japan) supplemented with 10% FBS at 38.5°C in an atmosphere of 5% CO2 in air. After maturation, metaphase II oocytes were enucleated using a beveled glass pipette, and donor cells were combined with enucleated oocytes. Fusion of the oocyte cell complex was induced by a single DC pulse (25 V/150 µm for 10 µsec, Electro cell fusion model LF101 [BEX, Tokyo, Japan]) in Zimmerman mammalian cell fusion medium. After electric stimulation, the fused oocytes were treated with 2.5 µg/ml cytochalasin D (Sigma, St. Louis, MO) plus 1 µg/ml cycloheximide (Sigma) for 1 h and 10 µg/ml cycloheximide alone for a further 4 h in CR1aa medium. Nuclear-transferred embryos were cultured in CR1aa medium for 48 h at 38.5°C in 5% CO2, 5% O2, and 90% N2. Cleaved embryos were cocultured with bovine cumulus cells in CR1aa medium supplemented with 5% FBS. On Day 7, the blastocyst was transferred nonsurgically to synchronized recipient Japanese black cows.

Animals and Sample Collections

Control pregnancies were derived by AI of Japanese black cows (Day 0 = day of insemination). Diagnosis of pregnancy was made from Days 30 to 60 of gestation by transrectal ultrasonography (7.5 MHz linear probe, SSD-1700; Aloka, Tokyo, Japan). Viability of the fetus was confirmed by a detectable heartbeat 2–3 days prior to slaughter. Fetuses with detectable and indiscernible heartbeats were grouped as motile (M) and immotile (IM), respectively. NT and AI cows were slaughtered around Days 30, 60, and 100 of gestation. The endometrial and placental tissues were collected and separated as detailed by Nikitenko et al. [35] and Regnault et al. [36] and illustrated schematically by Schauser et al. [37]. The endometrial tissue was separated into caruncular and intercaruncular areas for comparison during pregnancy. Similarly, fetal placental tissue from pregnancy was separated into cotyledonary and intercotyledonary tissues. The whole allantochorionic membrane was collected as fetal membrane on Day 30 of gestation. Collected fetal membrane, cotyledonary, intercotyledonary, caruncular, and intercaruncular specimens were immediately placed in liquid nitrogen and stored at -80°C prior to processing. In addition, intact placentomes were also collected and immediately embedded into O.C.T. compound (Tissue-Tek; Sakura, Tokyo, Japan) in cold isopentane and stored at -80°C until processed.

The committees of Animal Care and Experimentation of the National Institute of Agrobiological Sciences and National Institute of Livestock and Grassland Sciences approved this experiment.

Real-Time PCR

Total RNA was extracted from the placental and uterine tissues using ISOGEN (Nippon Gene, Kyoto, Japan) according to the manufacturer's instructions. Fifty nanograms of total RNA was reverse transcribed into cDNA and used for this real-time RT-PCR analysis. A quantitative detection method, TaqMan real-time RT-PCR [38], was used for analyzing mRNA levels in tissues as previously described [39]. Probes and primers were designed using the primer express software version 1.0 following manufacturer's instructions (Applied Biosystems, Foster City, CA). Briefly, the sequence of primers and probes for bPAG-1, bPAG-9, and bPRP-1 were chosen from regions of nucleotide sequence that provided the most variability from the other members of bPAG and bPRP families (Table 1). Real-Time RT-PCR detection was done using an ABI PRISM 7700 sequence detector and software version 1.7 (Applied Biosystems). The reaction mixture contained 1 µM primers, 0.25 µM TaqMan probe, 0.2 mM deoxynucleotide triphosphates, 5.5 mM magnesium chloride, and TaqMan buffer and was dispensed into a 96-well plate and amplified. The thermal cycling conditions included 2 min at 50°C and 10 min at 95°C. Thermal cycling proceeded with 40 cycles of 95°C for 15 sec and 60°C for 1 min. The standard curves for each gene were generated by serial dilution of plasmid containing bPRP-1 or bPL or bPAG-1 or bPAG-9 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA to quantify mRNA concentrations. A ratio of bPRP-1, bPL, bPAG-1, and bPAG-9 mRNA to GAPDH mRNA was calculated to adjust for any variations in the RT-PCR reaction.


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  		TABLE 1. Sequence of primers and probes used for real-time RT-PCR

In Situ Hybridization

Collected intact placentomes were immediately embedded into O.C.T. compound (Tissue-Tek; Sakura) in cold isopentane and stored at -80°C until processed. Embedded tissues were cut into 5-µm sections by a cryostat microtome (HM500 MICROM; Laborger GmbH, Heidelberg, Germany) and placed on poly-L-lysine coated slides (Matsunami, Tokyo, Japan). In situ hybridization of bPRP, bPL, bPAG-1, and bPAG-9 was performed as detailed elsewhere [17, 39]. Briefly the digoxigenin (Dig)-labeled single-strand cRNA probes were prepared using a Dig RNA labeling kit (Roche Molecular Biochemicals, Basel, Switzerland) according to the manufacturer's instructions. Then the cDNA fragments of bPL (GenBank accession no. J02840), bPRP-1 (GenBank accession no. J02944), and bPAG-1 (GenBank accession no. M73962) and bPAG-9 (GenBank accession no. AF020511) were subcloned into the HindIII-EcoRI sites of the pSPT 18 vector. The resulting plasmid was either linearized with HindIII followed by transcription with T7 RNA polymerase to generate the antisense probe or linearized with EcoRI followed by transcription with SP6 or T3 RNA polymerase to generate the sense probe. Dig-labeled ß-actin cRNA probe prepared under an identical procedure served as a control.

Statistical Analysis

All values are presented as mean ± SEM. Data were analyzed initially by nonparametric ANOVA and followed by either Tukey-Kramer multiple comparison test or Student t-test. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Significantly fewer (P < 0.05) placentomes were found in the NT group around Day 60 of gestation (28.3 ± 11.5, 34.3 ± 6.3, and 85.4 ± 5.9 in NT cows carrying immotile [IM, n = 4] and motile [M, n = 4] clones and AI [n = 5] cows, respectively). There was no significant difference in the fetal crown-rump length (CRL) between AI (6.3 ± 0.5 cm) and NT (M; 7.0 ± 0.5 cm) cows, but the size of fetus in the NT (IM; 3.4 ± 0.6 cm) group was significantly (P < 0.01) smaller than that of AI and NT (M) groups. There was no significant difference in either the fetal CRL or placentome number between AI (19.4 ± 0.9 cm and 93.6 ± 7.8, respectively) and NT (18.1 ± 1.8 cm and 78 ± 18.3, respectively) cows at Day 100 of gestation.

Expression of Trophoblast Cell-Specific Genes Around Day 30 of Gestation

There was no difference in the levels of bPRP-1 and bPAG-1 transcripts in the fetal membrane and endometrium of AI (n = 4) and NT (n = 4) cows at Day 30 of gestation (Figs. 1a and 2a). The expression levels of bPL and bPAG-9 in the fetal membrane were significantly (P < 0.01) higher in AI than NT cows at Day 30 of gestation (Figs. 1b and 2b).



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  		FIG. 1. Expression (mean ± SEM) levels of bPRP-1 (a) and bPL (b) transcripts in fetal membrane (FM), cotyledonary (COT), intercotyledonary (ICOT), caruncular (CAR), and intercaruncular (ICAR) tissues of AI and NT cows carrying motile and immotile fetuses. The whole allantochorionic membrane was collected as fetal membrane on Day 30 of gestation. a, bMeans with different letters are significantly different (P < 0.01)



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  		FIG. 2. Expression (mean ± SEM) levels of bPAG-1 (a) and bPAG-9 (b) transcripts in fetal membrane (FM), cotyledonary (COT), intercotyledonary (ICOT), caruncular (CAR), and intercaruncular (ICAR) tissues of AI and NT cows carrying motile and immotile fetuses. The whole allantochorionic membrane was collected as fetal membrane on Day 30 of gestation. a) The insets in the Days 30 and 60 panel are the same results shown on a different Y-scale. a,bMeans with different letters are significantly different (P < 0.01)

Expression of Trophoblast Cell-Specific Genes Around Day 60 of Gestation

There was no difference in the mean (± SEM) expression levels of bPRP-1, bPL, bPAG-1, and bPAG-9 in cotyledonary, intercotyledonary, caruncular, and intercaruncular tissues of AI (n = 5) and NT (M, n = 4) cows at Day 60 of gestation (Figs. 1 and 2). However, there was a large variation observed in the NT (M) group, especially with bPL expression. The transcription level of bPL was significantly (P < 0.01) higher in the cotyledonary and intercotyledonary tissues of the AI group than in the NT (IM, n = 4) group at Day 60 of gestation (Fig. 1b). The expression levels of bPRP-1, bPL, bPAG-1, and bPAG-9 were significantly (P < 0.01) higher in the caruncular tissue of AI cows, compared with NT (IM) cows at Day 60 of gestation (Figs. 1 and 2). The bPAG-9 expression was significantly (P < 0.01) higher in the intercotyledonary tissue of AI cows, compared with NT (M) and (IM) cows at Day 60 of gestation (Fig. 2b).

Expression of Trophoblast Cell-Specific Genes Around Day 100 of Gestation

There was a large variation seen in the different tissues of the NT (n = 3) group at this stage of gestation (Figs. 1 and 2). The level of bPL transcription in the intercaruncular area was significantly (P < 0.01) higher in NT cows, compared with AI (n = 4) cows.

In Situ Hybridization

There was no difference in the localization of bPRP-1, bPL, bPAG-1, and bPAG-9 in the placentomal and interplacentomal tissues of AI and NT (M) cows at Days 60 (Fig. 3) and 100 (data not shown) of gestation. Expression of bPL mRNA was low to undetectable in the cotyledonary tissue of NT (I/M) cows at Day 60 of gestation (Fig. 4). There was limited localization of bPRP-1, bPL, bPAG-1, and bPAG-9 in the caruncular tissue of NT (I/M) cows at Day 60 of gestation (Fig. 4).



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  		FIG. 3. Localization of bPRP-1, bPL, bPAG-1, and bPAG-9 mRNA in the placentomal and interplacentomal tissues of AI and NT (M) cows at Day 60 of gestation. Scale bar = 200 µm



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  		FIG. 4. Localization of bPRP-1, bPL, bPAG-1, and bPAG-9 mRNA in the placentomal and interplacentomal tissues of NT (IM) cows at Day 60 of gestation. Scale bar = 200 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The BNCs are considered to play a pivotal role in implantation, placentation, and fetomaternal communication in ungulates that is analogous to the role played by human extravillous trophoblast and rodent giant cells [18, 26, 27]. The BNCs produce multiple proteinaceous molecules similar to the giant cells of rodents [27–30]. Faulty gene expression during implantation and placentogenesis has been suggested to contribute to the pathologies observed in cloned offspring [12, 15, 19–21]. A common finding reported in cattle and sheep is atypical placentation including fewer placentomes [3, 7–11, 13, 14]. In the present study, fewer placentomes developed by Day 60 of gestation in both NT (M) and (IM) cows. However, there was no association between viability of the fetus and number of placentomes. This is in agreement with Hill et al. [13], who reported the birth of a viable cloned calf with only 12 functional placentomes. The fetal size in the NT (IM) group was significantly reduced, compared with the other two groups. The precise cause of stunted growth is unknown; fetomegaly is commonly reported with NT pregnancies [7, 12, 14]. This notwithstanding, our findings are in agreement with Hill et al. [10], who used ultrasonography to show that fetuses destined for death had reduced crown-rump length. Therefore, either alterations in the expression pattern of developmentally important genes or fetomaternal-placental asynchrony may be a likely cause. However, by the end of the first trimester, the fetal size and numbers of placentomes were comparable between AI and NT cows, suggesting a delay in placentogenesis in NT cows.

Two detailed studies have reported deviations from normal expression patterns for several imprinted genes in cattle [22, 24]. Two other studies reported that the expression of interferon-{tau}, a trophoblast gene originating from the mononucleate trophoblast cells, was heavily affected by the cloning procedure [23, 40]. However, this is the first study to quantitate the expression patterns between AI and NT pregnancies of four BNC-specific transcripts thought to play a crucial role in placentogenesis and fetomaternal communication. Although the precise functions of bPRP-1, bPL, bPAG-1, and bPAG-9 in the bovine are not known, they are believed to regulate placentogenesis and fetogenesis, mammogenesis, steroidogenesis, and immune activity [41–44]. They are all produced by the placental BNCs that appear just prior to implantation in the cytotrophoblast layer and play a crucial role in placentation in ungulates, including formation of the fetomaternal syncytium and production of placental hormones [27–30]. There was a significant difference in the levels of transcripts of bPL and bPAG-9 between the AI and NT groups during the peri-implantation period. Hill et al. [45] reported that NT cows expressed significantly higher levels of major histocompatibility complex (MHC)-1 during the peri-implantation period, compared with their natural cohorts. However, during normal pregnancy, trophoblast MHC-I expression is normally suppressed to prevent a maternal lymphocytic response [46]. The noted immunological reaction in the NT pregnancies [45] could be due to insufficiency of bPAGs at this stage, especially bPAG-9 because bPAGs induce the release of an alpha chemokine from the bovine endometrium [43].

The caruncular tissue of NT (IM) cows contained significantly less of all four BNC-specific transcripts at Day 60 of gestation. The real-time RT-PCR expression profiles paralleled the localization patterns of bPRP-1, bPL, bPAG-1, and bPAG-9 in the caruncular tissue of NT (I/M) cows at the same stage. Concurrent with the reported gross malformations of placenta in NT are microstructural aberrations of the anchoring chorionic villi [10, 17]. The latter leads to a disunion between the fetal and maternal units in NT cows [10, 17]. Plausibly poor apposition between the chorionic and endometrial epithelia directly affects the migration of BNCs, formation of fetomaternal syncytia, and exocytoses of secretory granules in NT pregnancies. In addition, fewer BNCs are reported to occupy the trophoblastic layer of the NT cows [17]. Thus, the fate of fetuses in the NT (IM) group could be a consequence of an asynchronous dialogue between the fetoplacental and maternal units culminating from the broad ultrastructural anomalies in the apposing trophoblastic layer that impede transit of trophoblastic-specific signal(s) to the maternal unit.

The precise cause of the marked variation in expression patterns of placentomal and interplacentomal bPRP-1, bPL, bPAG-1, and bPAG-9 genes in NT cows at Day 100 of gestation was not exhaustively investigated. It is plausible that a compensatory mechanism is activated in the NT group to meet the growing demands of the developing fetus at the expense of limited available placentomes. Earlier studies have reported that NT cows have poorly developed placentomes [8, 10, 17]. It is also established that the BNCs do migrate into the intercaruncular area of the bovine uterus, but the frequency is lower, compared with the caruncular region [46]. The fetus directly controls BNC production [47], and the abnormal induction seen in NT cows could be due to either anomalous migration of BNC or an inadequate fetal signal as a result of an unknown genetic aberration(s). In fact, both cotyledonary and intercotyledonary placenta are supplied by the same fetal blood supply [48]. Heyman et al. [34] found significantly higher peripheral levels of pregnancy-associated protein after Day 50 of gestation in NT cows. In cloned mice, specific aberrations in glycogen and spongiotrophoblast cells have been noted [16], but the authors did not investigate whether these anomalies led to alterations in the expression patterns of trophoblast-specific genes/proteins. In our study, we did not specifically compare and contrast the proliferation and differentiation of the BNCs between nonmanipulated and NT cows except for the comparison of genes expressed by the same cells. Interestingly, Wells et al. [8] found that NT cows had inadequate preparation of the birth canal at parturition as well as poor mammary gland development. It is well known that placental hormones have direct/indirect actions on mammogenesis during pregnancy and the cascade of events leading to parturition [49, 50]. The same deficiencies in birth canal preparation and mammogenesis observed in NT cows have also been noted in NT sheep [14]. The prolactin/growth hormone family members synthesized by the fetoplacental unit are implicated in regulation of mammogenesis, lactogenesis, and steroidogenesis during pregnancy [41, 42, 49]. Although we did not examine placentae from later in gestation, it is tempting to speculate that the aberrant transcription of trophoblastic cell-related genes persists in NT cows. The disparate patterns of the four BNC-specific transcripts appear to be indirect indicators of atypical differentiation and development of the extraembryonic membranes in cloned cattle, in particular the migration of BNCs.

In conclusion, well-orchestrated patterns of gene expression are pivotal to the establishment and progression of pregnancy. Our results suggest an aberration in the proliferation and differentiation of the trophoblast cells, especially the BNCs that may result from defective pregnancy-related gene transcriptional commands in NT cattle.


   FOOTNOTES
 
1 This research was supported by the Organized Research Combination System (ORCS) from the Education, Science, and Technology Agency and the Bio-oriented Technology Research Advancement Institution (BRAIN), Japan. O.V.P. was supported by an STA fellowship from JST. Back

2 Correspondence: Kazuyoshi Hashizume, Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan. FAX: 81 19 621 6212; kazuha{at}iwate-u.ac.jp Back

3 Current address: School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan Back

Received: 11 June 2003.

First decision: 7 July 2003.

Accepted: 1 December 2003.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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