Expression of receptor related to the polymorphism in the PGR gene in the rabbit reproductive tract1

R. Peiró,*2 A. Herrler,† M. A. Santacreu,* M. Merchán,‡ M. J. Argente,§ M. L. García,§ J. M. Folch,‡ and A. Blasco*

*Instituto de Ciencia y Tecnología Animal, Universidad Politécnica de Valencia, PO Box 22012, 46071 Valencia, Spain; †Department of Anatomy and Reproductive Biology, Medical School, RWTH University of Aachen, Wendlingweg 2, 52057 Aachen, Germany; ‡Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; and §Departamento de Tecnología Agroalimentaria, Universidad Miguel Hernández de Elche, 03312 Orihuela, Spain

ABSTRACT: The association of the 2464G > A SNP AA genotype in the oviduct (GG/AAPR-B = 0.81 and found in the promoter region of the rabbit progester- GG/AAPR-A = 0.73) and uterus (around 0.70 in both one receptor gene with progesterone receptor (PR) ex- isoforms). The GA genotype showed similar PR-A ex- pression was evaluated by Western blot analysis. This pression in both tissues and also similar PR-B expres- SNP was associated with 2 lines divergently selected sion in the oviduct to the GG genotype. Conversely, for uterine capacity, the high line selected to increase the GG genotype showed less PR-B expression than uterine capacity and the low line selected to decrease the GA genotype in the uterus (GG/GAPR-B = 0.79). uterine capacity. Two progesterone isoforms were ob- Similar expression of both PR isoforms was found in tained using a commercial monoclonal antibody: the the uterus at d 2 and 3 of gestation; meanwhile, an PR-B isoform described previously in rabbits, and the increase of both isoforms was observed in the oviduct. PR-A isoform, not described previously in rabbits. The Similar PR-A expression was observed in the ampulla GG genotype, the genotype more frequent in the high and isthmus; meanwhile, the PR-B expression in the line, showed less PR-B and PR-A expression than the isthmus was double that in the ampulla. Key words: oviduct, PGR genotype, progesterone receptor expression, rabbit, uterus

©2010 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2010. 88:421–427 doi:10.2527/jas.2009-1955

INTRODUCTION described in rabbits (Loosfelt et al., 1984), which has high homology with the PR-B isoform. Progesterone is required in mammals for maternal Different functions have been attributed to each PR support of conceptus, survival, and development. Most isoform. In vitro studies revealed that PR-B functions physiological effects of progesterone are applied by as a strong activator of transcription of several PR- its interaction with the specific progesterone receptor dependent promoters in which PR-A is inactive. In (PR). In humans, primates, pigs, rodent, and chicken, cultured cells, PR-A can repress the activity of PR-B, nuclear PR consist of 2 distinct isoforms, PR-A and as well as other nuclear receptors (Vegeto et al., 1993; PR-B, derived from a single gene independently regu- Hovland et al., 1998). Studies of knockout mice have lated by separate promoters (Kraus et al., 1993; Duffy also provided insight into different physiological func- and Stouffer, 1995). Up to now, only 1 isoform has been tions of each PR isoform for regulating female fertility (Mulac-Jericevic and Conneely, 2004). Selective abla- tion of the PR-A isoform shows uterine hyperplasia and 1 This study was funded with projects MCYT AGL2001-3068-C03 ovarian abnormalities, whereas ablation of the PR-B and AGL2005-07624-C03. The authors are grateful for the excellent assistance of Bärbel Bonn (Department of Anatomy and Reproduc- protein had no effect on these traits. tive Biology, Medical School, RWTH University of Aachen, Aachen, Five polymorphic positions were identified in the PR Germany), Sabine Eisner (Department of Anatomy and Reproduc- gene (PGR) in rabbits: 1 SNP in the promoter, 3 SNP tive Biology, Medical School, RWTH University of Aachen), and in exon 1 (5′UTR), and 1 silent SNP in exon 7. The Raquel Muelas (Departamento de Tecnologia Agroalimentaria, Uni- first 4 SNP segregated in 2 haplotypes. Using the SNP versidad Miguel Hernandez de Elche, Orihuela, Spain). 2 Corresponding author: [email protected] 2464G > A found in the promoter region, the G/G2464 Received March 11, 2009. genotype showed 0.5 kits and 0.5 implanted embryos Accepted September 28, 2009. more than the A/A2464 genotypes. The G/G2464 geno-

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Downloaded from jas.fass.org by Agustin Blasco on January 28, 2010. 422 Peiró et al. type also showed greater embryo survival (0.36 embry- the restriction enzyme Eco31I (Fermentas, Barcelona, os) and development than the A/A2464 genotype at d Spain), and the products were separated on 2% agarose 3 of gestation, whereas no effect was observed in these gels to confirm a 558-bp product. The PCR-RFLP as- traits at d 2 of gestation (Peiró et al., 2008). say yielded 2 bands of 416 and 142 bp (genotype GG), The objectives of this work are to detect differences a single 558-bp band (genotype AA), and all 3 bands in PR expression among PGR genotypes in the rabbit (genotype GA). oviduct and uterus and to assess when the differences appear. PR Expression

MATERIALS AND METHODS A total of 49 pregnant intact F2 does were slaughtered at d 2 or 3 of gestation. From 7 to 9 does were analyzed All experimental procedures were approved by the per genotype and day of gestation. Does were eutha- Polytechnic University of Valencia Research Ethics nized by intravenous injection of in a Committee. dose of 50 mg/kg of BW (Thiobarbital, B. Braun Medi- cal S. A., Barcelona, Spain) at least 1 mo after their 4th Animals parity, when does were not lactating. After euthanasia, the entire reproductive tract was removed. An F1 rabbit population was generated from the The oviduct and uterus were flushed with 5 and 10 reciprocal cross of the high (H) and low (L) lines of mL of 150 mM ammonium bicarbonate buffer, respec- a divergent selection experiment on uterine capacity tively. Oviducts were split into ampulla and isthmus, (UC) described by Argente et al. (1997). Both lines removing the middle piece and fimbriae. Uteri were were derived from a synthetic line (V line) selected for opened at the mesometrial side, and the endometrium 12 generations by litter size (LS) at weaning (Estany was sampled by scraping off the surface tissue. Ovi- et al., 1989). The H and L lines were selected for 10 ductal, as well as uterine, samples were submerged in generations, and then the selection was relaxed until 1.5 mL of 0.15 M ammonium bicarbonate buffer and the 17th generation. Uterine capacity was estimated as snap-frozen in liquid . Samples were stored at LS in unilaterally ovariectomized females by removing −20°C before analyses. Protein content was determined the left ovary of the doe before puberty via midventral by DC Protein Assay (Bio-Rad, Munich, Germany) incision between 14 and 16 wk of age (Blasco et al., according to Lowry et al. (1951). One hundred five 1994). An F2 population was constituted by crossing and 60 µg of protein were used in the oviductal and the divergent lines. The process is described in Peiró uterine samples, respectively, diluted in loading buf- et al. (2008). The F2 population was reared at the ex- fer [5% (vol/vol) β-mercaptoethanol; 30 mM Tris, pH perimental farm of the Universidad Miguel Hernández 6.8; 1.5 M urea; 7.5% (vol/vol) glycerol; 0.5% (wt/vol) (Orihuela, Spain), whereas the H and L lines and the SDS; 0.05% (wt/vol) Bromophenol Blue]. Samples were F1 population were reared at the experimental farm of heated to 100°C for 3 min and subjected to 1D SDS- the Polytechnic University of Valencia. Animals were PAGE using 5% stacking gels and 7.5% resolving gels fed a commercial diet, and the photoperiod used was under reducing conditions. For size determination, a 16 h light: 8 h dark. prestained molecular weight marker (PageRuler, Fer- mentas) was used. After electrophoretic separation, the Genotyping of the Progesterone proteins were transferred to a polyvinylidene diflouride Receptor Gene membrane (Millipore, Schwalbach, Germany) by semi- dry blotting (Kyhse-Andersen, 1984) for 30 min at 100 Blood samples, at least 3 mL, were collected from mA. After transfer, the membrane was blocked for 1 the marginal ear vein by venipuncture. Genomic DNA h with 5% (wt/vol) milk powder in Tris-buffered sa- was extracted from whole blood following the protocol line (TBS)/Tween-20 (0.1% wt/vol) at room tempera- of the ABI PRISM 6100 Nucleic Acid PrepStation (Ap- ture. A primary antibody against mouse PR was used plied Biosystems, Foster City, CA). A PCR-RFLP was (mouse anti-rabbit progesterone receptor; hPRa-1, Di- developed for further testing. Using the rabbit PGR anova, Hamburg, Germany) at a concentration of 33 gene sequence (GenBank accession No. X06623), the pg of protein per µL of TBS containing 5% milk pow- designed primers were forward 5′–GAA GCA GGT der. The monoclonal antibody recognized an epitope CAT GTC GAT TGG AG–3′ and reverse 5′–CGC present in both PR isoforms. The blot was incubated CTC TGG TGC CAA GTC TC–3′. These were used at 4°C for 12 h. After 3 washes in TBS/0.1% Tween, with 50 ng of genomic DNA in a 25-µL PCR, 0.5 µM the second antibody was added and left at room tem- each primer, 200 µM dNTP, 1.5 mM MgCl2, 1 × Taq perature for 1 h. After 3 washes, detection of the sec- reaction buffer, and 0.6 U of AmpliTaq Gold (Applied ond antibody [goat anti-mouse horseradish peroxidase Biosystems). Conditions were 95°C for 10 min, followed (HRP), DAKO, Hamburg, Germany; 1:200] was per- by 35 amplification cycles of 95°C, 30 s; 66°C, 60 s; and formed using enhanced chemiluminescence; ECLplus 72°C, 90 s. The program ended with a 15-min exten- (Amersham, Little Chalfont, UK) for ampulla and isth- sion at 72°C. The PCR fragment was digested with mus samples and enhanced chemiluminescence (ECL)

Downloaded from jas.fass.org by Agustin Blasco on January 28, 2010. Progesterone receptor isoforms of PGR genotypes 423 Table 1. Number of samples, and raw means (M) and SD for arbitrary densitometry units of progesterone receptor (PR)-B and PR-A in the oviduct and uterus

PR-B PR-A

Item No. M SD M SD

Oviduct 98 0.63 0.48 0.31 0.19 Uterus 46 3.32 1.37 0.42 0.24 reagents (Amersham) for uterine samples. The blot was being >1 when its posterior median is greater than 1 exposed to x-ray film (X-Omat UV Plus, Kodak, New and the probability of this ratio being <1 when its pos- York, NY) for 1 min. The intensities of the immunore- terior median is less than 1. Bounded flat priors were active bands were converted into digitalized signals us- used for all unknowns. Marginal posterior distributions ing a computerized densitometry system and quantified of all unknowns were estimated by Gibbs sampling. by a gel documentation program (Image Master 1D, After some exploratory analyses, we used 1 chain of Amersham). Signal intensities of PR isoforms were nor- 100,000 samples, discarding the first 20,000 and saving malized to those of β-actin and positive control (T47D every 10 thereafter. Convergence was tested using the cells) as ratios to produce arbitrary densitometry units Z criterion of Geweke and Monte Carlo sampling errors (ADU) of relative abundance. The protocol for the were computed using time-series procedures described detection of β-actin was as follows: monoclonal anti by Geyer (1992). β-actin (Clone AC15, Abcam Limited, Cambridge, UK: 1:50,000 in TBS/0.1% Tween; goat anti-mouse HRP RESULTS 1:5,000; ECL). The expression of 2 PR isoforms, PR-B and PR-A, Statistical Analyses was observed in the oviduct and uterus of pregnant rabbits at d 2 and 3 of gestation. The 2 isoforms where The model used to analyze PR expression in the ovi- distinguished by their different sizes, 110 kDa (PR-B) duct sample was and 81 kDa (PR-A; Figure 1). The PR-B expression in the uterus was greater (P < 0.05) than that of PR-A yijkl = µ + Di + Gj + Tk + (D × G)ij + (G × T)jk (Table 1). Features of the estimated marginal posterior distribu- + (D × T)ik + (D × G × T)ijk + eijkl, tions of ADU ratios for both PR isoforms in the oviduct and uterus are presented in Tables 2 and 3, respective- where yijkl is the ADU of each PR expression, Di is the effect of the time of gestation (with 2 levels: d 2 and 3 ly. Marginal posterior distributions were approximately symmetrical, and all Monte Carlo sampling errors were of gestation), Gj is the effect of PGR genotype (with 3 less than 0.005. Lack of convergence was not detected levels: GG, GA, and AA), and Tk is the effect of tissue (with 2 levels: ampulla and isthmus). The expression of by the Geweke test in any chain. PR in the uterus sample was analyzed using the same model excluding the effect of the tissue. PR Isoforms in the Oviduct The analysis was based on Bayesian methods. To compare levels of effects, we estimate the value of their An increase of both isoforms from d 2 to 3 was ob- ratio using the posterior median of this ratio (median). served in the oviduct because the median for the d2/d3 If the median is >1, this shows that the level in the ratio was less than 1 (Table 2; P = 100%). The PR-B numerator is greater than the other level, and the op- expression at d 3 of gestation was 2.4 times the expres- posite is true when the ratio is <1. To evaluate uncer- sion at d 2 of gestation, and the PR-A expression was tainty, we calculate the probability (P) of this ratio 1.6 times greater. In the oviduct, the PR-B expression

Figure 1. (A) Western blot analysis of progesterone receptor (PR)-B and PR-A in ampulla samples at 48 h of gestation. (B) Western blot analysis of β-actin in ampulla samples at 48 h of gestation. Cell extracts were loaded in 105-µg amounts per lane. Lanes: 1, standard (30 µg of T47D cells); 2, homozygous genotype; 3, heterozygous genotype. Marks to the right of gels indicate the positions of PR-B and PR-A; molecular mass markers in kilodaltons are shown on the left side of the gel. Color version available in the online PDF.

Downloaded from jas.fass.org by Agustin Blasco on January 28, 2010. 424 Peiró et al. Table 2. Features of the estimated marginal posterior Table 3. Features of the estimated marginal poste- distributions of the arbitrary densitometry unit (ADU) rior distributions of the arbitrary densitometry units ratio between day of gestation (d 2 and 3), tissue [am- (ADU) ratio between day of gestation (d 2 and 3) and pulla (A) and isthmus (I)], and genotypes of 2464G > genotypes of 2464G > A SNP in the promoter region A SNP in the promoter region of the progesterone (PR) of the progesterone receptor (PR) gene (GG, GA, and receptor gene (GG, GA, and AA) for the PR-B and AA) for the PR-B and PR-A isoforms in the uterus

PR-A isoforms 1 2 3 Item Median HPD95% P, % Item Median1 HPD 2 P,3 % 95% PR-B PR-B d2/d3 0.91 0.70, 1.12 80 d2/d3 0.42 0.29, 0.53 100 GG/AA 0.70 0.53, 0.95 100 A/I 0.50 0.38, 0.61 100 GG/GA 0.79 0.54, 1.05 92 GG/AA 0.81 0.58, 1.02 94 GA/AA 0.87 0.68, 1.17 78 GG/GA 1.02 0.74, 1.33 55 PR-A GA/AA 0.79 0.59, 1.03 95 d2/d3 1.12 0.76, 1.53 74 PR-A GG/AA 0.69 0.48, 1.02 97 d2/d3 0.61 0.45, 0.80 100 GG/GA 1.10 0.59, 1.70 65 A/I 1.05 0.86, 1.29 70 GA/AA 0.63 0.38, 0.95 99 GG/AA 0.73 0.51, 0.98 98 1Median: posterior median of the ADU ratio. GG/GA 0.93 0.66, 1.30 66 2 HPD95%, highest posterior density region at 95% of probability. GA/AA 0.78 0.56, 1.05 95 3P: P(ratio >1) when median >1 or P(ratio <1) when median <1. 1Median: posterior median of the ADU ratio. 2 HPD95%: highest posterior density region at 95% of probability. 3P: P(ratio >1) when median >1 or P(ratio <1) when median <1. isthmus (Figure 3). An interaction between the day of gestation and different parts of the oviduct was found in the isthmus was twice that in the ampulla. Similar only in the PR-B isoform. Differences between d 2 and PR-A expression was found in both tissues. 3 of gestation were greater in the isthmus than in the The AA genotype had greater (P = 94% and P = ampulla (Figure 4). Thus, similar differences in PR-A 98%, respectively) expressions of both PR than the GG expression between the ampulla and isthmus were ob- genotype. The PR-B expression in the AA genotype tained through gestation. was 1.2 times the expression in the GG genotype, and the PR-A expression was 1.4 times. The GG and GA PR Isoforms in the Uterus genotypes showed similar (P = 55% and P = 66%, re- spectively) amount of both isoforms in the oviduct. Similar expression for each PR isoform was found Differences among genotypes were similar at d 2 and from d 2 to 3 in the uterus, although in both cases the 3 of gestation for expression of both PR in the oviduct estimates had low precision (Table 3). Expression of (Figure 2). However, an interaction between the geno- both PR for the AA genotype were approximately 1.4 types and different parts of the oviduct was found. We times greater (P = 100% and P = 97%, respectively) assumed that there was an interaction when we found a than the expression for the GG genotype. Comparing difference between interaction effects greater than zero the GG and GA genotypes, different results for each with P > 0.90, or less than zero with P > 0.90. The isoform were observed. The PR-B expression in the GA difference in the PR-B expression between the GA and genotype was 1.3 times the expression in the GG geno- AA genotypes in the ampulla and isthmus was similar. type (P = 0.92); meanwhile, similar PR-A expression The GG genotype showed a different PR-A expression was obtained between the GG and GA genotypes, al- pattern regarding other genotypes in the ampulla vs. though the estimate had low precision.

Figure 2. Posterior median values of arbitrary densitometry units (ADU) of progesterone receptor (PR)-B and PR-A isoforms at d 2 and 3 of gestation for each genotype (GG, GA, and AA) in the oviduct.

Downloaded from jas.fass.org by Agustin Blasco on January 28, 2010. Progesterone receptor isoforms of PGR genotypes 425

Figure 3. Posterior median values of arbitrary densitometry units (ADU) of progesterone receptor (PR)-B and PR-A isoforms in different parts of the oviduct for each genotype (GG, GA, and AA).

In the uterus, an interaction between the genotypes is found 165 AA after the first one; meanwhile, in hu- and day of gestation was found. The GA genotype man and mice the second codon is located 164 AA af- showed different PR-A and PR-B expression patterns ter the first one and in chicken 128 AA after the first with respect to the homozygous genotypes, although codon (Tora et al., 1988; Schneider et al., 1991; Schott the estimates had low precision (Figure 5). et al., 1991), showing that not all species have the same protein length. In rabbits, only the PR-B isoform was previously found by Gutierrez-Sagal et al. (1993) at DISCUSSION 110 kDa, although 3 other bands were also obtained PR Isoforms at 65, 72, and 79 kDa, the last band having almost the same molecular weight as the PR-A isoform found in We found 2 PR isoforms in the rabbit reproductive this study. tract, as in other species. To our knowledge, this study is the first to describe the rabbit PR-A isoform. The PR Expression bands found in this experiment had around 110 and 81 kDa, showing a molecular weight similar to other spe- PR Isoforms in the Reproductive Tract. The cies of 110 and 79 kDa in chickens (Tora et al., 1988), intensity of the staining allowed semiquantitative es- 116 and 82 kDa in humans (Kastner et al., 1990), 115 timation of the protein expression. In the oviduct, an and 83 kDa in mice (Schott et al., 1991), 120 and 90 increase of both PR isoforms expression was found from kDa in primates (Duffy et al., 1997), and 116 and 92 d 2 to 3 of gestation. At d 3 of gestation, the majority kDa in cows (Ulbrich et al., 2003). of the embryos are in the isthmus (Tsutsumi and Hafez, The different isoforms, termed PR-B and PR-A, have 1974). When the expression of both PR isoforms was been described in rodents, chickens, primates, and hu- analyzed in the ampulla and isthmus, no differences mans. These isoforms are expressed from a single gene in the PR-A expression between tissues were found, as a result of transcription from 2 alternative promot- whereas the PR-B expression in the isthmus was double ers and translation initiation at 2 different AUG start that in the ampulla. A similar result was found by An- codons. Two different AUG codons also exist in the rab- zaldúa et al. (2007); they found that the PR-B expres- bit PGR gene. Based on the PGR gene rabbit sequence sion in the isthmus was greater than in the ampulla in (GenBank accession No. X06623), a second AUG codon the first days of gestation. Several results indicate that

Figure 4. Posterior median values of arbitrary densitometry units (ADU) of progesterone receptor (PR)-B and PR-A isoforms in different parts of the oviduct at d 2 and 3 of gestation.

Downloaded from jas.fass.org by Agustin Blasco on January 28, 2010. 426 Peiró et al.

Figure 5. Posterior median values of arbitrary densitometry units (ADU) of progesterone receptor (PR)-B and PR-A isoforms at d 2 and 3 of gestation for each genotype (GG, GA, and AA) in the uterus. there was selective modification of the histomorphologi- The GG genotype showed fewer PR expressions than cal characteristics and protein secretions of the different the AA genotype in the oviduct and also in the uterus regions of the oviduct. These changes could be due to at 48 and 72 h of gestation. It seems that PR expres- fetal-maternal crosstalk because pseudopregnant rab- sions were inversely related to early embryo survival bit females did not show these changes (Harper, 1994; and development in homozygous genotypes. Anzaldúa et al., 2002). Because a similar pattern of both isoforms PR-B The PR exhibits a differential expression pattern and PR-A has been observed, we cannot associate a in the oviduct and the uterus during early pregnancy particular isoform with early embryo survival and de- in rabbits (Anzaldúa et al., 2002). The 2 PR isoforms velopment. Taking into account that the SNP found have shown to be similar at d 2 and 3 of gestation in in the PGR gene is located in the promoter region of the uterus. Previous results showed that PR-B expres- the PR-B isoform, it seems that the PR-B isoform sion was not detected in the uterus at 4 h after mating, could be the isoform responsible for the differences in although it was detected at d 1, with an increase at d 2, early embryo survival and development between ho- followed by a decrease at d 3, and complete disappear- mozygous genotypes. There is no information in rab- ance at d 5 of gestation (Gutierrez-Sagal et al., 1993). bits about the relationship between PR-B and embryo The PR-B expression found in uterine epithelium and survival. In sheep, less PR-B expression in the oviduct stromal cells by Anzaldúa et al. (2007) also showed and uterus was related to greater embryo survival at a decrease from d 2 until d 3 of gestation. Both re- d 4 and 7 of gestation in synchronous females com- sults should be taken with caution because the number pared with asynchronous females (García-Palencia et of animals used was small. The differences in embryo al., 2007). Besides, it is known that PR is downregu- survival and development between homozygous geno- lated by progesterone in the uterus of the rabbit and types increased through gestation (Peiró et al., 2008), other mammalian species (Brenner et al., 1979, 1990; although we did not find an interaction between geno- Camacho-Arroyo et al., 1998; Bouchard, 1999; Ulbrich type and day of gestation. et al., 2003). Then, decreased PR expression should PR Expression Among Genotypes. The pro- be associated with greater progesterone concentration. gesterone receptor gene was proposed as a candidate Indirect evidence about the relationship between PR gene to explain differences in embryo survival between expression and embryo survival is provided by experi- 2 divergent lines selected for UC (Peiró et al., 2008). ments in which progesterone was supplemented in the The SNP located in the promoter region, which co- first stages of gestation. Progesterone supplementation segregates with 3 SNP in exon 1 (5′UTR) and 1 silent in the first stages of gestation was related to improve- SNP in exon 7, was associated with these lines; the ment of embryo survival and development in different GG genotype was the most frequent genotype in the species [Sheridan et al. (1986) in pigs; Proctor et al. H line, selected for high UC, whereas the AA geno- (2006) in humans; Mann et al. (2006), Carter et al. type was most frequent in the L line, selected for low (2008), and Morris and Diskin (2008) in cows; Ghaemi UC. Litter size and its components were studied in the et al. (2008) in mice]. F2 population, showing that the GG genotype had 0.5 To summarize, there are 2 different PR isoforms in more kits than the GA and AA genotypes. The differ- rabbits as in human and mice. Both isoforms are ex- ence in LS between homozygous genotypes was due to pressed in the oviduct and uterus in the first stages greater early embryo survival at d 3 and 7 and also to of gestation. The AA genotype showed greater expres- greater embryonic stage of development (Peiró et al., sion of both PR isoforms than the GG genotype in the 2008). The difference found in the first days of gesta- oviduct and uterus at 48 and 72 h of gestation, which tion in early embryo survival and development among could be related to the reduced survival observed in genotypes could be related to different PR expression. rabbits with the AA genotype vs. the GG genotype.

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