Improved annotation of the domestic pig genome through integration of Iso-Seq and RNA-seq data.

BACKGROUND: Our understanding of the pig transcriptome is limited. RNA transcript diversity among nine tissues was assessed using poly(A) selected single-molecule long-read isoform sequencing (Iso-seq) and Illumina RNA sequencing (RNA-seq) from a single White cross-bred pig. RESULTS: Across tissues, a total of 67,746 unique transcripts were observed, including 60.5% predicted protein-coding, 36.2% long non-coding RNA and 3.3% nonsense-mediated decay transcripts. On average, 90% of the splice junctions were supported by RNA-seq within tissue. A large proportion (80%) represented novel transcripts, mostly produced by known protein-coding genes (70%), while 17% corresponded to novel genes. On average, four transcripts per known gene (tpg) were identified; an increase over current EBI (1.9 tpg) and NCBI (2.9 tpg) annotations and closer to the number reported in human genome (4.2 tpg). Our new pig genome annotation extended more than 6000 known gene borders (5' end extension, 3' end extension, or both) compared to EBI or NCBI annotations. We validated a large proportion of these extensions by independent pig poly(A) selected 3'-RNA-seq data, or human FANTOM5 Cap Analysis of Gene Expression data. Further, we detected 10,465 novel genes (81% non-coding) not reported in current pig genome annotations. More than 80% of these novel genes had transcripts detected in > 1 tissue. In addition, more than 80% of novel intergenic genes with at least one transcript detected in liver tissue had H3K4me3 or H3K36me3 peaks mapping to their promoter and gene body, respectively, in independent liver chromatin immunoprecipitation data. CONCLUSIONS: These validated results show significant improvement over current pig genome annotations.

A survey of single nucleotide polymorphisms identified from whole-genome sequencing and their functional effect in the porcine genome.

Genetic variants detected from sequence have been used to successfully identify causal variants and map complex traits in several organisms. High and moderate impact variants, those expected to alter or disrupt the protein coded by a gene and those that regulate protein production, likely have a more significant effect on phenotypic variation than do other types of genetic variants. Hence, a comprehensive list of these functional variants would be of considerable interest in swine genomic studies, particularly those targeting fertility and production traits. Whole-genome sequence was obtained from 72 of the founders of an intensely phenotyped experimental swine herd at the U.S. Meat Animal Research Center (USMARC). These animals included all 24 of the founding boars (12 Duroc and 12 Landrace) and 48 Yorkshire-Landrace composite sows. Sequence reads were mapped to the Sscrofa10.2 genome build, resulting in a mean of 6.1 fold (×) coverage per genome. A total of 22 342 915 high confidence SNPs were identified from the sequenced genomes. These included 21 million previously reported SNPs and 79% of the 62 163 SNPs on the PorcineSNP60 BeadChip assay. Variation was detected in the coding sequence or untranslated regions (UTRs) of 87.8% of the genes in the porcine genome: loss-of-function variants were predicted in 504 genes, 10 202 genes contained nonsynonymous variants, 10 773 had variation in UTRs and 13 010 genes contained synonymous variants. Approximately 139 000 SNPs were classified as loss-of-function, nonsynonymous or regulatory, which suggests that over 99% of the variation detected in our pigs could potentially be ignored, allowing us to focus on a much smaller number of functional SNPs during future analyses.

Meta-analysis of genome-wide association from genomic prediction models.

Genome-wide association (GWA) studies based on GBLUP models are a common practice in animal breeding. However, effect sizes of GWA tests are small, requiring larger sample sizes to enhance power of detection of rare variants. Because of difficulties in increasing sample size in animal populations, one alternative is to implement a meta-analysis (MA), combining information and results from independent GWA studies. Although this methodology has been used widely in human genetics, implementation in animal breeding has been limited. Thus, we present methods to implement a MA of GWA, describing the proper approach to compute weights derived from multiple genomic evaluations based on animal-centric GBLUP models. Application to real datasets shows that MA increases power of detection of associations in comparison with population-level GWA, allowing for population structure and heterogeneity of variance components across populations to be accounted for. Another advantage of MA is that it does not require access to genotype data that is required for a joint analysis. Scripts related to the implementation of this approach, which consider the strength of association as well as the sign, are distributed and thus account for heterogeneity in association phase between QTL and SNPs. Thus, MA of GWA is an attractive alternative to summarizing results from multiple genomic studies, avoiding restrictions with genotype data sharing, definition of fixed effects and different scales of measurement of evaluated traits.

Genome-wide association with delayed puberty in swine.

An improvement in the proportion of gilts entering the herd that farrow a litter would increase overall herd performance and profitability. A significant proportion (10-30%) of gilts that enter the herd never farrow a litter; reproductive reasons account for approximately a third of gilt removals, with anestrous and failure to conceive the most common reasons for culling. Tools to select gilts for reproductive longevity through genomics or alternative phenotypes would be of great benefit to the producer. Ninety-one gilts that failed to display behavioral estrus by 240 days (cases) and 127 pubertal littermates (controls) were genotyped with the Illumina Porcine SNP60 Beadchip. One hundred and seventy-four SNPs with the most significant associations were genotyped in an additional 86 cases and 103 controls. Twelve of these associations were significant in the final analysis. The most significant (P < 1.5 × 10(-14) ) region associated with failure to attain puberty was on chromosome 4 surrounding the NHLH2 gene. Delayed pubertal development and age at first estrus have been associated with NHLH2 in mice. Because attainment of puberty is a complex trait, identifying genes that affect pubertal age would greatly contribute to our knowledge of reproductive development as well as overall fertility.

Validation and fine mapping of a QTL for ovulation rate on swine chromosome 3.

Ovulation rate (OR) is an important component of litter size, but mutation(s) in gene(s) underlying OR QTL have yet to be identified in pigs. Markers within an OR QTL on SSC3 were genotyped in three white composite lines selected for ten generations for increased OR or uterine capacity (UC), with one line being an unselected control. Numbers of corpora lutea (CL) and UC (number of fully formed fetuses) were collected at approximately 105 days of gestation, as well as ovary weight (OW), uterine length (UL) and uterine weight (UW) measurements at 160 d of age in generation 12 and 13 females from all three lines. Six microsatellites and ten single nucleotide polymorphisms (SNPs; 0-42 cM) were genotyped in pigs from all lines of generations 11 through 13. The allele frequencies of 24269.1, SW2429, 7907.2 and 7637.2 were different (P < 0.01) in the OR line compared to the control line. A significant (P < 0.05) association of CL with 24269.1 (additive effect 0.65 ± 0.32) was detected, and additive genotypic effects approached significance for markers at 28 through 35 cM (16963.2, 27514.1 and SWR1637). Haplotyping of 7637.2 and 16963.2 (31 through 32 cM) identified a significant additive association of haplotype 1 with CL (-0.62 ± 0.30). These markers were also associated with OW (24296.1 and SWR1637), UL (16963.2, 27514.1 and haplotypes of 7637.2/16963.2) and UW (haplotypes of 7637.2/16963.2). This study verifies an OR QTL on SSC3. However, based on the data, it was concluded that there may be two genes, at 13 through 18 cM and 28 through 35 cM, controlling OR on SSC3p.

Four genes located on a SSC2 meat quality QTL region are associated with different meat quality traits in Landrace × Chinese-European crossbred population.

Several quantitative trait loci (QTL) for different meat quality traits have been localized on the q arm of porcine chromosome 2 at position 55-78 cM. Association analyses were performed in a commercial Landrace × Chinese-European (LCE) crossbred population (n = 446) slaughtered at approximately 127 kg and an average age of 198 days with records for performance (growth, fat and meat accretion) and meat quality [intramuscular fat (IMF), Minolta L*, Minolta a*, Minolta b* and pH at 45 m]. Polymorphisms within positional candidate genes cloned from homologous regions on human chromosome 19, ubiquitin-like 5 (UBL5- AM950288:g.566G>A), resistin (RETN- AM157180:g.1473A>G causing substitution p.Ala36Thr), insulin receptor (INSR- AM950289:g.589T>C) and complement factor D (adipsin) (CFD- AM950287:g. 306C>T) were located at positions 62.1, 64.0, 68.0 and 70.7 cM respectively on the current USDA USMARC map of porcine chromosome 2 and had the following allele frequencies in the LCE: UBL5 566G - 0.57; RETN 1473G - 0.84; INSR 589C - 0.70; and CFD 306C - 0.73. The effects of alleles within the candidate genes on the recorded traits were estimated using an animal model. Significant effects (P < 0.05) were found for pH(45) in m. semimembranosus (m. sm.) (UBL5), IMF (RETN) and Minolta L* (RETN, CFD). Differences between phenotypic means of homozygotes at UBL5, RETN and either RETN or CFD explained 0.34 SD for pH(45) in m. sm., 0.47 SD for IMF and 0.68 SD for Minolta L* respectively. Suggestive effects (P < 0.10) on IMF (UBL5, CFD), Minolta a* (INSR, CFD) and Minolta b* (INSR) were also observed. Our results support the localization of further QTL for meat quality traits in this region and suggest that there are several genes affecting different meat quality traits.

Physiological and genomic insight into neuroendocrine regulation of puberty in gilts.

The timing of pubertal attainment in gilts is a critical factor for pork production and is an early indicator of future reproductive potential. Puberty, defined as age at first standing estrus in the presence of a boar, is brought about by an escape from estrogen inhibition of the GnRH pulse generator, which allows for increasing LH pulses leading to the onset of cyclicity. The biological mechanisms that control the timing of these events is related to decreasing inhibitory signals with a concomitant increase in stimulatory signals within the hypothalamus. The roles of gamma-aminobutyric acid, endogenous opioid peptides, and gonadotropin-inhibitory hormone in negatively regulating gonadotropin secretion in gilts is explored. Developmental changes in stimulatory mechanisms of glutamatergic and kisspeptin neurons are important for increased LH pulsatility required for the occurrence of puberty in pigs. Age at first estrus of gilts is metabolically gated, and numerous metabolites, metabolic hormones, and appetite-regulating neurotransmitters have been implicated in the nutritional regulation of gonadotropin secretion. Leptin is an important metabolic signal linking body energy reserves with age at puberty in gilts. Leptin acting through neuropeptide Y and proopiomelanocortin neurons in the hypothalamus has important impacts on the function of the reproductive neurosecretory axis of gilts. Age at puberty in swine is heritable, and genomic analyses reveal it to be a polygenic trait. Genome-wide association studies for pubertal age in gilts have revealed several genomic regions in common with those identified for age at menarche in humans. Candidate genes have been identified that have important functions in growth and adiposity. Numerous genes regulating hypothalamic neuronal function, gonadotropes in the adenohypophysis, and ovarian follicular development have been identified and illustrate the complex maturational changes occurring in the hypothalamic-pituitary-ovarian axis during puberty in gilts.

Relationships among calpastatin single nucleotide polymorphisms, calpastatin expression and tenderness in pork longissimus.

Genome scans in the pig have identified a region on chromosome 2 (SSC2) associated with tenderness. Calpastatin is a likely positional candidate gene in this region because of its inhibitory role in the calpain system that is involved in postmortem tenderization. Novel single nucleotide polymorphisms (SNP) in calpastatin were identified and used to genotype a population (n = 1042) of Duroc-Landrace-Yorkshire swine for association with longissimus lumborum slice shear force (SSF) measured at days 7 and 14 postmortem. Three genetic markers residing in the calpastatin gene were significantly associated with SSF (P < 0.0005). Haplotypes constructed from markers in the calpastatin gene were significantly associated with SSF (F-ratio = 3.93; P-value = 0.002). The levels of normalized mRNA expression of calpastatin in the longissimus lumborum of 162 animals also were evaluated by real-time RT-PCR and were associated with the genotype of the most significant marker for SSF (P < 0.02). This evidence suggests that the causative variation alters expression of calpastatin, thus affecting tenderness. In summary, these data provide evidence of several significant, publicly available SNP markers associated with SSF that may be useful to the swine industry for marker assisted selection of animals that have more tender meat.

Genetic variation in the mannosidase 2B2 gene and its association with ovulation rate in pigs.

Ovulation rate is an important phenotypic trait that is a critical component of litter size in pigs. Despite being moderately heritable in pigs, selection for increased ovulation rate is difficult because it is difficult to measure and is a sex-limited trait. A QTL for ovulation rate residing on the p-terminal end of pig chromosome 8 has been detected in a Meishan-cross resource population. Comparative analysis of this region yielded a positional candidate gene, mannosidase 2B2 (MAN2B2), for this QTL. The entire coding region of MAN2B2 was resequenced in the Meishan and White Composite founder animals of the resource population to identify SNPs. Eleven polymorphisms that alter the protein product of MAN2B2 were discovered and tested for statistical associations with ovulation rate in three generations of the resource population. The polymorphism located at position 1574 of the mRNA (D28521:c.1574A>G) was the most significant polymorphism tested (P = 0.00005) where the additive effect of the c.1574A allele was estimated to be -0.89 ova. This polymorphism was determined to be more significantly associated with ovulation rate than the breed-specific analysis conducted during the line-cross QTL discovery. The c.1574A>G marker was not associated with ovulation rate in an occidental population. Therefore, either MAN2B2 has a unique epistatic interaction within the Meishan-cross population or the c.1574A>G SNP is in linkage disequilibrium with the actual causative genetic variation in the Meishan-cross population.

Porcine single nucleotide polymorphisms and their functional effect: an update.

OBJECTIVE: To aid in the development of a comprehensive list of functional variants in the swine genome, single nucleotide polymorphisms (SNP) were identified from whole genome sequence of 240 pigs. Interim data from 72 animals in this study was published in 2017. This communication extends our previous work not only by utilizing genomic sequence from additional animals, but also by the use of the newly released Sscrofa 11.1 reference genome. RESULTS: A total of 26,850,263 high confidence SNP were identified, including 19,015,267 reported in our previously published results. Variation was detected in the coding sequence or untranslated regions (UTR) of 78% of the genes in the porcine genome: 1729 loss-of-function variants were predicted in 1162 genes, 12,686 genes contained 64,232 nonsynonymous variants, 250,403 variants were present in UTR of 15,739 genes, and 15,284 genes contained 90,939 synonymous variants. In total, approximately 316,000 SNP were classified as being of high to moderate impact (i.e. loss-of-function, nonsynonymous, or regulatory). These high to moderate impact SNP will be the focus of future genome-wide association studies.

Comparative and physical mapping of 111 previously reported and 105 new porcine microsatellites.

Here we report radiation hybrid mapping of 105 new porcine microsatellite markers on the IMpRH(7000) radiation hybrid panel. In addition, we searched flanking sequences of these markers, as well as 673 previously reported RH-mapped microsatellite markers, for orthology to human sequences. Eighty-seven new and 111 previously mapped sequences exhibited orthology to human sequences. Using a stringent sequence alignment, 25 microsatellite-flanking sequences were found to be highly similar to genic sequences, whereas 173 were similar to non-genic sequences in the human genome. Five markers were located near known breakpoints of synteny between human and swine.

Litter-of-origin trait effects on gilt development.

The preweaning litter environment of gilts can affect subsequent development. In a recent experiment designed to test the effects of diet on gilt development, litter-of-origin traits including individual birth weights, immunocrits (a measure of colostrum intake), sow parity, number weaned, and individual weaning weights were collected for approximately 1,200 gilts that were progeny of approximately 300 sows. Subsequently, BW, LM area, and backfat were measured at 100 d of age and at 28-d intervals until slaughter (260 d of age). From 160 d of age to slaughter, gilts were observed daily for estrus. At slaughter, the reproductive tract and 1 mammary gland were recovered. The reproductive tract was classified as cyclic or prepubertal; the number of corpora lutea was counted. Uterine horn lengths and ovarian dimensions were measured. Uterus and ovary samples from every 10th gilt were prepared for histological evaluation of uterine gland development and follicle counts, respectively. Mammary gland tissue protein and fat were assayed. Day of the estrous cycle at slaughter was calculated using the first day of the most recent standing estrus (d 0) recorded previous to slaughter. Each gilt development trait was analyzed for association with each litter-of-origin trait, after adjusting for dietary treatment effects. Uterine length, ovarian dimensions, mammary gland protein and fat, and uterine gland development were also adjusted for day of the estrous cycle at slaughter. All litter-of-origin traits were associated ( < 0.05) with growth traits. Top-down (backward elimination) multiple regression analysis indicated that BW and LM accretion in gilts was positively associated with immunocrit ( < 0.01), birth weight ( < 0.01), preweaning growth rate ( < 0.01), and parity ( < 0.01). Backfat accretion was positively associated with preweaning growth rate ( < 0.01), number weaned ( < 0.05), and parity ( < 0.05). Age at puberty was associated with birth weight (positive; < 0.01) and preweaning growth rate (negative; < 0.01). Total uterine length was positively associated with only birth weights ( < 0.05). Mammary gland protein was negatively associated with preweaning growth ( < 0.01). Mammary gland fat was positively associated with birth weight and number of piglets weaned ( > 0.05). These results indicate that colostrum consumption, birth weights, preweaning growth rate, number weaned, and parity are associated with gilt development traits during later life.

The coding sequence of the bovine ferrochelatase gene.

The amino acid code and surrounding regions in the bovine ferrochelatase gene were amplified by a combination of reverse transcriptase PCR and vectorette PCR and sequenced. The bovine code was 86% homologous to the human ferrochelatase code but was altered at a position corresponding to the presumed human initiator codon.

A microsatellite-based genetic linkage map for channel catfish, Ictalurus punctatus.

Microsatellite loci were identified in channel catfish gene sequences or random clones from a small insert genomic DNA library. Outbred populations of channel catfish contained an average of eight alleles per locus and an average heterozygosity of 0.70. A genetic linkage map of the channel catfish genome (N = 29) was constructed from two reference families. A total of 293 microsatellite loci were polymorphic in one or both families, with an average of 171 informative meioses per locus. Nineteen type I loci, 243 type II loci, and one EST were placed in 32 multipoint linkage groups covering 1958 cM. Nine more type II loci were contained in three two-point linkage groups covering 24.5 cM. Twenty-two type II loci remained unlinked. Multipoint linkage groups ranged in size from 11.9 to 110.5 cM with an average intermarker distance of 8.7 cM. Seven microsatellite loci were closely linked with the sex-determining locus. The microsatellite loci and genetic linkage map will increase the efficiency of selective breeding programs for channel catfish.

Relationships between day one piglet serum immunoglobulin immunocrit and subsequent growth, puberty attainment, litter size, and lactation performance.

Colostrum affects gut and uterine gland development in the neonatal piglet, suggesting that subsequent growth and reproductive performance may be affected. Measuring immunoglobulin in piglet serum using the immunoglobulin immunocrit on Day 1 of age provides a simple, inexpensive indication of the amount of colostrum acquired by the piglet in the first day of life. Relationships between serum immunoglobulin immunocrit measures and subsequent growth rates, age at puberty, incidence of puberty failure, litter size, and lactation performance were examined in pigs born and subsequently farrowing between 2009 and 2013. Immunoglobulin immunocrit measures were collected on 16,762 piglets on Day 1 of age. Of these piglets, BW measurements were available from 15,324 (7,684 males and 7,640 females) piglets at a range of ages from weaning to 200 d of age, allowing an assessment of growth rates. Age at puberty was recorded from a subset of 2,857 of the females after observing them for estrous behavior from approximately 170 to 250 d of age. To examine relationships between d 1 immunocrit and puberty failure, gilts with immunocrit measures that failed to reach puberty (n = 119) were matched with littermate gilts with immunocrit measures that achieved puberty (n = 167). Similarly, number born alive was collected on a subset (n = 799) of females from first to fourth parities for which d 1 immunocrits were measured on them as neonates. Finally, d 1 immunocrit effect on adult lactational competence was assessed by measuring litter average (offspring of 440 females) and litter average piglet preweaning growth rate (offspring of 774 females) in females where d 1 immunocrits were available from them as neonates. Results indicated that low d 1 immunocrits were subsequently associated with reduced growth (P < 0.01), increased age at puberty (P < 0.01), reduced number born alive (P < 0.05), reduced litter average immunocrit (P < 0.05), and reduced litter average preweaning growth rate during lactation (P < 0.05). This suggests that management efforts to improve the amount of colostrum ingested by neonatal piglets would result in beneficial changes in production efficiency, particularly for gilts destined for the breeding herd. It also suggests that the immunoglobulin immunocrit can be useful in monitoring colostrum ingestion to maximize the beneficial effects of colostrum on subsequent performance.

Implementing meta-analysis from genome-wide association studies for pork quality traits.

Pork quality plays an important role in the meat processing industry. Thus, different methodologies have been implemented to elucidate the genetic architecture of traits affecting meat quality. One of the most common and widely used approaches is to perform genome-wide association (GWA) studies. However, a limitation of many GWA in animal breeding is the limited power due to small sample sizes in animal populations. One alternative is to implement a meta-analysis of GWA (MA-GWA) combining results from independent association studies. The objective of this study was to identify significant genomic regions associated with meat quality traits by performing MA-GWA for 8 different traits in 3 independent pig populations. Results from MA-GWA were used to search for genes possibly associated with the set of evaluated traits. Data from 3 pig data sets (U.S. Meat Animal Research Center, commercial, and Michigan State University Pig Resource Population) were used. A MA was implemented by combining -scores derived for each SNP in every population and then weighting them using the inverse of estimated variance of SNP effects. A search for annotated genes retrieved genes previously reported as candidates for shear force (calpain-1 catalytic subunit [] and calpastatin []), as well as for ultimate pH, purge loss, and cook loss (protein kinase, AMP-activated, γ 3 noncatalytic subunit []). In addition, novel candidate genes were identified for intramuscular fat and cook loss (acyl-CoA synthetase family member 3 mitochondrial []) and for the objective measure of muscle redness, CIE a* (glycogen synthase 1, muscle [] and ferritin, light polypeptide []). Thus, implementation of MA-GWA allowed integration of results for economically relevant traits and identified novel genes to be tested as candidates for meat quality traits in pig populations.

Age at puberty, ovulation rate, and uterine length of developing gilts fed two lysine and three metabolizable energy concentrations from 100 to 260 d of age.

The objective of this study was to determine the effect of ad libitum feeding diets differing in standard ileal digestible (SID) lysine and ME concentrations that bracket those fed to developing gilts in U.S. commercial settings. Average SID lysine and ME concentrations in diets currently fed to developing gilts were obtained from a poll of the U.S. commercial swine industry. Crossbred Large White × Landrace gilts (n = 1,221), housed in groups, were randomly allotted to 6 corn-soybean diets in a 2 × 3 factorial arrangement formulated to provided 2 SID lysine and 3 ME concentrations. Gilts received grower diets formulated to provide 1.02% (control = survey average) or 0.86% (control minus 15%) SID lysine and 2.94, 3.25, or 3.57 (survey average ME ± 10%) Mcal of ME/kg from 100 d of age until approximately 90 kg BW. Then, gilts were fed finisher diet containing 0.85% (control = survey average) or 0.73% (control minus 15%) SID lysine and 2.94, 3.26, or 3.59 (control ± 10%) Mcal of ME/kg until 260 d of age. Gilts were weighed, and backfat thickness and loin muscle area were recorded at the beginning of the trial and then every 28 d. Starting at 160 d of age, gilts were exposed daily to vasectomized boars and observed for behavioral estrus. At approximately 260 d of age, gilts were slaughtered and their reproductive tract was collected. Each reproductive tract was examined to determine whether the gilt was cyclic, the stage of estrus cycle, ovulation rate, and uterine length. Data were evaluated for normality and analyzed using mixed model methods. Average age at puberty was 193 d of age with a range from 160 to 265 d. When all gilts on trial at 160 d of age were included in the analysis, 91.0% reached puberty as determine by observation of standing estrus. Differences between dietary treatments on age at puberty or measurements of the reproductive tract were not detected. Growth rates to 160 d were not limiting for attainment of puberty in response to daily boar stimulation from 160 d.

pH-dependent cytotoxicity of contaminants of phenol red for MCF-7 breast cancer cells.

The pH indicator phenol red (phenolsulfonphthalein) is present in most tissue culture media. Contaminants of this indicator have shown substantial estrogenic activity for estrogen-dependent cells in culture, including the human breast cancer-derived MCF-7 cell line. In the course of other studies, we observed that brief (1- to 4-h) incubations of these cells at 37 C in serum-free medium (Hanks' or Earle's Balanced Salts Solution) could be toxic to MCF-7 cells when the pH was increased above 7.4, but only if phenol red (10 micrograms/ml) was present in the medium. Because damaged/killed cells detached from the substratum (greater than 98% of detached cells stained with trypan blue), we used DNA assay of the cells remaining after treatment and wash (98% of the remaining cells were dye excluding) to further assess cytotoxicity. The MCF-7 cells were more susceptible to the cytotoxicity at lower cell densities, so further characterization of phenol red cytotoxicity was performed at cell densities of 1-10 micrograms DNA/2-cm2 well, or approximately 40,000-400,000 cells/ml medium. In the pH range of 7.0-8.2, 50% cell death was observed in the presence of phenol red at pH as low as 7.6-7.7, with nearly 100% of the cells killed by pH 8.0. Little effect was seen in phenol red-free medium at any part of the tested pH range or in medium that contained phenol red at pH less than or equal to 7.4. In time-course studies of cytotoxicity at pH 8.0 (phenol red, 10 micrograms/ml), greater than 50% cell damage could be observed after less than 1 h, and little cell recovery was observed if the pH was restored to 7.4. For phenol red samples from two major commercial sources, the concentration for half-maximal cytotoxicity (TD50) in dose-responses after 4 h at pH 8.0 showed TD50 values of 2 and 6 micrograms/ml, while the estrogenic activities, as half-maximal stimulation of estrogen-dependent proliferation, were identical at 2 micrograms/ml. Both the cytotoxic and estrogenic activities could be removed from the phenol red by extraction with diethyl ether. A number of contaminants of the commercial phenol red were detected by reverse phase C18 HPLC. Cytotoxicity and estrogen bioassays of each of the HPLC fractions indicated that the pH-dependent cytotoxicity was separate from the estrogenic activity and confirmed that neither activity was associated with the phenol red itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and endometrial expression of porcine high density lipoprotein receptor SR-BI during the estrous cycle and early pregnancy.

During rapid development of the fetus, levels of high density lipoprotein (HDL) are elevated in pregnant women. The receptor for HDL, scavenger receptor class B type I (SR-BI), mediates selective cholesteryl ester uptake and is highly expressed in the human placenta. Because of the rapid growth of uterus during early pregnancy and differences in placentation between swine and humans, we hypothesized that SR-BI may be expressed in porcine endometrium to take up HDL cholesterol. The objectives of this study were to obtain the full coding region for porcine SR-BI, determine endometrial expression of SR-BI mRNA during the estrous cycle and early pregnancy, and map the gene. By iterative screening of a porcine expressed sequence tag library, we obtained the full coding region of SR-BI. Endometrial expression of SR-BI in White composite gilts (n = 3-4 each) was determined by Northern blotting on Days 10, 13, and 15 cyclic gilts and Days 10, 13, 15, 20, 30, and 40 pregnant gilts. In cyclic gilts, endometrial expression of SR-BI did not change between Days 10 and 13, but increased (P < 0.01) between Days 13 and 15. In pregnant gilts, endometrial expression of SR-BI increased (P < 0.01) between Days 10 and 13, remained elevated until Day 30, and decreased (P = 0.015) on Day 40. The SR-BI gene was mapped to 46.3 cM on chromosome 14. These results show that endometrial expression of SR-BI changes during the estrous cycle and early pregnancy, and suggest that SR-BI takes up HDL for endometrial development during early pregnancy.