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.

Beef steers with average dry matter intake and divergent average daily gain have altered gene expression in the jejunum.

The objective of this study was to determine the association of differentially expressed genes (DEG) in the jejunum of steers with average DMI and high or low ADG. Feed intake and growth were measured in a cohort of 144 commercial Angus steers consuming a finishing diet containing (on a DM basis) 67.8% dry-rolled corn, 20% wet distillers grains with solubles, 8% alfalfa hay, and 4.2% vitamin/mineral supplement. From the cohort, a subset of steers with DMI within ±0.32 SD of the mean for DMI and the greatest (high) and least (low) ADG were chosen for slaughter and jejunum mucosa collection ( = 8 for each group). Dry matter intake (10.1 ± 0.05 kg/d) was not different ( = 0.41) but ADG was greater in the high-gain group (2.17 and 1.72 ± 0.02 kg/d for the high- and low-ADG groups, respectively; < 0.01). A total of 13,747 genes were found to be expressed in the jejunum, of which 64 genes were differentially expressed between the 2 groups (corrected < 0.05). Ten of the DEG were upregulated in the low-ADG group and 54 were upregulated in the high-ADG group. Gene ontology analysis determined that 24 biological process terms were overrepresented ( < 0.05), including digestion, drug and xenobiotic metabolism, and carbohydrate metabolism. Additionally, 89 molecular function terms were enriched ( < 0.05), including metallopeptidase activity, transporter activity, steroid hydrolase activity, glutathione transferase activity, and chemokine receptor binding. Metabolic pathways (28 pathways) impacted by the DEG ( < 0.05) included drug and xenobiotic metabolism by cytochrome P450, carbohydrate digestion and absorption, vitamin digestion and absorption, galactose metabolism, and linoleic acid metabolism. Results from this experiment indicate that cattle with average DMI and greater ADG likely have a greater capacity to handle foreign substances (xenobiotics). It is also possible that cattle with a greater ADG have a greater potential to digest and absorb nutrients in the small intestine.

GENOMICS SYMPOSIUM: Using genomic approaches to uncover sources of variation in age at puberty and reproductive longevity in sows.

Genetic variants associated with traits such as age at puberty and litter size could provide insight into the underlying genetic sources of variation impacting sow reproductive longevity and productivity. Genomewide characterization and gene expression profiling were used using gilts from the University of Nebraska-Lincoln swine resource population ( = 1,644) to identify genetic variants associated with age at puberty and litter size traits. From all reproductive traits studied, the largest fraction of phenotypic variation explained by the Porcine SNP60 BeadArray was for age at puberty (27.3%). In an evaluation data set, the predictive ability of all SNP from high-ranked 1-Mb windows (1 to 50%), based on genetic variance explained in training, was greater (12.3 to 36.8%) compared with the most informative SNP from these windows (6.5 to 23.7%). In the integrated data set ( = 1,644), the top 1% of the 1-Mb windows explained 6.7% of the genetic variation of age at puberty. One of the high-ranked windows detected (SSC2, 12-12.9 Mb) showed pleiotropic features, affecting both age at puberty and litter size traits. The RNA sequencing of the hypothalami arcuate nucleus uncovered 17 differentially expressed genes (adjusted < 0.05) between gilts that became pubertal early (<155 d of age) and late (>180 d of age). Twelve of the differentially expressed genes are upregulated in the late pubertal gilts. One of these genes is involved in energy homeostasis (), a function in which the arcuate nucleus plays an important contribution, linking nutrition with reproductive development. Energy restriction during the gilt development period delayed age at puberty by 7 d but increased the probability of a sow to produce up to 3 parities ( < 0.05). Identification of pleotropic functional polymorphisms may improve accuracy of genomic prediction while facilitating a reduction in sow replacement rates and addressing welfare concerns.

Linkage disequilibrium among commonly genotyped SNP variants detected from bull sequence.

Genomic prediction utilizing causal variants could increase selection accuracy above that achieved with SNPs genotyped by currently available arrays used for genomic selection. A number of variants detected from sequencing influential sires are likely to be causal, but noticeable improvements in prediction accuracy using imputed sequence variant genotypes have not been reported. Improvement in accuracy of predicted breeding values may be limited by the accuracy of imputed sequence variants. Using genotypes of SNPs on a high-density array and non-synonymous SNPs detected in sequence from influential sires of a multibreed population, results of this examination suggest that linkage disequilibrium between non-synonymous and array SNPs may be insufficient for accurate imputation from the array to sequence. In contrast to 75% of array SNPs being strongly correlated to another SNP on the array, less than 25% of the non-synonymous SNPs were strongly correlated to an array SNP. When correlations between non-synonymous and array SNPs were strong, distances between the SNPs were greater than separation that might be expected based on linkage disequilibrium decay. Consistently near-perfect whole-genome linkage disequilibrium between the full array and each non-synonymous SNP within the sequenced bulls suggests that whole-genome approaches to infer sequence variants might be more accurate than imputation based on local haplotypes. Opportunity for strong linkage disequilibrium between sequence and array SNPs may be limited by discrepancies in allele frequency distributions, so investigating alternate genotyping approaches and panels providing greater chances of frequency-matched SNPs strongly correlated to sequence variants is also warranted. Genotypes used for this study are available from https://www.animalgenome.org/repository/pub/;USDA2017.0519/.

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.

Genome-wide copy number variation in the bovine genome detected using low coverage sequence of popular beef breeds.

Copy number variations (CNVs) are large insertions, deletions or duplications in the genome that vary between members of a species and are known to affect a wide variety of phenotypic traits. In this study, we identified CNVs in a population of bulls using low coverage next-generation sequence data. First, in order to determine a suitable strategy for CNV detection in our data, we compared the performance of three distinct CNV detection algorithms on benchmark CNV datasets and concluded that using the multiple sample read depth approach was the best method for identifying CNVs in our sequences. Using this technique, we identified a total of 1341 copy number variable regions (CNVRs) from genome sequences of 154 purebred sires used in Cycle VII of the USMARC Germplasm Evaluation Project. These bulls represented the seven most popular beef breeds in the United States: Hereford, Charolais, Angus, Red Angus, Simmental, Gelbvieh and Limousin. The CNVRs covered 6.7% of the bovine genome and spanned 2465 protein-coding genes and many known quantitative trait loci (QTL). Genes harbored in the CNVRs were further analyzed to determine their function as well as to find any breed-specific differences that may shed light on breed differences in adaptation, health and production.

Genomewide association study of liver abscess in beef cattle.

Fourteen percent of U.S. cattle slaughtered in 2011 had liver abscesses, resulting in reduced carcass weight, quality, and value. Liver abscesses can result from a common bacterial cause, , which inhabits rumen lesions caused by acidosis and subsequently escapes into the blood stream, is filtered by the liver, and causes abscesses in the liver. Our aim was to identify SNP associated with liver abscesses in beef cattle. We used lung samples as a DNA source because they have low economic value, they have abundant DNA, and we had unrestricted access to sample them. We collected 2,304 lung samples from a beef processing plant: 1,152 from animals with liver abscess and 1,152 from animals without liver abscess. Lung tissue from pairs of animals, 1 with abscesses and another without, were collected from near one another on the viscera table to ensure that pairs of phenotypically extreme animals came from the same lot. Within each phenotype (abscess or no abscess), cattle were pooled by slaughter sequence into 12 pools of 96 cattle for each phenotype for a total of 24 pools. The pools were constructed by equal volume of frozen lung tissue from each animal. The DNA needed to allelotype each pool was then extracted from pooled lung tissue and the BovineHD Bead Array (777,962 SNP) was run on all 24 pools. Total intensity (TI), an indicator of copy number variants, was the sum of intensities from red and green dyes. Pooling allele frequency (PAF) was red dye intensity divided TI. Total intensity and PAF were weighted by the inverse of their respective genomic covariance matrices computed over all SNP across the genome. A false discovery rate ≤ 5% was achieved for 15 SNP for PAF and 20 SNP for TI. Genes within 50 kbp from significant SNP were in diverse pathways including maintenance of pH homeostasis in the gastrointestinal tract, maintain immune defenses in the liver, migration of leukocytes from the blood into infected tissues, transport of glutamine into the kidney in response to acidosis to facilitate production of bicarbonate to increase pH, aggregate platelets to liver injury to facilitate liver repair, and facilitate axon guidance. Evidence from the 35 detected SNP associations combined with evidence of polygenic variation indicate that there is adequate genetic variation in incidence rate of liver abscesses, which could be exploited to select sires for reduced susceptibility to subacute acidosis and associated liver abscess.

Correlation of the bovine cervical mucus penetration test with human sperm characterisitics in 1,406 ejaculates.

The bovine cervical mucus penetration test (BCMPT) was performed to determine its usefulness in screening the ability of sperm to successfully penetrate mucus in vitro. Ejaculates were obtained by masturbation from patients attending an infertility clinic. Routine semen analysis was performed using a microcomputerized multiple-exposure photography system. The BCMPT was performed. Overall, the average penetration of the mucus was 38 +/- 0.46 mm. Of the 1,406 ejaculates analyzed, 244 (17%) displayed a negative result (0-20 mm), 291 (21%) a questionable result (21-30 mm), and 871 (62%) a positive result (>30 mm). A highly significant (p < .001) correlation between mucus penetration distance and sperm MD (r = 0.541), MI (r = 0.484), count (r = 0.475), motility (r = 0.448), velocity (r = 0.400) and morphology (r = 0.369) was observed. Overall, the finding of an abnormal semen parameter resulted in a 34 +/- 5% accurate prediction of a negative or questionable BCMPT (<30 mm), while a normal semen parameter resulted in a 90 +/- 4% accurate prediction of a positive BCMPT (>30 mm). Sperm MD showed the strongest positive predictive value (98%), while morphology showed the greatest negative predictive value (50%). Of the 1,406 samples, 25 +/- 2% of the samples with normal semen parameters displayed a negative BCMPT. Conversely, 6 +/- 2% of samples with abnormal parameters showed a positive BCMPT. The BCMPT successfully identifies a significant subpopulation of patients as having an inadequate penetration of mucus with otherwise normal semen characteristics.

Results of the American Association of Bioanalysts national proficiency testing programme in andrology.

Proficiency testing samples for antisperm antibodies (ASAB), sperm count, morphology and vitality were mailed to participating laboratories. The majority participating utilized Immunobead ASAB procedures (81 versus 14% mixed antiglobulin reaction and 5% 'other'), and there was 95.6 +/- 1.2% agreement on the presence or absence of ASAB. The majority of laboratories utilized manual (79%) versus computer assisted semen analysis (CASA; 15%) methods. Approximately 64% used the haemocytometer and 26% used the Makler counting chambers for manual counts. Coefficients of variation (CV) in sperm counts ranged from 24 to 138%, with CASA displaying lower overall CV (53 +/- 8%) than manual methods (80 +/- 9%). A wide variation in the reports of percent normal morphology was noted (CVs calculated from arc sin transformed means ranged from 15 to 93%). Participants using American Society of Clinical Pathologists (ASCP) criteria reported sperm morphology values that were clustered in the 'normal' range (11 out of 12 samples), while those using strict criteria were clustered in the 'abnormal' range (10 out of 12 samples). Good agreement was observed in sperm vitality (overall mean CV = 18%). These data highlight the urgent need for improvement in overall quality of andrology testing and indicate that practical proficiency testing programmes can be made available on a large scale.

Epidermal growth factor activates extracellular signal-regulated protein kinases (ERK) in freshly isolated porcine granulosa cells.

We investigated the ability of EGF to stimulate the phosphorylation (i.e. activation) of extracellular signal-regulated kinases (ERKs) in freshly isolated porcine granulosa cells (pGC) held in suspension. pGCs were isolated from the ovaries of prepubertal pigs at slaughter, and equilibrated for 24 h at 37 degrees C in 12 x 75 mm culture tubes. The cells were then treated with 0-10 ng/ml EGF for 1-240 min. Treatments were terminated, and the total cell lysates were subjected to SDS-PAGE and Western analysis. The Westerns were blotted with anti-panERK and with anti-phosphoERK, antibodies that recognize all forms of ERKs and the phosphorylated (i.e. activated) forms of ERKs, respectively. Western blot analysis with the panERK antibody revealed a gel shift of ERKs, suggesting hyperphosphorylation after treatment with as little as 0.1 ng/ml of EGF. Phosphorylation of the ERKs was confirmed by using the phosphoERK antibody, which indicated increased phosphorylation of ERKs above control with 0.1 ng/ml EGF and maximal phosphorylation of ERK with 5-10 ng/ml EGF. Activation of ERK by EGF, as measured by both gel shift analysis and active ERK blotting, in the freshly isolated pGC was rapid, increasing above controls after 1 min of treatment, maintaining high levels through 40 min, and declining from 60 to 240 min. These data indicate that EGF stimulates active ERK in a time- and concentration-dependent manner in freshly isolated pGCs and that this experimental approach represents an effective manner with which to evaluate the role of EGF and the ERK signal transduction pathway in freshly harvested pGC.

Perinatal outcomes in a prospective matched pair study of pregnancy and unexplained elevated or low AFP screening.

Unexplained elevated or low maternal serum alpha-fetoprotein (MSAFP) is reported to indicate adverse perinatal outcome. We designed a prospective matched pair study to gain additional information about the type and frequency of adverse neonatal or maternal outcomes. Subjects were selected from 16,445 women who received second trimester MSAFP screening during the 4.5 year study period. For each unexplained elevated or low subject, a control patient in the second trimester of pregnancy was chosen by matching eight individual traits. After follow-up, 356 pairs were identified, for the unexplained low MSAFP group and 139 pairs for the unexplained elevated MSAFP group. Outcome information was obtained by way of a physician questionnaire. For the unexplained low MSAFP group, complications occurred no more often than for the paired controls and these pregnancies may not be considered at high risk for adverse outcome. Our study supports a strong association between unexplained elevated MSAFP and adverse neonatal outcomes of low birth weight, fetal death, preterm delivery, and fetal growth retardation. The highest frequency of adverse neonatal outcome occurred when the multiple of median value was > or = 4.0. Our study does not support an association between elevated MSAFP and maternal complications of hypertensive disorder, oligohydramnios, or placental abruption.

Mechanisms of hormone and growth factor action in the bovine corpus luteum.

The binding of hormones and growth factors to their cell surface receptors leads to an orderly cascade of events leading to activation of cytoplasmic effector molecules. The mechanism of action of luteinizing hormone involves the stimulation of multiple signal transduction effector systems including adenylyl cyclase and inositol phospholipid-specific phospholipase C (PLC). This results in the formation of second messengers that activate cAMP-dependent, Ca(2+)-dependent and lipid-dependent protein kinases. Prostaglandin F(2alpha) activates PLC which increases intracellular calcium and activates protein kinase C. This results in the activation of a series of protein kinases in the mitogen-activated protein (MAP) kinase cascade, leading to the activation of nuclear transcription factors c-fos and c-jun. Hormone responsive effector systems, therefore, operate by activating families of protein kinases which regulate cell metabolism, secretion, and gene transcription. Growth factors activate specific receptor protein tyrosine kinases which recruit additional signaling molecules (phospholipase Cgamma, phosphatidylinositol 3-kinase, Shc, Grb2, etc.) initiating a cascade of events mediated via MAP kinases. The signaling pathways activated by hormones interact or cross talk with the signaling pathways activated by growth factors. The diversity of cellular signaling mechanisms elicited by hormones and the potential for interactions with signals generated by growth factor receptor tyrosine kinases, may allow fine tuning of cellular responses during the life span of the corpus luteum.

Effects of epidermal growth factor on the tyrosine phosphorylation of mitogen-activated protein kinases in monolayer cultures of porcine granulosa cells.

We have examined porcine granulosa cells (pGCs) for the presence of immunodetectable mitogen-activated protein (MAP) kinases (extracellular signal-regulated kinases, ERK) and have further studied the effects of epidermal growth factor (EGF) on the activation of these kinases. Cell lysates prepared from untreated monolayer cultures of pGCs were subjected to Western immunoblotting analysis using monoclonal antibodies to ERK1, ERK2 and pan-specific ERK. MAP kinases were detected having mol wts of 87K (ERK87), 54K (ERK54), 44K (ERK1), and 42K (ERK2). Treatment of pGCs with increasing concentrations (1-10 ng/ml) of EGF for 10 min resulted in electrophoretic mobility shifts of ERK1 and ERK2 suggesting hyperphosphorylation. Immunoprecipitation with an antiphosphotyrosine antibody (PY20), followed by Western analysis using pan-ERK, revealed a marked concentration-dependent increase in tyrosine phosphorylation of ERK2 in response to EGF treatment. The mobility shift and tyrosine phosphorylation of ERK2 was observed as early as 1 min after treatment with 10 ng/ml EGF. In-gel myelin basic protein (MBP) kinase assays revealed significant MBP kinase activity associated with ERK1 and ERK2 in total cell lysates and ERK2 in PY20 immunoprecipitates. Although ERK1 displayed a moderate mobility shift in response to EGF, tyrosine phosphorylation of this MAP kinase was not appreciably increased by EGF. Furthermore, PY20 immunoprecipitates demonstrated minimal MBP kinase associated with ERK1 in response to EGF treatment. Electrophoretic migration, tyrosine phosphorylation, and MBP kinase activity of the ERK54 and ERK87 was not effected regardless of EGF concentration or duration of treatment. These data demonstrate for the first time that pGCs contain immunodetectable MAP kinases. EGF, in a concentration- and time-dependent manner, increases tyrosine phosphorylation and MBP kinase activity (i.e. activation) of ERK2, and to a lesser degree ERK1, suggesting that the activation of MAP kinase may mediate the mitogenic action of EGF in pGCs.

Biochemical characterization of bovine alpha-fetoprotein and comparison with human alpha-fetoprotein.

This study compares the molecular, charge and lectin microheterogeneity of bovine alpha-fetoprotein (bAFP) with human (h) AFP. Molecular weights of bAFP (81 kDa) and hAFP (69 kDa) were detected by Western immunoblotting. Marked crossreactivity was found between bAFP and hAFP by Western immunoblotting but no crossreactivity was noticed by radioimmunoassays. At least seven charge isoforms of bAFP and three isoforms of hAFP were consistently detected by chromatofocusing in a mixture of fetal bovine serum (FBS) and human cord blood (hCB), while only three isoforms of bAFP and hAFP were detected in a mixture of bovine (bAF) and human amniotic fluid (hAF). Using concanavalin A (Con A) chromatography, 50% of bAFP was Con A reactive and 50% non-reactive, while more than 98% of hAFP was Con A reactive in a mixture of FBS and hCB. However, in AFs, 72% of bAFP was Con A reactive, while 89% of hAFP was Con A reactive. These data indicate that marked differences exist in both the charge and lectin microheterogeneity of bovine and human AFP.

Heterogeneity of gonadotropins and levels of uncombined luteinizing hormone subunits in pituitaries of cryptorchid rams.

Pituitaries were collected from intact rams and rams that had been rendered bilaterally cryptorchid by surgery to examine the effects of cryptorchidism on gonadotropin heterogeneity, levels of uncombined luteinizing hormone (LH) subunits, and the apparent molecular sizes of LH and follicle-stimulating hormone (FSH). Cryptorchid rams had higher pituitary contents of LH and FSH as well as reduced testicular weights. The levels of uncombined LH subunits, their apparent molecular weights, and the apparent molecular weights of intrapituitary LH were similar in control and cryptorchid rams. However, the apparent molecular weight of intrapituitary FSH was slightly larger in cryptorchid rams. Cryptorchidism altered the pattern of gonadotropin heterogeneity by shifting the distribution of LH isoforms towards basic components and shifting the distribution of FSH isoforms towards acidic components. Thus, it appears that the altered gonadal feedback mechanisms resulting from cryptorchidism modify the pattern of both LH and FSH heterogeneity by shifting the distribution of isoforms in opposite directions.

Purified human alpha-fetoprotein inhibits follicle-stimulating hormone-stimulated estradiol production by porcine granulosa cells in culture.

We have investigated the effects of purified alpha-fetoprotein (AFP) on follicle-stimulating hormone (FSH)-stimulated estradiol production by porcine granulosa cells in monolayer culture. Granulosa cells isolated from small follicles of prepubertal pigs were cultured for 2 days in 5% fetal bovine serum for attachment and incubated for 3 days in medium containing androstenedione and various treatments. The media were then collected and assayed for estradiol by radioimmunoassay. Human AFP significantly (P < 0.05) and dose-dependently inhibited FSH-stimulated estradiol production with 313 ng/ml AFP returning FSH-stimulated estradiol to basal levels; human serum albumin was without effect. AFP purified from either term cord blood or midtrimester amniotic fluid dose-dependently inhibited estradiol production stimulated by the combination of FSH and insulin-like growth factor-I. Furthermore, 125 ng/ml AFP inhibited estradiol production stimulated by cholera toxin, forskolin and cAMP. In contrast, extracellular accumulation of cAMP and progesterone production was not inhibited by AFP. These data indicate that physiological concentrations of purified AFP significantly and dose-dependently inhibit FSH-stimulated estradiol production by porcine granulosa cells in culture. Since AFP is known to augment growth factor-mediated cell growth, these data suggest that AFP inhibits differentiated functions (such as aromatase) while enhancing the proliferation of porcine granulosa cells.

Purified human alpha fetoprotein inhibits growth factor-stimulated estradiol production by porcine granulosa cells in monolayer culture.

Purified alpha fetoprotein (AFP) synergizes with transforming growth factor alpha (TGF alpha) and insulin-like growth factor I (IGF-I) to enhance proliferation of porcine granulosa cells (pGC) in primary culture, suggesting a role for AFP in the modulation of growth factor-mediated cell growth. TGF alpha stimulates basal estrogen production by pGC and is in fact more potent than FSH in these cells. In this study, we investigated the effects of AFP on growth factor-stimulated estradiol (E2) production by pGC. Basal production of E2 was not altered by the addition of AFP. AFP dose-dependently inhibited TGF alpha-stimulated E2 production with statistically significant inhibition observed with 2.5 micrograms/ml. We have previously shown that the mitogenic effects of AFP are maximized with TGF alpha+IGF-I. E2 production was even more sensitive to AFP inhibition when the two growth factors were combined. Human serum albumin (HSA; 10 micrograms/ml) was without effect. AFP did not interfere with the E2 RIA, affect the uptake of or display specific in vitro binding of the androgen substrate. Furthermore, human AFP and HSA did not exhibit specific in vitro binding of E2, in contrast to purified rat AFP (positive control). These data indicate that physiological concentrations of purified AFP significantly and dose-dependently inhibit growth factor-stimulated E2 production by pGC in culture. Since AFP is known to increase TGF alpha+IGF-I mediated cell growth, these data suggest that AFP may be inhibiting the differentiated function (steroidogenesis) of pGC while enhancing the proliferation of these cells.

Human alpha-fetoprotein purified from amniotic fluid enhances growth factor-mediated cell proliferation in vitro.

Using a primary monolayer culture of porcine granulosa cells (pGC) as an in vitro cell proliferation assay, we have examined the growth-promoting activity of alpha-fetoprotein (AFP) purified from term cord blood and midtrimester amniotic fluid. Increasing concentrations (2.5-20%) of crude human cord blood (CB) increased pGC proliferation, while identical concentrations of crude amniotic fluid (AF) were ineffective. When the cell system was maximally stimulated, AF dose dependently decreased cell proliferation. AFP purified from AF and CB (1.25-5.0 micrograms/ml) was not mitogenic alone, but, in the presence of epidermal growth factor (EGF) + insulin-like growth factor (IGF-I) (10 ng/ml each), AFP dose dependently increased cell proliferation to nearly double that of EGF + IGF-I alone. The response of pGC to the proliferative effects of AF-AFP and CB-AFP were identical at each dose of AFP tested. These results indicate that although crude, pooled midtrimester AF does not display the mitogenic activity seen in cord blood, AFP purified from pooled AF significantly synergizes with growth factors to increase cell proliferation markedly.

Ovine luteinizing hormone. V. Significance of flow-through peaks observed during chromatofocusing as revealed by various methods of sample preparation and application.

In a previous study [Keel et al., Biol, Reprod., 36 (1987) 1102] the ovine luteinizing hormone (oLH) in pituitary extracts was chromatofocused on pH 10.5-7 gradients after equilibration in 25 mM triethylamine-HCl, pH 11.0, by gel permeation. Under these conditions, some immunoreactive oLH flowed through the columns unrestricted and this was interpreted to represent extremely basic isoforms. However, when selected flow-through peaks were re-chromatofocused, each was contaminated with other isoforms of oLH. In order to clarify this dilemma, various methods of sample preparation and application were systematically compared. Consistent with previous observations, variable amounts of the immunoreactive oLH in pituitary extracts equilibrated in triethylamine by gel permeation, dialysis, flow dialysis or ion-retardation chromatography eluted as flow-through peaks when chromatofocused. In contrast, when the ionic components in the pituitary homogenization buffer were removed by these methods as well as ultrafiltration and the proteins were applied to the resin in the elution buffer (1:45 Pharmalyte 8-10.5-HCl, pH 7.0), none of the immunoreactive oLH in pituitary extracts eluted as a flow-through peak. Thus, it appears that oLH eluting as a flow-through peak results from incomplete binding of the hormone to the chromatofocusing resin when applied in triethylamine.