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Selection of transgenic embryos prior to embryo transfer is a means to increase the efficiency of transgenic livestock production. Among transgenic reporters, cytoplasmic expression of green fluorescent protein (GFP) has features that make it ideal for transgenic embryo selection. The primary objective of this study was to assess cytoplasmic expression of a specially designed GFP reporter as a tool for transgenic bovine embryo selection. A second objective was to evaluate this reporter for studying transgenic mosaicism related to timing of integration of pronuclear microinjected DNA. Transgenic embryos produced by pronuclear injection showed a discrete pattern of GFP expression with clusters at 25, 50, and 100% of blastomeres expressing GFP. This pattern of mosaicism is interpreted to indicate that the integration of microinjected DNA occurred, not only at the pronuclear stage, but also in the subsequent cell divisions. Among the GFP-positive transgenic embryos, only in 21% did all the blastomeres show the green fluorescence. Using the fraction of positive blastomeres within an embryo, the timing of integration of microinjected DNA was estimated. The frequency of nonmosaic embryos expressing GFP is consistent with published germline transmission success rates of transgenic cattle derived from pronuclear microinjected embryos. These results indicate the possible application of GFP as a marker of transgenic embryos and graphically illustrate underlying complexities in DNA integration in embryos subjected to pronuclear microinjection.

A critical requirement for integration of retroviruses, other than HIV and possibly related lentiviruses, is the breakdown of the nuclear envelope during mitosis. Nuclear envelope breakdown occurs during mitotic M-phase, the envelope reforming immediately after cell division, thereby permitting the translocation of the retroviral preintegration complex into the nucleus and enabling integration to proceed. In the oocyte, during metaphase II (MII) of the second meiosis, the nuclear envelope is also absent and the oocyte remains in MII arrest for a much longer period of time compared with M-phase in a somatic cell. Pseudotyped replication-defective retroviral vector was injected into the perivitelline space of bovine oocytes during MII. We show that reverse-transcribed gene transfer can take place in an oocyte in MII arrest of meiosis, leading to production of offspring, the majority of which are transgenic. We discuss the implications of this mechanism both as a means of production of transgenic livestock and as a model for naturally occurring recursive transgenesis.

A longitudinal study was carried out on samples of bulk tank milk collected from 200 farms for 12 mo to evaluate SCC, plasmin and plasminogen activities, psychrotrophic bacteria count, and protein quality in milk and to examine the proteolytic effects of plasmin on milk proteins. Herds were selected randomly from client lists of two dairies to create four groups based on milk SCC of the month before the study; herds were reassigned monthly to one of four groups based on SCC for that month. Overall means were 3.73% fat, 3.13% protein by infrared analysis, 3.16% protein by Kjeldahl analysis, 2.42% casein percentage, 4.65% lactose, 5.43 cells/ml of log SCC, 1.13 U of plasmin, 45.6 U of plasminogen, and log 2.86 cfu/ml of psychrotrophic bacteria. The ANOVA showed a significant effect of month on all factors except SCC, which was fixed by the experimental design. Plasmin and plasminogen activities were high from December to May. Plasmin activities and psychrotroph counts were significantly higher for the high SCC group. Casein percentage and number were significantly higher for the low SCC group.

The serum concentration of beta-LG at wk 26 of pregnancy was determined in Holstein heifers of known genetic merit to evaluate use of a single blood sample as an index to predict their subsequent milk yield. Concentrations of beta-LG varied widely (16 to 1200 ng/ml) in the serum of the heifers at this stage of pregnancy. Subsequent measures of lactation performance showed that heifers with high beta-LG concentrations in pregnancy produced more milk than those with low beta-LG in serum. Previous studies have shown that the concentration of beta-LG in serum shows a log-linear increase during pregnancy, which is consistent with correlation to the growth of the mammary parenchyma. Therefore, the logarithm beta-LG concentration was used as the index in this study. The correlations of this index were .46, .54, and .48 for first lactation 305-d yields of milk, fat, and protein, respectively. The results suggest that serum concentrations of beta-LG estimated from a single blood sample at this stage of pregnancy can be used to predict milk yield. Analysis showed that an explanation of future milk yield was significantly improved by use of serum beta-LG and parent average. Serum beta-LG can be used as a physiological marker to prescreen heifers for lactation potential.

Transgenic mice were produced to study the production of bovine alpha-LA in their milk. A 7.6-kb fragment containing a bovine alpha-LA gene was purified and microinjected into pronuclear stage mouse embryos. This fragment contained 2.0 kb of 5' flanking region, the 1.7-kb coding region, and 2.7 kb of 3' flanking region. Out of 121 potential transgenic founder mice, 3 were identified as being transgenic by the polymerase chain reaction. Multiple mice from the second, third, and fourth generation from each line were milked, and the milk was analyzed using an ELISA assay and Western blots to determine the presence of bovine alpha-LA. Bovine alpha-LA was present at concentrations up to 1.5 mg of protein/ml of mouse milk. The high degree of expression variation between mice within each of the transgenic lines was a characteristic that has not been reported in other studies of transgene expression in milk. Production of bovine alpha-LA in the milk of these transgenic mice showed a high degree of variation both within a lactation and between mice within a line. The bovine alpha-LA concentration in a single line of transgenic mice exhibited as much as a 10-fold variation between mice. Variations as high as 3-fold were detected within a single lactation in the same mouse. These differences in expression appeared to be correlated with mouse milk production; bovine alpha-LA was higher on d 10 and 15 of lactation than on d 5. Transgenic mice that show variation in expression of a bovine gene might offer a unique system for studying quantitative traits in a laboratory model.

The alpha-lactalbumin (+15) polymorphism (a single base variation 15 basepairs 3' of the alpha-lactalbumin transcription start point) was examined for its usefulness as a genetic marker for Holsteins. The +15 polymorphism is located in a region of the gene that is potentially involved in the regulation of alpha-lactalbumin gene expression. Animals from two dairy herds and young sires from progeny-testing programs of four AI organizations were used in the analysis. A group of sons from a heterozygous sire were also evaluated. Each individual animal was genotyped at the alpha-lactalbumin (+15) locus, and differences of genotypes were investigated. Estimated differences among alleles were calculated for PTA for milk, kilograms of protein, protein percentage, protein dollars, kilograms of fat, fat percentage, and fat dollars. Animals having the alpha-lactalbumin (+15) AA (an adenine on both alleles at position +15) genotype had statistically higher PTA for milk, kilograms of protein, protein dollars, kilograms of fat, and fat dollars than did the alpha-lactalbumin (+15) BB (a cytosine, guanine, or thymine on both alleles at position +15) animals. The alpha-lactalbumin (+15) BB animals had higher protein and fat percentages than the alpha-lactalbumin (+15) AA animals. Animals that were heterozygous at this locus, alpha-lactalbumin (+15) AB, had intermediate values for all traits analyzed. These results indicate a potential marker or actual locus effect of the alpha-lactalbumin (+15) polymorphism in Holstein cattle.

Recent progress in recombinant DNA technology as well as in embryo manipulation and transfer has made the introduction of specific genes into the germline of animals relatively commonplace. With appropriate genetic constructs expression of the inserted genes in transgenic animals can be controlled in a tissue-specific and in a differentiation-specific manner; thus, it is now possible to consider alteration of the composition of milk produced by a lactating animal in any of a variety of ways. There is a growing list of foreign milk proteins that have been expressed, and one can envisage placing almost any protein gene of interest under the control of the cis-acting promoter and enhancer elements of a milk protein gene. Modification of milk composition can be extended not only to the proteins of commodity value but also, by manipulation of key metabolic enzymes, to fat, lactose, and other minerals in milk.

Exogenous daily oxytocin injections given immediately before milking increase milk production. To investigate the mechanism by which oxytocin increases milk production, oxytocin injections were given before and after milking, and saline injection was given before milking as a control. The experimental design was a replicated Latin square; two complete trials were performed: one with 12 cows (45 d) and another with 15 cows (95 d). In the first trial, the least squares means of milk production were 29.2, 29.3, and 28.3 kg for oxytocin injection before milking, oxytocin injection after milking, and saline injection before milking, respectively. In the second trial, the least squares means of milk production were 33.3, 32.9, and 32.4 kg for oxytocin injection before milking, oxytocin injection after milking, and saline injection before milking, respectively. Oxytocin before and after milking significantly increased milk production by 3%. The results suggest that increases in milk production may not be caused by removal of residual milk but by increased gland output of milk. The effect on milk plasmin activity, fat, protein, SCC, and lactose was nonsignificant and may indicate that effect of oxytocin is not manifested through an effect on cell remodeling.

We have found that xerographic paper (regular photocopy or laser printer paper) can be used as a transfer medium for protein blots for the immunodetection of low concentrations (a few nanograms) of bovine alpha S1-casein. With paper blotting, we could detect the protein with three times more sensitivity than with polyvinylidene difluoride. Blotting was achieved by transfer from sodium dodecyl sulfate-polyacrylamide gel electrophoresis to the methanol-wet paper. The blot was incubated with chicken anti-casein antibodies, sequentially peroxidase-labeled rabbit anti-chicken antibodies, and diaminobenzidine substrate. The blots were directly scanned and the pixel intensity of the band areas was integrated. An analysis of the scanned blots showed that the log of protein concentration was linearly related with the square root of an integrated pixel intensity (r2 greater than 0.96). This linear relationship was observed in a wide range of protein concentration from 5 ng to 15 micrograms. The coefficients of variation were 12.4% for intraassay and 15.7% for interassay. This new analytical procedure can be applied to estimate the concentration of proteins by immunological blotting.

Enzyme-linked immunosorbent assays for bovine alpha-lactalbumin and beta-lactoglobulin have been developed for measurements of serum and tissue culture samples. Either alpha-lactalbumin or beta-lactoglobulin antiserum was coated on ELISA plates. Biotinylated proteins were used in competition with unknown amount of proteins in samples. After unbound proteins were washed off, ExtrAvidin-peroxidase and tetramethylbenzidine were then used as a detection system. Crossreactivity of caseins or bovine serum albumin was less than .0001% in either alpha-lactalbumin or beta-lactoglobulin ELISA. Parallel curves from serial dilutions were obtained in serum and media samples. The additivity of alpha-lactalbumin and beta-lactoglobulin ELISA was validated in either serum or medium samples. The intraassay and interassay coefficients of variation for alpha-lactalbumin and beta-lactoglobulin ELISA were below 10% over 51 and 47 assays. The ELISA are useful in mammary gland biology studies for measuring milk whey protein in serum or culture media.

Serum concentrations of alpha-lactalbumin and beta-lactoglobulin in first pregnancy, parturition, lactation, involution, and second parturition in 37 Holstein cattle were determined and used as an index of mammary status and in predicting milk yield. During first pregnancy, serum alpha-lactalbumin increased in the last 3 mo and reached a peak at parturition (approximately 1100 ng/ml). Changes in alpha-lactalbumin could not be described by a simple exponential equation, whereas changes in serum beta-lactoglobulin were described by a single exponential from second trimester until 4 wk prepartum and reached a peak at parturition (approximately 460 ng/ml). By 2 wk after parturition, alpha-lactalbumin had dropped to approximately 140 ng/ml, and beta-lactoglobulin dropped to approximately 25 ng/ml. In late lactation, alpha-lactalbumin was approximately 70 ng/ml and beta-lactoglobulin approximately 20 ng/ml. Short-term elevations were found after cessation of milking in both alpha-lactalbumin and beta-lactoglobulin in serum. The concentrations of alpha-lactalbumin and beta-lactoglobulin at second parturition were similar to those at first parturition with no differences found between parity. Both alpha-lactalbumin and beta-lactoglobulin in serum were functionally associated with mammary growth and development. In heifers late in pregnancy, both serum concentrations of alpha-lactalbumin and beta-lactoglobulin were positively correlated with mature equivalent milk and fat yields in the subsequent lactation. Serum beta-lactoglobulin concentrations at 16 wk prepartum in heifers were highly correlated with the sum of first and second lactation milk (r = .60) and fat (r = .60) yields. The potential value of using serum beta-lactoglobulin as an index for prescreening of heifers for lactation potential is discussed.

In the membrane filter-deoxyribonucleic acid test, somatic cells are trapped on a special filter. A chemical determination of the DNA of the trapped material gives an estimate of the number of cells. The MF-DNA was evaluated and compared with the direct microscopic somatic cell counts (DMSCCs) in 10 laboratories. Cell levels in the 10 bulk milk samples analyzed ranged from 380,000 to 1,700,000/mL. Regression analysis gave a relation of 0.181 absorbance at 490 nm, when 2.5 mL of milk was analyzed and 5.0 mL reagent was used for color development. The 95% confidence limits on the slope were 0.158-0.204. Analysis of variance showed that both procedures had significant (P less than 0.001) systematic errors, but that only the DMSCC method showed a significant laboratory X sample interaction (P less than 0.001). The repeatability standard deviation of the MD-DNA procedure was 117,000 cells/mL (14%), and the reproducibility standard deviation was 198,000/mL (23%). This compared favorably with the DMSCC method which has a repeatability of 162,000/mL (19%) and a reproducibility of 187,000/mL (34%). The error of the MF-DNA was constant at all cell counts, whereas there was a tendency for the error to increase in the DMSCC method at high cell counts. This method has been adopted as official first action.

At a free Mg2+ concentration of 1.0 mM, myosin binds one Ca2+ per molecule when the Ca2+ concentration is 20 muM, a value in the concentration range expected during contraction of skeletal muscle. Mg2+ alters Ca2+ binding in a complex manner, not by simple competition. In the range from 20 to 100 muM Mg2+ it produces positive cooperativity between the high-affinity Ca2+ binding sites, in addition to shifting binding to higher Ca2+ concentrations. High-affinity Ca2+ binding is not significantly affected by the addition of ATP, increase in ionic strength to 0.1 and changes in temperature. Ca2+ binding did not increase actin-activated ATPase activity in the absence of regulatory proteins, but rather inhibited it.

Considerable discrepancies exist in the literature concerning the size and activity of bovine placental lactogen. Our bovine placental lactogen purification preparations were 20% as active as bovine PRL (bPRL) on a per weight basis when compared to bPRL in a lactogenic radioreceptor assay. To identify the active component in these preparations, the proteins were radioiodinated and bound to membrane receptors in the presence and absence of competing hormones, bPRL, and bovine GH (bGH). After centrifugation, membrane pellets were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gels were autoradiographed. Only one radioiodinated protein band was present. This protein was displaced in the presence of competing hormone and comigrated which a major component of the purification preparations. In radioreceptor assays the active component was as active as bPRL and bGH. From the migration of protein standards included with the radioiodinated purification preparations in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we estimate that the protein is 32,000 mol wt--about 10,000 mol wt larger than placental lactogens isolated in other species. The possibility that the active molecule was a precursor protein was investigated by examining proteins secreted by bovine placenta tissue cultures. The binding activity in these secretions, as well as in the purification preparations, eluted between ovalbumin (43,000 mol wt) and bPRL (22,000 mol wt) under nondenaturing conditions using high performance gel filtration chromatography. Analysis of this secreted protein, also by binding to membrane receptors, showed that the protein had the same molecular weight as that isolated from the purification preparations and was specifically displaced by the same hormones.

Bovine placental lactogen (bPL) was purified 4,200-fold from cotyledon homogenates by 1) salt precipitation and 2) ultrafiltration, followed by 3) gel filtration, 4) anion exchange, 5) hydroxylapatite, 6) chromatofocusing, and 7) final gel filtration chromatography. Purification was monitored by radioreceptor assay (RRA) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. During chromatofocusing, three distinct peaks of bPL were eluted: a small peak at pH 5.34 (bPL-1), a larger peak at pH 5.09 (bPL-2), and the largest peak at pH 4.84 (bPL-3). These three were further purified separately by the final gel filtration. The purified bPLs were compared with bovine (b) PRL (0.81 IU/mg) and bGH (30 IU/mg) by RRA. The bPRL-like activities were 67.1, 750, and 1,090 micrograms/mg, and the bGH-like activities were 74.4, 787, and 715 micrograms/mg for bPL-1, bPL-2, and bPL-3, respectively. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weights of all bPLs were the same, 31,100. In gel filtration under nondenaturing conditions, all bPLs had a 40,000 mol wt, with a Stokes radius of 2.70 nm. The isoelectric point (pI) of bPL-1 was higher than 5.85, the pI of bPL-2 was 5.52, and that of bPL-3 was 5.39. The amino acid compositions of bPL-2 and bPL-3 were virtually identical, except that bPL-3 had a few more acidic amino acids. Both bPL-2 and bPL-3 had an estimated 277 amino acid residues. Thus, bPL appeared to have both PRL- and GH-like activities equivalent to bPRL and bGH in the RRA on a mass basis, to be 50% larger than bPRL, bGH, and PLs in other species, and to occur in at least three different forms differing in their pI values and in their acidic amino acid compositions.

A new technique for obtaining a myofibril-like preparation from vertebrate smooth muscle has been developed. An actomyosin can be readily extracted from these myofibrils at low ionic strength and in yields 20 times as high as previously reported. The protein composition of all preparations has been monitored using dodecylsulfate-gel electrophoresis. By this method smooth muscle actomyosin showed primarily only the major proteins, myosin, actin and tropomyosin, while the myofibrils contained, additionally, three new proteins not previously described with polypeptide chain weights of 60000, 110000 and 130000. The ATPase activities of both the myofibrils and actomyosin preparations are considerably higher than previously described for vertebrate smooth muscle. They are sensitive to micromolar Ca2+ ion concentrations to the same degree as comparable skeletal and cardiac muscle preparations, even though troponin-like proteins could not be identified in these smooth muscle preparations. From the latter observation and the presence of Ca2+-sensitivity in tropomyosin-free actomyosin it is suggested that this calcium sensitivity is, as in some invertebrate muscles, a property of the myosin molecule.

Internal peptide fragments containing tryptophan residues are useful predictors of coding sequence for selection of restriction enzyme fragments or synthetic oligonucleotides to use in isolation of a cDNA or genomic clone. We describe a strategy to identify such fragments which uses an on-line photodiode array spectrophotometric analysis of tryptic fragment elution from an HPLC system to select peptides for amino acid sequence analysis. We applied this information to the identification and subsequent isolation of a cDNA corresponding to bovine placental lactogen from a bovine placental cDNA library which contains numerous closely related gene products.

Previous work has shown that, unlike other species, placental lactogen (PL) in the bovine (bPL) has a mol wt of approximately 32,000 and exists in several different forms with different isoelectric points. This study was carried out to develop a more rapid purification scheme, whereby the yield of bPL obtained was increased while at the same time the possibility of artifacts from a prolonged purification protocol was decreased. A procedure was developed in which a fraction enriched in bPL-containing granules was obtained after gentle disruption of the binucleate cells of the fetal cotyledon. The fetal portion of the placentomes from midpregnant cows was minced with scissors and vigorously stirred in order to remove and disrupt binucleate cells within the fetal villi. The supernatant from this step was fractionated by differential centrifugation followed by a four-step discontinuous Percoll gradient of 1.03-1.08 g/ml. A granule-enriched fraction was isolated from the 1.04 g/ml zone from which membrane-enclosed protein was released by freezing and thawing. Membranes and insoluble proteins were sedimented by high speed centrifugation to yield an extract which contained approximately 20% of the hormone initially in the tissue. Two subsequent chromatographic steps, gel filtration on Sephadex G-75 and high performance reversed phase chromatography with a C-4 column, resulted in a preparation of greater than 98% homogeneity. Two-dimensional gel electrophoresis of purified bPL revealed at least nine protein spots in the 31,000-33,000 mol wt range with isoelectric points ranging from 4.85-6.3. All forms exhibited parallel dilution curves in a RIA for bPL. It would appear, therefore, that multiple forms of bPL exist and that they are not artifacts of the prolonged purification protocol previously used.

The response of mammary myoepithelial cells to oxytocin was studied by monitoring the level of phosphorylation of the 20,000 mol wt light chain of myosin. Myoepithelial cells were obtained by collagenase dispersion of involuted rat mammary tissue. The cells were equilibrated with [32P]orthophosphate, and then stimulated with oxytocin. Phosphorylated proteins were separated by polyacrylamide gel electrophoresis, and incorporation of 32P into the proteins was detected by autoradiography. Nanomolar concentrations of oxytocin caused a 3-fold increase in the level of phosphorylation of the myosin light chain within 0.5 min. When the cells were incubated with oxytocin in a calcium-free medium, there was only a transient phosphorylation of myosin. However, readdition of calcium to these cells resulted in phosphorylation of the myosin light chain. The results suggest that calcium is involved in the intracellular events following stimulation of the cells with oxytocin.

Oxytocin binding activity of myoepithelial cell membranes from mammary tissue was measured under a variety of different experimental conditions. Mammary tissue from non-lactating rats bound oxytocin with a Kd of 9.2 +/- 1.6 nM (+/- S.E.) and indicates that receptors are retained by the myoepithelial cells in a non-lactating state. Ovariectomy of non-lactating rats did not depress the binding activity of the membranes. Administration of the estrogenic compounds estradiol-17 beta and diethylstibestrol at doses which affect uterine weight and are known to increase uterine oxytocin binding did not influence the binding activity of the myoepithelial cells. This indicates that the oxytocin receptors of the mammary gland are not under the same endocrine control as the uterine receptors.

Advances in genetic technology have made it possible to consider making substantial changes either in the composition of milk or in the production of entirely new products in milk. The technological capabilities that have given rise to the introduction and expression of new genes in animals are discussed. Examples are given of transgenic animals that express foreign proteins in their milk. Advantages of the mammary synthesis of proteins are discussed and potential alterations of milk composition and scenarios for introduction of new proteins are considered. Technological capabilities that either currently exist or are being developed are discussed along with the requirements for making it feasible to utilize the technology on a broad scale in dairy cattle.

Oxytocin (10 nM) stimulated the phosphorylation of the 20,000 mol wt myosin light chain in rat mammary myoepithelial cells from a basal level of 0.17 to 0.85 mol phosphate/mol light chain within 30 sec. Of the smooth muscle stimulants tested, oxytocin appears to be the only normal physiological stimulus for myosin phosphorylation in these cells. The roles of cAMP, cGMP, and calcium ions were investigated in the mode of action of oxytocin and the regulation of myosin phosphorylation. Although oxytocin had no effect on cGMP metabolism, there was an increase in the cAMP content of the treated myoepithelial cells. Further investigation suggested that the increase in cAMP levels in response to oxytocin was not directly involved in the regulation of myosin phosphorylation. Various agents known to interfere with calcium ion transport were used to study the role of calcium ions in the action of oxytocin and the regulation of myosin phosphorylation. The results indicate that the duration of the cellular response to oxytocin depends on an influx of extracellular calcium through calcium-specific channels in the plasma membrane.

The oxytocin-sensitive myoepithelial cells of the mammary gland form a system with characteristics of a potentially useful model for studying the mechanism of action of oxytocin and coupling phenomena of excitation-contraction. Our objectives were to develop a method for isolating mammary actomyosin, to determine the amount of actomyosin in the glands of lactating and nonlactating animals, and to investigate control of contractile protein interaction. Actomyosin in mammary glands represented a substantial portion of the soluble protein in the gland ranging from 9% of the total in lactating to 17% in weaned rats. The isolated actomyosin had a molecular composition like that of actomyosin of smooth muscle and the isolated actomyosin contained a light chain kinase that phosphorylated the 20,000 dalton light chain of myosin (L20). The kinase isolated as a component of actomyosin preparations did not show calcium control, but it did when isolated from mammary cytosol. Strips of involuted mammary tissue from rats developed tension when oxytocin was added to the bathing medium; thus, the myoepithelial cells appeared to retain their sensitivity to oxytocin even in nonlactating animals and may be a useful model for studying the action of oxytocin. We suggest that one of the final steps in the milk-ejection reflex is phosphorylation of myosin causing a contraction of the myoepithelial cells of the mammary gland.

Mammary tissue from five midpregnant heifers was transplanted subcutaneously into ovariectomized athymic mice (eight pieces/mouse). After a recovery period of 19 days, mice were injected daily for 5 days with buffer (50 mM NH4HCO3, pH 7.8) as control, 17 beta-estradiol (1 micrograms) plus progesterone (1 mg). Concurrently with the buffer or steroid hormone injections, mice were injected with bovine placental lactogen (0, 5, or 25 micrograms), bovine prolactin (0, 3.4, or 17.2 micrograms), or bovine growth hormone (0, 3.4, or 17.2 micrograms). All mice were injected with 2-bromo-alpha-ergocryptine (0.1 mg/day). Transplanted bovine mammary tissue was incubated for 4 hr in minimum essential medium containing 1 mu Ci/ml [3H]TdR. Two pieces were processed for autoradiography and the others were used for DNA assay and total [3H]TdR uptake. Bovine placental lactogen, prolactin, and growth hormone each increased [3H]TdR incorporation into DNA in a linear, dose-response manner. Addition of ovarian steroids to bPL resulted in a significant increase over protein hormones alone. Autoradiographic analysis indicated that the observed differences in DNA synthesis were due to hormonal effects on epithelial, rather than stromal, DNA synthesis. These results provide the first evidence of a mammogenic role of bovine placental lactogen.

The concentration of bovine placental lactogen (bPL) was determined in fetal placentomes, allantoic fluid, amniotic fluid, maternal and fetal plasma throughout pregnancy. In addition, chromatofocusing chromatography was used to separate the different forms of bPL found both in fetal serum and in placental homogenates in order to determine whether the different forms that have been reported to exist in the cotyledon are also found in the fetal circulation. Reproductive tracts were collected from cows between 109 and 247 days of pregnancy. The concentration of bPL in the fetal cotyledonary tissue was measured by both radioreceptor assay and radioimmunoassay, both assays showed that the concentration of bPL in the fetal portion of the placentomes remained constant throughout the period of pregnancy tested. The mass of the placenta increased approximately 10-fold during the period of study but the concentration of bPL in the maternal plasma was low (0.9 +/- 0.1 ng/ml) at all stages of pregnancy tested. The mean concentration of bPL (Mean +/- S.E.M.) in amniotic and allantoic fluid was 0.4 +/- 0.1 and 1.2 +/- 0.2 ng/ml respectively. Fetal blood contained the highest concentrations of bPL, from 11.6 to 18.4 ng/ml, and the concentration tended to decrease with advancing gestation (slope = 0.07, P = 0.001). Several forms of bPL were found in the fetal circulation; however, a higher percentage of forms with more acidic isoelectric points were found in the fetal serum than in placental homogenates. These results suggest that either some forms of bPL are more stable or that the hormone isolated from placental tissue is not representative of the final secreted product.

In a previous report we showed that purified bovine placental lactogen (bPL) exists in two isoforms in the 31,000-33,000 Mr range, each with at least five isoelectric variants differing in approximately 2 orders of magnitude in isoelectric points (pI) 4-6. The multiple isoelectric variants are unique to the bovine hormone. In an effort to determine the nature of these variants endo- and exoglycohydrolase digestions were conducted to determine if this hormone was glycosylated. Analysis of peptide/N-glycosidase F and endoglycosidase F digests of radioiodinated bPL on one-dimensional gel electrophoresis showed a Mr decrease from 31,000 to 24,000 and 33,000 to 26,000 for the two isoforms. Digestion with a mixture of neuraminidase plus mixed exoglycosidases resulted in a Mr decrease of 4,000. Digestion with neuraminidase resulted in a Mr decrease of 2,000. Further analysis of peptide/N-glycosidase F- and neuraminidase-treated bPL by two-dimensional gel electrophoresis showed the isoelectric variants shifted from pI 4.4-6.3 to 4.9-8.0. The sialic acid residues on the N-linkage are responsible for the pronounced acidic character of bPL, but do not account for the residual charge heterogeneity as the different isoelectric variants persist after sialic acid removal. The apparent Mr of the protein after removal of N-linked carbohydrate residues is similar to that of PRL and GH. These enzymatic digestion results demonstrate the presence of N-linked complex oligosaccharide residues attached to the beta-amide group of an asparagine residue. Analyses of the sugar content of the molecule were consistent with the presence of one biantennary N-linked and two O-linked carbohydrate chains.

The bovine placenta secretes at least one hormone with prolactin-like and placental growth-hormone-like activity. The cDNA for bovine placental lactogen was isolated from a bovine fetal cDNA library by virtue of its nucleotide sequence homology to bovine prolactin (70%) and identified as such from amino acid sequence obtained from the amino terminus and internal tryptic fragments from the isolated hormone. The cDNA predicts a preprohormone of 236 amino acids, with a signal peptide of 36 amino acids. A single consensus site for N-glycosylation marks a probable site of carbohydrate addition. The encoded hormone is quite distinct from the pituitary hormones, as well as the primate and rodent placental lactogens and other predicted bovine placental hormones. It is 51% similar to bovine prolactin in amino acid sequence, 30% similar to the protein predicted by bovine prolactin-related cDNA I, about 30% similar to the rodent predicted placental hormones, and only about 20% similar to human placental lactogen and bovine growth hormone. Despite its greater similarity to bovine prolactin, sequence homology in the region of 5' flanking sequences and first exon to bovine prolactin-related cDNA I suggests that bovine placental lactogen may share a common evolutionary origin with this other placentally expressed member of the prolactin gene family.

Computer image analysis programs developed by astronomers for celestial photometry were used for the analysis of autoradiographs of electrophoretic slab gels. Oxytocin-stimulated myosin light chain phosphorylation in mammary myoepithelial cells was studied by incubating cells with [32P]orthophosphate followed by oxytocin addition, polyacrylamide gel electrophoresis of cellular protein, and autoradiography of electrophoretic slab gels. After scanning and digitization of autoradiographs, [32P]phosphate incorporation into the myosin light chain was measured by the determination of radiation flux recorded on the X-ray film. Within seconds after oxytocin addition, the myosin light chain was fully phosphorylated. The large library of astronomical computer programs has applications for the quantitative analysis of both one- and two-dimensional electrophoretic gels.

Bovine placental lactogen (bPL) has been isolated from bovine trophoblast and characterized as a 32 K mol wt protein which exists in three different forms which differ in their isoelectric point values and their amino acid compositions. Two of the three forms have been shown to have both bovine GH (bGH)- and bovine PRL (bPRL)-like activities equal on a molar basis to bGH and BPRL in radioreceptor assays. It has been postulated that, in sheep, PL is delivered to the maternal circulation by the migration of fetal binucleate cells from the trophoblast across the fetal-maternal boundary into the uterine epithelium. To determine whether an analogous situation exists in the cow, antibodies to bPL were used to localize bPL in bovine placentomes and to measure its concentration in fetal and maternal sera. For cytology, bPL was localized on sections of placentomes from midgestation and term bovine placentas using an indirect immunoperoxidase technique. Stained binucleate cells were demonstrated throughout the trophoblast, often in close association with the microvillous boundary which separates the trophoblast from the maternal epithelium. In cross-sections of fetal villi, binucleate cells with cytoplasmic processes extending into and through the uterine epithelium were immunostained as well as cells within the plane of the uterine epithelium in close approximation or apposition to the maternal basement membrane. RIA demonstrated bPL to be present in maternal sera in concentrations of 1-2 ng/ml and in fetal sera at 5-12 ng/ml. These data are consistent with the hypothesis that binucleate cell migration accomplishes the delivery of bPL to the maternal circulation.

We studied the relationship of serum prolactin, estradiol-17 beta, and progesterone concentrations to plasma calcium, phosphorus, and free hydroxyproline concentrations, as well as to dry matter intake, in 14 aged dairy cows (mean of 4.5 parities), 7 of which became paretic, from 28 days before to 4 days after calving. Plasma calcium and phosphorus concentrations and dry matter intake decreased more at parturition in paretic cows than in nonparetic cows. Prolactin concentrations were not different between paretic and nonparetic cows, but were variable. Concentrations of estradiol were higher in paretic cows from 15 to 5 days before parturition, whereas hydroxyproline concentration was lower in paretic cows on days 10 through 3 before parturition. Progesterone concentration was lower in paretic cows and decreased earlier at parturition, compared with that in nonparetic cows. The findings suggested that high estradiol concentrations in late pregnancy inhibit bone resorption and predispose aged cows to parturient paresis. The earlier decrease in progesterone concentration at parturition and lower concentrations throughout late pregnancy might have contributed to the greater inappetence in paretic cows at parturition. The importance of prolactin in the pathogenesis of parturient paresis is not clear.

The biological function of bovine placental lactogen is unknown. However, bovine placental lactogen is known to bind prolactin receptors in rabbit mammary gland. This hormone was therefore cultured with rabbit mammary gland explants to confirm that it is lactogenic in this species. Mammary explants from pregnant heifers were also cultured with bovine placental lactogen to determine if the same preparation of hormone possessed similar lactogenic potential in homologous species. Bovine placental lactogen was tested over a range of concentrations from 10 to 500 ng/ml and was about 50 to 80% as potent as bovine prolactin when cultured with mammary explants from 19-d pregnant virgin rabbits. Lactogenic response was assessed by both the incorporation of [14C]acetate into lipid and by the synthesis of casein. Bovine placental lactogen displayed negligible lactogenic activity when cultured with mammary explants from 6 to 7-mo pregnant heifers. Lactogenic response was assessed using the same criteria as used with the rabbit mammary explants; in addition, accumulation of alpha-lactalbumin in the explants was also measured. Although bovine placental lactogen was lactogenic in the rabbit, the same hormone preparation was apparently not lactogenic in cattle. It is therefore vital to test for the biological activity of a hormone in a homologous system, because inappropriate conclusions may be drawn from the response obtained in a heterologous system.