M.F. McGrath

Cloning and in vivo expression of bovine growth hormone receptor mRNA.

A cDNA for the bovine growth hormone (bGH) receptor has been cloned out of a cDNA library prepared from liver of a pregnant Holstein heifer. The cDNA clone hybridizes to a single 4.5 kb mRNA species and shares a high degree of sequence homology with growth hormone receptors cloned from other species. Utilizing the bGH receptor cDNA as a probe, a relatively high level of bGH-receptor mRNA was detected in bovine liver. In comparison to liver values, lower concentrations of bGH-receptor mRNA were detected in bovine kidney, anterior pituitary, and mammary gland. Because specific binding sites for bGH have not been convincingly demonstrated in isolated cell membranes from whole bovine mammary tissue, mammary tissue from two pregnant heifers (separate experiments) was separated into fractions enriched for epithelium, stroma, and blood components. These fractions were then probed for growth hormone receptor mRNA using solution hybridization-nuclease protection assays performed on isolated RNA. The assay results indicated that a low level of bGH-receptor mRNA is relatively evenly distributed throughout the mammary tissues of the two cows studied. In contrast, experiments using a probe to bovine insulin-like growth factor-I (IGF-I) indicate that the IGF-I mRNA is localized in the stromal/blood component of the mammary gland. These data suggest a possible paracrine mechanism for bGH action in the mammary gland.

Regulation of bovine mammary growth by peptide hormones: involvement of receptors, growth factors and binding proteins

Abstract Involvement of the somatotropin (ST)/prolactin(PRL)/placental lactogen (PL) family and their mediators in regulating mammary growth in cattle was evaluated using both in vitro and in vivo models. Mitogenic activity of bovine mammary epithelial cells (BMEC) was not increased by ST, PRL or PL in vitro. Direct infusion of ST but not PRL into mammary glands of late pregnant beef cows increased mammary DNA. Exogenous treatment of steroid-primed dairy heifers with PL increased mammary DNA while PRL treatment resulted in lactogenesis but no detectable increase in mammary DNA. Insulin-like growth factor-I (IGF-I) directly increased BMEC DNA synthesis in vitro and infusion of IGF-I into mammary glands of late pregnant beef cows increased mammary growth. Epidermal growth factor and transforming growth factor-alpha increased the mitogenic response to IGF-I and are likely involved in paracrine regulation of mammary growth. IGF binding proteins are produced and secreted by BMEC and may be autocrine regulators of growth. Collectively, these data indicate that lactogenic hormone receptors are not involved in regulation of mammary growth in cattle. Increased mammary growth following ST and PL treatment was apparently mediated through local production or uptake of IGFs and/or alteration of IGF binding protein concentrations. Presently, it is not clear whether ST and PL act through local as well as distant somatotrophic receptors to alter availability and uptake of IGFs.

Comparative mitogenic and galactopoietic effects of IGF-I, IGF-II and DES-3-IGF-I in bovine mammary gland in vitro

Insulin-like growth factors (IGFs) I and II (IGF-I, IGF-II) and Des-3-IGF-I at physiological concentrations are potent mitogens of bovine undifferentiated mammary epithelial cells cultured in collagen in a serum-free medium. Des-3-IGF-I was found to be as potent as IGF-I, while IGF-II was significantly less active. All three factors acted either synergistically or additively with epidermal growth factor (EGF), cholera toxin and fetal calf serum (FCS). Indirect evidence indicates that despite its lower mitogenic activity the action of IGF-II is mediated through IGF-I receptors. The galactopoietic activity of Des-3-IGF-I and IGF-II was studied in an organ culture of bovine lactating mammary glands using lactogen-responsive fat synthesis as a test. Neither Des-3-IGF-I nor IGF-II exhibited galactopoietic activity nor did they affect the galactopoietic activity of prolactin.

Technical note: A rapid enzyme-linked immunosorbent assay blood test for pregnancy in dairy and beef cattle

The ruminant trophoblast produces pregnancy-associated glycoproteins (PAG) that can be detected in the blood of pregnant animals. The objective was to determine the accuracy of a rapid ELISA PAG-based test for the purpose of pregnancy detection in cattle. Blood was sampled from dairy cattle (539 Holstein cows, 173 Holstein heifers, 73 Guernsey cows, 22 Guernsey heifers, and 12 Jersey heifers) and crossbred beef cattle (145 cows and 46 heifers) that were >or=25 d after insemination (range = 25 to 45 d for dairy and 29 to 56 d for beef). Cattle were examined by ultrasonography for detection of pregnancy within 2 d of blood collection. Whole blood or plasma was incubated in a polystyrene tube coated with a monoclonal PAG antibody for 15 min. The tubes were then washed and subjected to sequential incubations with a biotinylated polyclonal PAG antibody (15 min, followed by wash), a horseradish peroxidase-streptavidin solution (15 min, followed by wash), and a peroxidase substrate. Tubes were visually assessed for color after 15 min (clear solution = PAG negative, not pregnant; blue solution = PAG positive, pregnant). Total assay time was approximately 90 min. The ultrasound examination was used as the standard for pregnancy diagnosis. The sensitivity (99.8 +/- 0.2%), specificity (91.7 +/- 1.4%), and negative predictive value (99.7 +/- 0.3%) for the PAG test used in dairy cattle were similar for different breeds and for cows and heifers. The positive predictive value for the test was greater in dairy heifers than in dairy cows (96.5 +/- 1.4% vs. 90.5 +/- 1.7%, respectively). In beef cattle, the sensitivity (100%), specificity (92.3 +/- 3.0%), positive predictive value (95.0 +/- 2.0%), and negative predictive value (100%) for the PAG test were similar for cows and heifers. The accuracy of the test was not different for dairy and beef cattle. In conclusion, the rapid ELISA pregnancy test based on PAG was highly sensitive and specific for pregnancy detection in dairy and beef cattle.

Effect of interval to resynchronization of ovulation on fertility of lactating Holstein cows when using transrectal ultrasonography or a pregnancy-associated glycoprotein enzyme-linked immunosorbent assay to diagnose pregnancy status

The objective of this study was to compare 2 strategies for resynchronization of ovulation based on nonpregnant diagnoses using transrectal ultrasonography or a pregnancy-associated glycoprotein (PAG) ELISA. Lactating Holstein cows (n = 1,038) were submitted for first postpartum timed artificial insemination (TAI) using a Presynch + Ovsynch protocol. After the initial breeding, cows were randomly assigned to initiate resynchronization 25 d (D25) or 32 d (D32) later. Pregnancy status of cows initiating Resynch 25 d after TAI was determined 27 d after TAI by using a PAG ELISA, whereas pregnancy status of cows initiating Resynch 32 d after TAI was determined 39 d after TAI using transrectal ultrasonography. Cows diagnosed as not pregnant continued the Resynch protocol by receiving an injection of PGF(2 alpha) 7 d after the initial GnRH injection and a second GnRH injection 54 h after the PGF(2 alpha) injection. Cows in both treatments were inseminated approximately 16 h after the second GnRH injection. Blood samples for analysis of progesterone (P(4)) were collected at the first GnRH injection of each Resynch protocol. Pregnancies per AI (P/AI) of nonpregnant cows initiating Resynch 25 vs. 32 d after first postpartum TAI did not differ 39 d after TAI and were 28.3 vs. 30.9% for D25 vs. D32 cows, respectively. Mean P(4) at the first GnRH injection of Resynch was greater for D32 than for D25 cows (3.67 +/- 0.22 vs. 2.83 +/- 0.22 ng/mL), indicating that the Resynch treatments were initiated at different stages of the estrous cycle. After blocking P(4) concentration into low (<1.0 ng/mL) or high (>or=1.0 ng/mL) classes, P(4) class was not found to affect P/AI 39 d after TAI. Early resynchronization was not found to affect P/AI 39 d after TAI; however, early resynchronization did decrease days between inseminations and the interval from the initial nonpregnant diagnosis to conception. Earlier detection of nonpregnant cows using the PAG ELISA in conjunction with a TAI resynchronization program may improve the rate at which cows become pregnant in a dairy herd compared with transrectal ultrasonography conducted at a later stage after TAI.

Isolation of Genomic Sequence Encoding a Biologically Active Bovine TGF- α Protein

AbstractGenomic clones encoding bovine TGF-α were identified by hybridization with probes derived from human TGF-α sequence. Nucleotide sequence of the clones predicts that mature bovine TGF-α is a 50 amino acid polypeptide which shares 96% and 92% homology with human and rat TGF-α, respectively. Bovine TGF-α with the predicted sequence was chemically synthesized and tested for activity. Synthetic bovine TGF-α competes in a radioreceptor assay with labelled mouse EGF with activity parallel to that of human TGF-α and mouse EGF. The mitogenic activity of bovine TGF-α is comparable to that of human EGF in causing proliferation of bovine mammary epithelial cells. An ∼ 5.0 kilobase RNA transcript is observed in polyadenylated RNA from MDBK cells by Northern blot analysis. The polymerase chain reaction detects the presence of a TGF-α transcript in many bovine tissues. These data indicate that bovine TGF-α may be a normal regulator of cell growth in the bovine animal.

CHANGES IN INSULIN AND SOMATOMEDIN RECEPTORS AND UPTAKE OF INSULIN, IGF-I AND IGF-II DURING MAMMARY GROWTH, LACTOGENESIS AND LACTATION

Receptors for insulin, IGF-I and IGF-II are present on growing, differentiating and lactating mammary epithelial cells. Scatchard analysis of these receptors indicate the presence of two receptor sites for insulin. In contrast, the type I and type II somatomedin receptors are composed of a single site in all physiological states examined. The type I somatomedin receptor cross-reacted with both insulin and IGF-II while the type II receptor only recognized IGF-II. Although the truncated form of IGF-I has a lower affinity for the type I receptor relative to native IGF-I it is more potent in stimulating mammary growth in vitro. This may be due to mediating effects of binding proteins. Mammary growth appears to be related to higher population of high affinity receptors for IGF-I in mammary tissue during pregnancy. Role of insulin, IGF-I and IGF-II during lactogenesis and galactopoiesis remains to be established.

Role of Bovine Placental Lactogen in Intercellular Signalling during Mammary Growth and Lactation

In dairy cattle, the majority of mammary growth takes place during pregnancy and as such, it might be expected that signals from the placenta would be involved in controlling mammogenesis. Placental lactogen (PL) is produced only during pregnancy and may be one of the endocrine factors that controls mammary growth. It is also generally recognized that mammary growth is controlled not only by endocrine signals, but also by paracrine and/or autocrine factors. The in vitro and in vivo mammogenic effects of some of these growth factors are discussed, along with possible interactions between the locally acting factors and endocrine hormones such as bovine PL (bPL). In addition, it is common for dairy cattle to be pregnant during much of lactation. Thus, the possible influences of bPL on lactation are also discussed.