Effect of continuous milking and bovine somatotropin supplementation on mammary epithelial cell turnover.

Objectives were to determine effects of continuous milking (CM) and bovine somatotropin (bST) administration on 1) mammary epithelial cell (MEC) proliferation, apoptosis, and ultrastructure during late gestation and early lactation, 2) expression of genes associated with proliferation, and apoptosis in mammary epithelial cells, and 3) milk yield and composition. Second-gestation, first dry-period cows were randomly assigned to either continuous bST throughout late gestation and early lactation (+bST; n = 4) or no bST (-bST; n = 4) administration. Within each animal, udder halves were randomly assigned to CM or a 60-d dry period (control) treatment. Daily milk yield and weekly milk composition were measured during the last 60 d of gestation in CM halves and from 1 to 30 d postpartum for both halves. Mammary biopsies were obtained at -20 +/- 7, -8 +/- 3, +1 +/- 0, +7 +/- 0, and +20 +/- 0 d (mean +/- standard error) relative to parturition. Prepartum half-udder milk yield was greater in +bST cows than in -bST cows (9.9 vs. 8.2 kg/d) and postpartum half-udder milk yields were dramatically reduced in CM halves compared with control halves (10.6 vs. 22.2 kg/d), regardless of bST treatment. Proliferation of MEC was reduced in CM halves at -8 d (2.7 vs. 5.4%). Apoptosis of MEC was elevated during early lactation for d +1 and +7 in control halves, but was only increased at d +1 in CM halves. Turnover of MEC was not affected by bST. Ultrastructure data indicated complete involution of the control half and lactation maintenance in CM glands (d -20). By d -8, control tissue contained alveoli in an immature secretory state, but CM tissue contained both lactating and immature alveoli. Postpartum ultrastructure parameters were similar between halves until d 20 when control tissue was composed of a homogeneous population of lactating alveoli, but CM tissue contained lactating, engorged, and resting alveoli. Expression of CCAAT/enhancer binding protein-beta (CEBP-beta), cyclin D1, and bcl(2) were up-regulated during late gestation, but did not differ between control and CM halves. Expression of alpha-lactalbumin was increased in CM halves during late gestation, but was not different in CM and control tissue after parturition. Other genes evaluated (bax, insulin-like growth factor binding protein 5, ATP-binding cassette 1, and p27) were not differentially expressed at any timepoints evaluated. Results indicate that CM reduced subsequent half-udder milk yield in primiparous cows through altered MEC turnover and secretory capacity. Negative effects of CM on the subsequent lactation were not alleviated by bST supplementation.

Use of gene expression microarrays for evaluating environmental stress tolerance at the cellular level in cattle.

Selecting domestic animals for tolerance to thermal stress (TS) has been counterproductive, because acclimation involves reducing or diverting metabolizable energy from production to balance heat gain and loss. Ideally, simultaneous selection for increased production and improved thermotolerance is desirable, but to accomplish this at the genomic level the genes associated with acclimation, adaptation, and thermo-tolerance need to be identified. We evaluated the effects of TS on mammary development and gene expression in vitro using a bovine mammary epithelial cell collagen gel culture system. Acute TS was characterized by inhibition and regression of the ductal branches. Gene expression profiling revealed an overall upregulation of genes associated with the stress response and protein repair. In contrast, genes associated with cellular and mammary epithelial cell-specific biosynthesis, metabolism, and morphogenesis were generally downregulated by TS. Future studies will examine the impact of acclimation and adaptation on gene expression to identify those genes associated with acquisition of thermal tolerance.

Ontogeny of stem cell factor receptor (c-kit) messenger ribonucleic acid in the ovine corpus luteum.

Stem cell factor (SCF) is a pleiotropic growth factor that is expressed by the ovine corpus luteum throughout its life span by both small and large steroidogenic cells. Determination of the action of SCF, however, requires localization of its receptor, c-kit; therefore, the objectives of the present study were to identify and localize c-kit within corpora lutea. Two cDNAs encoding different portions of the c-kit molecule were amplified by the polymerase chain reaction. The first was a 558-base pair (bp) cDNA encoding portions of the transmembrane and tyrosine kinase domains; the second was a 632-bp cDNA encoding most of the ligand-binding domain. Expression of c-kit was quantified by RNase protection assay of total cellular RNA collected on Days 3, 7, 10, 13, and 16 (n = 4, 4, 5, 4, and 4 per group, respectively) of the estrous cycle (Day 0 = estrus). The level of c-kit mRNA was low early in the luteal phase, reached (p < 0.05) maximum levels on Day 13, and then decreased (p < 0.01) on Day 16. On Day 3 (n = 4), c-kit was expressed in a cell-specific manner throughout the corpus luteum; identity of the specific cell types expressing c-kit could not be determined at this stage. On Day 14 (n = 4), c-kit did not appear to be expressed within large luteal cells but was prominently expressed in cells that surrounded large luteal cells and that possessed the morphological characteristics of small luteal cells and endothelial cells. Given the temporal regulation of c-kit expression within the corpus luteum, these data suggest that luteal SCF may act locally.

Regulation of amounts of mRNA for GnRH receptors by estradiol and progesterone in sheep.

Expression of GnRH receptors increases prior to the onset of the preovulatory surge of LH in sheep. Two experiments were conducted to investigate the interactions of progesterone (P) and estradiol (E) on amounts of mRNA for GnRH receptors and the number of receptors for GnRH. The first study was designed as a 2 x 2 factorial arrangement of treatments to investigate effects of removal of P and the presence of E. Ewes that had been ovariectomized (OVX) for at least 4 wk received one silastic implant containing E and two silastic implants containing P for 6 d to mimic concentrations of these steroids during the luteal phase of the estrous cycle. Anterior pituitary glands were collected (n = 4 animals/group): 1. Prior to implant removal and 12 h after removal of: 2. P only. 3. E only. 4. P and E. Regardless of whether or not E was present, amounts of mRNA for GnRH receptors (P = 0.87) and number of GnRH receptors (P = 0.43) were not different within 12 h after removal of P. In the second experiment, ewes were OVX on d10-12 of the estrous cycle (d0 = estrus), and immediately received silastic implants containing E and P as described above. Anterior pituitary glands were collected on d12 of the estrous cycle (n = 5), prior to implant removal (n = 5), and from the remaining ewes 24 h after removal of P only (n = 7) or removal of P and E (n = 6). Relative amounts of mRNA for GnRH receptors and the number of GnRH receptors were similar (P > 0.05) on d12 of the estrous cycle and prior to implant removal. Removal of both P and E did not affect (P > 0.05) amounts of GnRH receptor mRNA or number of GnRH receptors. However, the removal of P and the presence of E increased (P < 0.05) amounts of mRNA for GnRH receptors, but did not affect (P > 0.05) the number of GnRH receptors. We conclude that increased amounts of GnRH receptor mRNA require the removal of P and the presence of E.

Control of extracellular matrix remodelling within ovarian tissues: localization and regulation of gene expression of plasminogen activator inhibitor type-1 within the ovine corpus luteum.

Extensive extracellular matrix remodelling occurs within the lifespan of the corpus luteum, particularly during corpus luteum formation and regression. A major mechanism for the regulation of extracellular matrix remodelling is via local production of specific proteinase inhibitors, such as the serine proteinase inhibitor plasminogen activator inhibitor type-1 (PAI-1). The objective of the present study was to characterize the localization, ontogeny and regulation of PAI-1 expression within ovine corpora lutea. Urokinase binding activity was detected within medium conditioned by ovine luteal cells. Production of PAI-1 by ovine luteal cells was confirmed by immunoprecipitating it from labelled proteins in culture medium. mRNA encoding PAI-1 was present within developing (day 3), mature (day 10) and regressing (30 h after prostaglandin F2 alpha injection on day 10 after the onset of oestrus) corpora lutea as demonstrated by in situ hybridization. The ontogeny of PAI-1 mRNA expression was characterized within corpora lutea collected on days 3, 7, 10, 13 and 16 after the onset of oestrus (n = 4, 4, 4, 3 and 4, respectively). Expression of PAI-1 mRNA did not differ during the luteal phase (P = 0.06), although a trend for an increase in the amount of PAI-1 mRNA was observed on day 16. Expression of PAI-1 mRNA was also examined during luteal regression in corpora lutea collected 0, 6, 12, 24 and 36 h after injection of prostaglandin F2 alpha on day 10 after the onset of oestrus (n = 4 at each time). Relative PAI-1 mRNA concentrations changed significantly during luteolysis induced by prostaglandin F2 alpha (P = 0.0002). Administration of prostaglandin F2 alpha resulted in a transient sevenfold increase in PAI-1 mRNA 6 h after injection (P = 0.0001) but by 12 h the amounts had returned to values similar to those detected on day 10. We conclude that PAI-1 is a major secretory product of ovine luteal cells and that a transient increase in PAI-1 mRNA occurs during luteolysis induced by prostaglandin F2 alpha. PAI-1 probably plays a key local role in the control of extracellular proteolysis during the luteal phase.

Characterization of ovine stem cell factor messenger ribonucleic acid and protein in the corpus luteum throughout the luteal phase.

Stem cell factor (SCF) is a growth factor known to have profound effects on the proliferation, migration, differentiation, and survival of numerous cell types, including those of the ovary. The objectives of the present study were to identify and characterize expression of this growth factor in the ovine corpus luteum (CL). A 952-bp cDNA was amplified from Day 3 (Day 0 = estrus) ovine luteal total cellular (tc) RNA by reverse transcriptase-polymerase chain reaction and determined to encode SCF. Northern analysis of Day 10 luteal poly(A)+ RNA indicated one major transcript of approximately 6.5 kb. SCF mRNA was localized within Day 3 and Day 10 CL by in situ hybridization and was expressed throughout luteal tissue on both days examined. To asses expression throughout the luteal phase, SCF mRNA was quantified by ribonuclease protection assay in tcRNA collected on Day 3, 7, 10, 13, and 16; values did not differ across days (p > 0.10). Similarly, SCF mRNA was quantified in tcRNA isolated from pools of Day 10 large and small steroidogenic cells (n = 4 and 3, respectively); levels did not differ (p > 0.10) between cell types. In addition, SCF protein was detected in CL on Days 3 and 10, and was expressed in a cell-specific manner in cells with morphological characteristics of large and small luteal cells. These data indicate that SCF may be involved in communication among steroidogenic cells and/or between steroidogenic and nonsteroidogenic cells of the CL.

Differential gene expression within the ovine corpus luteum: identification of secreted protein acidic and rich in cysteine as a major secretory product of small but not large luteal cells.

Limited information is available regarding secretory proteins of the corpus luteum (CL), and the potential local role these proteins may play in control of luteal function. An ovine small luteal cell complementary DNA library was immunologically screened with a polyclonal antiserum generated against small cell secretory proteins. A relatively abundant complementary DNA (approximately 2.1 kb) encoding a calcium binding glycoprotein Secreted Protein Acidic and Rich in Cysteine (SPARC) was isolated. Production of SPARC protein by ovine luteal cells was confirmed by immunoprecipitating it from labeled culture medium. Expression of SPARC messenger RNA (mRNA) was examined within CL collected on days 3, 7, 10, 13, and 16 post estrus (n = 4, 4, 4, 3, and 4 respectively), and within pools of purified small (n = 3) and large (n = 4) luteal cells by Northern and dot blot analysis. Amounts of SPARC mRNA increased during the early luteal phase, peaked by day 7 (P < 0.05) and subsequently declined on days 10 and 13 (P < 0.05). SPARC mRNA content was significantly higher in the small than in the large cells (P < 0.003). In situ hybridization showed that SPARC mRNA was localized to the thecal layer of Graafian follicles and to day 3 and day 10 CL. Within CL, immunohistochemistry indicated that SPARC protein was associated with small luteal cells (spindle shaped, avg = 17 microM in diameter) but not with large cells. This specific localization to small cells was confirmed by colocalization of SPARC with 3 beta-hydroxysteroid dehydrogenase. We conclude that SPARC is a major secretory product of small steroidogenic luteal cells of the ovine CL. As SPARC is known to modulate many aspects of tissue reorganization, expression by small luteal cells may play a role in regulation of CL maturation.

Ontogeny and regulation of luteinizing hormone receptor messenger ribonucleic acid within the ovine corpus luteum.

Although LH regulates corpus luteum (CL) function and LH receptor (LHR) binding has been characterized within ovine CL, changes in LHR mRNA expression have not been documented. The objectives here have been to assess relative amounts of LHR mRNA in ovine CL throughout the estrous cycle and during luteolysis by ribonuclease protection analysis. In experiment 1, LHR mRNA was quantified within CL collected on Days 3, 7, 10, 13, and 16 post-estrus (n = 3, 4, 4, 3, and 4, respectively). LHR mRNA differed throughout the luteal phase (p < 0.01), with higher concentrations found during the midluteal phase (Days 10 and 13) than during the early (Day 3) or late (Day 16) luteal phase (p < or = 0.05). In experiment 2, LHR mRNA content was determined within pools of Day 10 small (n = 4) or large (n = 4) luteal cells. Relative amounts of LHR mRNA were not different (p = 0.1) in small (2.23 +/- 0.22 relative units) vs. large (1.62 +/- 0.22 relative units) luteal cells. In experiment 3, CL were collected at 0, 6, 12, or 24 h after injection of 15 mg prostaglandin F2 alpha) on Day 10 post-estrus (n = 4, 4, 3, and 4, respectively). LHR mRNA content fell during PGF2 alpha-induced luteolysis (p < 0.002), decreasing significantly by 6 h (1.76 vs. 1.10 relative units; p < or = 0.05). By 24 h, levels were approximately 60% of those detected within 0-h controls (1.76 vs. 0.79 relative units; p < 0.05). These results indicate that changes in LHR mRNA during the luteal phase correlates well with previously reported changes in LHR binding. After PGF2 alpha administration, LHR mRNA falls earlier than the reported drop in LHR binding sites occurs.