Uterine response to multiple inoculations with Arcanobacterium pyogenes and Escherichia coli in nulliparous ewes.

PROBLEM: Uterine infections seem more severe in nulliparous animals. Our objective was to determine whether intrauterine inoculation of nulliparous ewes with Arcanobacterium pyogenes and Escherichia coli would produce an antibody response and reduce the severity of subsequent infections. METHOD OF STUDY: Nulliparous ewes (n = 9/treatment) received (i) 'primary intrauterine inoculation' with phosphate-buffered saline (PBS) and 'secondary intrauterine inoculation' with PBS; (ii) primary PBS-secondary 75 x 10(7) cfu of A. pyogenes and 35 x 10(7) cfu of E. coli (PBS-Bacteria); (iii) primary bacteria-secondary PBS; or (iv) primary bacteria-secondary bacteria (Bacteria-Bacteria). RESULTS: Inoculations evoked an antibody response. Postmortem examinations 6 days after the secondary inoculation indicated that PBS-treated ewes did not develop uterine infections, but all bacteria-treated ewes did. Infections were either less severe or closer to resolution in Bacteria-Bacteria than they were in PBS-Bacteria ewes. CONCLUSIONS: Intrauterine inoculation of nulliparous ewes with A. pyogenes and E. coli evokes an antibody response that may help the uterus reduce the severity of subsequent infections.

Up-regulation of basal transcriptional activity of the cytochrome P450 cholesterol side-chain cleavage (CYP11A) gene by isoform-specific calcium-calmodulin-dependent protein kinase in primary cultures of ovarian granulosa cells.

Intracellular calcium ions (Ca2+) regulate steroidogenesis in the placenta, adrenal gland, testis, and ovary. Earlier data indicate that Ca2+/calmodulin-dependent protein kinase (CamK) may mediate Ca(2+)-dependent up-regulation of CYP11A (cholesterol side-chain cleavage). To examine this notion further, we assessed the expression and actions of isotype-specific CamK on in vitro transcription of the swine CYP11A gene promoter in primary cultures of ovarian granulosa-luteal cells. RT-PCR and oligodeoxynucleotide sequencing identified gene transcripts encoding CamKII and IV in granulosa and theca cells and corpora lutea. DNA sequence homology with the cognate human and rat genes was 97 and 94% (CamKII) and 96 and 88% (CamKIV), respectively. SDS-PAGE and isoform-specific immunoblotting corroborated expression of CamKII (approximately 52 kDa) and CamKIV (approximately 60 kDa) proteins. To monitor transcriptional control, granulosa-luteal cells were transfected transiently with a putative 5'-upstream regulatory region of the homologous CYP11A gene -2320 to +23 bp from the transcriptional start site driving luciferase (CYP11A/luc). Coexpression of constitutively active CamKIV elevated basal transcription by 3.5 +/- 0.2-fold (P < 0.001), whereas inactive mutant CamKIV and native CamKII had no effect. Forskolin, an activator of adenylyl cyclase, stimulated expression of CYP11A/luciferase by 4.5 +/- 0.9-fold (P < 0.001) and did not enhance transcriptional drive by exogenous CamKIV. Preliminary promoter-deletional analyses showed that a proximal 5'-fragment -100 to +23 bp, but not -50/+23 bp, retained full responsiveness to CamKIV (4.5 +/- 0.4-fold; P < 0.001). Threefold cotransfection of -100/+23 bp CYP11A/luciferase, active CamKIV, and a dominant-negative mutant of the cAMP-responsive element binding protein (10, 100, and 250 ng) inhibited CamKIV-stimulated transcriptional activity by 17, 47, and 48% (pooled sem+/- 2%) [P < 0.01]. The dominant-negative mutant of the cAMP-responsive element binding protein also repressed forskolin's stimulation of -100/+23 CYP11A/luciferase by 12, 38, and 52% (P < 0.01). Based on these ensemble outcomes, we postulate that endogenous CamKIV may serve as a Ca(2+)-dependent effector mechanism to maintain basal CYP11A gene expression in ovarian granulosa-luteal cells.

Uterine response to infectious bacteria in estrous cyclic ewes.

PROBLEM: Luteal-phase uteri are susceptible to infections, and PGE2 and exogenous progesterone can down-regulate, whereas PGF2alpha can up-regulate, uterine immune functions. METHOD OF STUDY: Uteri of follicular- or luteal-phase ewes were inoculated with either saline or bacteria (Arcanobacterium pyogenes and Escherichia colt). Vena caval blood was collected for the next 3 days, and progesterone, PGE2, and PGF2alpha were measured. The effects of 10(-7) M PGE2 (Experiment 1), 10(-7) M PGF2alpha (Experiment 2), 10(-7) M indomethacin (INDO), and diluent on proliferation of lymphocytes from the vena caval blood in response to mitogens was quantified. RESULTS: Experiment 1: Progesterone was greater (P < 0.01) in luteal than in follicular ewes (3.4 versus 0.4 ng/mL), and only luteal ewes inoculated with bacteria developed infections.Lymphocyte proliferation was least (P = 0.08) in follicular ewes (2.6 versus 4.5 pmol for follicular and luteal, respectively). Concanavalin A (Con A)-stimulated proliferation was less (P < 0.05) for ewes inoculated with bacteria and for cells cultured with diluent (5.9 versus 3.1 pmol for saline and bacteria, respectively) or with INDO (6.6 versus 2.8 pmol for saline and bacteria, respectively). Also, Con A-stimulated lymphocytes from ewes inoculated with bacteria tended to proliferate less (P < 0.1) when cultured with PGE2 (4.9 versus 3.7 pmol for saline and bacteria, respectively) or PGE2 + INDO (5.5 versus 3.8 pmol for saline and bacteria, respectively). Experiment 2: Progesterone was greater (P < 0.01) in luteal than in follicular ewes (6.5 versus 1.2 ng/mL), and only luteal ewes inoculated with bacteria developed infections. Con A-stimulated lymphocyte proliferation was greater (P < 0.001) for follicular ewes (4.1 versus 3.1 pmol for follicular and luteal, respectively). Proliferation of lymphocytes collected from follicular ewes was greater (P < 0.01) when cells were cultured with PGF2alpha (3.5 versus 2.7 pmol for follicular and luteal, respectively), but INDO did not affect unstimulated or mitogen-stimulated proliferation. CONCLUSIONS: Prostaglandin F2alpha enhanced lymphocyte proliferation, whereas bacterial inoculation and in vitro treatment with PGE2 suppressed lymphocyte proliferation. This may signify the involvement of bacterial products and prostaglandins in regulation of uterine immunity.

Modulation of the uterine response to infectious bacteria in postpartum ewes.

PROBLEM: Exogenous progesterone and prostaglandin E2 (PGE2) can downregulate uterine immune functions and render the uterus susceptible to bacterial infection. METHOD OF STUDY: Ewes were sham-ovariectomized (SHAM) or ovariectomized (OVEX) 9 days after parturition (day 0), and their uteri were inoculated with Arcanobacterium pyogenes and Escherichia coli on day 15. Vena caval blood was collected on day 14 and days 16-19, and uteri were collected on day 20. Ewes began receiving either canola oil (OIL) or progesterone in oil (PROG) on day 10. Lymphocytes from each blood sample were assigned to a 2 x 2 factorial array of in vitro treatments; 10(-7) M PGE2 and 10(-7) M indomethacin (INDO) were main effects. [3H]Thymidine incorporation (expressed in picomoles) was used to quantify proliferation. RESULTS: Progesterone was greater (P = 0.001) in PROG than in OIL ewes (3.6 versus 0.7 ng/mL), and only PROG ewes developed infections. Lymphocyte proliferation was least (P = 0.02) in PROG-OVEX ewes (4.1 versus 5.4, 5.7, and 5.8 pmol for OIL-SHAM, PROG-SHAM, and OIL-OVEX, respectively). Concanavalin A (Con-A)-stimulated proliferation was less (P < 0.01) for PGE2- and PGE2 + INDO-treated lymphocytes (7.5 and 8.3 pmol, respectively) than for control or INDO-treated cells (12.9 and 14.7 pmol, respectively). CONCLUSIONS: Progesterone treatment of postpartum ewes suppressed uterine immunity. In vitro PGE, treatment suppressed lymphocyte proliferation, regardless of PROG, and highlights a progesterone-independent level of regulation of uterine immune function.