ORIGIN OF UTERINE SUPPRESSOR CELLS AND CHARACTERIZATION OF SUPPRESSOR FACTOR

OBJECTIVES: Objective 1 - evaluate ovine bone marrow as a potential origin for endometrial suppressor cells. Objective 2 - determine whether estradiol - 17 beta (E2) affects suppressor cell activity in a direct manner. Objective 3 - obtain a more complete characterization of the suppressor factor.

APPROACH: For Objective 1, an experiment will be conducted to assess the transport and localization of injected fluorescent-labeled bone marrow cells to endometrial tissue of cyclic, pregnant and estrus ewes as well as estradiol (E)-treated ovariectomized ewes. Both small (<5.2Fm in diameter, which is equivalent in size to endometrial suppressor cells) and large bone marrow cells will be quantified within the tissue. In Objective 2, both tritiated E and leucine (in parallel cultures) will be incubated with fractionated ovine endometrial suppressor cell preparations to determine E binding and protein synthesis activity. If bound, the presence of nuclear estrogen receptor will be assessed with a monoclonal antibody using immunohistochemistry. For Objective 3, the suppressor macromolecule will be purified by Fast Performance Liquid Chromatography followed by physio-chemical evaluation of the molecule using a panel of enzymes and NMR and mass spectroscopy instrumentation. It is anticipated that the findings from this investigation will provide pertinent information for agricultural and medical laboratories working in the area of embryonic mortality and provide fundamental knowledge needed to determine whether immunosuppressive substances are necessary for the survival of the mammalian conceptus.

PROGRESS: 1999/01 TO 1999/12

Numbers of fluorescein isothiocyanate -labeled bone marrow (BM) cells of donor lambs were quantified within endometrial cell suspensions following their administration to ovariectomized (OVX; control- and estradiol-17B [E]-treated) and intact (estrus, d-14 cyclic and pregnant) ewes. The numbers of fluorescent cells were greater for the estrous and d-14 cyclic ewes than for both groups of OVX ewes. Fractionation of the endometrial cells with Percoll revealed that the majority of fluorescent cells were low-density (1.002 to 1.056 g/mL) cells. In coculture experiments, low-density cells from lamb BM not only suppressed the incorporation of thymidine into phytohemagglutinin-treated peripheral blood lymphocytes, but the cells also released suppressor factor into the culture medium. Suppressor activity tended to be reversed by a pan-specific neutralization antibody to transforming growth factor-B (TGF-B); however, the activity was unaffected by an antibody to TGF-B2. These findings suggest that ovine endometrial suppressor cells may represent a population of low-density BM-derived natural suppressor cells, and their trafficking and localization patterns may be dependent upon an ovarian factor(s). Further, suppressor activity does not appear to be mediated by TGF-B2.

IMPACT: 1999/01 TO 1999/12

In ruminant livestock, endometrial suppressor cells may be important in preventing the rejection of conceptus tissues by effector lymphocytes during early pregnancy. The findings from this investigation suggest that suppressor cells may originate from the bone marrow and that their trafficking through the peripheral blood and localization within the endometrium may be regulated, in part, by an ovarian factor(s). Elucidation of the regulatory mechanisms of these cells seems requisite in order to optimize their function.

PUBLICATIONS: 1999/01 TO 1999/12

1. Ireland, A.C. and E.C. Segerson. 1999. Ovine endometrial cells fail to lyse K-562 target cells: a preliminary investigation. Theriogenology 51:1431-1440.

2. Segerson, E.C. and P.K. Beetham. 2000. Suppressor activity of bone marrow cells and localization of fluorescent- labeled bone marrow cells within ovine endometrial tissue. J. Anim. Sci. (In Press)

PROJ NO: NCX-152-5-99-120-1 AGENCY: CSRS NC.X
PROJ TYPE: EVANS-ALLEN PROJ. STATUS: NEW
START: 01 OCT 1998 TERM: 30 SEP 2001 FY: 1999

INVESTIGATOR: Segerson, E. C.; Shirley, V. B.

PERFORMING INSTITUTION:
ANIMAL SCIENCE
NORTH CAROLINA A&T STATE UNIV
GREENSBORO, NORTH CAROLINA 27411