DISTRIBUTION OF TRANSFORMING GROWTH-FACTOR-ALPHA PRECURSORS IN THE MOUSE UTERUS DURING THE PERIIMPLANTATION PERIOD AND AFTER STEROID-HORMONE TREATMENTS

Temporal and cell-type specific distribution of transforming growth fa ctor alpha (TGF alpha) precursor (proTGF alpha) was examined in the mo use uterus during the periimplantation period, and after steroid hormo ne treatments of ovariectomized adult mice by immunohistochemistry usi ng antibodies that recognize the precursor forms of the growth factor. These studies were comple mented by immunoblot analysis of proTGF alp ha in separated uterine cell-type preparations. The specificity of the antibodies used in these studies was confirmed by use of pancreas or lactating mammary glands from transgenic mice in which mutated proTGF alpha, lacking recognition sites for proteolytic cleavages, was target ed for expression under a tissue-specific enhancer/promoter. Analysis of histochemical studies revealed accumulation of immunoreactive proTG F alpha primarily in luminal and glandular epithelial cells on Day 1 o f pregnancy or pseudopregnancy followed by little or no accumulation o n Days 2 and 3. However, immunoreactive proTGF alpha started to reappe ar in the luminal epithelium on the morning of Day 4 and became more p rominent in the afternoon. In pregnant mice, immunostaining persisted in these cells at the implantation sites during the time of attachment reaction (2130 h on Day 4), but disappeared by morning of Day 5. Immu nostaining appeared to be situated at the apical border of the luminal epithelium. No positive immunostaining could be detected in the nonre ceptive uterus on Day 5 or 6 of pseudopregnancy. Consistent with the i mmunohistochemistry results, Western blot analysis detected two specie s of precursor proteins (14.5 and 17 kDa) in isolated luminal epitheli al cell-enriched preparations on Day 4, but not on Day 5, of pseudopre gnancy. The results suggest that proTGF alpha accumulates in the lumin al epithelium of the receptive uterus prior to implantation. The effec ts of ovarian steroids on uterine accumulation of proTGF alpha were ex amined in ovariectomized adult mice by immunohistochemistry and immuno blotting. Whereas an injection of estradiol-17 beta (E(2)) or progeste rone (P-4) had little or a modest effect on epithelial accumulation of proTGF alpha, P-4 priming for several days resulted in distinct accum ulation of proTGF alpha in epithelial cells. The superimposition of an E(2) treatment on P-4 priming showed a biphasic response, with an ini tial gradual loss of immunostaining through 12 h followed by a return by 24 h of E(2) treatment. The combined hormone treatment schedule emp loyed here is similar to the situation of inducing implantation with E (2) in P-4-primed delayed implanting mice. The results suggest a parac rine/''juxtacrine'' role for this growth factor in implantation.