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Close association exists between melanocytes, the pigment melanin-producing cells in the body, and their neighboring keratinocytes. Keratinocytes are the pigment recipients and skin pigmentation is the result of this interaction. While the chemical basis of melanin production (melanogenesis) is well documented, the molecular mechanism of melanosome transfer needs to be elucidated. We are now providing first evidence that the protease-activated receptor 2 (PAR-2) expressed on keratinocytes, but not on melanocytes, is involved in melanosome transfer and therefore may regulate pigmentation. Activation of PAR-2 with trypsin or with the peptide agonist SLIGRL induced pigmentation in both two- and three-dimensional cocultures of keratinocytes and melanocytes, but not in cocultures that were spatially separated, indicating the need for intimate cell-cell contact. Topical application of SLIGRL on human skin transplanted on SCID mice resulted in a visible skin darkening. Histological examination revealed increased deposits of melanin in the keratinocytes. Inhibition of PAR-2 activation by RWJ-50353, a serine protease inhibitor, resulted in depigmentation and changes in expression of melanogenic-specific genes. Keratinocyte-melanocyte contact was essential for this depigmenting effect. Topical application of this inhibitor induced lightening of the dark skin Yucatan swine, which was confirmed by histochemical analysis. The results presented here suggest a novel mechanism for the regulation of pigmentation, mediated by the activation or inhibition of the keratinocyte receptor PAR-2.

Programmed cell death is a controlled process that leads to the elimination of single cells via apoptosis. Programmed cell death is fundamental to development, morphogenesis, and homeostasis. Proteases play a major role in the death process. We have previously shown that a serine protease, secreted by a keratinocyte cell line, can induce apoptosis in numerous cell lines. Here we show that serine proteases can induce cell death in vivo as well. Using a synchronized hair growth mouse model, we show that topical trypsin treatment following depilation induces cell death at the follicular papilla. This results in delaying hair growth and pigmentation. We speculate that trypsin might affect a receptor-mediated signaling pathway that leads to follicular papilla cell death. Dev. Dyn. 208:553–564, 1997. © 1997 Wiley-Liss, Inc.

An organ culture system was used to evaluate the response of human proliferative endometrium to progesterone stimulation. The glycogen content of proliferative tissue was increased as much as thirteenfold during organ culture in media containing progesterone. The steroid-induced increase in tissue glycogen was detectable after 16 hours and reached a maximum at 48 and 72 hours of culture. Progesterone induced a significant increase in glycogen at a media concentration of 1.6 × 10−6M and a maximal increase at 3.2 × 10−7to 3.2 × 10−6M. At higher media concentrations of progesterone (1.6 × 10−5M), glycogen levels failed to reach the maximum obtainable. The extent of the response correlated poorly with the initial glycogen content of the tissue and not at all with the initial content of high-affinity progesterone-binding sites in cytosol. Addition of estradiol-17β to medium (10−10M to 10−7) had no effect on the progesterone-induced increase in tissue glycogen. Delaying the addition of progesterone to the cultures for 24 hours resulted in a diminished glycogen response; the reduced response may be related to a rapid decrease in high-affinity progesterone-binding sites as measured in cytosol prepared from tissues cultured in the absence of progesterone. High-affinity progesterone-binding sites in endometrial cytosol were found to decrease rapidly during the first 24 hours of culture. The addition of cycloheximide or actinomycin D to the culture media inhibited the increase in tissue glycogen caused by progesterone. These results demonstrate that progesterone can induce an in vitro response in human proliferative endometrium similar to that seen in vivo. The response of the endometrium is reproducible and allows for comparisons between grouped data obtained by using tissues from several different donors.

The relative potency of six commonly used synthetic progestins has been evaluated in an organ culture system for human endometrium. The affinities of these progestins for endometrial progesterone receptor were also evaluated after removing the CBG-like protein by spheroidal hydroxylapatite chromatography. All six progestins induced an increase in tissue glycogen during culture at lower concentrations than did progesterone; only one (medroxyprogesterone acetate) had a relative affinity greater than progesterone. The relative potencies and affinities of the synthetic progestins were found to have the same relative order but to differ in relative magnitude.

The behaviour of bovine, equine and human blastocysts was studied in vitro by time-lapse videomicrography and computer imaging. This study revealed that cytoplasmic extensions of the trophectoderm [‘trophectoderm projections’ (TEP)] were expressed by embryos of all three species, prior to or during zona escape. Bovine and human blastocysts escaped their zonae with a combination of blastocoele expansion, collapse and re-expansion coupled with the penetration of the zona pellucida by TEP. In equine embryos, after several cycles of blastocoele expansion and collapse, trophectoderm ruptured the zona with the concomitant appearance of TEP. This study provides documentation that TEP are expressed by a diverse range of mammalian species, bringing the total number of species in which this phenomenon is found to six, since TEP are also known to be expressed by guinea-pig, hamster and rhesus monkey blastocysts, representing rodents, ungulates and primates. In all species studied, the dynamic nature (extension, retraction, and angular movement) of the TEP was similar, moving in an undulating manner with rapid cycles of extension and retraction. Because TEP appear to be a genera] feature of mammalian blastocysts, they are implicated in one or more key events in early development, namely zona escape, attachment and/or implantation.