In previous studies, the distribution of thyrotropes in the chicken pituitary gland has been analyzed by immunohistochemistry using heterologous antibodies. In this study, we examined the distribution of thyroid-stimulating hormone beta subunit-immunopositive (TSHbeta-ip) cells and the expression of TSHbeta mRNA in the pituitary glands of chicken embryos by immunohistochemistry using a specific antiserum to the chicken TSHbeta, in situ hybridization and RT-PCR. Immunohistochemical and morphometric analyses revealed that the TSHbeta-ip cells first appeared on embryonic day 10 (E10) in the pituitary gland and were mainly distributed in the cephalic lobe and that the cell density on E20 was almost 4 times greater than that on E10. The chicken TSHbeta-ip cells could be classified into two types based on morphological characteristics: round-shaped cells and club-shaped cells, which have long cytoplasmic processes. In situ hybridization analysis revealed that TSHbeta mRNA-expressing cells were expressed from E9 in the cephalic lobe and that the extent of TSHbeta mRNA-expressing cells coincided with that of TSHbeta-ip cells. RT-PCR also showed that TSHbeta mRNA was expressed from E9 and that Pit-1 mRNA was expressed from E5. These results clearly demonstrated that the expression of chicken TSHbeta mRNA starts on E9, that TSHbeta-ip cells appear on E10, mainly in the cephalic lobe, and that TSHbeta-ip cells can be classified into two cell types (round- and club-shaped cells).
DNA fragmentation is a hallmark of apoptosis that occurs in a variety of cell types; however, it remains unclear whether caspase‐3 is required for its induction. To investigate this, we produced caspase‐3 knockout Chinese hamster ovary (CHO)‐K1 cells and examined the effects of gene knockout and treatment with caspase‐3 inhibitors. Okadaic acid (OA) is a potent inhibitor of the serine/threonine protein phosphatases (PPs) PP1 and PP2A, which induce apoptotic cellular reactions. Treatment of caspase‐3−/− cells with OA induced DNA fragmentation, indicating that caspase‐3 is not an essential requirement. However, in the presence of benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp (OMe) fluoromethylketone (z‐DEVD‐fmk), DNA fragmentation occurred in CHO‐K1 cells but not in caspase‐3−/− cells, suggesting that caspase‐3 is involved in OA‐induced DNA fragmentation that does not utilize DEVDase activity. In the absence of caspase‐3, DEVDase activity may play an important role. In addition, OA‐induced DNA fragmentation was reduced but not blocked in CHO‐K1 cells, suggesting that caspase‐3 is involved in caspase‐independent OA‐induced DNA fragmentation. Furthermore, OA‐induced cleavage of caspase‐3 and DNA fragmentation were blocked by pretreatment with the wide‐ranging serine protease inhibitor 4‐(2‐aminoethyl)‐benzenesulfonyl fluoride hydrochloride. These results suggest that serine proteases regulate DNA fragmentation upstream of caspase‐3.
It has been reported that mammotropes in a rodent pituitary gland are derived from somatotropes via somatomammotropes (SMTs), cells that produce both growth hormone (GH) and prolactin (Prl). However, no studies have been done on the transdifferentiation of somatotropes in the chicken pituitary gland. In this study, in order to determine the origin of mammotropes, we studied detail property of appearance of chicken somatotropes, mammotropes and pit-1 cells and then evaluated the existence of SMTs in the chicken embryonic pituitary gland. Immunohistochemical analysis revealed that GH-immunopositive (GH-ip) cells appeared on embryonic day (E) 14 and were mainly distributed in the caudal lobe, while Prl-immunopositive (Prl-ip) cells appeared in the cephalic lobe of the pituitary gland on E16. In situ hybridization (ISH) and RT-PCR analysis showed that expression of GH and Prl mRNA starts at E12 in the caudal lobe and at E14 in the cephalic lobe respectively. Pit-1 mRNA was first detected on E5 by RT-PCR, and pit-1 mRNA-expressing cells were found in the cephalic lobe on E8. Then with the ontogeny of the chicken, these cells spread into both lobes. Using a double staining method with ISH and immunohistochemistry, we could not detect the existence of SMTs in the chicken embryonic pituitary gland even in the marginal region of either lobe. These results suggest that chicken somatotropes and mammotropes independently appear in different lobes of pituitary gland and that transdifferentiation from somatotropes to mammotropes is not the central route for differentiation of mammotropes in the embryonic chicken pituitary gland.
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