In order to characterize Sterling's triiodothyronine (T3) mitochondrial receptor using photoaffinity labeling, we observed two specific T3-binding proteins in the inner membrane (28 kDa) and in the matrix (43 kDa) of rat liver mitochondria. Western blots and immunoprecipitation using antibodies raised against the T3-binding domain of the T3 nuclear receptor c-Erb A alpha 1 indicated that at least the 43-kDa protein was c-Erb A alpha 1-related. In addition, gel mobility shift assays demonstrated the occurrence of a c-Erb A alpha 1-related mitochondrial protein that specifically binds to a natural or a palindromic thyroid-responsive element. Moreover, this protein specifically binds to a direct repeat 2 sequence located in the D-loop of the mitochondrial genome. Furthermore, electron microscopy studies allowed the direct observation of a c-Erb A-related protein in mitochondria. Lastly, the relative amounts of the 43-kDa protein related to c-Erb A alpha 1 were in good correlation with the known mitochondrial mass in three typical tissues. Interestingly, expression of a truncated form of the c-Erb A alpha 1 nuclear receptor in CV1 cells was associated with a mitochondrial localization and a stimulation of mitochondrial activity. These results supply evidence of the localization of a member of the nuclear receptor superfamily in the mitochondrial matrix involved in the regulation of mitochondrial activity that could act as a mitochondrial T3-dependent transcription factor.

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Eight-cell cow embryos were isolated and cultured in vitro in a medium enriched with 200 microCi of [5-3H]uridine for 20 min. Epon ultrathin sections of the embryos were investigated for the nucleolar morphology and for the appearance and localization of the sites of [5-3H]uridine incorporation by means of electron microscopic autoradiography. In addition to this, a general pattern of replicated embryonal DNA distribution was revealed by [methyl-3H]thymidine incorporation and light microscopic autoradiography. The essential phases of the transformation of the small nucleolus precursor body (NPB) into a vast, functionally fully active nucleolus, characterized by typical nucleolar substructural components, are taking place within the eight-cell stage. This process differed in its morphology from the nucleologenetic process in early embryogenesis of other mammals, especially of that in the mouse. The first sign of NPB transformation was the appearance of a large central vacuole followed later on by perinucleolar chromatin penetration into NPB, documented by both morphology and [3H]thymidine autoradiography. In some cases, concentration of dense fibrillar material forming clumps or stalks was seen in the central vacuole. The following rapid nucleolar development was characterized by the formation of secondary vacuoles concomitant with the onset of [5-3H]uridine incorporation into the dense fibrillar component and with the appearance of the first granules in the otherwise fibrillar structure of the nucleolus. During the late eight-cell stage, the still-rounded nucleolus developed features of a reticulated nucleolus known from somatic cells intensively synthesizing rRNA: a dense fibrillar component with associated labeling encircling fibrillar centers and a well-developed granular component. The labeled dense fibrillar component was observed mostly in the central area of the nucleolus; early embryonic NPB dense fibrous material not involved in transcription was disappearing rapidly. At the transition to the 16-cell stage the nucleoli lost their rounded shape because of the accumulation of a large amount of granular component, and they occupied a considerable part of the nucleus. In conclusion, the appearance of the nucleolar vacuole in eight-cell cow embryo is the starting point for following morphogenetic events linked with the onset of transcription.

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