The Histochemical Journal 33: 421–425, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. Alteration of the Bcl-2 : Bax ratio in the placenta as pregnancy proceeds M. De Falco 1 , L. De Luca 2 , F. Acanfora 2 , I. Cavallotti 2 , G. Cottone 3 , V. Laforgia 1 , B. De Luca 2 , A. Baldi 4 & A. De Luca 2,∗ 1 Department of Evolutive and Comparative Biology, University ‘Federico II’, Naples, Italy 2 Institute of Topographical Anatomy, School of Medicine, Second University of Naples, Via Luciano Armanni, 5, 80138 Naples, Italy 3 Laboratory of Cell Metabolism and Pharmacokinetics, Center for Experimental Research, Regina Elena Institute, Rome, Italy 4 Department of Biochemistry and Biophysics ‘F. Cedrangolo’, Section of Anatomical Pathology, Second University of Naples, Naples, Italy ∗ Author for correspondence Received 5 June 2001 and in revised form 25 September 2001 Summary The placenta is the primary site of nutrient and gas exchange between mother and foetus. During human placental development, proliferation, differentiation and apoptosis occur at different stages. In order to clarify some of the molecular mechanisms underlying these events, we investigated the pattern of expression of two members of the Bcl-2 family in human placenta samples and compared them to the level of apoptosis detected by the TUNEL method. In particular, we evaluated the expression of Bcl-2 and Bax and their ratio during the first and third trimester. We found that Bcl-2 was generally expressed at low levels during the entire gestational period. On the other hand, Bax was low during the first trimester but increased towards the end of gestation. In accordance with the change of ratio of these two molecules, the increase of apoptotic cells was observable in the third trimester. These data indicate that Bcl-2 and Bax are spatio-temporally regulated during placental development and that the different expression of the above mentioned genes is at least in part responsible for the delicate balance between cell proliferation and programmed cell death in the human placenta during pregnancy. Introduction The placenta has a dynamic and continuous self-renewal capacity and shows a high cell proliferation rate and a lack of cell contact inhibition during the entire gestational period. From these morphological characteristics, the term ‘pseudo- malignancy’ has been used to describe this tissue (Ohlsson et al. 1993). Most of the continuous remodelling of this tis- sue is due to apoptotic events. Therefore, regulation of the balance between cell proliferation and cell death is essential in the placenta in order to allow the necessary morphological and functional changes (Smith et al. 1997a, Lea et al. 1999, Levy & Nelson 2000). A large family of related protein products, which are able to balance mitogenesis and cell death stimuli, regulate apop- tosis. This well-defined class of genes is the Bcl-2 family (Kroemer 1997). The Bcl-2 family consists of intracellu- lar, membrane-associated proteins, and there is evidence that they may act by controlling mitochondrial permeability (Antonsson et al. 1997). Bcl-2 can inhibit apoptosis caused by a variety of physiological and pathological stimuli. However, its exact mechanism of action is mostly unknown. Several proteins have an amino acid sequence homology with Bcl-2, but some of them function as promoters of cell death rather than suppressors of apoptosis (Oltvai & Korsmeyer 1994). One of these is Bax, a protein with an extensive amino acid homology with Bcl-2, and it functions to counter the effects of Bcl-2 on cell survival. Moreover, Bax has been shown to bind the C-terminal domain of Bcl-2, forming heterodimers with it (Oltvai et al. 1993). It has been proposed that the ratio of Bcl-2 to Bax expression determines whether a cell is protected or sent to programmed cell death (Antonsson et al. 1997). The Bax gene is ubiquitously expressed (Krajewski et al. 1994) and represents a protective factor whose expression is acti- vated by different stimuli such as radiation (Zhan et al. 1994). In addition, Bax is a well known downstream mediator of p53 following sublethal damage of DNA (Sang et al. 1995). We investigated the expression of two Bcl-2 family mem- bers in sections of human placentas obtained in the first and third trimesters and contrasted this expression to the level of apoptosis as investigated by TUNEL. This analysis provides further information on the role of this family of proteins in the development of the human placenta. Materials and methods Normal tissues Placentas were collected from therapeutic abortions in the first trimester or immediately after spontaneous delivery at