Analysis of microRNAs and their precursors in bovine early embryonic development.

In animals, the maternal-to-embryonic transition (MET) occurs in the first days of early development and involves the degradation of maternal transcripts that have been stored during oogenesis. Moreover, precise and specific control mechanisms govern the adequate synchronization of the MET events to promote the activation of the embryonic genome. These mechanisms are not well understood, but it is believed that microRNAs (miRNAs) could be one of the mechanisms involved. After a microarray screening study, we analysed the expression of specific miRNA during oocyte maturation and early embryo development until preimplantation stages. Two differentially expressed candidates were selected for further analysis. Mature and precursor forms of miR-21 and miR-130a were quantified by qRT-PCR in pools of 20 oocytes at GV (germinal vesicle), GV breakdown and metaphase II stages as well as in pools of embryos at the 2-cell, 4-cell, 8-cell and blastocyst stages. The results showed a linear increase during the 1-8 cell stage for the mature forms of miR-130a and miR-21 (P < 0.05 and P < 0.003, respectively) and for the precursor form of miR-130a (P < 0.002). To see if this increase was due to minor transcriptional activity, 2-cell embryos were exposed to α-amanitin for 30-34 h. Results showed a significant decrease in miR-21, pre-miR-21, miR-130a and SRFS3 in α-amanitin-treated embryos (P < 0.05). Considering the potential regulatory role of these miRNA, the bovine genome was screened to identify putative targets with a 3'UTR exact seed match. This study suggests that miRNAs could be important players in the MET, as expression profiles suggest a potential regulation role during early development steps.

Aromatase expression in Xenopus oocytes: a three cell-type model for the ovarian estradiol synthesis.

In contrast to the classical model describing the synthesis of androgens and estrogens as restricted to somatic cells, a previous study demonstrated that Xenopus laevis oocytes participate in androgen synthesis. The objective of our study was to determine whether Xenopus oocytes are also involved in estrogen synthesis. More precisely, we analyzed aromatase expression by in situ hybridization and RT-QPCR and measured aromatase activity. Aromatase, the enzyme responsible for estrogen synthesis, appears to be expressed and active not only in the follicular cells but also in the vitellogenic oocytes. During late oogenesis, aromatase oocyte expression and activity decreased concomitantly with the trend observed in surrounding follicular layers. In order to investigate the role of estradiol-17ß (E(2)), we studied its effect on oocyte meiotic resumption. It appears that, as in Rana pipiens, E(2) inhibited the follicle-enclosed maturation of Xenopus oocytes, likely through inhibition of LH-induced maturation-inducing steroid synthesis. In addition, E(2) exerted a slight enhancing action on denuded oocyte maturation whose biological significance remains unclear. Together, our results demonstrate that Xenopus oocyte significantly participates in ovarian E(2) synthesis and this may be a common feature of vitellogenic vertebrates.

Immunohistochemical analysis of presenilin 2 expression in the mouse brain: distribution pattern and co-localization with presenilin 1 protein.

Missense mutations of presenilin 1 (PS-1) and presenilin 2 (PS-2) genes cause the majority of early-onset familial forms of Alzheimer's disease (AD). We previously characterized the distribution of the PS-1 protein in the mouse brain by immunohistochemistry using an antibody directed against an epitope located in the large hydrophilic loop [Moussaoui, S., Czech, C., Pradier, L., Blanchard, V., Bonici, B., Gohin, M., Imperato, A. and Revah, F., Immunohistochemical analysis of presenilin 1 expression in the mouse brain, FEBS Lett., 383 (1996) 219-222]. Similarly, we now report the distribution pattern of PS-2 protein in the mouse brain. For these experiments we used a polyclonal antibody raised against a synthetic peptide corresponding to the amino-acid sequence 7-24 of the predicted human PS-2 protein. The specificity of the antibody was evidenced by its ability to recognize PS-2 protein in immunoprecipitation studies and by antigen-peptide competition. In the mouse brain, PS-2 protein was present in numerous cerebral structures, but its distribution in these structures did not correlate with their susceptibility to AD pathology. In all examined structures of the gray matter, PS-2 protein was concentrated in neuronal cell bodies but it was not detected in the glial cells of the white matter. The regional distribution pattern of PS-2 protein was almost identical to that of PS-1 protein. Moreover, PS-2 protein co-localized with PS-1 protein in a large number of neuronal cell bodies. In terms of subcellular localization, PS-2 immunostaining was present almost exclusively in neuronal cell bodies while PS-1 immunostaining was also present in dendrites. This could be explained by the different epitopes of the antibodies and the known proteolytic processing of both presenilins in vivo [Tanzi, R.E., Kovacs, D.M., Kim, T.-W., Moir, R.D., Guenette, S.Y. and Wasco, W., The presenilin genes and their role in early-onset familial Alzheimer's disease, Alzheimer's disease Rev., 1 (1996) 91-98]. Within neuronal cell bodies, the immunostaining of PS-2 protein, as well as that of PS-1 protein, had a reticular and granular appearance. This suggests in agreement with previous observations on PS-1 and PS-2 in COS and H4 cells [Kovacs, D.M., Fausett, H.J., Page, K.J., Kim, T.-W., Moir, R.D., Merriam, D.E., Hollister, R.D., Hallmark, O.G., Mancini, R., Felsenstein, K.M., Hyman, B.T., Tanzi, R.E., Wasco, W., Alzheimer-associated presenilins 1 and 2: neuronal expression in brain and localization to intracellular membranes in mammalian cells, Nature Med., 2 (1996) 224-229] that these proteins are situated in intracytoplasmic organelles, possibly the endoplasmic reticulum and the Golgi complex.

Immunohistochemical analysis of presenilin-1 expression in the mouse brain.

At least 22 different mutations associated with early-onset familial Alzheimer's disease (AD) in various kindreds have been reported to occur in a recently identified gene on chromosome 14, presenilin 1 (PS-1) (Sherrington et al. (1995) Nature 375, 754-760 [1] and reviewed by Van Broeckhoven (1995) Nat. Genet. 11, 230-231 [2]). In order to study the localization of PS-1 in the brain, we raised a polyclonal antiserum specific to a fragment of the predicted protein sequence of PS-1. PS-1 immunostaining was found intracellularly, in the perikaria of discrete cells, mostly neurons, appearing as thick granules, resembling large-size vesicles. These granules were located in the periphery of cell bodies and extended into dendrites and neurites. PS-1 expression was found to be broadly distributed throughout the mouse brain, not only in structures involved in AD pathology, but also in structures unaltered by this disease.