Differences in neuronal numbers, morphology and developmental apoptosis in mice nigra provide experimental evidence of ontogenic origin of vulnerability to Parkinson’s disease

Parkinson disease (PD) prevalence varies by ethnicity. In an earlier study we replicated the reduced vulnerability to PD in an admixed population, using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-susceptible C57BL/6J, MPTP-resistant CD-1 and their F1 crossbreds. In the present study we investigated if the differences have a developmental origin. Substantia nigra was evaluated at postnatal days 2 (P2), P6, P10, P14, P18, and P22. C57BL/6J mice had smaller nigra and fewer dopaminergic neurons than the CD-1 and crossbreds at P2, which persisted through development. A significant increase in numbers and nigral volume was observed across strains till P14. A drastic decline thereafter was specific to C57BL/6J. CD-1 and crossbreds retained their numbers from P14 to stabilize with supernumerary neurons at adulthood. The neuronal size increased gradually to attain adult morphology at P10 in the resistant strains, vis-à-vis at P22 in C57BL/6J. Accordingly, in comparison to C57BL/6J, the nigra of CD-1 and reciprocal crossbreds possessed cyto-morphological features of resilience, since birth. The considerably lesser dopaminergic neuronal loss in the CD-1 and crossbreds seen at P2, P14 and thereafter was complemented by attenuated developmental cell death. The differences in programmed cell death were confirmed by reduced TUNEL labelling, AIF and caspase-3 expression. GDNF expression aligned with the cell death pattern at P2 and P14 in both nigra and striatum. Earlier maturity of nigra and its neurons appear to be better features that reflect as MPTP-resistance at adulthood. Thus variable MPTP-vulnerability in mice and also differential susceptibility to PD in humans may arise early during nigral development.

A mitochondrial mechanism is involved in apoptosis of Robertsonian mouse male germ cells

The aim of this study was to determine whether the intrinsic mechanism of apoptosis is involved in the death of germ cells in Robertsonian (Rb) heterozygous adult male mice. Testes from 5-month-old Rb heterozygous CD1×Milano II mice were obtained and compared with those from homozygous CD1 (2n=40) and Milano II (2n=24) mice. For histological evaluation of apoptosis, TUNEL labelling and immunohistochemistry were used to localise Bax and cytochrome c. Expression of calbindin D28k (CB), an anti-apoptotic molecule, was also analysed by immunohistochemistry and immunoblotting. Testicular ultrastructure was visualised by electron microscopy. Morphology and cell associations were abnormal in the Rb heterozygous seminiferous epithelium. An intense apoptotic process was observed in tubules at stage XII, mainly in metaphase spermatocytes. Metaphase spermatocytes also showed Bax and cytochrome c redistributions. Mitochondria relocated close to the paranuclear region of spermatocytes. CB was mainly expressed in metaphase spermatocytes, but also in pachytene spermatocytes, spermatids and Sertoli cells at stage XII. The co-localisation of CB and TUNEL labelling was very limited. Sixty per cent of metaphase spermatocytes were apoptotic and calbindin negative, while 40% were calbindin positive without signs of apoptosis. Ten per cent of the Bax- and cytochrome c-positive cells were also calbindin positive. These data suggest that apoptosis of the germ cells in heterozygous mice occurs, at least in part, through a mitochondrial-dependent mechanism. Calbindin overexpression might prevent or reduce the apoptosis of germ cells caused by Rb heterozygosity, which could partially explain the subfertility of these mice.

151A SIMPLE AND FAST METHOD FOR CONCURRENT DIFFERENTIAL STAINING AND TUNEL LABELLING OF BOVINE BLASTOCYSTS

Currently techniques of TUNEL labelling for detection of apoptosis and differential staining for counting the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells are used separately for assessment of embryo quality in different species. The majority of these techniques are antibody-based, and time-consuming, frequently giving inconsistent results. Here we report on the development of a simple and fast method for simultaneous differential staining and TUNEL labelling of bovine embryos. Cleaved embryos produced from in vitro-matured and fertilized oocytes were cultured to the blastocyst stage in synthetic oviductal fluid culture medium (SOF) supplemented with 4mgmL−1 BSA and 5% FCS. Embryos were partially permeabilized in 0.5% Triton X-100 solution containing 2μMmL−1 TOTO-3 dye (Molecular Probes, Eugene, OR, USA) for 30s. TOTO-3 is a cell-impermeant nucleic acid dye; thus only permeabilized cells are stained red. The embryos were then quickly washed in PBS containing 3mgmL−1 PVA, fixed for 15min at RT in 4% paraformaldehyde containing 10μgmL−1 Hoescht, and TUNEL-labelled using a Cell Death Kit (Roche Applied Science, East Sussex, UI) for 30min at 37°C in a humid chamber. The embryos were then treated with RNase A (50UmL−1) for 30min at 37°C, washed and mounted in a small drop of glycerol on a glass slide. RQ1-DNase (3UmL−1)-treated embryos were used as a positive control. After three-dimensional reconstruction using a Leica TCS SP2 confocal microscope, we determined the number of ICM (blue), TE (red) and apoptotic nuclei (green). Only peripheral cells of the blastocysts were labelled red, indicating that TE cells were permeabilized by the short exposure to the detergent Triton. ICM cells were consistently stained blue by the cell permeant dye Hoechst. Apoptotic nuclei were found in both types of cells. More consistent differential staining was observed in hatched blastocysts (n=30) than in zona-enclosed blastocysts (n=35); also, more apoptotic nuclei were observed. No differences were found in the consistency of the technique for embryos grown with or without FCS. When compared to dual staining without Tunel, no differences in cell number (74±22) , and ICM/TE ratio (0.28±0.06) were detected, indicating that the Tunel procedure does not affect the labeling of the DNA. Preliminary observations also indicate that this method can be successfully applied to porcine and ovine embryos. This technique has the advantage of being fast and can be applied for assessment of embryo quality. It can also be used to determine the time and origin of ICM and TE differentiation while monitoring the degree of apoptosis in different culture systems and in different species. This work was in part supported by Department of Environment Food and Rural Affairs (Defra) UK.

Apoptosis in the early bovine embryo

Cell death occurs during early development in vivo and in vitro, although little is known about the mechanism of blastomere death and the relation to embryonic loss. Apoptosis, characterised by chromatin condensation, DNA fragmentation and membrane blebbing, occurs without damage to surrounding cells in contrast to necrosis. Bovine oocytes and in vitro fertilised embryos (total n = 449) were analysed for (1) DNA fragmentation using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and (2) morphological features of apoptosis. TUNEL labelling was detected in immature and mature oocytes (7%, n = 57 and 23%, n = 60, respectively), and at least one cell of 8- to 16-cell embryos (5%, n = 57), morulae/early blastocysts (79%, n = 39) and expanded/hatched blastocysts (100%, n = 48). In contrast, TUNEL labelling was not detected in zygotes (n = 61), 2-cell embryos (n = 46) or 3- to 7-cell embryos (n = 81). Chromatin condensation, nuclear fragmentation, absence of neighbouring cell destruction and extrusion of cells was frequent among advanced stage embryos. Although not detected during early cleavage under standard conditions, TUNEL labelling indicative of apoptosis was induced by treatment with 10 μM staurosporine for 30 h in 95% of cleavage stage embryos (n = 59). Determination of the expression and localisation of the p53 tumour suppressor gene using reverse transcription polymerase chain reaction and whole-mount immunofluorescence revealed that although p53 transcripts were present throughout early development, nuclear localisation of p53 protein could not be detected in any blastocyst suggesting p53-independent apoptosis. This study has shown that apoptosis is dependent on embryonic developmental stage after standard culture. This suggests that bovine embryos become more capable of accommodating damaged or abnormal cells as development proceeds.