Analysis of different cell death processes of prepubertal rat oocytes in vitro

Abstract

The processes of cell death were studied in vitro in populations of oocytes isolated from prepubertal rats. In order to identify apoptosis, the externalized phosphatidylserine was recognized with Annexin-V coupled to FITC and the fragmentation of DNA was demonstrated by means of electrophoresis. Oocytes were tested for autophagy by means of the incorporation of monodansylcadaverine and monitoring Lc3-I/Lc3-II by western blot. The expression of mRNA marker genes of autophagy and of apoptosis was studied by means of RT-PCR in pure populations of oocytes. Some oocytes expressed at least one of the following markers: caspase-3, lamp1 and Lc3. Some oocytes were positive to Annexin-V or to monodansylcadaverine. However, most of them were simultaneously positive to both markers. The relative frequency of oocytes simultaneously positive to markers of apoptosis and autophagy did not change in the different ages studied. The transformation of Lc3-I in Lc3-II was present in all populations of oocytes studied. The mRNAs for caspase-3, lamp1 and Lc3 were present in all populations of oocytes analyzed. Our results demonstrate that oocytes of rats from new born to prepubertal age are eliminated by means of three different cell death processes: apoptosis, autophagy and a mixed event in which both routes to cell death participate in the same cell.

Z-cells and oogonia/oocytes in the advanced process of autophagy are the dominant altered cells in the ovaries of hypothyroid newborn rats

“…Also, the ratio between apoptosis and autophagy oogonia/oocyte that may lead to cell death was inverse in this study (for the control and hypothyroidism) when compared to those published previously for "normal" animals [20]. It is possible to hypothesize that the majority of cells that started the apoptotic pathway switched into necrosis forming Z-cells.…”

“…Autophagic cell death has been reported in cancer cells, induced by histone deacetylase inhibitors [26] and in cells genetically engineered to lack some of the apoptosis/autophagy regulatory pathways [27]. However, autophagic and apoptotic cell death, as well as their combination are engaged in the elimination of degenerated nurse cells in the Drosophila virilis ovary [28] and prepubertal rat oocytes in vitro [20]. Based on previous data, one possible outcome of advanced autophagy could be the oogonia/oocyte death.…”

Autophagy is a cell survival program for female germ cells in the murine ovary

“…A non-apoptotic mechanism for loss of primordial follicle pools has been highlighted in two recent studies. Atretic oocytes from P1 to P28 wild-type rat ovaries were described as having apoptotic markers of active caspase-3 and positive staining for TUNEL as well as the autophagic markers of increased numbers of autophagosomes (Escobar et al 2008a(Escobar et al , 2008b. Of particular interest was that hallmark apoptotic characteristics, pyknotic nuclei and membrane blebbing were never observed.…”

“…In the pre-pubertal mouse ovary, apoptotic characteristics (nuclear condensation, caspase 3 activation, PARP1 'poly (ADP-ribose) polymerase 1' C/C and Becn1 A cell survival program for female germ cells cleavage, and DNA fragmentation) were not correlated with primordial follicles that were lost from atresia (Tingen et al 2009). In vitro analyses of murine prepubertal oocytes showed that both apoptotic and autophagic processes were involved in the cell death of the oocytes (Lobascio et al 2007, Escobar et al 2008a, 2008b. The importance of autophagy in follicular atresia has been demonstrated across multiple species including Drosophila, teleostei, caecilians, stingray, dove, quail, sheep, rats, and humans (Rosales--Torres et al 2000, Velentzas et al 2007, Gaytan et al 2008, Santos et al 2008, Zarnescu 2004.…”

Morphological and molecular effects of cortisol and ACTH on zebrafish stage I and II follicles

“…Interestingly, an increase of nucleosomes (histone-complexed DNA fragments) in the blood was observed in the same study (Burkhardt et al 2011), which is in agreement to our observation of an increased DNA damage. In an attempt to characterize the induced vacuolization by ACTH, fluorescence staining was applied using MDC as specific dye for autophagosomes (Biederbick et al 1995, Escobar et al 2010 and acridine orange for acidic compartments (red emission) or nucleic acids (green emission) (Okuthe 2013, Notaro et al 2014. ACTH treatment led to a significantly reduced incorporation of MDC into the ooplasm of stage II oocytes, which could be indicative of a lower presence of autophagosomes or an increased removal rate.…”