Mitonucleons formed during differentiation of Ishikawa endometrial cells generate vacuoles that elevate monolayer syncytia: Differentiation of Ishikawa domes, Part 1

Abstract

In 1998, we published a paper (Fleming et.al, 1998) describing some aspects of Ishikawa endometrial epithelial cell differentiation from monolayer cells into cells forming fluid-filled hemispheres called domes. The process begins with the dissolution of membranes within discrete regions of the monolayer. Nuclei from fused cells aggregate and endogenous biotin in particulate structures assumed to be mitochondria increase throughout the resulting syncytium. Endogenous biotin is also the distinguishing feature of a membrane that surrounds aggregates of multiple nuclei in a structure called a mitonucleon. The current paper includes additional observations on structural changes accompanying Ishikawa differentiation. Vacuoles form in the heterochromatin of the mitonucleon and within the biotin-containing double membrane surrounding heterochromatin. With the formation of vacuoles, the mitonucleon can be seen to rise along with the apical membrane of the syncytium in which it formed. The small vacuoles that form within the heterochromatin result in structures similar to “cells with optically clear nuclei” found in some cancers. The second larger vacuole that forms within the membrane surrounding the heterochromatin transforms the cell profile to one that resembles “signet ring” cells also observed in some cancers. Eventually the membrane surrounding the massed heterochromatin, generated three to four hours earlier, is breached and previously aggregated nuclei disaggregate. During this process heterochromatin in the mitonucleons undergoes changes usually ascribed to cells undergoing programmed cell death such as pyknosis and DNA fragmentation (Fleming, 2016b). The cells do not die, instead chromatin filaments appear to coalesce into a chromatin mass that gives rise to dome-filling nuclei by amitosis during the final three to four hours of the 20 hour differentiation (Fleming, 2016c).

Megamitochondria Initiate Differentiation of Monolayer Cells into Detached Dome Cells That Proliferate by a Schizogony-Like Amitotic Process

“…An early distinguishing characteristic of dome-forming Ishikawa syncytia is a dramatic increase in the number of mitochondria as detected by streptavidin peroxidase binding to the endogenous biotin of mitochondrial enzymes. Figure 1(a) shows a syncytium within the first 4 hours after initiation of dome formation as previously described [4]. Most of the biotin stain is associated with particulate structures, but it is also possible to detect a transparent membrane staining for biotin wrapped around a nuclear aggregate.…”

“…The biotin stain in domes has also been shown to be associated with mitochondrial enzymes containing biotin [3], demonstrating that the intimate association of fused mitochondria with chromatin early in differentiation likely explains the existence of "intranuclear inclusions" in endometrium and in cancer [10]. tures that could arise from activity of electron transport elements [11] engaged perhaps in anaerobic metabolism in the fused mitochondria creating the gas vacuoles that pressure a chromatin bolus [4]. In a previous paper, it was shown that the formation of the large central gaseous vacuole begins with the merging of multiple gas vacuoles within the fused mitochondria early in the process [12].…”

“…The megamitochondrial structures called mitonucleons form within 5 to 7 hours in syncytia created by cell fusions throughout the monolayer [3]. Apical and basal syncytial membranes containing mitonucleons detach from the petri dish, remaining in contact with the monolayer by cuboidal cells around their circumference and elevating as fluid accumulates under the basal membrane forming a hemicyst [4] [5]. In some clones of Ishikawa endometrial cells, hemispheres grow out into gland-like structures within two to three weeks after initial induction of domes [6].…”

“…Mitonucleons are detected as large (often 15 to 30 microns) structures in syncytia stained by a streptavidin assay for endogenous biotin. An overall increase in endogenous biotin throughout the syncytium results from increased biogenesis of mitochondria [4] [5] [6], and within that increasing population, a subset of mitochondria fuses around aggregates of multiple nuclei (8 or more nuclei in each aggregate; 3 to 4 aggregates within each syncytium). Since observing the role of mitonucleons in dome formation, we have also documented the apparently spontaneous formation of mitonucleons in single multinucleated Ishikawa monolayer cells that detach from the monolayer to become hollow spheroids [7].…”

“…One truth about at least some megamitochondria is that mitochondrial christae are lost or rearranged [9] resulting in the shut-down of measurable electronic potential, essential for oxidative phosphorylation. The authors of that study assumed that that this was a terminal condition, but anaerobic metabolism must continue in mitonucleons since they are characterized by the formation and expansion of gas vacuoles [4], assumed to contain CO 2 , the constant abundant gaseous product of catabolism by anaerobic, and well as aerobic, cycles. As we have shown, mitonucleons are explicitly formed around aggregated chromatin that is then compressed by the expanding gas vacuole into a pyknotic state [3].…”