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Mitonucleons formed during differentiation of Ishikawa
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endometrial cells generate vacuoles that elevate monolayer
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syncytia: Differentiation of Ishikawa Domes, Part 1
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Honoree Fleming
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Castleton State College, Castleton Vt., Dean of Education, retired
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founder CancerCellsinVitro.com
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Castleton, Vermont
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USA
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Emails: honoree.fleming@castleton.edu or
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honoree@cancercellsinvitro.com
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Abstract
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In 1998, we published a paper (Fleming et.al, 1998) describing some aspects of
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Ishikawa endometrial epithelial cell differentiation from monolayer cells into cells
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forming fluid-filled hemispheres called domes. The process begins with the
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dissolution of membranes within discrete regions of the monolayer. Nuclei from fused
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cells aggregate and endogenous biotin in particulate structures assumed to be
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mitochondria increase throughout the resulting syncytium. Endogenous biotin is also
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the distinguishing feature of a membrane that surrounds aggregates of multiple nuclei
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in a structure called a mitonucleon. The current paper includes additional
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observations on structural changes accompanying Ishikawa differentiation. Vacuoles
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form in the heterochromatin of the mitonucleon and within the biotin-containing
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double membrane surrounding heterochromatin. With the formation of vacuoles, the
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mitonucleon can be seen to rise along with the apical membrane of the syncytium in
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which it formed. The small vacuoles that form within the heterochromatin result in
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structures similar to “cells with optically clear nuclei” found in some cancers. The
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second larger vacuole that forms within the membrane surrounding the
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heterochromatin transforms the cell profile to one that resembles “signet ring” cells
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also observed in some cancers. Eventually the membrane surrounding the massed
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heterochromatin, generated three to four hours earlier, is breached and previously
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aggregated nuclei disaggregate. During this process heterochromatin in the
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mitonucleons undergoes changes usually ascribed to cells undergoing programmed cell
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death such as pyknosis and DNA fragmentation (Fleming, 2016b). The cells do not die;
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instead chromatin filaments appear to coalesce into a chromatin mass that gives rise
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to dome-filling nuclei by amitosis during the final three to four hours of the 20 hour
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differentiation (Fleming, 2016c).
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Introduction
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Endometrial epithelial cells lining the uterine cavity proliferate and differentiate in
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response to the hormones estradiol and progesterone in preparation for implantation
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of a fertilized egg in humans. More than 30 years ago, researchers including this
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author, began working with cultures of human endometrial epithelia hoping that some
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aspects of this process could be studied in vitro (Fleming, 1999).
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Endometrial cancer cell lines retain some of the characteristics of their in vivo
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counterparts and have the distinct advantages of predictability and ready availability
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for experimentation. But some cell lines retain more of the organ appropriate
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characteristics than other lines and that proved to be true for the Ishikawa line.
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Started from a well-differentiated adenocarcinoma and found to contain estradiol and
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progesterone receptors (Nishida et al. 1985) the cells were shown capable of
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functions characteristic of normal endometrial cells such as enhanced proliferation in
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response to estradiol and tamoxifen (Holinka et al.,1986a). Holinka and colleagues
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also demonstrated that placental alkaline phosphatase became elevated in these cells
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in response to hormones (1986b). Having received these cells from Dr. Erlio Gurpide’s
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laboratory through Dr. Chris Holinka, we discovered their capacity to form fluid-filled
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multicellular hemispheres and decided to study what we believed might be an
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example of epithelial differentiation.
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To characterize the process we needed to find conditions that predictably resulted in
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dome formation (Fleming, 1995), determine what factors enhanced the process
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(Fleming et al. 1998), look for the synthesis of new proteins (Fleming et.al. 1995;
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Fleming 1999), and finally examine the structural changes underlying differentiation.
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Progesterone and a large factor in fetal calf serum stimulate dome formation in
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confluent monolayers of Ishikawa cells, with dimethyl sulfoxide (DMSO) and fatty
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acids enhancing the process (Fleming et al. 1995; 1998; 1999). The process occurs
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over a 16 to 20 hour period starting with the formation of syncytia in the first 4 to 6
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hours when stimulating factor contained in fetal bovine serum is added to confluent,
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quiescent monolayers. The development of elevated predomes from syncytia occurs
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over the next 6 to 8 hours, and finally mature domes appear after 4 to 6 more hours.
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Clones could be isolated that made more and larger domes including a clone that
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routinely extended domes into everted gland-like structures (Fleming et al., 1998;
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Fleming, 1999; Fleming 2016c).
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The early research demonstrated a role for mitochondria whose numbers increase
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dramatically in newly formed syncytia. The most unexpected involvement of
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endogenous biotin was its presence in membranes that envelop aggregated syncytial
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nuclei early in the differentiation in transient structures we have called mitonucleons.
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But endogenous biotin associated with nuclei had actually also been discovered for
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nuclei of some biopsied cancer tissues (Tsujimoto, Noguchi, and Taki, 1991; Yokoyama
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et al., 1993; Tanaka et. al., 1998; Gamachi et al. 2003). It turns out that the process
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of differentiation being studied in Ishikawa cells relates not only to what occurs in
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cycling endometrium but also may explain the apparent presence of nuclear
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endogenous biotin in some cancers. This paper explores the possible significance of
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endogenous biotin linked to mitochondrial carboxylases in mitonucleons in
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differentiating Ishikawa cells.
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Results and Discussion
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Fig. 1 shows a syncytium formed in an Ishikawa monolayer in response to the stimulus
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to differentiate (Fleming, 1995). Levels of stainable endogenous biotin (salmon), not
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detectable when fusion initially occurs, increase dramatically in syncytia and
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ultimately characterize a membrane that envelops nuclear aggregates in transient
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structures (Fleming et.al. 1998) called mitonucleons (Fleming, 2014). Initially, it is
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possible not only to detect multiple nuclei within the mitonucleon, but even to
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approximate the number. As few as 4 and as many as 10 individual nuclei have been
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seen within mitonucleons.
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Fig. 1 Syncytium six hours after the addition of
fresh medium with fetal calf serum to elicit
dome formation in a confluent, quiescent
monolayer of Ishikawa endometrial cells.
Endogenous biotin increases dramatically in
syncytia including in a membrane that envelops
aggregated nuclei. Chromatin stains blue with
hematoxylin and eosin. Endogenous biotin stains
salmon using avidin linked to peroxidase
together with AES. For a brief period, it is
possible to detect the presence of multiple
nuclei within the enveloping membrane (white
arrow). (bar=50
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Relatively rapidly, the mitonucleon becomes opaque so that individual nuclei can no
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longer be detected as shown in fig.2, suggesting that a second membrane, at least,
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has been elaborated around the structure. The chromatin will become compressed
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between the double membrane and the apical membrane of the syncytium as a
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vacuole within the double membrane grows in size. The “nucleus-like” mitonucleon
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stains maroon, the salmon stain of endogenous biotin in the enveloping membranes
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overlaying the blue hematoxylin stain of the enveloped heterochromatin.
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The involvement of mitochondria in this differentiation adds to a growing list of
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functions for these organelles shown to be more than ovoid powerhouses over the last
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20 years. They have been shown to be diverse with regard to size, structure,
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placement in the cell, extent of polarization and perhaps even functions (VanBlerkom
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et al., 2002; Liesa, Palacin, Zorzano, 2009; Wang et al., 2012). Mitochondria have
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been shown to be involved in apoptosis (Karbowski and Youle, 2003) and it is
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theorized that the perinuclear positioning of mitochondria may be relevant to the
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quality of “stemness.” (Bavister, 2006; Lonergan and Bavister, 2007; Rehman, 2010)
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Subplasmalemma mitochondria have also been identified in mouse oocytes
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(VanBlerkom et al. 2002) Finally, in cells treated with microtubule-active drugs
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researchers observed “perinuclear clustering of mitochondria i.e. mitochondria
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encircling the aggregated chromatin of the nucleus that had lost the nuclear
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membrane” (Kedzior et. al. 2004) These effects may be related to what has been
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observed during Ishikawa differentiation, although there is no mention of a membrane
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enveloping multiple nuclei as is true for the mitonucleons that form during Ishikawa
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differentiation.
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Fig. 2 Aggregated nuclei in multiple mitonucleons
elevating with the apical membrane.
The mitonucleon structure becomes opaque due
most probably to the elaboration of a second
membrane around each of the nuclear
aggregates. Mitonucleons together with apical
membrane of the syncytium begins to elevate.
Plane of focus for mitonucleon is above that of
the monolayer.
Bar = 50 microns
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On the other hand, numerous reports of membranes generated from mitochondria do
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exist. These include vesicles that form in HeLa cells and appear to participate in
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communication within the cell (Neuspiel et al 2008; Andrade-Navarro MA, Sanchez-
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Pulido L, McBride HM., 2009; Sugiura A. et al., 2014), as well as mitochondrial outer
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membranes that form autophagosomes in starved rat kidney cells (Hailey et al.,
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2010). Ding and collaborators have shown that, following administration of an
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electron transport uncoupler, whole mitochondria can become spheres engulfing
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various cytosolic components including other mitochondria (Ding et.al. 2012). And, in
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an intriguing result from non-differentiating Ishikawa cells, it was recently shown that
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a membrane staining for biotin can also envelop chromosomes under certain
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circumstances in Ishikawa monolayers. (Fleming, 2014)
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Within 2 to 3 hours, the mitonucleons begin to elevate with the syncytial apical
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membrane as shown in fig. 2. Three to four mitonucleons in the center of the
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syncytium only come into sharp focus above the monolayer, surrounded by apparent
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“folds” indicating that the elevation may have been higher before the structure was
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fixed and stained. The pervasiveness of mostly particulate material staining for
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endogenous biotin throughout the syncytium is also clear at this stage of the
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differentiation. Fig. 3 shows that the heterochromatin of elevating mitonucleons
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contains vacuoles. This photomicrograph was taken of a living culture and the
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vacuoles only come into focus above the plane of the monolayer cells, indicating
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elevation of the syncytium. The vacuoles are occluded when enveloping membranes
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are stained for endogenous biotin as in fig. 2.
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Fig. 3a Focusing on a monolayer in the midst of
which a predome containing mitonucleons is
beginning to elevate.
Vacuoles form in neighboring monolayer cells as
well as in the mitonucleons, but vacuoles in the
mitonucleons appear to be larger and have a
dramatic effect on the surrounding
heterochromatin. Bar=25 microns
Fig. 3b Focusing on the elevating apical
membrane of the elevating predome.
Vacuoles appear to be compressing the
surrounding heterochromatin in a structure found
in endometrium during pregnancy and in certain
kinds of tumors where they have been called
“optically clear” nuclei.
Bar=25 microns
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As the bubble-like vacuoles enlarge within and compress the heterochromatin, the
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resulting structure (fig.3) comes to resemble “optically clear nuclei” identified in
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normal tissue (Mazur MT, Hendrikson MR, Kempson RL, 1983) as well as in cancer, and
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found to be associated with endogenous biotin. Such structures have been of interest
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to oncologists since Hapke and Dehner (1979) first identified them in a papillary
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carcinoma of the thyroid. Aside from the “odd” look of vacuolated nuclei, Yokoyama
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and his colleagues (1993) reported the surprising result that endogenous biotin,
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thought to localize exclusively to mitochondria, could be found associated with
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optically clear nuclei. In a thorough review of tissues containing optically clear
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nuclei, Gamachi and his colleagues (2002) showed not only that endogenous biotin
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could be detected in 27 samples of tissue containing optically clear nuclei, but also
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demonstrated that the endogenous biotin is specifically associated with
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mitochondrial enzymes, pyruvate carboxylase and propionyl carboxylase. These
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otherwise unexpected results make sense if optically clear nuclei in cancerous tissues,
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as well as those that form in endometrium in response to pregnancy (Mazur MT,
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Hendrikson MR, Kempson RL, 1983), arise in the manner of the structures shown in
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fig. 1, that is to say nuclei become enveloped by membranes containing mitochondrial
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carboxylases and perhaps other mitochondrial proteins.
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Fig. 4a Focusing on membrane telescoping upward
from a syncytium in a living culture that has not
been fixed or stained.
The apical membrane of the predome extends
upward during the next phase of the
differentiation. The nucleon is clearly flattened
against the rising membrane. Bar=25 microns
Fig. 4b Predome with two protrusions, fixed and
stained.
Upward extending membranes collapse back into
the syncytium where the framework of the
extension can take on the appearance of a cell
membrane.
Bar = 25 microns
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Additionally, a large central vacuole begins to form within the double membrane
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elaborated around the aggregated nuclei compressing the heterochromatin even
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further against the elevating apical membrane (fig. 4a). This vacuole appears to be
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responsible for significant elevation of the apical membrane, the full extent of which
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can only be appreciated by focusing above the monolayer in unfixed, living cultures.
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Even then, it is clear that the boundaries of the protrusion are not all in focus. The
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protrusions collapse if the predome is fixed and stained as in fig.4b. The collapsed
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vacuole looks like the annulus of a “ring” whose signet stone is the heterochromatin,
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now quite pyknotic.
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The other prominent feature of the “rings” in fig. 4b includes dark particulate
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structures, contained within the double membrane surrounding the vacuole. Similar
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structures can be observed in fig 4a, outside of, but accompanying the nucleon
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compressed against the elevating apical membrane. Subsequent deployment of
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microtubule-like structures (Fleming, 2015b) suggests that these may be centriole-like
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structures. While only a single protrusion can be detected in fig. 4a, multiple
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protrusions, seen in fig 4b, are more the rule than the exception. It appears that
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each mitonucleon is capable of generating a vacuole that will elevate a portion of the
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apical membrane.
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The fixed and stained predome in fig. 5 provides insight into how mitonucleons
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disassemble. The membrane protrusion on the left in fig. 6 resembles the protrusions
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in fig. 4b, except that at least three pyknotic nuclei appear to make up the “signet
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stone” suggestive that the nuclear aggregate formed several hours earlier is coming
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apart. Numerous particulate nucleoli-sized structures can be observed in the space
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Fig.5 Fixed and stained apical membrane
protrusions showing signs that the mitonucleon is
falling apart.
The heterochromatin in the left-most mitonucleon
looks pyknotic, almost wafer like in profile,
although the aggregated nuclei appear to be
disaggregating. Subtle but detectable changes
have occurred in the second mitonucleon possibly
due to a breach of the membrane that originally
encircled chromatin. Dark structures appear to be
“leaking” out of the surrounding membrane and
the chromatin has begun to spread.
Bar=50 um
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between the inner and outer vacuolar membranes. While in the neighboring
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mitonucleon, those structures are leaking into the syncytial cytoplasm (arrow) as the
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mitonucleon double membrane begins to break down.
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The chromatin also appears to be “spreading” out from its pyknotic state, suggestive
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of critical changes in the chromatin as the enveloping membrane that stained for
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endogenous biotin disassembles. The fate of the chromatin in these structures is
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discussed in the accompanying paper (Fleming, 2015b).
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Fig. 6 Bright-field micrography focusing on the
three protrusions in the apical membrane of a
predome approximately half way through the
process of differentiation.
An arrow points to heavily chromatic material in
the protrusion furthest to the right elevating with
the apical membrane. The chromatic material in
the left-most protrusion appears to be spreading
in the syncytial envelope, around the projection.
Chromatin is not visible in the projection in the
center. As the accompanying paper describes,
the deconstruction of chromatin into 10 micron
fibers can produce what appears to be a nuclear
clearing, as in the central protrusion. The fibers
then coalesce into a mass of chromatin found in
the apical-basal membrane at the base of
protrusions.
Bar = 25 microns
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Bright field micrography of an unfixed structure allows visualization of the surfaces of
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the predome protrusions resulting from mitonucleon activity. Protrusions early on are
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characterized by dense heterochromatin moving up with the apical membrane, and
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flattened against it by the central vacuole (black arrow in fig 6). In the left-most
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protrusion in fig. 5, by contrast, a mass of spreading refractive chromatin (white
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arrows) formerly confined in a mitonucleon appears to be spreading, resembling the
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mass of chromatin that forms following fragmentation of disaggregated nuclei
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(Fleming, 2016b). The fact that nothing can be seen in the middle protrusion may
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indicate that this is the stage in differentiation when chromatin is deconstructed into
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10nm fibers (Fleming, 2016b) It is tempting to speculate that the punctile
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discontinuities in the protrusion itself are involved in the accumulation of fluid under
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the structure. The mitonucleons in fig. 5 appear to be at three different stages. The
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mitonucleon on the right, intact and elevated is the earliest stage. The mitonucleon
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in the middle may be at the stage of chromatin deconstruction. The 10nm filaments
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characteristic of that stage come together once again at the base of apical membrane
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protrusions, which appears to be what is being seen in profile in the protrusion to the
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far left. A complete description of those stages can be found in the second paper of
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this series (Fleming 2015b)
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Commonality of structures in differentiating endometrial epithelia and
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in cancer tissue
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Structures closely resembling those derived from mitonucleons in differentiating
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Ishikawa cells are frequently observed in cancer tissue. More than one term has been
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used to describe vacuolated nuclei resembling structures in fig. 2 including optically
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clear, ground glass or empty nuclei. It was suggested that these structures might
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serve as a diagnostic criterion for papillary carcinoma of the thyroid gland more than
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30 years ago (Hapke MR, Dehner, LP, 1979) The structures have since been found in
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many other cancers of which the following are representative: colonic tubular
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adenocarcinoma (Sasaki et al. 1999), ovarian borderline endometrioid tumor (Li et.al.
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2002), pancreatoblastoma (Hasegawa et al., 2003), and adenocarcinoma of the gall
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bladder (Kimura et.al. 2005).
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Another cell structure associated with cancer is the “signet ring cell.” More than 2800
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references are listed in the Medline data base as relevant to the descriptor “signet
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ring cell carcinoma,” from many different organs including stomach, colon, lung, and
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ovary, with the earliest reference discussing their appearance in bladder cancer
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(Rosas-Uribe and Luna, 1969). Some cancers are even named for this particular cell
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structure such as signet-ring cell melanoma (Grilliot, Goldblum, Liu; 2012) and signet
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ring cell carcinoma of the testis (Williamson et al., 2012)
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On the other hand papers have also appeared reminding pathologists that not all
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signet ring cells are neoplastic (Iezzoni and Mills, 2001). In Ishikawa differentiation,
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these structures are derived sequentially from mitonucleons. An obvious question
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then is why haven’t mitonucleons per se been identified in tissue cross sections. One
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possible reason is that the structures are relatively non-descript without the vacuoles.
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Their dense chromatin and meager cytoplasm may look like “small cells” or even
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“bare nuclei” (Wright, Leiman, Burgess; 1998).
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The sequential appearance of “optically clear nuclei” followed by “signet ring cells”
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in differentiating Ishikawa epithelia cells demonstrates that these structures
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represent morphological stages in a differentiation program for epithelial cells. Such
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a possibility would not, of course, be obvious from visual micro-inspection of a tumor
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at a single point in time, the necessary approach when cancerous tissue is excised. It
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has actually been understood for some time that tumors can be classified as poorly or
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well- differentiated and that the prognosis of the latter is usually better than that of
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the former. A preponderance of cells with optically clear nuclei or of signet ring cells
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in a biopsy may represent gradations between poorly and well differentiated.
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Membrane elevation during Ishikawa Differentiation
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The physiologically significant event during the first 10 hours of Ishikawa dome
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differentiation is the elevation of syncytia containing mitonucleons. Vacuole
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formation appears to be the driving force. The rapid rise and fixation-dependent
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collapse of the apical protrusion suggests that the central vacuole is filled with
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material readily generated and easily dispersed such as a gas. The simplest, albeit
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unorthodox, explanation is that the mitochondrial-like membranes enveloping
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aggregated nuclei contain the metabolic enzymes necessary to generate CO2, and are
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oriented so that CO2 accumulates both within the nuclear compartment and within
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the double membrane surrounding the aggregated nuclei. The stimulus to
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differentiate was found to be most effective when delivered with fresh medium to a
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quiescent monolayer, which, of course, contains glucose. It is useful to note that
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numerous vacuoles also appear to be generated in surrounding cells but not into
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heterochromatin, rather at the borders of the cells that are not differentiating (an
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example of that can be seen in fig. 3a)
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Gas vesicles, commonplace in planktonic microorganisms such as cyanobacteria where
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they facilitate vertical migrations (Walsby, 1994), are not generally a feature of
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animal cells. But that does not mean that gasses could not build up in an unusual
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structure such as the mitonucleon. Furthermore the century-old dogma that all
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lipophilic gasses, such as CO2, are so highly soluble in lipid bilayers that they always
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move freely in and out of cell membranes is being refined as a result of some clever
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Fig. 7 Mature domes four days
after the addition of fetal calf
serum under conditions that
stimulate dome formation.
As long as the domes are elevated,
they stain brightly, although not
uniformly, for endogenous biotin
associated with mitochondrial
carboxylases. Perhaps these are
vanguard mitochondria.
Bar = 100 microns
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experimentation as reviewed by Endeward et al. (2014). Approximately 2 decades
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ago, research using a single stomach gland demonstrated that while CO2 was
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transported across basal membranes as rapidly as might be predicted by the theory of
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“free movement,” transport across the apical membrane (Waisbren et al. 1994) was
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at least two orders of magnitude slower. Similarly, Endeward and Gros (2013)
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demonstrated that CO2 permeability for guinea pig colon membrane is more than 100
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times slower than CO2 permeability for human red cells. Subsequent research has
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begun to unravel the significance of cholesterol in diminishing CO2 transport through
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membranes, as well as the effects of proteins, particularly the water channel protein
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aquaporin 1, on the permeability of CO2 through membranes (Itel et.al. 2012).
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Nakhoul et al. (1998) demonstrated that aquaporin-1 (AQP-1), which facilitates
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transport of H20 across a membrane (Preston et. al. 1992), also affects CO2
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permeability in Xenopus oocytes leading to the controversial proposition that protein
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gas channels such as aquaphorin 1 facilitate exchange of CO2 with H20. Similar
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reports have appeared based on other systems (Talbot et. al. 2015).
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Several other aquaphorins have been found, although it is still debated whether they
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have a physiological significance. The system being proposed for dome formation
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might provide an example of significance. Do some of the cavities that form in vivo
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start out as gas-filled cavities with aquaphorins facilitating the exchange of gas for
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fluid? Even with the dissolution of the mitonucleons, mature domes continue to
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contain significant amounts of endogenous biotin, linked to carboxylases (Fleming,
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1998), and lying close to the apical membrane surface so that domes stain brightly as
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in fig. 7. The abundant staining diminishes when domes collapse. This fact could be
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explained if CO2 generation provides for ongoing refreshment of dome fluid in
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exchange for that CO2. The loss of stainability and the flattening of domes appear to
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occur at the same time, although it is important to note that wholesale death of the
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cells, as might be evident by holes in the monolayer, is not seen. The organelles
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responsible for the staining may be similar to “vanguard mitochondria” which have
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been shown to occupy a circumferential domain immediately subjacent to the plasma
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membrane (Van Blerkom and Davis, 2006) in mouse, as well as human, oocytes where
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they are believed to have specialized function early development, perhaps during
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blastocyst formation.
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Two different kinds of vacuolization are seen early in dome differentiation and have
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been described in this paper: small vacuoles that form in the enveloped
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heterochromatin itself and a large vacuole forming within the double layer of the
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mitonucleon membrane. Two well-known cell types, frequently but not exclusively
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found in cancer, are cells with optically clear nuclei and signet ring cells whose
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profiles are characterized by these two kinds of vacuoles. Additionally, research
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describes a phenomenon in excised human tissue prepared for light microscopy called
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“pseudolipomatosis” a name that describes variably sized optically clear spaces first
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found in colon cross sections by Snover et al. (1985) The name arises from the fact
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that although the vacuoles look to be filled by lipids, they are not. It has been
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assumed that they are artefactually introduced during tissue preparation. Deshmukh-
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Rane and Li-cheng Wu (2009) looked for, and found, such vacuoles in 100% of the 50
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specimens of endometrial tissue they examined. Our results suggest that the vacuoles
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may, like vacuolization in differentiating Ishikawa cells, be of some physiological
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importance.
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The results suggesting a physiological role for vacuolization in differentiating Ishikawa
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cells raise some interesting question. Do any gasotransmitters, short-lived in aqueous
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solutions, mix with CO2? Does building pressure in the central vacuole contribute to
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chromatin pyknosis or to the break-down of the mitonucleon double membrane? What
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is the effect of the release of CO2 upon breakdown of the mitonucleon? Does the
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mixing of significant amounts of CO2 with fluid elevate the pH for a short period of
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time and lead to chromatin fragmentation? Buoyancy and cavity formation are
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essential to the differentiation of domes described in this paper and gas vacuoles, not
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previously considered relevant to mammalian cells may be involved. Furthermore,
324
the biochemistry of gas vacuoles may turn out to be interesting beyond buoyancy and
325
cavity formation.
326
Materials and Methods
327
Ishikawa cells were cultured (Fleming 1995) in phenol red-free, Minimum Essential
328
(MEM) supplemented with 2 mM glutamine, 100U/ml penicillin, 0.1 mg/ml
329
streptomycin, and .25 mg amphotericin B (GIBCO, Grand Island, NY). The cells,
330
obtained from Dr. Erlio Gurpide at Mt. Sinai Hospital in New York, were originally
331
derived from an endometrial adenocarcinoma line developed by Nishida et al. (1985),
332
who demonstrated the presence of receptors for both estradiol and
333
progesterone. Cells seeded at an approximate density of 5 x 105 cells/cm2, were
334
grown for 1 -2 weeks in MEM containing 5% calf serum (CS), and then transferred to
335
medium containing 1% calf serum. Cultures left in MEM with 1% CS could survive for
336
an additional 3-5 days with little proliferation. Assays for dome formation were done
337
in confluent cultures, although differentiation has been observed to occur, to a
338
limited extent, in nonconfluent cultures.
339
Differentiation was initiated with the addition of l0-15% fetal bovine serum (FBS).
340
Multiple dishes were fixed and stained for biotin and./or for chromatin at different
341
times during differentiation. Structures were viewed using an Olympus inverted stage
342
microscope at powers of 100X, 200X and 400X. As indicated in the text,
343
differentiating structures were sometimes examined, and pictures taken without
344
fixing and staining the cultures.
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Other photomicrographs were taken of cells fixed by adding 4% paraformaldehyde in
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phosphate buffered saline (PBS) to the culture dish. After 10 min, the cells were
347
washed gently four times with 5-10 ml PBS. A solution of 1% Triton X-100 was added to
348
the cells to permeabilize the membrane. Again after 5 min, the culture was washed
349
with successive changes of PBS. After washing, cells were exposed to a1:200 dilution
350
of Extravidin-conjugated horse-radish peroxidase (HRP) (Signa) for 30 min. After
351
further washing, a solution of 3-amino-9-ethylcarbazole (AEC), prepared by dissolving
352
20 mg of AEC in 2.5 ml of dimethylformamide and diluting with 47.5 ml of 50 mM
353
potassium acetate adjusted to pH 5.0, was added to the cells together with .25%
354
H2O2. This solution"was incubated at 37"C for 45 min to allow color to develop. The
355
AEC solution was removed, and the cultures were examined and then stored in the
356
presence of PBS at 4"C. If avidin linked to peroxidase is not added to the cultures,
357
there is no reaction. If avidin without peroxidase is added first to the cultures,
358
followed by avidin-linked to peroxidase, staining is not observed. Staining does not
359
occur if avidin-HRP is not added to the cultures prior to AEC indicating that an
360
endogenous peroxidase is not responsible for the staining. To ensure that avidin was
361
reacting with biotin, we stained domes using streptavidin linked to horseradish
362
peroxidase as well as primary antibody to biotin and secondary antibody-linked to
363
horseradish peroxidase. Staining occurred under all circumstances, indicating that
364
avidin does indeed react with biotin that is endogenously present in the cell in
365
significant amounts.
366
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