From Laboratory to Application – Differences between Small and Large Battery Cells

The effect of LH on the intracellular free Ca2+ concentration ([Ca2+]i) was investigated in highly purified small and large bovine luteal cell populations. Luteal cells were obtained from midcycle corpora lutea dispersed with collagenase and separated by flow cytometry into large and small cells. Resting levels of Ca2+ were higher (P less than 0.05) in the large than small cells [314 +/- 25 nM (n = 5) vs. 186 +/- 13 nM (mean +/- SE; n = 13) for large and small cells, respectively]. LH rapidly increased [Ca2+]i in both small and large cells loaded with the fluorescent Ca2+ probe fura-2. In the small cells, [Ca2+]i was immediately increased 2- to 6-fold (from 176 +/- 8 to 468 +/- 8 nM; n = 5) after adding LH. The LH induced [Ca2+]i rise occurred in two phases: an initial peak due to intracellular Ca2+ mobilization and a secondary rise due to Ca2+ influx from extracellular sources. Preincubation of the small cells with EGTA reduced the initial phase and abolished the secondary rise in [Ca2+]. Both forskolin and 8-bromo-cAMP increased [Ca2+]i in the small cells. In contrast, only a single phase of [Ca2+]i rise was observed in LH-treated large cells, and the response was 1.5- to 2-fold greater than the resting Ca2+ levels [314 +/- 25 vs. 435 +/- 60 nM (n = 4), for resting vs. LH-treated values, respectively]. The addition of both LH and prostaglandin F2 alpha (PGF2 alpha) to the large cells resulted in increases in [Ca2+]i that were greater than those induced by each hormone separately (2.0-fold for LH and 2.7-fold for PGF2 alpha vs. 7- to 9-fold in the presence of both hormones). These findings demonstrate that LH induces rapid increases in intracellular [Ca2+]i that differ in magnitude and profile between the small and large bovine luteal cells. Furthermore, LH and PGF2 alpha interacted to promote increases in [Ca2+]i in the large cells, that were higher than the sum of [Ca2+]i induced by each hormone separately.

The genetic events involved in the transformation of chronic lymphocytic leukemia (CLL) to Richter's syndrome (RS) are poorly understood. Frequently large cells are seen in the bone marrows of patients with CLL and evidence of RS. Using a laser-capture microdissection we analyzed small and large leukemic bone marrow cells from 19 patients with RS for loss of heterozygosity (LOH) on chromosome 11 (D11S2179 at the ATM gene), 17 (D17S938 and D17S1852 at the TP53 site), and 20 (Plc1, D20S96, D20S110, and D20S119). Megakaryocytes were also isolated and used as a control for normal cells. Four of 15 (27.7%) informative cases showed LOH in small cells in the ATM gene while seven (46.7%) showed LOH in large cells. Six of 15 (40%) informative cases had LOH in chromosome 17 in small cells, and eight (53%) showed LOH in large cells. Eleven of 19 informative cases (61.1%) showed LOH in chromosome 20 in large cells, and eight (42.1%) showed LOH in small cells. RS cases with LOH at chromosome 20 were associated with marginally shorter survival rates (P = 0.08). Our data suggest that there are significant molecular differences between large and small cells in patients with CLL. Further analysis of the genes on these chromosomes may provide new insight into our understanding of the transformation of small CLL cells to large (Richter) cells.

Enriched fractions of small and large luteal cells were incubated for 2 h with 1 or 10 microM calcium ionophore, A23187: unstimulated secretion of progesterone and viability in small cells were not affected but these measures were decreased (P less than 0.01) for unstimulated large cells and were significantly correlated (P less than 0.05). This effect in large cells was independent of extracellular calcium. Therefore, incubations of the two cell types were made in the presence of increasing concentrations of a protein kinase C activator, phorbol 12-myristate 13-acetate (TPA). Secretion of progesterone and viability were not augmented in unstimulated small cells, but TPA prevented (P less than 0.05) the full stimulation of secretion of progesterone by LH. Secretion of progesterone in unstimulated large cells was inhibited (P less than 0.01) by TPA (100 nM and 10 microM), although viability was unaffected. The non-tumour promoting phorbol ester, 4 alpha-phorbol didecanoate, had no effect on large cells. Extracellular calcium was not required for the observed effect of TPA. Sphingosine, an agent inhibitory to protein kinase C activity, inhibited (P less than 0.01) secretion of progesterone in small and large cells, and also reduced (P less than 0.01) cell viability. These values were significantly correlated (P less than 0.05) in both cell types. The above observations suggest that protein kinase C may invoke negative regulation on progesterone production in unstimulated large and hormone-stimulated small luteal cells of sheep. Since sphingosine significantly reduced viability in small and large cells and ionophore selectively inhibited viability in large cells, the ability of these agents to influence calcium-mediated intracellular regulation of steroidogenesis is still uncertain.

1. Semi-isolated preparations of the nine-celled cardiac ganglion of the crab, Portunus sanguinolentus, were studied electrophysiologically, using simultaneous recording from extracellular and two or three intracellular electrodes. Nine penetrations of small cells were achieved. 2. Three large (80 x 120 micron) cells lie near the anterior end of the 5-mm main trunk; two large and four small (less than 50 micron) cells at the posterior end. Large-cell axons pass along the main trunk and then exit to innervate cardiac muscle; small-cell axons do not leave the ganglion. 3. The semi-isolated ganglion produces spontaneous electrical activity organized into regularly patterned, rhythmic bursts of large- and small-cell impulses recurring at rates of 0.3-0.6/s and lasting 500-800 ms. Small impulse activity commences and ends each burst. Small cells fire trains during the burst, but impulses are not synchronized among them. Large-cell trains are synchronous, are at about one-half the frequency, and have fewer impulses than small-cell trains. 4. Intracellular recordings from small cells show a slow, pacemaker depolarization from a maximum membrane potential of -54 mV leading with only a slight inflection at ca. -50 mV to a depolarized plateau at ca. -40 mV; nonovershooting impulses are superimposed on this but cease before it repolarizes. Impulses, therefore, arise at a site distant from the soma and do not invade it. Deflections suggesting synaptic potentials are not seen. 5. Intracellular recordings from large cells show complex depolarizations corresponding to extracellularly recorded bursts. These represent excitatory postsynaptic potentials (EPSPs) corresponding with individual small-cell impulses, attenuated, non-overshooting spikes, and an underlying slow depolarization; usually no pacemaker depolarization is apparent between bursts. Chemically mediated transmission is probable for the EPSPs because they show delay, increase in amplitude with hyperpolarization, sometimes show facilitation, and are reduced in saline having one-third Ca, 3 x Mg. 6. EPSPs, impulses, and the slow depolarization occur synchronously among the large cells. Potentials recorded from posterior cells are attenuated and slower than those of the anterior cells. This is interpreted to reflect sites of occurrence more distant from the soma in the posterior than in the anterior cells. Impulses do not invade the somata. 7. Intracellular recordings from large-cell axons 4 mm from the soma show overshooting action potentials arising sharply from a base line. EPSPs are absent or highly attenuated and there is little underlying depolarization (less than 2 mV). 8. Current passing with electrodes intracellular to two cells has established directly that all large cells are electrotonically coupled and that an anterior cell and a small cell are coupled. Changes of burst rate during current passing into any large cell indicate that all large cells and small cells are electrotonically coupled. 9...

Inflammation is associated with increased body mass and purportedly with increased size of adipose cells. We sought to determine whether increased size of adipose cells is associated with localised inflammation in weight-stable, moderately obese humans. We recruited 49 healthy, moderately obese individuals for quantification of insulin resistance (modified insulin suppression test) and subcutaneous abdominal adipose tissue biopsy. Cell size distribution was analysed with a multisizer device and inflammatory gene expression with real-time PCR. Correlations between inflammatory gene expression and cell size variables, with adjustment for sex and insulin resistance, were calculated. Adipose cells were bimodally distributed, with 47% in a 'large' cell population and the remainder in a 'small' cell population. The median diameter of the large adipose cells was not associated with expression of inflammatory genes. Rather, the fraction of small adipose cells was consistently associated with inflammatory gene expression, independently of sex, insulin resistance and BMI. This association was more pronounced in insulin-resistant than insulin-sensitive individuals. Insulin resistance also independently predicted expression of inflammatory genes. This study demonstrates that among moderately obese, weight-stable individuals an increased proportion of small adipose cells is associated with inflammation in subcutaneous adipose tissue, whereas size of mature adipose cells is not. The observed association between small adipose cells and inflammation may reflect impaired adipogenesis and/or terminal differentiation. However, it is unclear whether this is a cause or consequence of inflammation. This question and whether small vs large adipose cells contribute differently to inflammation in adipose tissue are topics for future research. ClinicalTrials.gov NCT00285844.

Recent studies have proposed that release of calcium from the sarcoplasmic reticulum (SR) modulates the spontaneous activity of the sinoatrial node (SAN). Previously we have shown that several calcium regulatory proteins are expressed at a lower level in the centre of the SAN compared with the periphery. Such differences may produce heterogeneity of intracellular calcium handling and pacemaker activity across the SAN. Selective isolations showed that the centre of the SAN is composed of smaller cells than the periphery. Measurements of cytosolic calcium in spontaneously beating cells showed that diastolic calcium, systolic calcium, the calcium transient amplitude and spontaneous rate were greater in larger (likely to be peripheral) cells compared with smaller (likely to be central) SAN cells. The SR calcium content was greater in larger cells, although SR recruitment was more efficient in smaller cells. The sodium-calcium exchanger and sarcolemmal calcium ATPase had a lower activity and the exchanger was responsible for a larger proportion of sarcolemmal calcium extrusion in smaller cells compared with larger cells. Ryanodine had a greater effect on the spontaneous calcium transient in larger cells compared with smaller cells, and slowed pacemaker activity in larger cells but not smaller cells, thus abolishing the difference in cycle length. This study shows heterogeneity of intracellular calcium regulation within the SAN and this contributes to differences in pacemaker activity between cells from across the SAN. The smallest central cells of the leading pacemaker region of the SAN do not require SR calcium for spontaneous activity nor does disruption of the SR alter pacemaking in these primary pacemaker cells.

Voltage-gated calcium currents in whole-cell patch-clamped bullfrog dorsal root ganglion cells: effects of cell size and intracellular solutions

Interactions of prostaglandins with subpopulations of ovine luteal cells. I. Stimulatory effects of prostaglandins E 1, E 2 and I 21

Specific monoclonal antibodies to granulosa and thecal cell surface antigens were produced and used to determine the contributions of theca and granulosa cells to the bovine corpus luteum (CL). Binding of each antibody was examined on collagenase-dispersed luteal cells from 18 cycling and 14 pregnant heifers by indirect immunofluorescence. The percent binding of the large luteal cells to granulosa antibody (GrAb) declined (P less than 0.01) as the age of the CL advanced: 77 +/- 6, 47.5 +/- 3, and 30 +/- 2 for Days 4-6, 10-12 and 16-18, respectively. Further reduction in binding of GrAb to large cells occurred between 50 and 100 days of pregnancy and no labeling was seen thereafter. Fourteen percent of the small luteal cells were bound by GrAb on Days 4-6 of the cycle, and none were labeled during subsequent stages. In contrast, when thecal antibody (TAb) was used, the proportions of large cells that were labeled increased (P less than 0.01) between Days 4-6 (10 +/- 1.3%) and 10-12 (46 +/- 3%). The percentage of large cells bound by TAb then remained unchanged until midpregnancy, declined as pregnancy advanced, and disappeared during late gestation. A majority of small luteal cells were bound by TAb throughout the estrous cycle: 70 +/- 4%, 69 +/- 3% and 58 +/- 6% at Days 4-6, 10-12, 16-18, respectively. Labeling of small cells by TAb occurred throughout pregnancy but declined (P less than 0.05) as gestation advanced. These studies suggest that the large cells of the early cyclic CL are derived from granulosa cells, while most of the small cells are of thecal origin. Small cells develop into large cells as the age of the CL increases. Granulosa-derived cells disappear during early pregnancy, while cells of thecal origin persist throughout pregnancy.

Differential incorporation of tritiated proline and leucine by neurons of the dorsal column nuclei in the cat

Action of the opioid agonist FK 33-824 on porcine small and large luteal cells from the mid-luteal phase: effect on progesterone, cAMP, cGMP and inositol phosphate release.

The seid mouse, characterization and potential uses: Edited by M.J. Bosma, R.A. Phillips and W. Schuler. Current toplcs in microbiology and immunology, vol. 152. Springer, Berlin, 1989, 263 pp. DM132

Are the large cells derived from cultured DEAD box polypeptide 4 (DDX4)-positive oogonial stem cells (OSCs), isolated from the ovarian cortex of non-menopausal and menopausal women, oocyte-like cells? Under appropriate culture conditions, DDX4-positive OSCs from non-menopausal and menopausal women differentiate into large haploid oocyte-like cells expressing the major oocyte markers growth differentiation factor 9 (GDF-9) and synaptonemal complex protein 3 (SYCP3) and then enter meiosis. The recent reports of OSCs in the ovaries of non-menopausal and menopausal women suggest that neo-oogenesis is inducible during ovarian senescence. However, several questions remain regarding the isolation of these cells, their spontaneous maturation in vitro, and the final differentiation state of the resulting putative oocytes. DDX4-positive OSCs were obtained from 19 menopausal and 13 non-menopausal women (who underwent hysterectomy for uterine fibroma, ovarian cyst, or other benign pathologies) and cultured for up to 3 weeks. Large and small cells were individually isolated and typed for early and late differentiation markers. Ovarian cortex fragments were processed by immuno-magnetic separation using a rabbit anti-human DDX4 antibody and the positive populations were measured by assessing both FRAGILIS and stage-specific embryonic antigen 4 (SSEA-4) expression. After 3 weeks in culture, large oocyte-like cells were individually isolated by DEPArray based on PKH26 red staining and cell size determination. GDF-9 and SYCP3 as final, and developmental pluripotency-associated protein 3 (DPPA3) as primordial, germline markers were measured by droplet digital PCR. The haploid versus diploid chromosomal content of chromosomes X and 5 was investigated using fluorescence in situ hybridization (FISH). SSEA-4+ and FRAGILIS+ subsets of DDX4-positive populations were present at lower mean levels in menopausal (SSEA-4+: 46.7%; FRAGILIS+: 47.5%) than in non-menopausal (SSEA-4+: 64.9%; FRAGILIS+: 64.8) women (P < 0.05). A comparison of the women's age with the ratio of DDX4-positive cells/cm3 of ovarian cortex revealed an inverse correlation with OSC number (P < 0.05). Once cultured, cells from both groups differentiated to form large (up to 80 μm) mature oocyte-like cells with typical oocyte morphology. Despite the higher numbers of these cells in cultures from non-menopausal women (37.4 versus 23.7/well; P < 0.001), the intra-culture percentages of large oocyte-like cells did not differ significantly between the two groups. Single large oocyte-like cells isolated from non-menopausal and menopausal women expressed equivalent levels of GDF-9 (e.g. 2.0 and 2.6 copies/μl RNA, respectively) and SYCP3 (e.g. 1.2 and 1.5 copies/μl RNA, respectively) mRNA. The remaining small cells isolated from the cultures expressed large amounts of DPPA3 mRNA (e.g. 5.0 and 5.1 copies/μl RNA, from menopausal and non-menopausal women, respectively), which was undetectable in the large oocyte-like cells. FISH analysis of the large and small cells using probes for chromosomes X and 5 revealed a single signal in the large cells, indicative of chromosome haploidy, whereas in the small cells two distinct signals for each chromosome indicated diploidy. Not applicable. Our study demonstrated the final differentiation of OSCs, collected from the ovarian cortex of adult women, to oocyte-like cells. However, because the rate of differentiation was low, a major role of the stem cell niche housing these OSCs cannot be ruled out. Since the ability of OSCs to generate mature oocytes in vitro is highly variable, the viability of these cells in the ovarian cortex of non-menopausal and menopausal women may well be determined by the stem cell niche and the woman's concurrent reproductive state. Our study showed that the oocyte-like cells obtained by OSC differentiation in vitro, including those from the OSCs of menopausal women, express markers of meiosis. This model of ovarian neo-oogenesis will contribute to the development of approaches to treat female infertility. The study was funded by Italian Association for Cancer Research (IG grant 17536), and from the Apulia Region ('Oncogenomic Project' and 'Jonico-Salentino Project'). All Authors declare no competing interests.

Morphological and biochemical characterization of small and large bovine luteal cells during pregnancy

In a recent paper, we proposed a model for the dependence of roughness on the resolution that was determined by an objective of an optical profiler, when surface grain diameter was smaller than the resolution. However, the model does not take into account the effect of waviness beneath surface grains whose wavelengths are larger than the resolution. The model is generalized by introducing terms that include the waviness. The area of a large cell with resolution determined by a low-magnification (SX) objective is adjusted to R2-times the area of a small cell with resolution determined by the high-magnification (LX) objective, where R is the ratio of the large cell area defined by the resolution of the SX objective to the small cell area defined by the resolution of the LX objective. If the wavelengths of the waviness and the diameters were random, the optical height in the large cell and R2-times the small cell would be divided into an invariable portion that is common to both large and small cells, and into the variable portions that are inherent to large and small cells. Finally, a new model is obtained as a result of multiplying the old model by a root term that takes the effect of waviness into account. The new model agrees well with the experimental results.