THE KINETICS OF GRANULOSA CELLS IN DEVELOPING FOLLICLES IN THE MOUSE OVARY

This investigation describes the kinetics of the granulosa cells in medium-sized follicles type 3b, 4 and 5a in ovaries of 28-day-old Bagg mice. the method of labelling with ³H-thymidine followed by high resolution autoradiography is used in the experimental work, which consist of determining percentage labelled mitosis (PLM-) and continuous labelling (CL-) curves. In order to analyse the data by computer two alternative hypotheses A and B are set up. Both include the assumptions of no cell loss, exponential growth and a resting compartment Q. In hypothesis A cells from Q re-enter the mitotic cycle via the normal DNA-synthesis compartment S_p. Hypothesis B includes beside compartment S_p a special DNA-synthesis compartment S_q where only cells from Q are synthesizing DNA, and these cells re-enter the mitotic cycle via the G₂ compartment. the mean transit time in S_q is considered to be longer than the mean transit time in S_q. On the basis of the hypothesis mathematical expressions for the PLM- and CL-curves are obtained, and by means of a computer the theoretical curves are fitted to the experimental values: thereby all relevant cell kinetical parameters are estimated. Hypothesis B seems to give the best fit between the theoretical and experimental curves. the estimated parameters are: mean cycle times, μ_c= (56.1 hr, 56.1 hr and 22.3 hr for type 3b, 4 and 5a respectively), doubling times, T _D= (96.4 hr, 118.6 hr and 59.1 hr) and the proportion of cells in Q, p _Q = (0.60, 0.71 and 0.69).     

THE EFFECT OF ESTRADIOL ON THE CELL KINETICS IN THE UTERINE AND CERVICAL EPITHELIUM OF NEONATAL MICE

The effect of estradiol-17β on the length of the various phases of the cell cycle was studied in the neonatal mouse uterine, and cervical epithelium. A double labelling method was used, and in addition labelled mitoses were counted. In the uterus proper, estradiol shortens the length of the total cell cycle, T_c, from 17-9 hr to 15-7 hr, and the duration of S phase, T_s, from 6–7 to 5-1 hr 6 hr after estradiol treatment. 12 hr after estradiol treatment, T_c is shortened to 7-4 hr and T_s to 4–5 hr. The shortening of T_c at 12 hr is mainly due to an effect on T_G1, which is shortened from 8–55 hr in untreated animals to 1–8 hr in estradiol treated animals. The T_c of cervix epithelium cells in untreated animals was found to be 21-8 hr. After treating the mice for 6 hr with estradiol the T_c was now increased to 47 hr and further to 61 -2 hr following 12 hr treatment with the hormone. T_s increases from 8-3 hr to 15-2 hr following 6 hr estradiol treatment, and to 15-4 hr after 12 hr treatment. The effect is most pronounced in T_G1, which is lengthened from 10–95 hr in untreated animals to 28-1 hr and 43 hr, respectively, in animals treated for either 6 or 12 hr with estradiol.     

ANALYSIS OF INTESTINAL CELL PROLIFERATION AFTER GUANETHIDINE-INDUCED SYMPATHECTOMY

Neonatal administration of guanethidine-sulfate results in an alteration of the cell proliferative pattern of the small intestinal epithelium of the young adult rat. Sympathectomy with guanethidine has previously been shown to depress mitotic, labelling, and total cellular migration indices while increasing the generation cycle time (T_C) of small intestinal crypt cells as measured by a stathmokinetic method. The present study showed that the G₁, S and G₂ phases of the crypt cell cycle are altered by sympathectomy, G₁ accounting for most of the increase in T_C. In addition, the percentage of [³H]-thymidine labelled crypt cells is reduced and the duration of crypt cell transit is lengthened by guanethidine-induced sympathectomy.     

ANALYSIS OF THE GROWTH KINETICS OF A HUMAN LYMPHOMA CELL LINE

The growth kinetics of an established human lymphoma cell line were analyzed by a variety of techniques utilizing various cell inocula (5 x 10⁴ - 5 x 10⁵ cells) dispensed into 60 mm diameter dishes. Techniques included pulse-labeled mitosis (PLM), continuous labeling with ³H-TdR, time-lapse photography (TLP), cell counts by electronic particle counter, and DNA histography obtained by pulse cytophotometry (PCP). There were no significant differences among values determined for any kinetic parameters as a function of cell concentration. the average doubling time of exponentially growing cells, regardless of cell inoculum, was 44.1 hr. the generation time determined by PLM was 31.1 hr with a SD of 4.7 hr. Transit times for each stage were: T_G1= 10.6 hr, T_s= 9.9 hr, T_G2= 9.9 hr, and T_m= 0.7 hr. Repeated experiments using continuous labeling with ³H-TdR demonstrated a T_G2 of 6.3 hr. the longer value determined by PLM is possibly due to the technical manipulations of this procedure which may delay pulse-labeled cells from resuming cell cycle transit. Hence, values for cell cycle stages were recalculated to give T_G1= 14.1 hr, T_s= 9.9 hr, T_G2 = 6.3 hr, and T_m= 0.7 hr. These results were used to compute the size of each cell cycle stage compartment pool and corresponded very closely to values defined directly by PCP. TLP analysis considered only cells that produced colonies of at least thirty-two cells. Generation times ranged from 8 to 89 hr and showed a positive skewness. the average value measured for 330 divisions was 34.5 hr with a SD of 13.2 hr. Thus, the variance predicted by curve fitting of the PLM data did not correlate with that defined by time-lapse photography nor did it encompass the range in generation times observed directly by TLP. There was a positive correlation between sister-sister cell generation times (+0.66) but no relation was noted for mother-daughter values.     

Cell Synchrony Techniques. II. Analysis of Cell Progression Data

Abstract CHO cells which have been sorted by mitotic detachment, centrifugal elutriation and fluorescence activated cell sorting have been followed for up to 14 hr by flow cytometry to examine their progression characteristics. Mathematical modelling techniques were used to provide quantitative estimates of the cell-cycle parameters. Mitotic detachment gives an 11.2-hr cycle time with mean transit times T_G1, T_s and T_G2M equal to 3.2, 5.6 and 2.4 respectively. Cells prepared by central elutriation in an early G₁ state have a 14-hr cycle time with T_G1, T_s and T_G2M of 5.7, 6.0 and 2.3 hr. Populations prepared by centrifugal elutriation enriched in early S and late S and G₂M have transit times of 2.7, 5.9 and 1.6 hr and 4.9, 6.7 and 2.1 hr with cycle times of 11.2 and 13.2 hr respectively. Cell sorting for a G₁ population gives transit times of 9.8, 8.0 and 3.6 for an overall 21.4-hr cycle time.     

ON THE EXISTENCE OF A Go-PHASE IN THE CELL CYCLE

The model is based on the assumption that the cell cycle contains a G_o-phase which cells leave randomly with a constant probability per unit time, γ. After leaving the G_o-phase, the cells enter the C-phase which ends with cell division. The C-phase and its constituent phases, the‘true’G₁-phase, the S-phase, the G₂-phase and mitosis are assumed to have constant durations of T, T₁T_s, T₂ and T_m, respectively. For renewal tissue it is assumed that the probability per unit time of being lost from the population is a constant for all cells irrespective of their position in the cycle. The labelled mitosis curve and labelling index for continuous labelling are derived in terms of γ, T, and T_s. The model generates labelled mitosis curves which damp quickly and reach a constant value of twice the initial labelling index, if the mean duration of the G_o-phase is sufficiently long. It is shown that the predicted labelled mitosis and continuous labelling curves agree reasonably well with the experimental curves for the hamster cheek pouch if T has a value of about 60 hr. Data are presented for the rat dorsal epidermis which support the assumption that there is a constant probability per unit time of a cell being released from the Go-phase.     

CHARACTERISTICS OF THE ISOPRENALINE STIMULATED PROLIFERATIVE RESPONSE OF RAT SUBMAXILLARY GLAND

A simple stochastic model has been developed to determine the cell cycle kinetics of the isoprenaline stimulated proliferative response in rat acinar cells. The response was measured experimentally, using ³H-TdR labelling of interphase cells and cumulative collections of mitotic cells with vincristine. The rise and fall of the fraction of labelled interphase cells and of metaphase cells is expressed by the product of the proliferative fraction and a difference of probability distributions. The probability statements of the model were formulated and then compared by an iterative fitting procedure to experimental data to obtain estimates of the model parameters. The model when fitted to the combined fraction labelled interphase (FLIW) and fraction metaphase (FMW,) waves gave a mean G_is transit time of 21-2 hr, mean G_is+ S transit time of 270 hr, and mean G_is+ S + G₂ transit time of 35-8 hr for a single injection of isoprenaline, where G_is is the initiation to S phase time. When successive injections of isoprenaline were given at intervals of 24 and 28 hr the corresponding values after the third injection were 12-4 hr, 20-8 hr and 25-7 hr respectively. The variance of the G_is phase dropped from 18-1 to 1–3 while the other variances remained unchanged. The estimated proliferative fraction was 0–24 after a single injection of isoprenaline, and 0–31 after three injections of the drug. Independently determined values of the proliferative fraction, obtained from repeated ³H-TdR injections, were 0–21 and 0–36 respectively.     

CELL CYCLE STUDY UP TO THE TIME OF HATCHING IN THE EMBRYOS OF THE SEA URCHIN,HEMICENTROTUS PULCHERRIMUS*

Cell cycles have been analyzed in 10 divisions up to the time of hatching in the embryos of the sea urchin, Hemicentrotus pulcherrimus. In the first 5 cleavages, division synchrony is very high. On the average, T_GC= 55.4 min, T_G1= 0 min, T_s= 12 min, T_G2=±0 min, T_M= 42 min. In the remaining 5 cleavages, T_GC becomes longer: 70 min for the 7th to 246 min for the 10th cleavage. G₁ and G₂ become definitely recognizable and become longer along with T_s. T_M stays more or less constant. Plots of the changing lengths of the four compartments (G₁, S, G₂, M) on the Y-axis against T_GC (X-axis) can be fitted to the following 4 regression equations; T_G1= 0.28T_GC - 19.7, T_s= 0.609T_GC - 15.2, T_G2= 0.104T_GC - 4.72 and T_M= 0.007T_GC+ 39.6.     

STUDIES ON THE POPULATION KINETICS OF THE WALKER CARCINOMA BY AUTORADIOGRAPHY AND PULSE CYTOPHOTOMETRY

The proliferation parameters of the Walker carcinoma were estimated from both in vivo and in vitro measurements. The transplantable Walker carcinoma 256 was grown in male inbred BD1 rats. During exponential growth, 5-6 days after transplantation, a PLM curve was performed, yielding estimates of Tc ? 18.0 hr, Ts ? 6.4 hr, T_G2+M? 4.1 hr. With the double labelling technique in vitro under 2.2 atm oxygen we obtained: Tc ? 18.2hr, Ts ? 8.2 hr, T_G2+M? 2.0hr. From pulse cyto-photometry DNA content histograms the fractions of cells in the cell cycle phases were calculated using a computer program: f_G1? (47.6 ± 1.1)%, f_s? (34.1 ± 1.0)%, f_G2+M? (18.3 ± 1.5)%. These fractions remained constant between the fifth and the twelfth day after transplantation. At that time the tumour growth had already slowed down appreciably. The growth fraction determined by repetitive labelling was 0.96 on the fifth and 0.93 on the seventh and eleventh day. The cell loss factor was φ? 17% during exponential tumor growth and increased to about 100% between the tenth and twelfth day. The agreement of the cell kinetic data determined by autoradiography from solid tumours in vivo (PLM, continuous labelling) and autoradiography as well as pulse cytophotometry from in vitro experiments (excised material) was satisfactory.     

EVIDENCE OF RAPID AND SLOW PROGRESSION OF CELLS THROUGH G2 PHASE IN MOUSE EPIDERMIS: A COMPARISON BETWEEN PHASE DURATIONS MEASURED BY DIFFERENT METHODS

Percentage labelled mitosis (PLM) measurements were initiated at four different times during a 24-hr period and continued for 24 hr in hairless mouse epidermis. Estimates of G₂ and S phase durations (mean T_G2 and mean T_S) were calculated. A significant number of labelled mitoses (10–20%) was seen after 30 min in all four PLM measurements and the estimated mean T_G2 varied from 1.4 to 2.5 hr and was in agreement with values from PLM measurements in other epithelial tissues. These mean T_G2 values were much shorter than expected from [³H]TdR double labelling experiments and from a multiparameter cell kinetic study in hairless mouse epidermis and did not reflect the circadian variations seen in these studies. the differences in estimates of phase durations can be explained by postulating two G₂ cell populations; one with a rapid and another with a slow rate of cell cycle progression. the cells with the higher rate are mainly registered by the PLM method, whereas those with the lower rate largely escape detection by this method. T_G2 estimates from PLM measurements in mouse epidermis therefore do not reflect the phase duration of the entire G₂ population. It is also concluded that circadian variations in T_S can not be accurately registered by the PLM method.     

PROLIFERATION KINETICS OF AN EXPERIMENTAL ASCITES TUMOUR OF THE MOUSE

The cell cycles of an experimental ascitic tumour of the C3H mouse (NCTC 2472) were determined at various times after the intraperitoneal injection of 10⁶ cells. It was found that, contrary to results in solid NCTC 2472 tumours, obtained with the same NCTC cells, the duration of the cell cycle and its phases lengthened with the age of the tumour while the growth fraction remained relatively constant. G₁ was the first phase to lengthen, while later T_s and T_G2 increased also. The amount of DNA per cell was determined by cytospectrophotometry. This method provides data on the evolution during growth of the relative number of cells in each phase of the cell cycle.     

Cell kinetic studies on the JB-1 ascites tumour of the mouse

Abstract. The FLM method, modified by double labelling with [³H]- and [¹⁴C]-thymidine, has been applied to the 4-day old JB-1 ascites tumour of the mouse. It results in well separated waves of purely [³H]- and purely [¹⁴C]-labelled mitoses, which show a remarkable asymmetry with long tails to the right. The following values for the mean transit times of the cells have been derived from this FLM curve, for a tumour age of 4–6 days: T_C= 32.5 hr, T_S= 16.7 hr, T_G1= 3.7 hr, T_G1= 11.0 hr and T_M= 1.1 hr. A further evaluation of the FLM curve, however, is difficult, due to the non-stationary growth of the tumour. A number of other experimental findings (growth curve, decrease of the labelling and mitotic index with increasing tumour age, two single-labelled FLM curves starting 4 and 6 days after tumour inoculation) indicate that the cell cycle time increases during the experimental period of the double-labelled FLM curve (about 2 days). A lengthening of the cycle time should result in an increasing enlargement of the areas under the waves of the modified FLM curve. However, such an increase in area has not been found; the areas are constant. All the results of the present cell kinetic studies would be consistent if it were postulated that the cell cycle time lengthens with increasing tumour age up to about 4 days after inoculation, then remains relatively constant at between 4 and 6 days and thereafter increases again. Short-term double labelling experiments suggest that this is actually the case. Under the assumption of nearly constant phase durations during the 5th and 6th day of tumour growth further conclusions can be drawn from the modified FLM curve. In particular, it follows that the transit times of the cells through successive cycle phases are uncorrelated and the variances of the transit times through a cycle phase are proportional to the duration of this phase.     

STUDIES ON THE MECHANISM OF DIURNAL VARIATION OF PROLIFERATIVE INDICES IN THE SMALL BOWEL MUCOSA OF THE RAT

In the rat small bowel mucosa significant variation was found in both the labelling and the mitotic indices with time of day. The zenith and the nadir of labelling and mitotic activity coincided at 15.00 and 02.00 hours respectively. Small changes were found in the ‘cut-off’ position, but this variation in proliferative compartment size was insufficient to account for the comparatively wider fluctuations in proliferative indices. Measurements of the rate of entry into mitosis, using metaphase arrest with vincristine at three widely separated times during the day, showed no significant change. Changes in the growth fraction or in the birth rate as measured cannot account for diurnal variation in the proliferative activity of the small bowel mucosa. We propose a hypothesis which involves diurnal fluctuations in the transit times through G₁ and through G₂.     

NMR relaxation study of water protons in Syrian hamster fetal cells at 300 MHz

TheT ₁ andT ₂ relaxation times of water protons in two cell types in culture derived from Syrian hamster fetuses (normal primary or secondary fetal cells vs BP6T tumor cells derived from the normal cells transformed by carcinogens) were measured at 7.05 Tesla magnetic field (proton frequency =300 MHz). TheT ₁/T ₂ ratios and the correlation time, τ _c , calculated from theT ₁/T ₂ ratio of cellular water protons, are significantly different in these two fibroblastic cell types of the same biological origin and with similar morphologies and growth rates in culture.     

SOLUTE CONCENTRATIONS IN THE PHLOEM AND APEX OF THE ROOT OF ZEA MAYS

Plasmolytic studies utilizing a graded series of mannitol solutions (0.1–1.4 M in 0.1 M increments) were conducted on adventitious roots of Zea mays to determine solute concentrations of cell types at various locations in the root. Results indicated that mature sieve-tube members had the highest solute concentration as determined by their C₅₀ (the estimated mannitol concentration plasmolyzing an average of 50% of a given cell type) of any cell type in the root. In tissue 12 cm from the tip, C₅₀ values calculated for proto- and metaphloem sieve-tube members were 1.15 and 1.19 M, respectively, while in tissue 0.5 cm from the root tip, values for the same cell types were 0.68 and 0.46 M, respectively. The C₅₀ values for sieve elements in tissue 5 cm from the tip were intermediate (1.08 and 1.11 M). Although the companion cells generally plasmolyzed at nearly the same concentrations of mannitol as the sieve elements, their C₅₀ values were slightly lower than adjacent mature sieve elements. The lowest C₅₀ (0.35 M) for any cell type examined was associated with meristematic cells in tissue 0.1 cm from the root tip. Taken collectively, the results indicate that positive concentration gradients exist between mature sieve tubes and meristematic cells of the root apex of maize.     

Effect of basal lamina on progesterone production by chicken granulosa cells in vitro — influence of follicular development

Experiments were conducted in vitro to study the regulation of progesterone production in chicken granulosa cells by homologous basal lamina isolated from preovulatory follicles of chicken ovary. The majority of components of the basal lamina (90–95% by weight) were solubilized with guanidine-HCl (and designated fraction 1); the remaining components were solubilized with β-mercaptoethanol containing guanidine-HCl (and designated fraction 2). The ability of fraction 1 to regulate progesterone production in granulosa cells obtained from the largest (F₁, mature), third largest (F₃, growing), fifth to seventh largest (F_5–7, growing) follicles and a pool of small yellow follicles (SYF, immature) of chicken ovary was assessed. Granulosa cells isolated from SYF follicles were in the least differentiated (undifferentiated) and those obtained from F₁ follicles were in the most differentiated state. The ability of fraction 1 to regulate progesterone production by chicken granulosa cells was influenced both by the state of cell differentiation and the form of the matrix material (whether solid or liquid). When fraction 1 was added as liquid to the incubation mixture, it promoted progesterone production by granulosa cells at all stages of differentiation; however, it caused a greater relative increase in the amount of progesterone produced by undifferentiated (SYF) and differentiating (F₃) granulosa cells than by differentiated (F₁) ones. In the presence of the liquid-form of fraction 1, luteinizing hormone (LH) stimulated progesterone production in differentiated (F₁) and differentiating (F_5–7) granulosa cells. Similarly, follicle-stimulating hormone (FSH) stimulated progesterone production by differentiating (F₃) and undifferentiated (SYF) granulosa cells in the presence of the liquid-form of fraction 1 protein. In culture wells that had been pre-coated with fraction 1 (solid-form), progesterone production by less differentiated (SYF, F_5–7) granulosa cells was enhanced, whereas progesterone production by differentiated (F₁) cells was reduced. The solid-form of fraction 1 augmented LH-stimulated progesterone production by less differentiated (F_5–7) granulosa cells however, it attenuated LH-induced progesterone production in differentiated (F₁) cells. FSH-promoted progesterone production in granulosa cells from immature follicles (SYF) was augmented by solid-form of fraction 1 whereas the effect of FSH on cells obtained from older follicle (F₃) was suppressed by solid-form of fraction 1. In experiments in which gonadotropin action was attenuated by solid-form of fraction 1, the amount of progesterone produced in the presence of maximally inhibiting concentrations of fraction 1 protein was greater than control values (no fraction 1, no gonadotropin). These results show that basal lamina of the ovarian follicle can regulate progesterone production by granulosa cells. The data demonstrate that the interactions between the components of basal lamina and LH or FSH on granulosa cell function were dependent on the stage of follicular development and were influenced by the form of the matrix material. It is concluded that the basal lamina of the chicken ovarian follicle is biologically active and regulates granulosa cell function.     

Interrelationship among specific rates of cell growth,substrate consumption,and metabolite formation in some simple microbial reactions producing primary metabolites

Applying the carbon balance principle, the interrelationship between ν = μ/Y + m (μ is the specific growth rate of microorganism, v is the specific substrate consumption rate) and π = Aμ B (Luedeking–Piret eqyuation, π is the specific metabolite formation rate) has been established for three types of simple microbial reactions. Equations for the kinetic parameters A and B have been proposed for each of the three types of microbial reactions, Expresses in terms of γ_x, γ_s and γ_p (carbon contents of dry cell, mass, major carbon energy source, and metabolite) as well as the parameters Y and m. Values of both A and B calculated from the proposed equations were compared with their experimental data for lactic acid fragmentation, aerobic SCP production, and alcohol fermentation. The estimated values agreed with the observed ones with reasonably small deviations.     

A minimal physiologically based pharmacokinetic model to investigate FcRn-mediated monoclonal antibody salvage: Effects of Kon,Koff, endosome trafficking,and animal species

ABSTRACT

Manipulation of binding affinity between monoclonal antibodies (mAbs) and the neonatal Fc receptor (FcRn) has been leveraged to extend mAb half-life; however, the steps required for success remain ambiguous and experimental observations are inconsistent. Recent models have considered the time course of endosomal transit a major contributor to the relationship between FcRn affinity and antibody half-life. Our objective was to develop a minimal physiologically based pharmacokinetic model to explain how changes in IgG-FcRn association rate constant (K_on), dissociation rate constant (K_off), and endosomal transit time [T_(w)] translate to improved IgG clearance across mice, monkeys and humans. By simulating mAb clearance across physiological values of K_on, K_off, and T_(w), we found that lowering K_off improves clearance only until the dissociation half-life reaches endosomal transit time. In contrast, K_on influenced clearance independently of T_(w).The model was then applied to fit 66 mAb plasma profiles across species digitized from the literature, and clearance of mAb (CL_IgG) and vascular fluid-phase endocytosis rate (CL_up) were estimated. We found that CL_IgG scaled well with body weight (allometric exponent of 0.90). After accounting for mAbs with significant FcRn binding at physiological pH, CL_up was allometrically scalable (exponent 0.72). For the antibodies surveyed, K_on was more highly correlated with CL_IgG across all species. The relationship between K_off and K_D with CL_IgG was largely inconsistent. Taken together, this model provides a parsimonious approach to evaluate endosomal transit kinetics using only mAb plasma concentrations. These findings reinforce the idea that endosomal transit kinetics should be considered when modeling FcRn salvage.