147
J. Anat. (1977), 123, 1, pp. 147-156 With 3 figures Printed in Great Britain
Studies on the postnatal growth of the rat adrenal cortex NICHOLAS WRIGHT AND DRAGO VONCINA* Department ofPathology, University of Newcastle upon Tyne
(Accepted 22 December 1975) INTRODUCTION
For over ninety years the mode of adrenocortical cell renewal has been debated. There are basically two schools of thought: the proponents of the migration hypothesis (Gottschau, 1883) maintain that cells are produced mainly in the outer layers of the cortex, from which they migrate in a centripetal manner. With the advent of a zonal theory of steroid production in the adrenal cortex (Deane, Shaw & Greep, 1948) interest in adrenocortical cytogenesis was revived, and Race & Green (1955) proposed that each anatomical zone was responsible for the maintenance of its own cell number. Despite the increase in technical sophistication in recent years the problem remains unresolved. The technique of following the movement of glomerulosal cells labelled with tritiated thymidine (3HTdR) with time after injection (Ford & Young, 1963) can be criticized on the grounds of thymidine re-utilization from adrenocortical cells that die (Wyllie, Kerr & Currie, 1973). In this paper we describe an alternative approach. The rates of growth of the several adrenocortical zones are measured, and at the same time the rate of cell production in these zones is also obtained. If cells are indeed migrating from the glomerulosa, then cell production should exceed that needed for its own growth. Similarly, if the reticularis is receiving cells from without, its cell production rate should not be sufficient to account for its rate of growth. If, however, each zone is responsible for the maintenance of its own structural integrity, then in each case cell production rates should balance the respective rates of growth. The model which has been used is the postnatal growth phase in the rat: growth is rapid and the proliferative rates are sufficiently high for them to be measured without too much difficulty (Wright, Appleton & Morley, 1973). MATERIALS AND METHODS
Experiment 1 Male Wistar rats of ages ranging from 3 to 120 days were killed by cervical dislocation at 09.00 hours. After fixation in Carnoy's fluid for 24 hours the adrenals were weighed on an analytical balance, and the mean value of the left and right adrenals was calculated in each animal. Serial transverse paraffin sections were cut at a thickness of 5 ,um and stained with haematoxylin and eosin. From these sections the area of the cortex and medulla was estimated by scanning and point counting every tenth section at x 100 magnification. The cortex was then divided into * Present address: Department of Pathology, University of Ljubljana, Yugoslavia. IO-2
148 NICHOLAS WRIGHT AND DRAGO VONCINA glomerulosa, fasciculata and reticularis, and the areas obtained for these in exactly the same manner (see Fig. 3). Then, from the Delisse principle (Aherne, 1967) (1) alA = v/V, where v is the volume and a the area of any adrenal component (for example, the glomerulosa), Vis the total volume and A the total area of the gland. Then, assuming equality of density for all adrenal tissues a/A = w/W, (2) where w is the weight of any adrenal component, and W the weight of the gland. In this way the volume of any defined portion of the adrenal gland can be calculated for each time interval. To avoid the subjective procedure of fitting a hand-drawn curve, the data has been fitted by the Gompertz method. This function has found wide usage in describing the growth (particularly the postnatal growth) of many organs and tumours. It is particularly valuable since, although exponential modes of growth are allowed initially, the function is self-retarding and fits the slowing in growth rate so often observed in these tissues. The Gompertz function has the general form Vt = VO exp [A/a(I - e-t)], (3) where Vt and VO are the volumes at 0 and time t respectively, a is a constant, and A is the initial specific growth rate. This simplifies to Vt
or
=
ea+bpt
(4)
loge Vt = a + bpt, (5) where a, b and p are constants; a is related to the final height achieved by the curve (the asymptotic value of Vt is ea), b is related to the height grown by the curve, and p is related to the growth rate. In this simplification
V0 = eabg x
=
-logep,
blogep. The derivative of equation (5) is the specific growth rate, which at time t is A
=
1/V.dV/dt
blogep.pt. (6) The curves are fitted by computer using a least squares method, and the programme also generates a, b and p values with standard errors. =
Experiment 2 Ten male Wistar rats aged 14 days were injected intraperitoneally with 1 ,Ci/g body weight of tritiated thymidine (Radiochemical Centre, Amersham, England) at 09.00 hours. The animals were killed 1 hour later and autoradiographs were prepared as previously described (Wright, 1971). The adrenal cortex was divided into bands,
149
Postnatal growth of the adrenal cortex 10.0 r
100 0 _
100 0
E
E
E
0-1
0~~~~~~~~ E 10i0oo Whole gland
100
E
/
>
>2
1.0
(Gompertz)
0
10
0
80 120 40 Time (days)
Time (days)
Medulla
(Gompertz) 0.1
80 120 40 Time (days)
0
100.0 r
100 0
10*0
10*0
10.0 r bO
E
w
0
E
E
-a
Z. fasciculata and Z. reticularis
E
1.0
1*0
Z. Glomerulosa
0
I
0
I
0.1
80 40 Time (days)
120
0
100.0
E
1*0
(combined)
(Gompertz) 0
Z. fasciculata (Gom pertz)
40 80 120 Time (days)
01
0
40 80 120 Time (days)
r
10*0
0
Eo -.
reticularis (Gompertz)
I
I,,,,
z.
-a
1*0
01
0
40 80 120 Time (days)
Fig. 1. The growth curves for the various portions of the adrenal cortex. The lines are fitted by the Gompertz method. In the zona fasciculata the open circles are the values obtained before the zona reticularis can be considered separately.
150
NICHOLAS WRIGHT AND DRAGO VONCINA 60 x
a)
-0
4*0
0
30
C
-o 2-0 20_ 1.0
0Capsule
15
e-ZG
LI
45
30
ZF
60 Medulla *R
Fig. 2. The labelling index measured in contiguous bands, each three cells in thickness, throughout the adrenal cortex of the pre-pubertal male rat (ZG, zona glomerulosa; ZF, zona fasciculata; ZR, zona reticularis).
each three cells in thickness, counting from the capsule inwards to the corticomedullary junction. In each band 2000 nuclei were counted to determine the flash labelling index; this enabled the distribution of labelling indices to be plotted across the cortex. In this procedure, only sections containing medulla and showing clearly the relationship between the various cortical zones were analysed. RESULTS
Rates ofgrowth in the adrenal gland The growth curves for the several portions of the adrenal cortex are shown in Figure 1. In the whole gland the initial portion (about the first 13 days) of the curve is practically a straight line; the plot is semi-logarithmic, indicating that over this period growth occurs in an exponential manner. Thereafter there is a gradual decrease in the growth rate, until the curve becomes horizontal. The curve obtained for the whole cortex describes a similar course, with an exponential form of growth initially, followed by a gradual decrease in growth rate as adulthood is reached. In the medulla the situation is different; the growth curve shows exponential conditions over the first 15 days, followed by a relatively abrupt decrease in the growth rate. Similar formats are apparent for the various zones of the cortex, i.e. an initial exponential phase followed by slowing of growth. Since the reticularis is not readily discernible until the 12th postnatal day, the curve for the fasciculata and reticularis is first drawn with the zones combined (1 E); after 12 days a growth curve for the reticularis could be constructed, together with a separate curve for the fasciculata. In drawing the fasciculata curve, it has been assumed that the readings prior to 12 days are applicable solely to the fasciculata. Consequently after 12 days the fasciculata curve flattens more noticeably, since the reticularis is then considered separately. The curve for the reticularis starts at 12 days, and again the growth rate slows after a short period of near exponential growth. Since it is intended to analyse the growth situation at 14 days, equation (6) can be used to calculate the specific growth rate (kg) by putting t = 14 days. The growth
-Pstnm
growth ofthe adrenal cortex
151
Table 1. Specific growth rates and Gompertz constants for the adrenal cortex of the male rat Zona glomerZona Zona ulosa fasciculata reticularis
Whole gland
Whole cortex
Z. fasciculata+ Z. reticuMedulla laris
0-0030 0-0039 0 0051 0 0042 0-0043 Growth rate (mg/ 0-0030 0-0045 mg/h or cells/ cell/h) 233 177 137 165 163 261 Doubling time (hours) 153 Gompertz data 0-886 2-746 2-344 3-306 3-275 a 0-317 3*176 -3 59 -4-82 -2-95 -3-83 -3-89 b -3-96 -4-13 0 352 0 449 0-591 0 473 0-453 p 0-188 0*475 0 101 0-092 0-174 0 095 0 097 0 074 0-096 S.E. a 0-24 0-97 0-22 0-21 0-31 0-49 S.E. b 0-20 0 046 0-035 S.E. p 0-062 0045 0-040 0-038 0-074 All growth rates and doubling times are quoted for 14 days of age, but can be calculated from equations (6) and (7) for any age.
rates for the gland and its component parts are summarized in Table 1, where values for a, /3 and p with S.E. are also given from the Gompertz analysis. Knowing the growth rate, the doubling time (td) for each population can be calculated from the
relationship, td = n2kg
(7)
and doubling times are shown in Table 1, again at 14 days.
Distribution of labelling indices in the adrenal cortex at 14 days Figure 2 shows the flash labelling index (L) measured in contiguous bands, each three cells in thickness, throughout the cortex. Maximum proliferative activity is apparent in the peripheral cell layers; the outer cells of the glomerulosa have a labelling index of between 4 and 5 %, and peak labelling of 6-5 % is reached whilst still within the glomerulosa. From this peak value, there follows a steady fall in I. through the fasciculata, and the cells of the reticularis and inner fasciculata show I, values of well below 1 %. In Figure 2 the anatomical zonation is indicated beneath the diagram, and the overall labelling index can be calculated for each portion of the cortex. For glomerulosa, L, is 5-14 % (00514), for fasciculata 1 66 % (00166), and for the reticularis 023 % (00023). These labelling index measurements indicate that most cell production takes place in the outer layers of the cortex, notably the glomerulosa. Calculation of cell birth and death rates Although maximum labelling indices are found in the glomerulosa, it is apparent that a measurable rate of cell proliferation also occurs in the inner portions of the cortex. But is the birth rate sufficient to account for the rate of growth of the reticularis noted in Table 1 ? Similarly, is the rate of cell birth in the glomerulosa wholly con-
152
NICHOLAS WRIGHT AND DRAGO VONCINA
B
A Capsule
Capsule Zona glomerulosa
Zona fasciculata
Zona reticularis
Medulla (x 1 00)
( x 63)
Fig. 3. To show the division of the adrenal cortex into its component zones at two time periods. A is at 14 days of age, and, for comparison, B is the cortex at 90 days. The area of each zone was measured by point counting every tenth section at x 100 magnification, and the respective volume calculated from the Delisse principle (magnification as shown: Harris's haematoxylin).
cerned with the growth of that zone, or is there excess cell production, perhaps to feed the inner zones by migration? These points can be checked by calculating the respective birth rates and comparing them with the measured growth rates. The birth rate (kb) can be calculated from the equation kb
=
Islts
(8)
which will hold for exponential growth, since the duration of the phase of DNA synthesis (t8) is short compared with the cell cycle time, and also occurs near the end of the cell cycle (Wright, 1971). The labelling index is known from Figure 2, and in each case t8 is known accurately from previous fraction of labelled mitoses experiments also at 14 days (Wright, 1971). These values were 7 4, 8-6 and 9-2 hours for the glomerulosa, fasciculata and reticularis respectively. Thus the birth rate for the glomerulosa is 0-0069 cells/cell/hour (or 6-9 cells/1000 cells/hour), and for the reticu-
Postnatal growth of the adrenal cortex
153
Table 2. Birth rates, cell loss rates and cell loss factors for the adrenal cortex of the male rat at 14 days Zona glomerulosa
Zona fasciculata
Zona reticularis
0 0069 0 0039 0-57
0 0044 0 00052 0 12
0 00025 -0 0048 -19-2
Birth rate (cells/cell/h) Cell loss rate (cells/cell/h) Cell loss factor 0
laris, 0-00025 cells/cell/hour (0 25 cells/1000 cells/hour). Now the growth rate (kg) as calculated from equation (6) and the growth curves (Table 1) is 0 0030 cells/cell/ hour (3 0 cells/1000 cells/hour) for the glomerulosa, which is less than half the birth rate; cell production in the glomerulosa outstrips the rate of growth, and in the absence of cell death in the zone (see Discussion) cells must be migrating inwards. In the reticularis, however, the growth rate of 0-0051 cells/cell/hour (5'1 cells/ 1000 cells/hour) is much more than the birth rate (0-00025 cells/cell/hour); consequently the low rate of cell birth is insufficient to account for the measured growth rate, and cells must be migrating into the reticularis from the outer zones. The size of these fluxes can also be calculated. The cell loss rate (kb) is given by kL = kb-kg (9) which for the glomerulosa is
kL = 0-0069 - 0-0030 = 0 0039 cells/cell/hour, or 3 9 cells/1000 cells/hour, which means that each hour, for every 1000 cells in the glomerulosa about 4 migrate inwards. Similarly, in the reticularis kL = 0-00025 - 0-0051 = -00048 cells/cell/hour.
This means that there is in effect no cell loss, but a cell gain rate of 0-0048 cells/cell/ hour; for every 1000 cells in the reticularis, about 5 cells are added each hour by cell migration from the outer zones. The magnitude of these migration rates can perhaps be more easily appreciated by calculating the cell loss factor 0 (Steel, 1968). This is simply the ratio of the cell loss rate (kL) to the cell birth rate (kb), 0
=
kLlkb
(10)
and for the glomerulosa, 0 is 0 57; this means that for every 100 cells produced by cell division in the glomerulosa, 57 cells migrate into the fasciculata, again assuming no cell death. For the reticularis, 0 is - 19'73, indicating that for every 100 cells produced by cell division in the reticularis, 1973 or nearly 2000 cells enter it by migration from the outer zones. Thus at 14 days of age, the main factor causing growth of the reticularis is cell migration. In the fasciculata, the birth rate is 0 0044 cells/cell/hour, and the growth rate 0-0039 cells/cell/hour. These values are close and
154
NICHOLAS WRIGHT AN-D DRAGO VONCINA
indicate a net cell efflux (by migration) of 0 00052 cells/cell/hour; 0 5 cells are lost for every 1000 fasciculata cells, by migration into the reticularis. The cell loss factor qS is 0- 12; for every 100 cells produced, about 12 migrate. If these values are accurate, then they go some way to explaining why, with only 4 cells migrating per 1000 cells per hour in the glomerulosa, 5 cells per 1000 cells per hour are being received by the reticularis. These values are summarized in Table 2. DISCUSSION
These studies allow several tentative conclusions concerning the modes of cell renewal in the pre-pubertal adrenal cortex. The distribution of labelling indices in the outer cortex supports the concept of increased cell production rate in this portion of the cortex. It is also evident that the inner zones do show measurable 1, values, even in the reticularis, and it has been suggested that these proliferative rates in the inner zones are capable of maintaining structural homeostasis (Race & Green, 1955). The aim of these experiments was to establish whether proliferative rates in the inner zones can explain the increases in zonal volumes noted at the reference point at 14 days. The 0 values in the reticularis suggest that a net gain of cells occurs over and above those cells produced by cell division; further, this influx would appear to be large. A sizeable cell loss occurs from the glomerulosa. It is concluded that the glomerulosa is feeding cells to the fasciculata; the fasciculata adds to these cells by cell division within its substance, and augments the migration of cells into the reticularis. It should be noted that, at 14 days, growth in the glomerulosa is exponential (see Fig. 1) and cell loss would not be expected to be a major flux parameter; nevertheless, even during this period such loss could be demonstrated. The measurements made of cell migration from the glomerulosa are not applicable if any degree of cell loss occurs in this zone. Wyllie and co-workers (Wyllie, Kerr, Macaskill & Currie, 1973; Wyllie, Kerr & Currie, 1973) have studied the distribution of cell loss by apoptosis in the pre-pubertal gland; apoptosis appears to be confined to the inner cortical cell layers, notably the reticularis and is not a feature in the
glomerulosa. The growth curves all seem to follow the Gompertz model quite closely. The doubling time for the whole cortex and the whole gland at 14 days is the same (see Table 1); this is probably because the contribution of the medulla at this time is insufficient to decrease the doubling time of the whole gland, bearing in mind the limitations of the technique. The apparent continuity between the separate readings for the fasciculata and the earlier combined readings (Fig. 1) might suggest that the earlier readings are applicable to the fasciculata only. However, after 12 days the reticularis splits only a small portion from the fasciculata, which would not greatly disturb the fasciculata curve at this time. In calculating growth rates from the weight measurements, two assumptions are incurred; firstly that equal densities of tissues are found throughout the gland, and secondly, that when converting growth rates in mg to growth rates in cells, that there are equal cellular densities as between the various zones. While the first of these may be reasonable, it is probable that cellular density is higher in the glomerulosa.
Fostnatal growth of the adrenal cortex
155 If this is so, then the growth rate in the glomerulosa would be underestimated when expressed as cells/cell/hour, compared with the other zones. The measurements of labelling index in bands across the cortex has confirmed that maximum proliferative activity resides in the outer zones. This method of analysing cortical labelling indices, though infinitely more laborious than straightforward zonal indices (see, for example, Reiter & Hoffman, 1967), is preferred, since greater insight into variation within zones is obtained. The cause of the decrease in labelling index with increasing distance from the capsule is debatable; Ford & Young (1963) reported that the cell cycle time lengthened in the inner zones, but Wright (1975) considered that the cell cycle time remained substantially constant and that it was the growth fraction which fell across the cortex. The results allow a tentative model of adrenocortical cytogenesis: Zona glomerulosa ... stem cell compartment Zona fasciculata
... dividing transit compartment
Zona reticularis
... dividing transit compartment
cell death The glomerulosa must act as a stem cell compartment for the rest of the cortex: if we accept a migration theory, then by definition glomerulosa cells have no input, and may be regarded as stem cells. The fasciculata is then a dividing transit compartment, receiving cells from the stem cell compartment, and subsequently feeding the reticularis. Cells then die in the reticularis, but this is also regarded as a dividing transit compartment, since there is cell proliferation and the transit is towards cell death (Wyllie et al. 1973). In conclusion, although it is evident that a measurable proliferative rate is present at all levels of the cortex, such an observation is not incompatible with cells migrating in a centripetal manner, and maintaining a decreasing degree of proliferative activity as they progress. The present results would appear to support a migration hypothesis of adrenocortical cytogenesis. SUMMARY
In the male rat the volumes of the several zones of the adrenal cortex were measured using a point-counting technique at various ages. The resulting growth curves were fitted by the Gompertz method, and the specific growth rate calculated for each zone at 14 days of age. Also at 14 days the flash labelling index was measured in contiguous bands each three cells in thickness across the cortex; maximum indices were found in the outer cortical layers. Calculations of the birth rate for each zona were compared with the growth rates. In the zona glomerulosa, the birth rate exceeded the growth rate, giving a cell migration rate of 4 cells/1000 cells/hour. In the zona reticularis there was a net cell gain of 5 cells/1000 cells/hour; in this zone, for every 100 cells born by cell division, nearly 2000 were added by cell migration.
156
NICHOLAS WRIGHT AND DRAGO VONCINA
The results support the migration hypothesis of adrenocortical cytogenesis. REFERENCES AHERNE, W. A. (1967). Methods of counting discrete tissue components in microscopical sections. Journal of the Royal Microscopical Society 87, 493-508. BARTON, A. D. & LAIRD, A. K. (1969). Analysis of allometric and non-allometric differential growth. Growth 33, 1-16. DEANE, H. W., SHAW, J. H. & GREEP, R. 0. (1948). The effect of altered sodium or potassium intake on width and cytochemistry of the zona glomerulosa of the rat's adrenal cortex. Endocrinology 43, 133-153. FORD, J. K. & YOUNG, R. W. (1963). Cell proliferation and displacement in the adrenal cortex of young rats injected with tritiated thymidine. Anatomical Record 146, 125-153. GoTrsCHAU, M. (1883). Structur und embryonale Entwicklung des Nebennieren bei Saugethieren. Archiv fur Anatomie und Entwicklungsgeschichte 9, 412-458. RACE, C. J. & GREEN, R. F. (1955). Studies on zonation and regeneration of the adrenal cortex of the rat. Archives ofPathology 59, 578-586. REITER, R. J. & HOFFMAN, R. A. (1967). Adrenocortical cytogenesis in the adult male golden hamster. A radioautographic study using tritiated thymidine. Journal of Anatomy 101, 723-729. STEEL, G. G. (1968). Cell loss from experimental tumours. Cell and Tissue Kinetics 1, 193-207. WRIGHT, N. A. (1971). Cell proliferation in the prepubertal male rat adrenal cortex; an autoradiographic study. Journal of Endocrinology 49, 599-609. WRIGHT, N. A. (1975). Studies in the control of cell proliferation-in mammalian tissues. Ph.D. Thesis. University of Newcastle upon Tyne. WRIGHT, N. A., APPLETON, D. R. & MORLEY, A. R. (1974). Effect of dexamethasone on cell population kinetics in the adrenal cortex of the prepubertal male rat. Journal of Endocrinology 62, 527-536. WYLLIE, A. H., KERR, J. F. R. & CURRIE, A. R. (1973). Cell death in the normal neonatal adrenal cortex. Journal of Pathology 111, 255-261. WYLLIE, A. H., KERR, J. F. R., MACASKILL, I. A. M. & CURRIE, A. R. (1973). Adrenocortical cell deletion; the role of ACTH. Journal of Pathology 111, 85-94.
J. Anat. (1977), 123, 1, pp. 147-156 With 3 figures Printed in Great Britain
Studies on the postnatal growth of the rat adrenal cortex NICHOLAS WRIGHT AND DRAGO VONCINA* Department ofPathology, University of Newcastle upon Tyne
(Accepted 22 December 1975) INTRODUCTION
For over ninety years the mode of adrenocortical cell renewal has been debated. There are basically two schools of thought: the proponents of the migration hypothesis (Gottschau, 1883) maintain that cells are produced mainly in the outer layers of the cortex, from which they migrate in a centripetal manner. With the advent of a zonal theory of steroid production in the adrenal cortex (Deane, Shaw & Greep, 1948) interest in adrenocortical cytogenesis was revived, and Race & Green (1955) proposed that each anatomical zone was responsible for the maintenance of its own cell number. Despite the increase in technical sophistication in recent years the problem remains unresolved. The technique of following the movement of glomerulosal cells labelled with tritiated thymidine (3HTdR) with time after injection (Ford & Young, 1963) can be criticized on the grounds of thymidine re-utilization from adrenocortical cells that die (Wyllie, Kerr & Currie, 1973). In this paper we describe an alternative approach. The rates of growth of the several adrenocortical zones are measured, and at the same time the rate of cell production in these zones is also obtained. If cells are indeed migrating from the glomerulosa, then cell production should exceed that needed for its own growth. Similarly, if the reticularis is receiving cells from without, its cell production rate should not be sufficient to account for its rate of growth. If, however, each zone is responsible for the maintenance of its own structural integrity, then in each case cell production rates should balance the respective rates of growth. The model which has been used is the postnatal growth phase in the rat: growth is rapid and the proliferative rates are sufficiently high for them to be measured without too much difficulty (Wright, Appleton & Morley, 1973). MATERIALS AND METHODS
Experiment 1 Male Wistar rats of ages ranging from 3 to 120 days were killed by cervical dislocation at 09.00 hours. After fixation in Carnoy's fluid for 24 hours the adrenals were weighed on an analytical balance, and the mean value of the left and right adrenals was calculated in each animal. Serial transverse paraffin sections were cut at a thickness of 5 ,um and stained with haematoxylin and eosin. From these sections the area of the cortex and medulla was estimated by scanning and point counting every tenth section at x 100 magnification. The cortex was then divided into * Present address: Department of Pathology, University of Ljubljana, Yugoslavia. IO-2
148 NICHOLAS WRIGHT AND DRAGO VONCINA glomerulosa, fasciculata and reticularis, and the areas obtained for these in exactly the same manner (see Fig. 3). Then, from the Delisse principle (Aherne, 1967) (1) alA = v/V, where v is the volume and a the area of any adrenal component (for example, the glomerulosa), Vis the total volume and A the total area of the gland. Then, assuming equality of density for all adrenal tissues a/A = w/W, (2) where w is the weight of any adrenal component, and W the weight of the gland. In this way the volume of any defined portion of the adrenal gland can be calculated for each time interval. To avoid the subjective procedure of fitting a hand-drawn curve, the data has been fitted by the Gompertz method. This function has found wide usage in describing the growth (particularly the postnatal growth) of many organs and tumours. It is particularly valuable since, although exponential modes of growth are allowed initially, the function is self-retarding and fits the slowing in growth rate so often observed in these tissues. The Gompertz function has the general form Vt = VO exp [A/a(I - e-t)], (3) where Vt and VO are the volumes at 0 and time t respectively, a is a constant, and A is the initial specific growth rate. This simplifies to Vt
or
=
ea+bpt
(4)
loge Vt = a + bpt, (5) where a, b and p are constants; a is related to the final height achieved by the curve (the asymptotic value of Vt is ea), b is related to the height grown by the curve, and p is related to the growth rate. In this simplification
V0 = eabg x
=
-logep,
blogep. The derivative of equation (5) is the specific growth rate, which at time t is A
=
1/V.dV/dt
blogep.pt. (6) The curves are fitted by computer using a least squares method, and the programme also generates a, b and p values with standard errors. =
Experiment 2 Ten male Wistar rats aged 14 days were injected intraperitoneally with 1 ,Ci/g body weight of tritiated thymidine (Radiochemical Centre, Amersham, England) at 09.00 hours. The animals were killed 1 hour later and autoradiographs were prepared as previously described (Wright, 1971). The adrenal cortex was divided into bands,
149
Postnatal growth of the adrenal cortex 10.0 r
100 0 _
100 0
E
E
E
0-1
0~~~~~~~~ E 10i0oo Whole gland
100
E
/
>
>2
1.0
(Gompertz)
0
10
0
80 120 40 Time (days)
Time (days)
Medulla
(Gompertz) 0.1
80 120 40 Time (days)
0
100.0 r
100 0
10*0
10*0
10.0 r bO
E
w
0
E
E
-a
Z. fasciculata and Z. reticularis
E
1.0
1*0
Z. Glomerulosa
0
I
0
I
0.1
80 40 Time (days)
120
0
100.0
E
1*0
(combined)
(Gompertz) 0
Z. fasciculata (Gom pertz)
40 80 120 Time (days)
01
0
40 80 120 Time (days)
r
10*0
0
Eo -.
reticularis (Gompertz)
I
I,,,,
z.
-a
1*0
01
0
40 80 120 Time (days)
Fig. 1. The growth curves for the various portions of the adrenal cortex. The lines are fitted by the Gompertz method. In the zona fasciculata the open circles are the values obtained before the zona reticularis can be considered separately.
150
NICHOLAS WRIGHT AND DRAGO VONCINA 60 x
a)
-0
4*0
0
30
C
-o 2-0 20_ 1.0
0Capsule
15
e-ZG
LI
45
30
ZF
60 Medulla *R
Fig. 2. The labelling index measured in contiguous bands, each three cells in thickness, throughout the adrenal cortex of the pre-pubertal male rat (ZG, zona glomerulosa; ZF, zona fasciculata; ZR, zona reticularis).
each three cells in thickness, counting from the capsule inwards to the corticomedullary junction. In each band 2000 nuclei were counted to determine the flash labelling index; this enabled the distribution of labelling indices to be plotted across the cortex. In this procedure, only sections containing medulla and showing clearly the relationship between the various cortical zones were analysed. RESULTS
Rates ofgrowth in the adrenal gland The growth curves for the several portions of the adrenal cortex are shown in Figure 1. In the whole gland the initial portion (about the first 13 days) of the curve is practically a straight line; the plot is semi-logarithmic, indicating that over this period growth occurs in an exponential manner. Thereafter there is a gradual decrease in the growth rate, until the curve becomes horizontal. The curve obtained for the whole cortex describes a similar course, with an exponential form of growth initially, followed by a gradual decrease in growth rate as adulthood is reached. In the medulla the situation is different; the growth curve shows exponential conditions over the first 15 days, followed by a relatively abrupt decrease in the growth rate. Similar formats are apparent for the various zones of the cortex, i.e. an initial exponential phase followed by slowing of growth. Since the reticularis is not readily discernible until the 12th postnatal day, the curve for the fasciculata and reticularis is first drawn with the zones combined (1 E); after 12 days a growth curve for the reticularis could be constructed, together with a separate curve for the fasciculata. In drawing the fasciculata curve, it has been assumed that the readings prior to 12 days are applicable solely to the fasciculata. Consequently after 12 days the fasciculata curve flattens more noticeably, since the reticularis is then considered separately. The curve for the reticularis starts at 12 days, and again the growth rate slows after a short period of near exponential growth. Since it is intended to analyse the growth situation at 14 days, equation (6) can be used to calculate the specific growth rate (kg) by putting t = 14 days. The growth
-Pstnm
growth ofthe adrenal cortex
151
Table 1. Specific growth rates and Gompertz constants for the adrenal cortex of the male rat Zona glomerZona Zona ulosa fasciculata reticularis
Whole gland
Whole cortex
Z. fasciculata+ Z. reticuMedulla laris
0-0030 0-0039 0 0051 0 0042 0-0043 Growth rate (mg/ 0-0030 0-0045 mg/h or cells/ cell/h) 233 177 137 165 163 261 Doubling time (hours) 153 Gompertz data 0-886 2-746 2-344 3-306 3-275 a 0-317 3*176 -3 59 -4-82 -2-95 -3-83 -3-89 b -3-96 -4-13 0 352 0 449 0-591 0 473 0-453 p 0-188 0*475 0 101 0-092 0-174 0 095 0 097 0 074 0-096 S.E. a 0-24 0-97 0-22 0-21 0-31 0-49 S.E. b 0-20 0 046 0-035 S.E. p 0-062 0045 0-040 0-038 0-074 All growth rates and doubling times are quoted for 14 days of age, but can be calculated from equations (6) and (7) for any age.
rates for the gland and its component parts are summarized in Table 1, where values for a, /3 and p with S.E. are also given from the Gompertz analysis. Knowing the growth rate, the doubling time (td) for each population can be calculated from the
relationship, td = n2kg
(7)
and doubling times are shown in Table 1, again at 14 days.
Distribution of labelling indices in the adrenal cortex at 14 days Figure 2 shows the flash labelling index (L) measured in contiguous bands, each three cells in thickness, throughout the cortex. Maximum proliferative activity is apparent in the peripheral cell layers; the outer cells of the glomerulosa have a labelling index of between 4 and 5 %, and peak labelling of 6-5 % is reached whilst still within the glomerulosa. From this peak value, there follows a steady fall in I. through the fasciculata, and the cells of the reticularis and inner fasciculata show I, values of well below 1 %. In Figure 2 the anatomical zonation is indicated beneath the diagram, and the overall labelling index can be calculated for each portion of the cortex. For glomerulosa, L, is 5-14 % (00514), for fasciculata 1 66 % (00166), and for the reticularis 023 % (00023). These labelling index measurements indicate that most cell production takes place in the outer layers of the cortex, notably the glomerulosa. Calculation of cell birth and death rates Although maximum labelling indices are found in the glomerulosa, it is apparent that a measurable rate of cell proliferation also occurs in the inner portions of the cortex. But is the birth rate sufficient to account for the rate of growth of the reticularis noted in Table 1 ? Similarly, is the rate of cell birth in the glomerulosa wholly con-
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NICHOLAS WRIGHT AND DRAGO VONCINA
B
A Capsule
Capsule Zona glomerulosa
Zona fasciculata
Zona reticularis
Medulla (x 1 00)
( x 63)
Fig. 3. To show the division of the adrenal cortex into its component zones at two time periods. A is at 14 days of age, and, for comparison, B is the cortex at 90 days. The area of each zone was measured by point counting every tenth section at x 100 magnification, and the respective volume calculated from the Delisse principle (magnification as shown: Harris's haematoxylin).
cerned with the growth of that zone, or is there excess cell production, perhaps to feed the inner zones by migration? These points can be checked by calculating the respective birth rates and comparing them with the measured growth rates. The birth rate (kb) can be calculated from the equation kb
=
Islts
(8)
which will hold for exponential growth, since the duration of the phase of DNA synthesis (t8) is short compared with the cell cycle time, and also occurs near the end of the cell cycle (Wright, 1971). The labelling index is known from Figure 2, and in each case t8 is known accurately from previous fraction of labelled mitoses experiments also at 14 days (Wright, 1971). These values were 7 4, 8-6 and 9-2 hours for the glomerulosa, fasciculata and reticularis respectively. Thus the birth rate for the glomerulosa is 0-0069 cells/cell/hour (or 6-9 cells/1000 cells/hour), and for the reticu-
Postnatal growth of the adrenal cortex
153
Table 2. Birth rates, cell loss rates and cell loss factors for the adrenal cortex of the male rat at 14 days Zona glomerulosa
Zona fasciculata
Zona reticularis
0 0069 0 0039 0-57
0 0044 0 00052 0 12
0 00025 -0 0048 -19-2
Birth rate (cells/cell/h) Cell loss rate (cells/cell/h) Cell loss factor 0
laris, 0-00025 cells/cell/hour (0 25 cells/1000 cells/hour). Now the growth rate (kg) as calculated from equation (6) and the growth curves (Table 1) is 0 0030 cells/cell/ hour (3 0 cells/1000 cells/hour) for the glomerulosa, which is less than half the birth rate; cell production in the glomerulosa outstrips the rate of growth, and in the absence of cell death in the zone (see Discussion) cells must be migrating inwards. In the reticularis, however, the growth rate of 0-0051 cells/cell/hour (5'1 cells/ 1000 cells/hour) is much more than the birth rate (0-00025 cells/cell/hour); consequently the low rate of cell birth is insufficient to account for the measured growth rate, and cells must be migrating into the reticularis from the outer zones. The size of these fluxes can also be calculated. The cell loss rate (kb) is given by kL = kb-kg (9) which for the glomerulosa is
kL = 0-0069 - 0-0030 = 0 0039 cells/cell/hour, or 3 9 cells/1000 cells/hour, which means that each hour, for every 1000 cells in the glomerulosa about 4 migrate inwards. Similarly, in the reticularis kL = 0-00025 - 0-0051 = -00048 cells/cell/hour.
This means that there is in effect no cell loss, but a cell gain rate of 0-0048 cells/cell/ hour; for every 1000 cells in the reticularis, about 5 cells are added each hour by cell migration from the outer zones. The magnitude of these migration rates can perhaps be more easily appreciated by calculating the cell loss factor 0 (Steel, 1968). This is simply the ratio of the cell loss rate (kL) to the cell birth rate (kb), 0
=
kLlkb
(10)
and for the glomerulosa, 0 is 0 57; this means that for every 100 cells produced by cell division in the glomerulosa, 57 cells migrate into the fasciculata, again assuming no cell death. For the reticularis, 0 is - 19'73, indicating that for every 100 cells produced by cell division in the reticularis, 1973 or nearly 2000 cells enter it by migration from the outer zones. Thus at 14 days of age, the main factor causing growth of the reticularis is cell migration. In the fasciculata, the birth rate is 0 0044 cells/cell/hour, and the growth rate 0-0039 cells/cell/hour. These values are close and
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NICHOLAS WRIGHT AN-D DRAGO VONCINA
indicate a net cell efflux (by migration) of 0 00052 cells/cell/hour; 0 5 cells are lost for every 1000 fasciculata cells, by migration into the reticularis. The cell loss factor qS is 0- 12; for every 100 cells produced, about 12 migrate. If these values are accurate, then they go some way to explaining why, with only 4 cells migrating per 1000 cells per hour in the glomerulosa, 5 cells per 1000 cells per hour are being received by the reticularis. These values are summarized in Table 2. DISCUSSION
These studies allow several tentative conclusions concerning the modes of cell renewal in the pre-pubertal adrenal cortex. The distribution of labelling indices in the outer cortex supports the concept of increased cell production rate in this portion of the cortex. It is also evident that the inner zones do show measurable 1, values, even in the reticularis, and it has been suggested that these proliferative rates in the inner zones are capable of maintaining structural homeostasis (Race & Green, 1955). The aim of these experiments was to establish whether proliferative rates in the inner zones can explain the increases in zonal volumes noted at the reference point at 14 days. The 0 values in the reticularis suggest that a net gain of cells occurs over and above those cells produced by cell division; further, this influx would appear to be large. A sizeable cell loss occurs from the glomerulosa. It is concluded that the glomerulosa is feeding cells to the fasciculata; the fasciculata adds to these cells by cell division within its substance, and augments the migration of cells into the reticularis. It should be noted that, at 14 days, growth in the glomerulosa is exponential (see Fig. 1) and cell loss would not be expected to be a major flux parameter; nevertheless, even during this period such loss could be demonstrated. The measurements made of cell migration from the glomerulosa are not applicable if any degree of cell loss occurs in this zone. Wyllie and co-workers (Wyllie, Kerr, Macaskill & Currie, 1973; Wyllie, Kerr & Currie, 1973) have studied the distribution of cell loss by apoptosis in the pre-pubertal gland; apoptosis appears to be confined to the inner cortical cell layers, notably the reticularis and is not a feature in the
glomerulosa. The growth curves all seem to follow the Gompertz model quite closely. The doubling time for the whole cortex and the whole gland at 14 days is the same (see Table 1); this is probably because the contribution of the medulla at this time is insufficient to decrease the doubling time of the whole gland, bearing in mind the limitations of the technique. The apparent continuity between the separate readings for the fasciculata and the earlier combined readings (Fig. 1) might suggest that the earlier readings are applicable to the fasciculata only. However, after 12 days the reticularis splits only a small portion from the fasciculata, which would not greatly disturb the fasciculata curve at this time. In calculating growth rates from the weight measurements, two assumptions are incurred; firstly that equal densities of tissues are found throughout the gland, and secondly, that when converting growth rates in mg to growth rates in cells, that there are equal cellular densities as between the various zones. While the first of these may be reasonable, it is probable that cellular density is higher in the glomerulosa.
Fostnatal growth of the adrenal cortex
155 If this is so, then the growth rate in the glomerulosa would be underestimated when expressed as cells/cell/hour, compared with the other zones. The measurements of labelling index in bands across the cortex has confirmed that maximum proliferative activity resides in the outer zones. This method of analysing cortical labelling indices, though infinitely more laborious than straightforward zonal indices (see, for example, Reiter & Hoffman, 1967), is preferred, since greater insight into variation within zones is obtained. The cause of the decrease in labelling index with increasing distance from the capsule is debatable; Ford & Young (1963) reported that the cell cycle time lengthened in the inner zones, but Wright (1975) considered that the cell cycle time remained substantially constant and that it was the growth fraction which fell across the cortex. The results allow a tentative model of adrenocortical cytogenesis: Zona glomerulosa ... stem cell compartment Zona fasciculata
... dividing transit compartment
Zona reticularis
... dividing transit compartment
cell death The glomerulosa must act as a stem cell compartment for the rest of the cortex: if we accept a migration theory, then by definition glomerulosa cells have no input, and may be regarded as stem cells. The fasciculata is then a dividing transit compartment, receiving cells from the stem cell compartment, and subsequently feeding the reticularis. Cells then die in the reticularis, but this is also regarded as a dividing transit compartment, since there is cell proliferation and the transit is towards cell death (Wyllie et al. 1973). In conclusion, although it is evident that a measurable proliferative rate is present at all levels of the cortex, such an observation is not incompatible with cells migrating in a centripetal manner, and maintaining a decreasing degree of proliferative activity as they progress. The present results would appear to support a migration hypothesis of adrenocortical cytogenesis. SUMMARY
In the male rat the volumes of the several zones of the adrenal cortex were measured using a point-counting technique at various ages. The resulting growth curves were fitted by the Gompertz method, and the specific growth rate calculated for each zone at 14 days of age. Also at 14 days the flash labelling index was measured in contiguous bands each three cells in thickness across the cortex; maximum indices were found in the outer cortical layers. Calculations of the birth rate for each zona were compared with the growth rates. In the zona glomerulosa, the birth rate exceeded the growth rate, giving a cell migration rate of 4 cells/1000 cells/hour. In the zona reticularis there was a net cell gain of 5 cells/1000 cells/hour; in this zone, for every 100 cells born by cell division, nearly 2000 were added by cell migration.
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The results support the migration hypothesis of adrenocortical cytogenesis. REFERENCES AHERNE, W. A. (1967). Methods of counting discrete tissue components in microscopical sections. Journal of the Royal Microscopical Society 87, 493-508. BARTON, A. D. & LAIRD, A. K. (1969). Analysis of allometric and non-allometric differential growth. Growth 33, 1-16. DEANE, H. W., SHAW, J. H. & GREEP, R. 0. (1948). The effect of altered sodium or potassium intake on width and cytochemistry of the zona glomerulosa of the rat's adrenal cortex. Endocrinology 43, 133-153. FORD, J. K. & YOUNG, R. W. (1963). Cell proliferation and displacement in the adrenal cortex of young rats injected with tritiated thymidine. Anatomical Record 146, 125-153. GoTrsCHAU, M. (1883). Structur und embryonale Entwicklung des Nebennieren bei Saugethieren. Archiv fur Anatomie und Entwicklungsgeschichte 9, 412-458. RACE, C. J. & GREEN, R. F. (1955). Studies on zonation and regeneration of the adrenal cortex of the rat. Archives ofPathology 59, 578-586. REITER, R. J. & HOFFMAN, R. A. (1967). Adrenocortical cytogenesis in the adult male golden hamster. A radioautographic study using tritiated thymidine. Journal of Anatomy 101, 723-729. STEEL, G. G. (1968). Cell loss from experimental tumours. Cell and Tissue Kinetics 1, 193-207. WRIGHT, N. A. (1971). Cell proliferation in the prepubertal male rat adrenal cortex; an autoradiographic study. Journal of Endocrinology 49, 599-609. WRIGHT, N. A. (1975). Studies in the control of cell proliferation-in mammalian tissues. Ph.D. Thesis. University of Newcastle upon Tyne. WRIGHT, N. A., APPLETON, D. R. & MORLEY, A. R. (1974). Effect of dexamethasone on cell population kinetics in the adrenal cortex of the prepubertal male rat. Journal of Endocrinology 62, 527-536. WYLLIE, A. H., KERR, J. F. R. & CURRIE, A. R. (1973). Cell death in the normal neonatal adrenal cortex. Journal of Pathology 111, 255-261. WYLLIE, A. H., KERR, J. F. R., MACASKILL, I. A. M. & CURRIE, A. R. (1973). Adrenocortical cell deletion; the role of ACTH. Journal of Pathology 111, 85-94.
