ultrastructure of the endocrine cell types in the adenohypophysis of the teleost, poecilia latipinna...

Cell Tiss. Res. 167, 125-146 (1976) Cell and Tissue Research �9 by Springer-Verlag 1976

Ultrastructure of the Endocrine Cell Types in the Adenohypophysis of the Teleost, Poecilia/at/p/nna-a Morphometric Model

Michael Benjamin

Department of Cellular Biology and Histology, St. Mary's Hospital Medical School, Paddington, London, England

Summary. A morphometric model providing detailed quantitative information on the ultrastructure of the adenohypophysial endocrine cells has been developed for Poecilia latipinna. The model consists of various morphological components quantified in terms of volumes, surfaces or numbers. For prolactin and growth hormone cells, appropriate results are expressed relative to the average volume of that cell type. The difficulties of quantifying EM data on pituitary glands, together with the various sources of error to which the data are clearly open, have been discussed. Some practical applications of quantitative EM to problems in fish pituitary research are outlined.

Key words: Adenohypophysis - Teleosts (Poecilia latipinna) - Cell types - Ultrastructural morphometry.

Introduction

Fundamental to modern Cell Biology is the notion that most specialised cells contain the majority of organelles (Bloom and Fawcett, 1968; Weibel, 1972); cells differ quantitatively rather than qualitatively. Considering the vast number of organelle profiles visible in electron microscope (EM) sections, even within small areas of tissue (that may not represent the whole tissue), the frequent use of the subjective terms 'more' and 'less' in fine structural studies is a very dubious way of describing organelles. An objective and quantitative approach to electron microscopy is needed, such as that advocated by Weibel (1969). There have been relatively few such EM studies on pituitary glands. Weatherhead and Whur (1972) quantified the ultrastructural changes in the pars intermedia of Xenopus laevis when the animals were adapted to different backgrounds, while Thornton and Howe (1974) have presented a similar study on the pars

Send offprint requests to." Michael Benjamin, Department of Cellular Biology and Histology, St. Mary's Hospital Medical School, Paddington, London W.2., England.

I thank Dr. J.N. Ball for supplying the fish and Mr. L. Ethridge for technical assistance.

126 M. Benjamin

intermedia of the eel, Anguilla anguilla. In a recent series of papers, Kobayashi (1974a, b, c) quantified his EM studies on the mouse pars intermedia. Benjamin (1974a) used a morphometric approach in his study of the adenohypophysial cell types of the freshwater stickleback, Gasterosteus aculeatus form leiurus, and in subsequent papers specifically on the prolactin and ACTH cells of this fish (Benjamin, 1974b; Benjamin and Ireland, 1974).

One of the main purposes of the present investigation is to further develop quantitative EM techniques that are suitable for studying fish adenohypophyses. The chosen animal is the sailfin molly, PoeciIia latipinna, whose pituitary gland has recently been the subject of an extensive qualitative account (Batten et al., 1975). Both the account of Batten et al. (1975) and the study presented here, also form part of a wider, experimental scheme of study on the hypothalamo- neurohypophysial system in this fish.

Materials and Methods

Eight, adult, female mollies (Poecilia latipinna), 50-60 mm in length (weight ca. 2 g) were obtained from a dealer in Florida, U.S.A. and acclimatized for a month to standard conditions in the Sheffield laboratory as described previously (Batten et al., 1975). All fish had oocytes in late vitello- genesis (stage 4, Ball and Baker, 1969) and were the same animals as those on which the qualitative account of GtH cells was based (Batten et al., 1975).

Electron Microscopy. All fish were sacrificed between 1,400 and 1,500 h, by decapitation. The pituitaries were rapidly dissected out under fixative (Karnovsky's, 1965; glutaraldehyde in 0.2 M cacodylate buffer) and fixed for 2 h at 5 ~ C and pH 7.2. After a brief rinse in cacodylate buffer, the material was post-fixed in Palade's osmium tetroxide for 1 h at 5 ~ C, rinsed several times in distilled water and then block stained in 5% aqueous uranyl acetate for l h. The material was dehydrated in graded alcohols and embedded in TAAB resin. Blocks were sectioned on a Cambridge Huxley microtome using glass knives. For identifying cell types, adjacent thick (1.0 ~tm) and thin (silver interference colour) sections were cut. The thick sections were stained with a hot, alkaline solution of 0.5% azur II in 1% borax and compared with the thin sections used for EM that were stained in lead citrate (Reynolds, 1963) and uranyl acetate. The thin sections were mounted on 100-mesh, collodion-coated, copper grids, and examined on a Miles M R 60C microscope that was calibrated at each recording sessions with a line grating (2,160 lines/mm).

f

Nuclei

Mitochondria

RER

Golgi apparatus

Cytoplasmic ground substance

Mature secretory granules

t Nuclei RER

Mitochondria

Dense bodies

Multivesicular bodies

Acanthosornes

Free ribosomes

Fig. 1. Morphometric model of an adenohypophysial endocrine cell

Morphometry of Poecilia Pituitary 127

Table 1. Cell components selected for point-counting volumetry in order to provide additional data for the 'reconstruction drawings' (mean values not quoted in this paper)

1. Parallel arrays of RER around the nucleus 2. Small isolated pieces of RER without dilated cavities 3. Dilated pieces of RER without dense contents 4. Dilated pieces of RER with dense contents 5. Parallel arrays of RER in the main cell 'body' 6. Parallel arrays of RER next to the cell membrane 7. Curvilinear whorls of RER 8. Small Golgi vesicles 9. Large dilated Golgi saccules

10. Flattened Golgi cisternae 11. Multivesicular bodies 12. Dense bodies 13. Free ribosomes 14. Translucent vesicles 15. Cytoplasmic ground substance excluding free ribosomes

Morphometric Model. For the purposes of the morphometric analysis, only the seven endocrine cell types described by Batten et al. (1975) were considered. The analysis did not include the stellate cells that occur throughout the pars distalis, or the so called 'Z' cells of the proximal pars distalis (Batten et al., 1975).

The cell components chosen for analysis are shown in Fig. 1. Volume estimations of rough endoplasmic reticulum (RER) included the cisternae, its bounding membranes and their associated ribosomes. Nuclear volume estimates included the nuclear envelope and its ribosomes. The 'Golgi apparatus' included flattened cisternae, dilated vacuoles and associated vesicles-their membranes and their cavities. The term 'mature secretory granule' excludes those small secretory granules that are restricted to the Golgi area and have a very large halo between the central, dense material and the boundary membrane. The 'cytoplasmic ground substance' included non membrane-bound structures such as ribosomes and microtubules. The volumes of the various components shown in Table 1 were also estimated in order to provide the necessary additional raw data for the 'reconstruction drawings' (see below). These additional volumes are not quoted in numerical form in this paper.

A variety of reference systems have been included in the model. For every cell type, the volumes of the various components are expressed as percentages of the total cell and the total cytoplasmic volume. The numbers of the various organelle profiles (Fig. 1) were expressed as the numbers per unit area of cytoplasm. The nuclear surface is expressed as a volume-to-surface ratio (v/s ratio) and membranes of the RER as lam 2 of membrane per Ixm 3 of cytoplasm. But for prolactin and STH cells, the various volume and number estimates together with the RER surface estimate are expressed in relation to an 'average cell volume'.

Sampling. For any one cell type, 4-6 pituitaries were used, and 30 electron micrographs were taken at an initial, nominal magnification of x 7,500. The final, nominal magnification of the printed micrographs was x 15,000. Calibration of the EM showed that the initial magnification was x 6,170. The final magnification of the printed micrographs was thus x 12,340. Possible errors introduced by differential shrinkage of the photographic paper (Weibel, 1969) were found to be negligible in the present study. For practical reasons simple random sampling of the electron micrographs was adopted throughout, rather than the admittedly preferable method of systematic random sampling advocated by Ebbesson and Tang (1967) and Weibel (1969). Simple random sampling was achieved by defocussing the microscope and then moving the specimen grid an arbitrary distance. It was only after the new position was established that refocussing was permitted (Elias et al., 1971).

128 M. Benjamin

Fig. 2. Typical electron micrograph characterising a sample field of GtH cells. The test system is superimposed on the micrograph. Volumetric points falling onto mitochondria are circled. Magn. x 12,340

Morphometric Procedures. The relative volume of the cells occupied by the various cell components was determined by the point-counting method of Weibel (1969), the v/s estimates on the nuclei were determined by combined point and intersection counts (Chalkley et al., 1949), and the RER membrane profile concentrations were determined by the method of Loud et al. (1965).


At light microscope level, nuclear volume was determined for prolactin and STH cells only. The major (a) and minor (b) axes of nuclear profiles in 1.0 lam TAAB sections were measured under a 10 • magnifier equipped with a centimetre scale showing 0.1 mm subdivisions. Each nuclear profile was then considered to be a circle whose diameter was (a +b)/2. An estimate of mean nuclear diameter was obtained from measurements on 150 profiles per gland, using the Giger and Riedwyl (1970) transformation. Mean nuclear volume was then assumed to be that of a sphere of this diameter. From the EM estimates of nucleus: cytoplasm volume ratios, it was possible indirectly to measure prolactin and STH cell volumes. Thus for these two cells, the point-counting data were also expressed as 'absolute volumes' on a 'per cell' basis.

The numbers of certain organelle profiles per unit area of cell cytoplasm were also determined (Fig. 1). Because of their small size and high profile numerical density, ribosomes were enumerated in a slightly different manner from the remaining organeUes. The method used is essentially that of Mayhew and Williams (1974). A number of circles were impressed onto PVC sheeting using "Letraset" instant lettering. The diameter of the circles used (7.5 mm) was equivalent to a diameter of 608 nm and an area of 0.29 lam z on the printed micrograph. These circles were superimposed systematically on to micrographs in areas of the cytoplasm free of the other cytoplasmic components. Free ribosomes were counted in five such areas for each micrograph and for each cell type. Both single ribosomes and those occurring as polysomes were counted.

In no instance was profile numerical density translated into the actual number of organelles per unit volume of cytoplasm, since the present purpose of quantifying EM information was merely to reduce the subjective element in describing cell types and not to relate morphologic to biochemical or physiological data.

The chosen test system for point-counting volumetry, and for estimates of membrane profile concentrations of RER, consisted of a quadratic lattice of lines, the cross-points of which served as markers for point-counting, and the lines for intersection counts (Fig. 2). The distance between the lines on the printed micrograph was 0.8 ~tm, each point on the test system representing the centre of an area equivalent to 0.5 rtm z.

The series of'reconstruction drawings' are an attempt to visualise the quantitative data presented in numerical form. Details of the method of constructing such drawings have been described in a previous paper (Benjamin, 1974a). The secretory granules were drawn to scale, the raw data being provided by the histograms of profile diameters given previously by Batten et al. (1975), while the shape of the nucleus was based on the point and intersection counts described in the present paper.

Statistics. The statistical methods used have been documented in a previous paper (Benjamin, 1974a). However in the present case there is no need for an arc-sine angular transformation of the profile numerical density data, the nuclear v/s ratios or the RER membrane profile concentrations before assessing significant differences.

Results

A de ta i l ed q u a l i t a t i v e d e s c r i p t i o n o f the a d e n o h y p o p h y s i a l cell types in P. latipinna, i l lus t r a t ed by n u m e r o u s e l e c t r o n m i c r o g r a p h s has a l r e a d y b e e n p r e s e n t e d

(Ba t t en et al., 1975). T h u s in the c u r r e n t p a p e r a t t e n t i o n is d i r ec t ed en t i r e ly

to p r e s e n t i n g p u r e l y m o r p h o m e t r i c d a t a o n the seven e n d o c r i n e cell types o f

the a d e n o h y p o p h y s i s .

Volume Estimations. T h e resul t s o f the p o i n t - c o u n t i n g v o l u m e t r y a re s u m m a r - ised in T a b l e s 2-3 . F ig . 3 r ep r e sen t s the v o l u m e t r i c c o m p o s i t i o n o f p r o l a c t i n

a n d g r o w t h h o r m o n e cel ls e x p r e s s e d in r e l a t i o n to t he e s t i m a t e d t o t a l cell v o l u m e s . I t is p a r t i c u l a r l y n o t e w o r t h y t h a t a l t h o u g h g r o w t h h o r m o n e cel ls c o n t a i n a g r ea t e r r e l a t ive v o l u m e o f s ec re to ry g r a n u l e s ( T a b l e 2), the l a rge r p r o l a c t i n cell

v o l u m e m e a n s t h a t in a b s o l u t e t e r m s (i.e., o n a ' p e r c e l l ' basis) , it is t he p r o l a c t i n cel l t h a t has the g r e a t e r s ec re to ry g r a n u l e v o l u m e . W h e n the R E R m e m b r a n e

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Table 2.

Percentage

of the total cell volume occupied by various cell components

(means ~

s.e.).

Cell

types whose mean values did not differ significantly

(P <

0.05%)

are underlined.

Non-adjacent

groups are joined

by dotted lines

PRL

ACTH

GtH

TSH

PI i

PI 2

Nucleus

19.9 ~

2.08

39.7 ~

2.43

15.7 !

1.46

36.0 Z

2.82

Golgi

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secretory

qranules

Mitochondria

RER

Cytoplasmic

ground

substance

2.1!

0.39

30.1!1.80

2.2!0.29

3.7~0.39

40.5+1.58_

2.4~0.52

7.4 !

0.92

4.0L

~.59

6.8+0.65-

40.3+2.03-

GH

17.9 -+ 2.00

2.1

+ 0.

53

36.7 +- 1.79

2.4 +

0.28

4.7 1

0.46

35.4

+ 1.30

4.8t0.!~

18.0

+1.

27-

3.11

~.&

3_

23.2

!2.2

33.4 - + 1.17

2.0 - +

0.34

15.8

- +

1.69

3.7 +

0.37

8.7 - +

1.1

1

33.9

~

1.38

18.1

+

2.11

1.9 -+ 0.47

36.2

-+

1.

82

2.3 +

0.62

5.0

t 0.59

+ 36.3 -

1.72

22.8 - + 2.03

3.2

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19.5

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- 1.

03

+

3.0 -

0.30

8.6

! o.58

+ 41.6 -

1.21

Table 3.

Percentage

of the total cytoplasmic

volume occupied by various cell components

(means !

s.e.).

Cell types whose mean values did not differ significantly

(P <

0.05%)

are underlined.

Non-

adjacent groups are joined by dotted lines.

Golgi

apparatus

Mature

secretory

oran~l@s

Mitochondria

RER

Cytoplasmic

ground

~ub~t&ncc

PRL

ACTH

GH

GtH

TSH

PI 1

PI 2

2.5 -+ 0.46

38.1!1.74

3.0~0.40

4.6~0.52

50.2~1.49

3.9 +

0.83

13

.oL

1.1

6.1~0.72

+ _10.6 -

1.11

65.3+1.58-

2.4!

0.59

42.9!2.07

3.1~0.38

5.8+0.59_

43.3 +

1.40

_ 5

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t o.

&5_

20.9

-+ 1.50

3.7~0.39

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39.8 +

1.41

3.7+0.63_

22.3~2.27

7.2~1.37

13.8

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82

50.5 +

2.43

2.0!0.46

_44.0+1.88-

2.8 +

0.73

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0.62

25.0 -+ 0.92

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O = a


Fig. 3. Bar diagrams of prolactin (PRL) and growth hormone cells (GH) relating the composition of each cell type to its total cell volume. [] nucleus; [] Golgi apparatus; [] mature secretory granules; �9 mitochondria; [] RER; [] cytoplasmic ground substance

5OO

450

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350

300

2:50

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150

100

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Any variation in the percentage volume of a cytoplasmic component will also reflect any variation shown by another component, especially one as large as the nucleus. Thus expressing the point-counting data on the various cytoplasmic components as percentages of the total cell (Table 2) rather than the total cytoplasmic volume (Table 3), means that the relative magnitude of the standard errors will vary accordingly. Fig. 12 shows the standard error expressed as a percentage of the mean value for mature secretory granules when this organelle is expressed both ways. In addition the significance of differences between the means can depend on which method is chosen for expressing the percentage volumes. For example, comparing the information for mature secretory granules in Tables 2 and 3, shows that when the volume estimates are expressed as percentages of the whole cell, there is a significant difference between prolactin and growth hormone cells. However when the same point-counting data are expressed in relation to the total cytoplasmic volume, there is no significant difference between these 2 cells with regard to their mature secretory granules.

Number Estimations. The profile numerical densities of various organelles expressed per unit area of cytoplasm are shown in Table 4. Essentially the

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organelles chosen for analysis are either ones that are easy to count, or ones that cannot be accurately determined by point-counting volumetry under the somewhat limiting conditions of the present 'single-stage analysis '

It should be noted that the data given in Table 4 represent estimated numbers of organelle profiles within a given area, and are not to be confused with the numbers of organelles within a given vo lume


0.4

o 0"3

~ 0"2 !- ~ 0-1

PRL

I I ACTH GH GtH TSH PI 1 PI

Fig. 11. Nuclear volume: surface ratios of the endocrine cell types. Standard errors are indicated. Cell types where mean values did not differ significantly (P< 0.05%) are underlined

12

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PRL ACTH GH GtH TSH PI 1 PI 2

Fig. 12. The standard error expressed as a percentage of the mean value when the volume of mature secretory granules is considered relative to the total cytoplasmic volume ([]) and the total cell volume (Fl)

Fig. 13. The membrane profile concentrations of RER. Cell types where mean values did not differ significantly (P< 0.05%) are underlined. Non-adjacent groups are joined by dotted lines

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140 M. Benjamin

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Surface Estimations. The mean nuclear v/s ratios for the various cell types together with their standard errors are shown in Fig. 11. Volume-to-surface ratios are depicted in the 'reconstruction drawings' (Figs. 4-10) by the degree of departure of the nucleus from a theoretically round body. In studying the 'reconstruction drawings' it is important to bear in mind that their primary purpose as far as the nucleus is concerned, is to reflect its v/s ratio and not to represent accurately the shape of the nucleus as it would appear in an 'average' cell. To combine both purposes in a single drawing is impossible if the various cell components are to be represented within a single outline, the area of which is constant for the different cell types. A useful comparison


of shape coefficients could be obtained by comparing the nuclear v/s ratio with that of a sphere of equal volume, as demonstrated by Cope and Williams (1973).

The membrane profile concentrations of RER in the different cell types are shown in Fig. 13.

Discussion

The present paper presents a quantitative approach to the basic ultrastructure of the adenohypophysial endocrine cells in P. latipinna, based broadly along the lines of a recent study on Gasterosteus aculeatus form leiurus (Benjamin, 1974a). It furnishes base-line data on the cell types from fish in a defined physiological state. This data could be used for comparing cell types in fish subjected to a variety of experimental conditions or examined in different physiological states. In addition, the data can be used to compare functionally similar cell types in different fish.

The heterogenous array of cell types in a pituitary gland makes it a difficult subject for quantitative EM. It would be as well to state these difficulties and the various sources of error to which the quantitative data are clearly open. Some of the errors have been mentioned previously in ' Materials and Methods'.

A most pressing difficulty concerns the need to distinguish between the absolute amounts of organelles in a cell and the amounts of these organelles expressed relative to each other (i.e., as percentage values). In describing an organelle in one cell type as being 'more numerous' than in another, the electron microscopist may sometimes forget whether (s)he is referring to the 'absolute' or the 'relative' amounts of that organelle. This distinction becomes important when comparing cells of different sizes (Benjamin, 1974 a). To compare the'absolute' values for an organelle in different cells, one must measure their cell volumes. This is precisely where the difficulty lies in pituitary morphometry. In a previous paper on the pituitary of G. aculeatus form leiurus (Benjamin, 1974a) a compromise solution was proposed, based on comparing the relative sizes of the various cell types as seen in 8 lain paraffin wax sections, stained by special pituitary techniques. The relative sizes of the cell profiles were assumed to be in direct proportion to their relative volumes. There are several criticisms against this method. Firstly, it still does not provide data specifically on the absolute amounts of organelles, it merely allows the reader to compare the ratios of these absolute amounts. Secondly cell outlines are difficult to determine accurately and the clarity of these outlines varies according to the staining affinities of each cell. Finally, cell volumes were measured on paraffin wax sections after Bouin fixation and related to EM data on glutaraldehyde-osmium tetroxide fixed material.

The method adopted in the present paper is to measure nuclear profile diameters (as nuclear outlines are easier to determine than cell outlines) and then to calculate the nuclear volume from a sufficiently large sample, assuming each nucleus to be approximately spherical. By relating nuclear volume estimates to nuclear-cytoplasmic ratios (determined at EM level), it is possible to measure

142 M. Benjamin

absolute cell volumes and not just the ratio of cell volumes to each other. Estimating nuclear volumes from profile diameters using the Giger and Riedwyl (1970) transformation, assumes a negligible section thickness compared with the nuclear diameter. Consequently such estimates cannot be made on 8 ~tm or even 5 ~tm paraffin wax sections. 0.5-1.0 ~tm epoxy resin sections are better. At least this means that the volumes are estimated on cells fixed by the same method as used for EM, but it does raise the difficulty of unambiguously identifying lots of cells in 'thick' epoxy resin sections, where using rather specialised pituitary staining techniques may be difficult for many research workers. Also, in comparative studies, the assumption that adenohypophysial nuclei are spherical, may give significant errors if the various nuclei differ in shape.

There are possibly other ways of determining adenohypophyseal cell volumes that may be suitable for particular projects, depending on the accuracy required and the cell type(s) investigated. If very thick (15-20 tam) paraffin wax sections are cut, it may be presumed that the largest profile diameters are equivalent to the actual diameters of the unsectioned nuclei. This removes any need for a Giger and Riedwyl (1970) transformation and allows special pituitary staining techniques to be used. Difficulties will obviously arise if there are several different size classes, making it impossible to determine what are the 'largest diameters'. Again, light microscopy fixatives and embedding media must be used.

The whole procedure of estimating cell from nuclear volume could be done at EM level by photographing sufficient nuclear profiles, measuring their areas and then calculating the diameters of circles of equivalent area. Again, after a Giger and Riedwyl (1970) transformation, nuclear volume could be calculated, assuming approximately spherical nuclei. In this method there would presumably be no difficulty in identifying the cells, the negligible section thickness would suit a Giger and Riedwyl (1970) transformation, but the time and cost of taking a great number of photographs may prove prohibitive.

Another pressing difficulty of which the author is acutely aware, is how to select cells for analysis. Ideally one should use a method of systematic random sampling such as that advocated by Ebbeson and Tang (1967) and Weibel (1969), rather than the simple random sampling method used in the present study. However, in an organ as small as a fish pituitary, the complexity of different cell types means that any one cell type is relatively rare (at least compared with cells from organs particularly favoured for stereological studies e.g., liver). Systematic random sampling of all cells in a fish pituitary gland would mean viewing an unyieldy number of EM sections before a representative number of cells could be examined. The only easy exception would be with prolactin cells, as these are localised in one large mass in the rostral pars distalis and are sufficiently numerous to make systematic random sampling quite fea- sible. Practical difficulties have also led other authors to compromise in their sampling technique. In a study of mouse peritoneal macrophages, Williams and Mayhew (1973) biased their cell selection towards 'equatorial profiles' so that they could be certain of correctly identifying their cells. Hecker et al. (1974) were aware of the need to use randomly orientated sections in their stereological studies, but were unable to fulfill this condition.

There is also the problem of defining an appropriate sample unit for statistical


analysis. This difficulty has also been recognised by Weibel (1969). Should the morphometric parameters be calculated for every microscopic field, with these estimates averaged and analysed statistically? Or should statistics be per- formed on morphometric data using one animal rather than one micrograph as the statistical unit? As suggested by Weibel (1969), there is no answer to this question at the moment. In the present paper the micrograph has been adopted as the unit, but in retrospect using the animal as the unit would have reduced the number of calculations needed for acceptable morphometric data, and also indicated variation among individuals.

The volume estimations of RER and Golgi are open to error; they are probably underestimates because of apparent 'membrane loss' in tangential sections (Loud, 1967). However, for comparative purposes the figures are still perfectly reliable. Again the small size of ribosomes makes their number difficult to estimate in thin sections. But after all, as Mayhew and Williams (1974) point out, such errors are inevitable consequences of trying to extrapolate a three-dimensional object from a two-dimensional image.

All the quantitative information presented in this paper necessarily refers to 'average'.cells. The 'reconstruction drawings' are attempts to visualise these average cells. As pointed out by Mayhew and Williams (1974) average cell data, whilst useful, indeed necessary for statistically comparing different popula- tions, disguise to some extent the inherent heterogeneities of the cells. In connec- tion with fish pituitary glands, Schreibman et al. (1973) wisely comment that "too often in descriptions of prolactin cells, there is a morphological uniformity implied ~hat may be misleading...". Their comments could justifiably be extended to other cell types. However it should be remembered that in the present morphometric study, the standard errors of mean values reflect this heterogeneity wherever it is present. For instance the qualitative observations of Batten e ta l . (1975) that TSH cells are extremely variable and GH cells relatively constant in their morphology, are reinforced by comparing the standard errors of the organelles within these cells with similar values for the other adenohypophyseal cells.

Despite these difficulties ultrastructural morphometry is potentially a power- ful tool in fish pituitary research. It may thus be useful to outline briefly some possible applications. It would seem an ideal way of directly relating morphological to physiological data. In particular, changes in secretory granule content could be correlated with changes in pituitary hormone levels. The granule content (expressed as 'volumes' or 'numbers') can be easily monitored by quantitative microscopy, while hormone levels could be estimated by polyacrylamide gel electrophoresis or radioimmunoassay. Such an approach would help consid- erably in 'bridging the gap' between morphology and physiology.

Quantitative EM, in allowing a meticulous study of the differential composition o f a cell type, also detects minute quantitative changes. It should thus be possible to study the movements of parts of the cell's membrane-network during a secretory cycle. Such an approach may well provide new insights into the role of membranes in the synthesis, intracellular transport and release of secretory proteins within fish pituitaries. The prolactin cell would seem to be most amenable to morphometry because of its localisation in one part of

144 M. Benjamin

the pituitary. In this respect fish offer considerable advantages over mammals, where there is no discrete prolactin cell zone.

Olivereau (1966) has strongly emphasised the need to combine qualitative and quantitative data when studying teleost endocrine glands. Her remarks mainly refer to light microscopy and histochemical studies, but it is interesting to note that she also appreciated the potential value of applying quantitative EM techniques to pituitary studies. She pointed out that at the time she was writing, quantifying EM data had been limited to measuring the dimensions of secretory granules. Pooley (1971), Benjamin (1974a), Batten et al. (1975) and Olivereau (1966) have all remarked that granule size does not always give a certain means of identifying a cell type. Other features of a cell must be taken into account. Kaul and Vollrath (1974), although not quantifying other cell components besides secretory granules in their study of the goldfish (C. auratus) pituitary, have at least made an interesting attempt to quantify the electron density of the secretory granules from the various cell types, as well as their profile sizes.

Boddingius (1975) offers the general criticism against pituitary morphologists that most authors pay little attention to the exact distribution of cells throughout the adenohypophysis. He points out that although electron microscopy has contributed much to our knowledge of adenohypophysial cell types, conventional light microscopical studies have often been abandoned. Consequently, certain morphological studies for which light microscopy is essential, easily escape attention at present. It should be an easy matter to devise quantitative methods that would adequately characterise the distribution of cells at LM level. Several authors have previously attempted to measure the number of particular cells in a pituitary gland (e.g., Montemurro, 1964; Yoshimura and Ishikawa, 1967; Kathuria, 1972). Obviously the greater the variety of quantitative methods used the better.

It is interesting to compare the quantitative data on Poecilia with the data previously presented for the adult, winter stickleback (Benjamin, 1974a). Some of the detailed similarities are remarkable. In both fish the ACTH and TSH cells have a large percentage of their total cell volume occupied by nuclei- although this is more clear-cut in Poecilia than in Gasterosteus. TSH cells contain a greater 'relative' volume of mitochondria than the other PPD cell types in both fish, and it is the ACTH cells that are the mitochondrial-rich cells in the RPD. However in each cell type except GtH cells, there is a lower percentage of the total cell volume occupied by mitochondria in Poecilia than in the compa- rable cells of Gasterosteus. In both animals the GtH cells have the greatest relative volume of RER; and the prolactin, growth hormone and PI 1 cells contain the greatest relative volume of secretory granules. In addition, a high percentage of the cell is occupied by 'cytoplasmic ground substance' in ACTH and PI 2 cells in either animal. These two sets of figures are not directly compara- ble though, for in Poecilia the term 'cytoplasmic ground substance' includes non-membrane-bound organelles such as free ribosomes and microtubules.

There are significant differences between the prolactin cells in Poecilia and Gasterosteus. These may be in part accounted for, by the ambient salinity to which the animals were adapted. Poecilia were kept in 1/3 SW, while Gasterosteus


were killed immediately after collection from FW. Hence the larger relative volume of the prolactin cell occupied by Golgi apparatus, RER and mitochondria in Gasterosteus compared with Poecilia, together with the increased relative volume of secretory granules in Poecilia, may largely reflect the decreased syn- thetic activity of the prolactin cells in Poecilia. However it must be remembered that the two species may well differ from each other with regard to prolactin involvement in osmoregulation. Hence there is no reason to expect a close agreement in ultrastructure between these two species even if they were kept in waters of similar salinity.

References

Ball, J.N., Baker, B.I. : The pituitary gland: anatomy and histophysiology. In: Fish physiology (Hoar, W.S. and Randall, D.J., eds.), vol. II, p. 1-110. New York: Academic Press 1969

Batten, T., Benjamin, M., Ball, J.N. : Ultrastructure of the adenohypohysis in the teleost Poecilia latipinna. Cell Tiss. Res. 161,239-261 (1975)

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Pooley, A.S.: Ultrastructure and size of rat anterior pituitary secretory granules. Endocrinology 88, 400~11 (1971)

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Weibel, E.R., Bolender, R.P.: Stereological techniques for electron microscopic morphometry. In: Principles and techniques of electron microscopy (ed. by Hayat, M.A.), vol. 3, p. 239-296. New York-Cincinnati-Toronto-London-Melbourne: van Nostrand Reinhold Company 1973

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Received November 29, 1975

ultrastructure of the endocrine cell types in the adenohypophysis of the teleost, poecilia latipinna...

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