Cell Tiss. Res. 152, 93--102 (1974) �9 by Springer-Verlag 1974

Seasonal Changes in the Prolactin Cell of the Pituitary Gland

of the Freshwater Stickleback, Gasterosteus aculeatus, form leiurus*

Michael Ben jamin

Department of Zoology, University College of Wales, Aberystwyth, Wales

Received March 29, 1974

Summary. The relative volume of the RPD region of the pituitary gland of Gasterosteus aculeatus form leiurus was greater in animals collected in the spring than in winter. A morpho- metric analysis of the prolactin cells from spring animals showed only slight changes in cell ultrastrueture compared with winter animals. However, in spring the prolactin cells apparently formed and released more secretory granules. Three distinct sites for the release of secretory granules are described. A preliminary study of the response of prolactin cells from spring animals to 70 % sea water did not reveal any large scale changes in cell ultrastructure. The significance of this finding is discussed. I t is concluded that although the freshwater stickle- back cannot be regarded as "physiologically hypophysectomised:" with regard to prolactin secretion in the winter, it still shows a seasonal change in prolactin cell ultrastructure.

Key words: Prolactin cell - - Freshwater stickleback - - Seasonal changes - - Seawater adaptation - - Morphometry.

Introduction

There is now considerable evidence t h a t the p i t u i t a r y g land of several te leost fishes is concerned wi th osmoregula t ion (Ball, 1969 a, b; Olivereau and Ball, 1970; Lam, 1972). Two of the hormones conce rned - -p ro l ac t i n and A C T H - - a r e secreted b y cells in the R P D (rostral pars distalis) , bu t one or more hormones secreted b y cells in the pars in t e rmed ia also seem impor t an t .

I f m ig ra to ry s t icklebacks (Gasterosteus aculeatus form trachurus) t h a t no rma l ly overwinter in the sea are to be kep t alive in freshwater , t hey mus t be given pro- lac t in in ject ions (Lam, 1968, 1969a, b; Lam, and Hoar , 1967; L a m and Leather - land, 1969a, b, 1970; Lea the r l and and Lam, 1969a, b). I t seems t h a t these winter s t icklebacks cannot synthesise or release prolact in , and have thus been regarded as "phys io logica l ly hypophysec tomized" . This has been confirmed b y Lea the r l and (1970) in an u l t r a s t ruc tu r a l s t u d y of the pro lac t in cells of mig ra to ry s t icklebacks a t dif ferent t imes of the year .

I n a morphomet r i c s t u d y of the p i t u i t a r y cell t ypes in the f reshwater st ickle- back, Gasterosteus aculeatus form leiurus (previous paper) i t was found t h a t the pro lac t in cells of this an imal bo th formed and released secre tory granules in the winter . The present inves t iga t ion is an a t t e m p t to ex tend these ini t ia l observa t ions b y examining the pro lac t in cell u l t r a s t ruc tu re of an imals collected in the spring.

* This work formed part of a thesis submitted for the degree of Doctor of Philosophy in 1973 and for which the author was in receipt of an S.R.C. studentship. My thanks are due to Dr. M.P. Ireland for his support and supervision throughout the course of the work.

94 M. Benjamin

I n addition, by serial sectioning of the pi tu i tary glands of animals collected at four different times of the year, information on the changes in the relative volume of the R P D has been obtained.

Materials and Methods

Most of the methods used have been described in detail in the previous paper. However in the morphometric study, only the relative volumes of the organelles were estimated and not their absolute volumes. For light microscopy the brains with pituitaries attached were dis- sected out and fixed in Bouin's fluid, dehydrated and embedded in 52~ paraffin wax. I t was found in preliminary investigations that 52~ paraffin wax caused less shrinkage of the material than 58~ wax. 5-7 pituitaries of fish collected at each time of year were serially sectioned at 8 [zm and stained with Alcian blue-PAS-orange G. Every fifth section was traced on high quality paper from the screen of a projection microscope. These paper outlines of the pituitary glands were weighed. The RPD region was then cut out and weighed. Consequently it was possible to estimate the relative volumes of the RPD and the whole pituitary gland at different times of the year.

Results

Light Microscopy There was a slight increase in the relative volume of the P R D in spring animals

compared with this volume in winter animals of the same length (Fig. 1). However the prolactin cell size remained approximate ly the same (mean area in spring

30.2 ~= 3.7 btm 2 ; mean area in winter : 28.0 ~= 1.9 btm2). There was no difference in the staining properties of the prolactin cells at any t ime of the year.

Electron Microscopy The Prolactin Cell in Spring. Table 1 summarises the information yielded by

the morphometr ic s tudy on the relative volume of the prolactin cell occupied by various organelles. Fig. 3 is a reconstruction drawing of this cell, based on the above information, the surface-area: volume ratio of the nucleus (0.55 • 0.11) and the secretory granule profile diameters (Fig. 2). The structure of this prolactin cell was therefore similar in m a n y respects to the prolaetin cell of the winter stickleback described in the previous paper. However there were a larger number of immature secretory granules, an increase in the amount of R E R tha t had dilated cavities, and a decrease in the number of free ribosomes and the amoun t of perinuclear R E R . The volume of the cell occupied by the GoJgi appara tus and also the number of mature secretory granules were not significantly different f rom the values found in winter animals described in the previous paper.

As well as the increased number of immature secretory granules in the Golgi apparatus, there were considerably more exocytosed secretory granules, a l though this aspect was not analysed quanti tat ively. There was no obvious budding of the prolactin cell, and indeed the only form of granule release was when secretory granules were exocytosed. There were 3 distinct sites for the release of secretory granules, both in winter and spring animals.

1. In to the intercellular space next to chromophobes (Fig. 4). 2. At the periphery of the pi tu i tary gland in the prolactin cell region was a

wide intercellular space usually adjacent to a superficial capillary network (Fig. 5). Secretory granules were often released into this area.

Seasonal Changes in Stickleback Prolactin Cell

36

95

,,z

APR JUL AUG DEC

Fig. t. Percentage of the total pituitary volume occupied by the RPD at different times of the year

40 t

u c-

o"

20

g 50 150 250 350

D i a m . ( n m ) Fig. 2. Frequency distribution of the secretory granule profile diameters in the prolactin cell

of sticklebacks collected in the spring

3. I n t o the pseudofoll icle areas. These are areas t h a t p r e sumab ly corresponded to the noncel lular , PAS-pos i t ive areas seen a t LM level (Fig. 6). A t EM level, the pseudofoll icles con ta ined masses of e lectron-dense ma te r i a l in the in te rce l lu la r spaces. Acan thosomes were p rominen t in the pseudofoll icles (Fig. 7).

A t the sites of release, the cell membrane f requen t ly had a fuzz coat (Fig. 8). There were acan thosomes (coated vesicles) in bo th the areas of fo rma t ion and the areas of release of the sec re to ry granules.

96 M. Benjamin

Table 1. Percentage of the prolactin cell volume occupied by various organelles (means •

Nucleus 20.8 4-2.07

Nueleolus 0.i5 4-0.08

Golgi (a) small vesicles 7.4 4-0.96 (b) large dilated vacuoles 1.7 4-0.39 (c) flattened cisternae 0.13 4- 0.06 (d) total Golgi 9.2 4- 1.08

Immature secretory granules 0.80 •

Acanthosomes 0.12 4-0.03

Multivesicular bodies 0.09 4-0.03

Dense bodies 0.24 4-0.07

RER membrane profiles (a) isolated small pieces without dilated cavities 6.7 4-1.2 (b) isolated small pieces with dilated cavities 3.8 • 1.17 (c) perinuclear arrays 3.4 4-0.56 (d) eurvilinear whorls 0.00 4-0.00 (e) total RER 14.2•

Mature secretory granules 14.6 4-1.05

Mitoehondria 4.0 •

Free ribosomes 13.3 4-0.92

Cytoplasmic ground substance 22.4 4-0.98

Significant differences between these values and those of winter animals (previous paper) are underlined (p > 0.05)

Response to 70% Seawater. Sticklebacks collected in the spring, acclimatised in the laboratory to freshwater, and then kept in 70 % seawater for 1 week had well granulated prolactin ceils of the same size as cells in control animals kept in freshwater for 1 week. Similarly the volumetric percentage of the R P D to the total p i tu i tary (27.2 • 1.9%) was nearly the same as tha t f rom animals kept in freshwater for 1 week (28.9 • 1.6%).

At EM level both the sticklebacks kept in freshwater and those kept in 70 % seawater showed abundant signs of secretory granule format ion and release.

Discussion

The increase in the relative volume of the R P D in spring fish compared with winter fish, and the ul t rastructural differences between the prolactin ceils at these times of year would indicate tha t prolactin ceils f rom spring fish are slightly more active than ceils f rom winter fish. Thus, a l though the pi tu i tary gland of the fresh- water stickleback cannot be considered as "physiologically hypophysec tomised" in the winter (previous paper) unlike its migra tory relative (Leatherland, 1970), there is still a seasonal change in the ul trastructure of its prolactin cell. Although

Seasonal Changes i~ Stickleback Prolactin Cell 97

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:. @ ~5 0o., ,,:,o:::, %++++o @ : i : + + :... . . . . ,

%0~ ,,o oo

0o, i I oo: ~~ o++, , , ~

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A reconstruction do+wing of a t+++t+ceiu cell from +n znimml collecte4 +a the spring

Lea the r l and (1970) d id not provide quan t i t a t i ve in format ion on the re la t ive volume of the R P D a t different t imes of the year , he never theless descr ibed a large increase in the ex ten t of this region in spr ing fish. He also descr ibed a s ignif icant increase in th~ ~ize Q[ the p~ak~+ctin ve~s ~n ~}~ swing , The re ~ a s ~9 such ~et~ siL~ increase in f reshwater ~ticklebacks. W h e n eva lua t ing the significance of this seasonal change in size of m ig ra to ry s t ick leback pro lac t in cells, i t mus t be remem- bered t h a t Lea thc r ]a~d ' s (1970) "cell i ndex" is a r a the r suspect measurement of cell si2e. The cell volume cannot be de te rmined by megsuring the m a x i m u m cell

7 C~11 T ~ s l~es . 1 5 2

98 M. Benjamin

Fig. 4. Exocytosis of a secretory granule (-~) from a prolactin cell (pro) into the intercellular space next to a chromophobe (chr). x 32000

Fig. 5. The wide intercellular space (is) at the periphery of the pituitary gland in the pro- lactin cell region (pro). A red blood corpuscle (rbc) is present in the lumen of the capillary (cap)

outside the prolactin cell. X 48000

Fig. 6. PAS-positive areas (-+) among the prolactin cells in the RPD. The ACTH cells border, the prolactin cells dorsally. Alcian blue-PAS-orange G

Fig. 7. A portion of a pseudofolliele in the prolactin cell region. Note the masses of extra- cellular material to which numerous acanthosomes are a t tached (ac). --> points to exocytosed

secretory granules, x 32 000

7*

100 M. Benjamin

Fig. 8. An exocytosed secretory granule (esg) in the intercellular space. The cell membrane on the prolactin cell side bears a fuzzy coating of spine like projections (+). • 62000

length and cell width. The whole area occupied by the cell in section must be accurately measured.

Leatherland (1970) put forward the hypothesis that the secretory activity of the prolactin cells in migratory sticklebacks is under a dual control. He postulated that the synthesis and release of prolactin are independently controlled; synthesis depends on the photoperiod and release of secretory granules on the environmental salinity. Although the freshwater stickleback investigated in the present paper does not migrate to the sea in the winter, it is still subiect to the same seasonal changes in the photoperiod as is the migratory animal. As its prolactin cell both synthesised and released secretory granules in the winter, Leatherland's (1970) hypothesis would not seem applicable to the freshwater stickleback.

As the prolactin cells of freshwater sticklebacks kept in 70% seawater for 1 week still synthesised and released secretory granules, and as there were no large scale responses of the pars intermedia or ACTH cells to 70% seawater (Benjamin, 1973) it is tempting to suggest that the freshwater stickleback belongs to that group of teleosts in which the pituitary gland is not important in osmo- regulation. However although high salinities did not stop the synthesis or release of prolactin, it still remains tha t these prolactin cells had the cytological charac- teristics of active cells in freshwater. In the adult, winter stickleback (previous paper) the prolaetin ceils were the most active cells in the pituitary. Hypophysec- tomy, and not the response of pituitary cells to seawater, is obviously the only way to assess the importance of the pituitary gland to osmoregulation. Unfortuna- tely hypophysectomy is technically extremely difficult in sticklebacks. I t is interesting to note that Cook and Overbeeke (1969) found no change in the fine structure of the prolactin cells from adult sockeye salmon (Oncorhynchus nerka) during their anadromons migration. McKeown and Overbeeke (1969) have con- sidered it possible that in the prolactin cell of the adult sockeye salmon, the physio-

Seasonal Changes in Stickleback prolactin Cell 101

logical changes under the natural conditions of anadromous migration are less drastic than in other teleosts, and thus do not alter morphological features.

Freshwater sticklebacks collected from a land-locked, highly saline lagoon had a greatly reduced RPD region that only occupied 8.0 ~: 0.94% of the total pituitary volume (Benjamin, 1973). In addition the prolactin cells of these animals were highly chromophobic. Thus it is possible that only long-term adaptation to sea water would alter the cytology of the prolactin cells.

According to Leatherland (1970) secretory granules are released from the pro- lactin cells of the migratory stickleback by exocytosis into intercellular spaces adjacent to chromophobes. In addition he described a budding of the prolactin cell cytoplasm similar to that found by Weiss (1965) in the platyfish. I t is curious that neither Foll6nius (1968) in his study of an unnamed form of stickleback, nor Leatherland (1970) in his study of the migratory form, associated the pseudo- follicles with granule release. In the freshwater stickleback it seems the pseudo- follicles are active sites for exchange of materials between the prolactin cells and intercellular spaces, for acanthosomes were common in these areas. As the pro- lactin cell region is poorly vascularised in this fish, it is quite feasible that these areas act as "substitute blood vessels" for the exchange of materials. Possibly the fuzz coating around exocytosed secretory granules represents an acantho- some forming. Acanthosomes could be concerned with recycling the additional material added to the cell membrane during exocytosis. Benedeczky and Smith (1972) have also seen a fuzz coating around the part of the plasma membrane contributed by the secretory granule during exocytosis in the adrenal medulla of the golden hamster. If indeed these acanthosomes are concerned with recycling the plasma membrane, this would explain their prominence in the prolactin cells described in the previous paper as these cells showed abundant signs of secretory granule release.

I t is hard to see how either Weiss (1965) or Leatherland (1970) could describe cell budding as a means of granule release on the basis of conventional transmission EM. Serial sectioning is essential to establish that buds are not connected above or below the plane of section.

References

Ball, J. N.: Prolactin and osmoregulation in teleost fishes: a review. Gcn. comp. Endocr., Suppl. 2, 10-25 (1969a)

Ball, J. N.: Prolactin (fish prolactin or paralactin) and growth hormone. In: Fish physiology (Hoar, W. S., and D. J. Randall, eds.), col. II, p. 207-240. New York-London: Academic Press 1969b

Benedeczky, I., Smith, A.D.: Ultrastruetural studies on the adrenal medulla of golden hamster: origin and fate of secretory granules. Z. Zellforsch. 124, 367-386 (1972)

Benjamin, M.: Studies on the pituitary gland and saccus vasculosus of teleost fishes. Ph. D. Thesis. Wales (1973)

Cook, H., Overbeeke, A. P. van: Ultrastrueture of the eta cells in the pituitary gland of adult migratory sockeye salmon (Oncorhynchus nerka). Canad. J. Zool. 47, 937-941 (1969)

Foll6nius, E.: Analyse de la structure fine des diff$rents types de cellules hypophysaires des poissons t~l~ost6ens. Path. et Biol. 16, 619-632 (1968)

Lain, T. J.: Effect of prolactin on plasma electrolytes of the early-winter marine threespine stickleback, Gasterosteus aculeatus, form trachurus, following transfer from sea- to fresh water. Canad. J. Zool. 46, 1095-1097 (1968)

102 M. Benjamin

Lain, T. J.: The effect of prolactin on osmotic influx of water in isolated gills of the marine threespine stickleback Gasterosteus aculeatus L., form trachurus. Comp. Biochem. Physiol. 81, 909-913 (1969a)

Lain, T. J.: Effect of prolactin on loss of solutes via the head region of the early-winter marine threespine stickleback (Gasterosteus aculeatus L., form trachurus) in fresh water. Canad. J. Zool. 47, 865-869 (1969b)

Lain, T. J . : Prolactin and hydromineral regulation in fishes. Gen. Comp. Endocr., Suppl. 8, 328 338 (1972)

Lam, T. J., Hoar, W. S.: Seasonal effects of prolactin on freshwater osmoregulation of the marine form (trachurus) of the stickleback Gasterostcus aculeatus. Canad. J. Zool. 4~, 509- 516 (1967)

Lam, T. J. , Leatherland, J. F.: Effect of prolaetin on freshwater survival of the marine form (trachurus) of the three-spine stickleback, Gasterosteus aculeatus, in the early winter. Gen. Comp. Endocr. 12, 385-387 (1969a)

Lain, T. J., Leatherland, J. F.: Effects of prolactin on the glomerulus of the threespine stickle- back, Gasterosteus aculeatus L., form trachurus, after transfer from seawater to fresh water, during the late autumn and early winter. Canad. J. Zool. 47, 245-250 (1969b)

Lam, T. J. , Leatherland, J. F.: Effect of hormones on survival of the marine form (trachurus) of the threespine stickleback (Gasterosteus aculeatus L.) in deionized water. Comp. Biochem. Physiol. ~3, 295-302 (1970)

Leatherland, J. F. : Seasonal variation in the structure and ultrastrueture of the pituitary of the marine form (Trachurus) of the threespine stickleback, Gasterosteus aculeatus L. Z. Zell- forsch. 104, 301-317 (1970)

Leatherland, J. F., Lain, T. J. : Prolaetin and survival in deionized water of the marine form (trachurus) of the threespine stickleback, Gasterosteus aculeatus L. Canad. J. Zool. 47, 989-995 (1969a)

Leatherland, J. F., Lain, T. J . : Effect of prolactin on the density of mucous cells on the gill filaments of the marine form (trachurus) of the threespine stickleback, Gasterosteus acu. leatus L. Canad. J. Zool. 47, 787-792 (1969b)

McKeown, B. A., Overbeeke, A. P. van.: Immunohistochemical localization of ACTH and prolactin in the pituitary gland of adult migratory sockeye salmon (Oncorhynchus nerka). J. Fish. Res. Bd. Canada 26, 1837-1846 (1969)

Olivereau, M., Ball, J. N.: Pituitary influences on osmoregulation in teleosts. In: Hormones and the environment. Mem. Soc. Endocr. 18, 57-82 (1970)

Weiss, M.: The release of pituitary secretion in the platyfish, Xiphophorus maculatus (Guenthcr). Z. Zellforsch. 68, 783-794 (1965)

Dr. Michael Benjamin Department of Cellular Biology and Histology, St. Mary's Hospital Medical School Paddington, London W2 England