Acta Zoologica, Vol. 62, No. 2, pp. 121-128, 1981 Printed in Sweden

0001-7272/81/02012 l-O8$02.00/0 @ 1981 The Royal Swedish Academy of Sciences

Factors Associated with the Seasonal Variation in the Incidence of Pituitary Cysts in Nine-Spined Sticklebacks, Pungitius pungitius L. Michacl Ben jamin

Department of Anatomy, University College, Cardiff, Wales, U.R. i Received August 19, 1980)

Abstract Benjamin, M . 1981. Factors associated with the seasonal variation in the incidence of pituitary cysts in nine-spined sticklebacks, Pungitius pungitius L. (Department of Anatomy, University College, Cardiff, Wales, U.K.) - Acta

Cysts among the prolactin cells in the pituitary gland of the nine-spined stickleback, Pungitius pungitius L., are most common during and shortly after periods of high mortality that mark the end of the reproductive season. Fish are oldest a t this time of year. Nevertheless, cysts may occur in breeding as well as spent fish, and their presence does not relate to changes in oocyte development. They can be induced by starving animals in February, but not in September-October. Starved fish always have small prolactin cells and nuclei. I t is suggested that cysts form in older fish in response to stress, be this natural or induced. No correlation was noticed between the condition factor of fish caught in the wild, and the incidence of pituitary cysts.

Michael Benjamin, Department of Anatomy, University College, Cathays Park, Cardiff CFl I X L , Wales, U.K.

~ 0 0 1 . (Stockh.) 62(2): 121-128.

Introduction

In the pituitary gland of the nine-spined stickle- back (Pungitius pungitius L.) there are cysts in the prolactin zone of the rostral pars distalis (Benjamin 1978, 1979, 1981, Benjamin and Williams 1979a, b ) . These are cavities of various sizes that are usually filled with fluid. They are often lined by a basal lamina and a neighbouring layer of non-granulated (stellate) cells (Benja- min 1981), but may also be irregular cavities without any definite lining (Benjamin and Wil- liams 197913). Cysts usually develop within areas of non-granulated cells that sheath blood vessels passing through the rostral pars distalis (RPD) on their way to its periphery (Benjamin 1981). As they are virtually absent from animals <20

mni in length, they are of no embryological significance (Benjamin and Williams 1979a), and thus differ fundamentally from the colloid- filled cysts described in Xiphophorus maculatus by Schreibman (1966). Yet there is no close relation between cyst incidence and body length in larger fish. Pituitary size seems a better guide, as cysts are more common in fish with large pituitaries (Benjamin 1981). This is probably related to the high vascularity of the RPD in large pituitaries and the importance of blood vessels in the development of cysts (Benjamin 1981). As the remaining prolactin cells are usually small and inactive, cysts may not be holocrine stores of prolactin (Benjamin 1979).

Although cysts can be found in freshwater animals throughout the year, they are more

122 MichaeE Benjamin

common in June-August, towards the end, and just after, the breeding season (Benjamin and Williams 1979a). They can be induced experi- mentally by transferring adult fish to seawater (Benjamin 1978). These and the other findings pose several important questions, which the pres- ent paper tries to answer. Can animals with cysts have gonads filled with mature eggs or sperm, or are they restricted to spent fish? As the breeding season of teleosts is commonly a time of increased stress, is the high incidence of cysts in June-August related to high mortalities? Can cysts be induced experimentally by simulating conditions of stress (e.g. starvation) outside the breeding season, and is cyst incidence related to the condition factor of fish collected from the wild?

Materials and Methods

Length-frequency studies. Nine-spined sticklebacks (Pungitius pumgitius L.) of both sexes were col- lected from freshwater reens at Marshfield, Gwent, throughout the year. Because of the difficulty of fishing in weed-choked areas, a hand-net of 7 mm mesh was used. The populations were considered randomly sampled because all fish could escape through the net with equal ease (though few did). Fish were measured from the tip of the snout to the base of the tail, and lengths recorded to the nearest millimetre. 200-250 animals were used for each of the four length-frequency studies (Fig. 1 ) and the fish were always caught on two occa- sions one month apart. Otoliths extracted from samples of fish collected in July, November and December, were used to age the animals according to the method of Jones and Hynes ( 1950).

Starvation experiments. Adult fish, 30-38 mm in length, were acclimatised to aerated, laboratory- tapwater for one week before experiments. The animals were then transferred to 10 1 plastic tanks and either fed daily on live Tubifex, or deprived of food for 3.5-4 weeks. Although 50 % of the starved animals died before the end of the experi- ments (conducted in February and in September- October), equal numbers of fed and starved fish were finally killed (total number of animals, 52).

Light microscopy procedures. All light micro- scopy procedures for stickleback pituitaries have been described previously (Benjamin 1978). 6 p m serial sections were stained with alcian blue-PAS --orange G. Gonads were similarly processed, but stained with haematoxylin and eosin.

Quantitntiue methods. The nuclear diameters of prolactin cells were measured on photographic prints at a magnification of x 815, using 20 nuclei

per animal. Prolactin cell nuclei in a standard area were counted to assess cell size; the fewer the nuclei, the larger the cells. The relative volumes of the rostra1 pars distalis (RPD), proximal pars distalis (PPD) and pars intermedia ( P I ) were estimated by tracing every tenth section of a pituitary gland on to good quality paper. The weights of the paper outlines were proportional to the relative volumes of the different regions. Be- cause of the intimate association of the adeno- and neurohypophyses, the RPD, PPD and PI were measured together with their overlying or inter- mingled neural tissue.

The percentages of various size classes of oocytes were estimated on fish > 3 0 mm in length, caught in June, according to the method of Olivereau and Olivereau (1979).

The ‘condition factor’ of 83 fish ( > 3 0 mm in length) killed in February, was calculated from the following formula (Weatherley 1972) :

100xWeight (9) Length (cm)3

Condition Factor ( K ) =

Numerical data was assessed with Student’s t-test, and all mean values were expressed f S.E.

Results

Length-frequency studies. Measurements of stick- lebacks throughout the year showed that large fish (30 mm or more in length) were rarest in June-August and commonest in March-May (Fig. 1 ) . Thus, between the last March-May collection and the first in June-August, many fish disappeared from the population. Similarly, a large number of small fish (25 inm or less in length) appeared at the same time. As this period (mid-May to early July) is towards the end of the breeding season, the small animals are prob- ably first year fish and the large animals presum- ably died. Significant numbers of dead fish were netted at this time of year, supporting the latter conclusion. The shift towards larger fish in Sep- tember-November reflects the rapid growth of young fry. Thus animals 30 mm or more in length should on average be older in June- August than in September-November. Otolith readings confirmed this, and showed that none of the fish examined, survived into a third winter.

Starvat ion experiments . The basic morphology of the pituitary gland in Pungitius has been described previously (Benjamin 1978). Pituitary cysts within the RPD are usually fluid-filled spaces, varying considerably in size, that originate near blood vessels and enlarge at the expense of

neighbouring cells. For further details of the morphology of cysts, the reader is referred to Benjamin and Williams (1979b) and Benjamin (1981). A typical cyst among the prolactin cells of the RPD is illustrated in Fig. 2.

When animals were starved for 4 weeks from the beginning of February onwards, there was a marked increase in the incidence of cysts. They were present in the pituitaries of 69 % of starved fish, but in only 19 % of fed fish. In both groups of animals, cysts varied considerably in size-the smallest were 10-20 pin in diameter, whereas the largest virtually obliterated the R P D and reached diameters > 200 pm. The prolactin cells and their nuclei were smaller in starved than fed animals, and their cytoplasm often showed les; affinity for orange G (Figs. 2-5). The propor- tions of the RPD, PPD and P I were similar in both groups (Fig. 6 ) , but the whole pituitary was markedly smaller in starved animals (Fig. 8 ) .

When the experiment was repeated in Septem- ber-October, there was no difference in the number of animals with pituitary cysts (30 % of fish in both groups had cysts). However the prolactin cells and their nuclei were again smaller in starved animals (Figs. 4 - 5 ) , and their cyto- plasm stained less with orange G. The relative volume of the pituitary gland occupied by the RPD and by the PI, was slightly smaller in starved animals (Fig. 7 ) . The high relative volume of the PPD (Fig. 7 ) can be largely attributed to the smaller pituitaries of starved animals (Fig. 8) and the shrinkage of the other two regions.

Condition factors, prolactin cells and cyst incidence. Of 83 fish killed in February whose condition factors were recorded, 15 had RPD cysts (i.e. 18 %). There was no significant dif- ference between the mean condition factor of fish with (1.01 f 0.02) and without (1.02 f 0.01) cysts. When the nuclear diameters of prolactin cells from those 20 fish with the highest condi- tion factors (mean value 1.15 * O . O l ) were com- pared with those from the 20 fish with the lowest condition factors (mean value 0.90 f O.OO), the mean values were also similar (3.8 f0.04 pin and 3.8 f 0.06 pni respectively).

Pituitary-gonadal axis of fish withlwithout cysts. By examining at autopsy the gonads of fish killed early in July, it was clear that animals with and without pituitary cysts (as seen in sectioned pituitaries) could both have gonads filled with mature eggs or sperm. By the end of July, there were very few fish in breeding condi- tion. The distribution of oocyte areas in fish with

Pituitary Cysts in Pungitius 123

1 DM. - Feb.

Length (mm)

L 151 Mar. - May

i ~ ~ w n i a a s ~ i n 3 7 ~

Length (mm)

June - Aug.

Length (mm)

F i g . 1 . Measurements of fish lengths throughout the year.

and without pituitary cysts was similar (Figs.

In July fish, the PPD was packed with ventral- ly-placed gonadotrophs, yet the relative volume of this region was similar in animals with (mean value 55.5 k2.5 %) and without (mean value 53.6 52.7 %) RPD cysts. The gonadotrophs were invariably heavily stained and showed no evidence of degeneration. As their nuclear diame-

9-10).

9 ~ Arta zool. 1981 : 2

124 Michael Benjamin

Figs. 2 and 3. Sagittal sections of the pituitary gland from an animal starved for 4 weeks in February (Fig. 2 ) and from a fed, laboratory- control (Fig. 3 ) . Note the srnall cyst (arrow) among the prolactin cells (PRL) in Fig. 2. At its periphery are several blood vessels fBV) . The prolactin cells and their nuclei are smaller in Fig. 2, and their cytoplasm has less affinity for orange G. Alcian blue-PAS-orange G. Turquoise filter.

Outlines 9.1 *0*73 g) RPD cysts (P<O.O5), and 36 % of the Population had cysts.

Discussion

I t has been shown previously that pituitary cysts in the RPD of Pungitius are most common in June-August (Benjamin and Williams 1979a).

ters have been reported previously, the reader is Evidently this is also a period of high mortality referred elsewhere for details (Benjamin 1980). that marks the end of the stickleback breeding The whole pituitary was slightly larger in fish season at Marshfield. A similar high mortality with (mean weight of pituitary outlines 10.5 rate may be inferred from the data of Jones and f 1.12 g) than without (mean weight of pituitary Hynes (1950) on a population of Pungitius in

Pituitary Cysts in Pungitius 125

4

5 o w 0 starved

ill -*-- Fig. 4. The nuclear diameters of prolactin cells in fed and starved animals (means * S . E . ) . ** p < <0.01.

North-West England, for large fish again dis- appeared at a similar time. In both the present study and that of Jones and Hynes (1950), fish are oldest a t this time of year. Nevertheless, RPD cysts may be found in breeding as well as spent fish, and the similar distribution of oocyte size classes in animals with and without cysts, sug- gests that they do not produce changes in the reproductive state.

There are strong parallels between the present work and that of Robertson and Wexler (1957, 1962a, b) . These authors reported widespread degenerative changes, including the formation of

RPD PPD PI

PPD

6

7

Sept-Oct

PI

Fig. 5. The number of prolactin cell nuclei per unit area, in fed and starved animals (means *S.E.) . ** p < 0.01 ; * p < 0.05. Figs. 6 and 7. The relative volumes of the RPD, PPD and PI (expressed as a percentage of the whole gland) in fed and starved animals in Febru- ary (Fig. 6 ) and September-October (Fig. 7 ) . Means f S.E. ** p < 0.01 ; * p < 0.05.

126 Michael Benjamin

8 9

“1 10

Feb hpr- Oct

Figs. 9 and 10. The distribution of oocyte areas in July fish with (Fig. 9 ) and without (Fig. 10) pituitary cysts. Each figure was based on a study of 6 fish ( ,30 mm in length) and 300 oocytes. Fig. 8. Pituitary-outline weights (an indication of

the size of the gland) in fed and starved animals in February and in September-October. Means f S.E. * p < 0.05.

cysts, in the pituitary glands of breeding and spent salmon and trout. As in Pungitius, the changes were most pronounced during periods of high mortality and were characteristic of older populations of fish. Yet it is important to note that while pituitary cysts in Pungitius are de- generative changes (Benjamin 1979, 1981, Ben- jamin and Williams 1979a), they usually affect only the prolactin zone of the RPD. In particular, the cysts do not appear a t the expense of gonado- trophs, nor were there other signs of degenera- tion in these cells e.g. pycnotic nuclei, intra- cellular vacuoles etc. In salmon and trout (Ro- bertson and Wexler 1957, 1962a, b) , pituitary degeneration was more general, and certainly affected the gonadotrophs. Other workers also considered pituitary cysts as typical of senile or post-spawning fish (Honma 1966, Honma and Tamura 1965, Yoshie and Honma 1978). Again, some of the cavities described affected the PPD, and were thus more predictably associated with reproductive state.

Evidently, pituitary cysts in Pungitius can be induced experimentally by starving animals in

February. I t is proposed that depriving stickle- backs of food, simulates one of the characteristic stresses of the breeding season. I t is certainly associated with a high mortality rate. One should also note that Wootton (1976) comments on the disrupted feeding activity of breeding Pungitius. All this does not necessarily imply that pituitary cysts cause the death of fish. It is possible they indicate harmful effects elsewhere in the body.

Robertson and Wexler (1962a) were able to accentuate degenerative changes in trout pitui- taries by keeping post-spawning animals in cap- tivity. They also considered stress an important factor in producing these changes. Indeed they concede that prolonged starvation may be asso- ciated with pituitary degeneration. Bage and Fernholm (1975) described pituitary cysts in migrating river lampreys, Lampetra fluviatilis. These animals stop feeding during their migra- tion, and their gut atrophies. Honma and Matsui (1973) reported an RPD cyst in a starved Japanese eel, Anguilla japonica.

I t is difficult to explain why cysts should form in starved Pungitius at one time of year but not another. Yet there are parallels with previous attempts to induce pituitary cysts by transferring adult sticklebacks to seawater (Benjamin 1978,

Pitui tary Cysts in Pungitius 127

Benjamin and Williams 1979a). I t seems they can be induced early in the year (January- February), but not later (July onwards). Perhaps age is an important factor, for the length-fre- quency data and otolith readings suggest that fish >30 inm in length, are likely to be older in January-February than e.g. in September- October. In a short-lived fish such as the stickle- back, this difference of a few months may be particularly significant. I t seems reasonable to expect stress to have a greater influence on older fish. On the other hand, perhaps the mere stress of keeping fish in captivity increased cyst inci- dence and masked any additional effect of starving them in September-October. At this time of year, 30 % of fed fish had cysts, and this is higher than one would expect from animals in the wild. I t has already been remarked that Robertson and Wexler (1962a) induced various pathological changes, including pituitary cysts, merely by keeping post-spawning trout in cap- tivity.

Although fed and starved fish were not weighed, it is certain that starved animals lose weight and that their condition factors decrease. I t was thus worth looking for a correlation between a low condition factor and a high cyst incidence in natural populations of sticklebacks, at a time when starving animals induces cysts. None existed. There are probably too many other variables influencing the condition factor of Pungitius besides food intake-notably gonadal development.

Why prolactin cells should be inactive when fish are starved, is uncertain. The accompanying stress may interfere primarily with osmotic or ionic regulation at various body sites e.g. the integument, gills, kidneys etc. As prolactin is important in the hydro-mineral regulation of teleosts (Ball 1969a, b, Ball and Baker 1969, Qlivereau and Ball 1970, Schreibman et al. 1973), changes in these organs may subsequently affect the prolactin cells. If indeed this is the case, it emphasizes the similarity between in- ducing RPD cysts by starving fish and by trans- ferring them to seawater (Benjamin 1978). Both could be essentially osmoregulatory stresses. Fur- thermore, it is possible that the breeding season of Pungitius (i.e. immediately before cysts are naturally most common), is a time when fish change their osmoregulatory behaviour, for this is certainly the case with Casterosteus (Koch and Heuts 1943).

Prolactin has also been implicated in regulating the lipid metabolism of tt-leosts (Joseph and

Meier 1971, Meier et al. 1971, Sage and de Vlaming 1975) and is a controlling agent in the annual fattening cycles of fish. However, pro- lactin seems both to deplete and enhance body lipids, according to when it is administered. This makes it difficult to apply these findings to the present results, where there is presumably a decrease in lipid stores after 4 weeks starvation. Evidently, starvation does not influence prolactin synthesis or release in the rat (Akikusa 1971).

Acknowledgements

I would like to thank Mr. P. F. Hire for his help with the photography, and Mrs. D. Hayman for preparing many of the sections.

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