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Endocrine aspects of ageing in the guppy, Lebistes reticulatus (Peters)
Exp. Geront. Vol. 4. pp. 17-25. Pergamon Pren 1969. Printed in Great Briudn
ENDOCRINE ASPECTS OF AGEING IN THE GUPPY, LEBISTES RETICUL.4TUS (PETERS) III. THE TESTIS A. D. WOODH~D* and SALLYELLETT Zoological Society of London and Fisheries Laboratory, Lowestoft, Suffolk
(Received22 ffuly 1968) INTRODUCTION IN RECENTpapers we have described some ageing changes in the thyroid gland and the interrenal gland of the guppy, Lebistes reticulatus (Peters) (Woodhead and Ellett, 1966, 1967 a, b). The present paper reports the histological changes occurring in the testis with advancing age. The fish were taken from a laboratory population which had been maintained to extreme old age by Dr. Alex Comfort, University College, London, for his researches into ageing. Details of the keeping conditions of the fish have been given by Comfort (1960); the fish used here had not been subjected to any radical changes in environmental conditions during their lifespan, and were those designated "normal growers" by Comfort (1960). MATERIALS AND METHODS Fourteen males, ranging in age from 2½ months to 59 months, were studied. The fish were killed by placing them in ice to anaesthetize them, and the bodies fixed in iced Bouin or Carnoy's fluid (Comfort, 1961). They were then embedded in paraffin wax, and serially sectioned at 10/~ throughout their entire length. The series were mounted, and stained with haematoxylin and eosin. The growth in length of the gonad with age, in relation to the growth in length of the body, was estimated by counting the number of sections upon which testicular tissue appeared. Since the thickness of the sections was known (10#), the extent of the testis could be obtained. This value was expressed as a percentage of the body length, measured from the tip of the snout to the base of the caudal peduncle. The growth in width of the testis was also measured, readings being taken from ten sections about the mid-point of the gonad; the mean value obtained was expressed as a percentage of the body width. The cells comprising the germinal tissue were classified in order of their development as follows: (a) Primary spermatogonia: large faintly staining cells with a large rounded central nuclens, having a single central darkly staining nudeolus and a heavy chromatin network. (b) Secondary spermatogonia: morphologically similar to the primary spermatogonia, but smaller, with less abundant cytoplasm. The nucleolus was less prominent. e Present address: Heron Island Research Station, Great Barrier Reef, Australia. 17
18
A. D. WOODHEAD AND SALLY ELLETT
(c) Primary spermatocytes: cells of approximately the same dimensions as the secondary spermatogonia, but the chromatin threads of the nucleus stained less intensely and had become aggregated into distinct small droplets. (d) Secondary spermatocytes: smaller than the primary spermatocytes, with the chromatin of the nucleus more evenly dispersed, and fewer aggregations. This stage occurred less frequently than other stages of development, and may represent a shorter phase of spermatogenesis. (e) Spermatids: relatively small rounded cells, with a thin ring of cytoplasm surrounding an elliptically-shaped nucleus. (f) Spermatozoa: developed from the spermatids, with an elongated compact nucleus and a cytoplasmic tail of approximately 32/z in length (varying between 27 and 36/~). Maturation of the fish testis does not always proceed simultaneously throughout the gonad, and spermatogenesis may be further advanced in the central than in the peripheral areas. This was the case in the guppy, and in order to obtain a representative assessment of the state of gonadal maturity, every twentieth section along the length of the testis was examined. The cell population of the lobules were classified as "ripe"--those containing spermatids and spermatozoa---or "unripe"--those with earlier stages of cell development. The findings were expressed as the percentage of ripe lobules in the testis. The amount of connective tissue and germinal tissue in the testis was assessed by projecting an image of a section on to a screen, upon which a vertical line had been drawn, and marking off the extent of each component. Measurements were made every 100/~ along the testis, and the results were expressed as the percentage of connective tissue in the testis. Measurements were made of the thickness of the testis wall, taking 25 measurements from sections near the centre of the testis. RESULTS
The testis in young fish The testis, covered by a thin layer of peritoneum, was seen to be subdivided into large numbers of lobules supported by connective tissue extensions of the tunica albuginea. Each lobule was composed of numerous cysts of germ cells, which apparently divide and develop through successive cell generations before producing mature spermatozoa (Fig. 1). In the fully ripe testis, the cyst walls have ruptured, and spermatozoa were seen free in the lumen of the lobule, which communicated with the efferent duct of the testis. In young males, ranging in age from 2½ to 8 months, the tunica albuginea was composed of one or two layers of connective tissue cells, and measured less than 5/z in thickness (except in one individual). The lobular walls were tightly packed with cysts which contained cells at all stages of spermatogenesis, the most frequently occurring cell stage in unripe lobules being the primary spermatocyte. Generally the lobules at the periphery of the testis were unripe, whilst towards the centre of the gonad the number of lobules containing spermatids and spermatozoa was increased. Cysts in different stages of development were found in some of the lobules, but in the majority of lobules the phases of development were synchronous. In the ripe unruptured cysts, the heads of the spermatozoa were oriented towards the cyst wall; after rupture, bundles of spermatozoa
FIc. 1. Photomicrograph of the testis of the guppy, showing various cell types : (a) Secondary spermatogonia; (b) Primary spermatocytes; (c) Secondary spermatocytes ; (d) Spermatids ; (e) Spermatozoa. Magnification × 469.
facing p. 18
b FIG.
5.
Photomicrograph
of the testis wall: (a) in a guppy 59 months. Magnification x 490.
aged 8 months;
(b) in a guppy
aged
ENDOCRINE ASPECTS OF AGEING IN THE GUPPY, LEBIS~ES RgTICUIMTU$ (PETERS)
19
were seen lying closely packed around the lobule walls. The central lumen of the lobules usually contained a fluid which was faintly eosinophilic. There was some variability in the percentage of ripe lobules in the testis of young males. Thus in two guppies aged 2½ months the proportion of ripe lobules was 34 per cent and 46 per cent, and in one of 3½ months it was 70 per cent.
Ageing changes in the testis The length of the testis in relation to body length. There was no evidence of any significant variation in the length of the testis in relation to body length during life; correlation between these two factors was significant only at the 50 per cent level. In nine fish less than 2 years old the testis extended over 20-30 per cent of the standard body length, and in four older fish it had increased only to 31-34 per cent. The width of the testis. There was no evidence of a change in testis width withincreasing age. A regression analysis of age against testis width, expressed as a percentage of the body width, showed significance at the 10 per cent level only. Changes in the numbers of ripe lobules with age. During the lifespan all the males showed an increase in the percentage of lobules containing spermatids and spermatozoa, which was significant at the 1 per cent level (Fig. 2). The relationship obtained was Y - ~ 0.63X + 45.45 where X = age in months and Y-~-percentage of ripe lobules. The correlation coefficient 'r' was 0.75. 100
.~-
75 • / I
i /
--
50
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i
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t
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I
I
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I
I
I
I
I
5
10
15
20
25
30
35
~0
~5
50
55
60
Age in months
Fzo. 2. The percentage of lobules containing spermatozoa or spermatids in the testis of the guppy with increasing age. The regression is shown by a broken line.
20
A. D. WOODHEAD AND SALLY ELLETT
In seven fish less than 1 year old, the proportion of ripe lobules (27-70 per cent) was more variable than in seven older fish (50-87 per cent). Unripe lobules were generally found at the periphery of the testis. In fish older than 30 months, the spermatozoa in some of the lobules appeared degenerate (these are discussed below), and the number of these lobules increased with advancing age. However, all cell stages of spermatogenesis were represented in the testis throughout life, and even in the testes of senile fish cysts of spermatogonia and spermatocytes were seen, although these were fewer in number. Changes in the amount of connective tissue in the testis with age. There was a pronounced increase in the amount of connective tissue in the testis with advancing age (Fig. 3). In fish of 2~L8 months the connective tissue between the lobules was sparse, and an average value of 10 per cent was obtained. The first indication of an increase in the amount of stroma was seen in a fish of 13½ months of age, where areas of thickened stroma were found at the periphery of the testis. When tested, this increase was found to be significant at the I per cent level. 5045 ~- 4O ._= 35 / ,,.e
/
3O
•
4~
•
~
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& ~'15 L,.
10
I 5
I 10
I 15
I 20
I I 25 30 Age in months
I 35
1 40
I 45
i 50
I 55
i 60
FIc. 3. T h e percentage of connective tissue in the testis o f the g u p p y at various ages. regression is s h o w n b y a broken line.
The
Linear regression analysis gave a relationship: Y ~ 0.38X + 12.10 where X ~ age in months and Y ~ percentage connective tissue in the testis. The correlation coefficient 'r' was 0.76. Proliferation of the connective tissue was usually greater at the outer areas of the testis, where isolated cysts containing spermatids and spermatozoa lay embedded in the
ENDOCRINE ASPECTS OF AGEING IN THE GUPPY, LEBISTE8 RETICUL,4TU,,q (PETEI~S)
21
thickened stroma. In young guppies the centre of the testis remained relatively free from infiltration. Multiplication of connective tissue continued with age, gradually extending into large areas, particularly at the periphery of the testis. A comparison of the percentage of stroma at a point one-tenth of the way along the length of the testis (from the anterior end) gave values of 12 per cent in two fish less than 2 years old, and 52 per cent in two fish older than 2 years. However, at the centre of the testis (half-way along the length) the values were 13 and 14 per cent, respectively. Eventually, in senile fish, extensive thickening had taken place throughout the testis, until the stroma had gradually spread to occupy much of the space which had previously been filled with spermatogenic tissue. In association with this there was a decrease in the amount of germinal tissue in the testis with age; the testes of fish of less than 1 year old contained approximately 87 per cent germinal tissue, compared with 68 per cent in fish of 30 months and over. 15-
13 12 17 --I0 =c:l9 B
,....-. I
~7
1 t /
~5
I
..... /
~5
I
4
3 2 I 0
I 5
I 10
I 15
i 20
I I I 25 30 35 Age in months
I 40
I L,5
I 50
55
I 60
FIG. 4. T h e increase in thickness of the testis wall with age. T h e regression is shown by a broken line.
Changes in thickness of the testis ~oall with age. The tunica albuginea thickened with age, until, in the older fish, it was frequently twice its original thickness; this increase was significant at the 5 per cent level (Fig. 4). A linear regression analysis of the thickness of the testis wall on age gave the relationship: Y = 0.08X + 3.51 where X = age in months and Y -k- thickness of tunica albuginea (~). The correlation coefficient 'r' was 0.51.
22
A. D. WOODHEAD AND SALLY ELLETT
In young fish the cells of the connective tissue sheath were elongated and thin, with a long narrow nucleus, and the covering was composed of one or two layers of cells. In older fish the walls had become thickened by proliferation of the connective tissue elements, and were composed of six or seven layers of cells. Some thickening of the tunica albuginea was undoubtedly due to contraction of the connective tissue cell fibres, which appeared shorter and thicker than in young fish, but the increase in the number of the cells accounted for the larger part of the recorded increase in thickness. The appearance of the testis wall in young and senile guppies is shown in Figs. 5(a) and
(b). Abnormalities of spermatogenesis with age The material available for this study was limited, so that it was not possible to follow in detail the chronological sequence of ageing events in the testis. However, several features were regularly recorded in the testes of old fish. Degeneration of the cells in the early phases of spermatogenesis was recorded very infrequently. In one guppy, nuclear fragmentation had taken place in the secondary spermatocytes, and, in the same individual, multinucleate cells, possibly also spermatocytes, were present; but both of these types were few in number. Generally cells in the earlier phases of spermatogenesis showed few morphological differences even in the most senile fish, compared with young individuals. Some abnormalities of the later stages of spermatogenesis were apparent in fish of 13½ and 16 months of age, where cysts with fragmented spermatozoa were sporadically recorded; their numbers increased with advancing age. In a few cysts only fragments of pycnotic nuclei remained. A further consistent change was one in which the stroma surrounding some of the cysts containing spermatozoa had proliferated and become arranged in closely packed concentric layers around the cyst. Another prominent change was one in which the cells of the cyst wall had become tall, in some cases almost obliterating the cyst cavity and the spermatozoa contained within. Other atypical cysts were seen far less frequently. In some of those which contained atypical spermatozoa, the fluid contained in the central cavity was basophilic or colourless, and was represented by a much shrivelled mass which appeared to have been formed by the solidification of the fluid and its gradual shrinkage. This change was not regarded as a fixation artefact. These features of the senile testis were usually found at its outer edges and in the proximal region. Whilst atretic cysts were common in senile guppies, complete degeneration of the testis was not encountered in any fish of this series, and often the centre of the testis of senile fish appeared normal and contained apparently normal spermatozoa. Pigmentation of the tunica albuginea of the testis was regularly seen in older fish, and accumulation of pigment also occurred in the stroma. Melanin deposits were generally localized in large cells with ragged, irregular outlines. Cells with a heavily staining nucleus and a yellow-brown pigmented cytoplasm were also found in young males, but occurred much more commonly in senile fishes; groups of these cells were sometimes associated with deposits of melanin. The yellow-brown cells were distinguishable from normal interstitial tissue and did not have the appearance of adipose cells. Adipose tissue was present in the testes of males of all ages, but in older fish there appeared to be more adipose cells associated with the sperm duct than were seen in young fish.
ENDOCRINE ASPECTS OF AGEING IN THE GUPPY, L E B I S T E S R E T I C U L A T U S (PETERS)
23
The interstitial tissue of the testis of the guppy Since the slides of the guppies had been stained with a general purpose stain, no detailed study of the interstitial cells of the testis could be made. These cells were seen embedded in the stroma, and usually abutting the walls of the lobules of all the testes examined. They were large and rounded, and had a central nucleus with a single prominent nucleolus; their cytoplasm was abundant and showed faintly eosinophilic granules. Their histological appearance in senile males was very similar to that in young fish. DISCUSSION The testes of Lebistes show well marked ageing changes, their gradual involution being characterized by a replacement of spermatogenic tissue with connective tissue, by a thickening of the tunica albuginea, and by an increasing incidence of atretic cysts. The first signs of ageing were seen in a fish of 13½ months, in which the testis wall had thickened and augmentation of connective tissue had occurred. Ageing changes were regularly established in fish over 30 months old, and progressively increased with advancing age, although the extent of these changes in the testis showed considerable individual variation. However, there seemed to be little change in the interstitial cells throughout life. We have only found one previous histological study of the senile testis in fish, made by Rasquin and Halter (1951) on characins, Astyanax mexicanus. These authors reported an increased formation of insoluble calcified "concentrations" in the testis of Astyanax, which they believed were similar to those found in the ageing mammalian prostate gland. The basophilic concentrations seen in some lobules of the testis of senile Lebistes appear to have arisen by solidification or condensation of sperm duct fluid. Since our material had already been fixed and stained, we could not employ the techniques used by Rasquin and Hafter to show the presence of calcium in these concentrations. No assumptions can therefore be made concerning the resemblances between these structures in the testes of Lebistes and Astyanax. However, the concentrations were not as numerous in senile Lebistes as in Astyanax. Rasquin and Hafter also described the infiltration of connective tissue into the gonad of senile Astyanax, although this did not occur throughout all the testis, a large part of which was still producing spermatozoa. Although apparently normal spermatozoa were present in the testis of aged guppies, it seems likely that the progressive increase in the number of atretic lobules with age might occasion a progressive decline in reproductive performance, and it is notable that Comfort (personal communication) finds an early and rapid decline in male fertility in Lebistes. Atretic lobules were not described in Astyanax, but Rasquin and Halter reported the presence of cells with hyperchromatic nuclei and yellow cytoplasm. A prominent component of the interlobular tissue of Astyanax was adipose cells; these were also found in all the guppies examined and became more abundant with age. Removal of the pituitary gland in bony fishes results in the gradual regression of the gonads, which become reduced in size (Atz, in Pickford and Atz, 1957). The later stages of spermatogenesis are arrested, and those present at the time of operation gradually disappear, until the reduced testis finally consists of cysts of spermatogonia. Barr (1963) found that the testes of hypophysectomized plaice, Pleuronectes platessa L., were eventually composed of a few isolated cysts of spermatogonia lying in a very thick connective tissue stroma. Whilst some of the ageing changes in the testis of Lebistes may be
24
A. D. WOODHEAD AND SALLY ELLETT
due to a decrease in the output of pituitary gonadotrophins, so that spermatogenic tissue was lost and progressively replaced by connective tissue, the resemblances between the aged testis in Lebistes and the atrophied testis of hypophysectomized teleosts were not great. T h e r e was no reduction in size of the testis with age in Lebistes, nor did spermatogenesis cease; even in senile individuals apparently normal spermatozoa were produced. Considerable differences in the extent of involution of the testis with age were seen in individual Lebistes, and in this respect the findings were closer to those obtained b y Rasquin and Rosenbloom (1954) who kept characins (.4styanax mexicanus) in darkness for prolonged periods. T h i s treatment, which was claimed to produce a state of "physiological" hypophysectomy, had variable effects on the testis, some fish having m u c h reduced testes and little spermatogenic activity, whilst others were but little affected. T h e present results appear to us to indicate endocrine disfunction with age, rather than a f o r m of "physiological" hypophysectomy.
,4cknowledgememts--We wish to thank Dr. ALEX COMFORT, University College, London, for generously allowing us to use his guppy material for this study. Dr. H. A. COLE, Director of the Fisheries Laboratory, Lowestoft, and Dr. I. W. ROWLANDSof the Wellcome Institute of Comparative Physiology, The Zoological Society of London, gave us considerable support and encouragement throughout the work. We are grateful to the Natural Environment Research Council and The Nuffield Foundation for grants for this study. This paper was written whilst one of us (A.D.W.) held a Research Fellowship of the University of Queensland at Heron Island Research Station, Australia.
REFERENCES
ATZ, J. W. (1957) In The Physiology of the Pituitary Gland of Fishes (Edited by PICKFORD,G. E. and ATZ, J. W.), p. 178-270. New York Zoological Society, New York. BARR, W. A. (1963) Gem. comp. Endocrinol. 3, 216-225. COMFORT, A. (1960) Gerontologia 4, 177-186. COMFORT, A. (1961) Gerontologia S, 209-222. RASQUIN, P. and HA~r~, E. (1951)ft. Morph. 89, 397-404. R_~SQUIN, P. and ROSENBLOOM,L. (1954) Bull..din. Mus. nat. Hist. 104, 359-426. WOODHEAD,A. D. and ELLETT, S. (1966) Exp. Geront. 1, 315-330. WOODHEAD,A. D. and ELLETT, S. (1967a) Exp. Geront. 2, 73-77. WOODHEAD, A. D. and ELLETT, S. (1967b) Exp. Geront. 2, 159-171. S u m m a r y - - A histological study was made of the testes of guppies ranging in age from 2½ to 59 months. The testis showed a gradual loss of spermatogenic tissue and a progressive infiltration of connective tissue with age. The connective tissue sheath of the testis had thickened considerably in old fish. Atrophic and degenerate cysts were also found in senile fish, but these usually occurred side by side with apparently normal cysts. Even in extreme old age apparently normal spermatozoa were produced by the testis. Some of the changes in the senile testis of Lebistes resembled those occurring in the testes of hypophysectomized fish, and it is possible that these may result in part from a decline in the output of pituitary gonadotrophins in old age. R t s u m G Les testicules de guppies (Lebistes du Vtntzuela) ~gts de deux mois et demi A 59 mois ont fait l'objet d'une 6tude histologique. On y a relevt, ~tmesure du vieillissement, une perte graduelle de tissu spermatog~ne et une infiltration progressive de tissu conjonctif. La gaine de tissu conjonctif du testicule s'~paississait
ENDOCRINE ASPECTS OF AGEING IN THE GUPPY, L E B I S T E S RETICUL.4TUS (PETERS)
consid~rablement chez les poissons ~g~s. Les poissons ~g~s pr~sentaient aussi des kystes atrophiques et d~g6n~r~s, mais ceux-ci voisinaient g~n~ralement avec des kystes apparemment norm_aux. Les testicules produisaient des spermatozoides apparemment normaux jusqu'~ u n {tgeextr~mement avanc6. Certaines modifications des testlcules s~niles de Leb/stes ressemblaient ~ celles qui se manifestent dans les tes= ticules de poissons ayant subi l'hypophysectomie et il est possible qu'elles soient, dans une certaine mesure, la consequence d'une diminution de la production de gonadotrophines bypophysaires au cours de la vieillesse.
Z u s a m m e n f a s s u n g - - E i n e histologische Untersuchung an Testes von Guppies im Alter yon 2½ bis 59 Monaten wurde durchgefdhrt. Die Testes zeigten einen aU= m~_lichen Rtickgang des sperrnatogenen Gewebes u n d fortschreitende Infiltration mit Bindegewebe mit dem Alter. Die bindegewebige Hiille der Testes war bei alten Fischen betr~chtlich verdickt. Bel senilen Fischen fanden sich auch atrophische u n d degenerative Zysten. Diese traten aber gew~hnlich gemeinsam n-tit offenbar normalen Zysten auf. Auch im extrem hohen Alter produzierten die Testes offenbar norrnale Spermatozoen. Einige der VerLnderungen in senilen Testes von Lebistes ~.nelten denen in Testes hypophysektomierter Fische. Es ist m6glich, daft diesc zum Tell durch Abfall der Gonadotropinproduktion der Hypophyse in h6hcren Alter entstehen.
Pc3mMe---I'IpOBO,/~r/IOCb I"HCTOJIOrlFICCXO0HgY.Jlo~oBaml¢ COMelqrlm~COBp]hl~ ~ B Bo3pa~"e OT 2~ ~O ~ Mg~I~[eB. COMeEtI-mKH1 I O I ~ 3 ~ IIoc'roI~HHOe yMOH]bnle]H[]~ec~epMaTOremaoi~
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o 6 o a o ~ a CCMeum.-xoe 6~Laa caa~Ko y r o m a c a a . Y craph~tx
psi6 6sI.qH Taxze O6HapyxeH~ aTpocbHpovm~n.xe a nereHepRp3nonme traCTS, HO OH~ O6MKHOBeHHOBcTpe'laJmCl,pK/~OMC HHCTaMHHOpMaY~HOrOBK~a. ~ B CoBceM HpeK= aOHHOMcTapOMBoapacre CHepMaTO3OKn.h)HopMaJIbHOFO BK~aB~dpa6aTHBaymc],BceMeHHX= Kax. HeXOTOpHe~ H 3 M C H e ~ B ycTapeBm~c c e M e ~ a x Lebistes HanoMmtaymTagOBbIe B CeMet-mt4gaxpl~i6i i o ~ B e p ~ c ~ l rHIIO~H~KTOMHH,IIO~TOMy~I~TaeTCRBO3MOXH]dM,qTO OHHBSIZBaH~OTqaCTHCHHZeHXeMB~eJ~eim~ nn3TwrapHoro roHa~oTpomma B crapocTH.
25
ENDOCRINE ASPECTS OF AGEING IN THE GUPPY, LEBISTES RETICUL.4TUS (PETERS) III. THE TESTIS A. D. WOODH~D* and SALLYELLETT Zoological Society of London and Fisheries Laboratory, Lowestoft, Suffolk
(Received22 ffuly 1968) INTRODUCTION IN RECENTpapers we have described some ageing changes in the thyroid gland and the interrenal gland of the guppy, Lebistes reticulatus (Peters) (Woodhead and Ellett, 1966, 1967 a, b). The present paper reports the histological changes occurring in the testis with advancing age. The fish were taken from a laboratory population which had been maintained to extreme old age by Dr. Alex Comfort, University College, London, for his researches into ageing. Details of the keeping conditions of the fish have been given by Comfort (1960); the fish used here had not been subjected to any radical changes in environmental conditions during their lifespan, and were those designated "normal growers" by Comfort (1960). MATERIALS AND METHODS Fourteen males, ranging in age from 2½ months to 59 months, were studied. The fish were killed by placing them in ice to anaesthetize them, and the bodies fixed in iced Bouin or Carnoy's fluid (Comfort, 1961). They were then embedded in paraffin wax, and serially sectioned at 10/~ throughout their entire length. The series were mounted, and stained with haematoxylin and eosin. The growth in length of the gonad with age, in relation to the growth in length of the body, was estimated by counting the number of sections upon which testicular tissue appeared. Since the thickness of the sections was known (10#), the extent of the testis could be obtained. This value was expressed as a percentage of the body length, measured from the tip of the snout to the base of the caudal peduncle. The growth in width of the testis was also measured, readings being taken from ten sections about the mid-point of the gonad; the mean value obtained was expressed as a percentage of the body width. The cells comprising the germinal tissue were classified in order of their development as follows: (a) Primary spermatogonia: large faintly staining cells with a large rounded central nuclens, having a single central darkly staining nudeolus and a heavy chromatin network. (b) Secondary spermatogonia: morphologically similar to the primary spermatogonia, but smaller, with less abundant cytoplasm. The nucleolus was less prominent. e Present address: Heron Island Research Station, Great Barrier Reef, Australia. 17
18
A. D. WOODHEAD AND SALLY ELLETT
(c) Primary spermatocytes: cells of approximately the same dimensions as the secondary spermatogonia, but the chromatin threads of the nucleus stained less intensely and had become aggregated into distinct small droplets. (d) Secondary spermatocytes: smaller than the primary spermatocytes, with the chromatin of the nucleus more evenly dispersed, and fewer aggregations. This stage occurred less frequently than other stages of development, and may represent a shorter phase of spermatogenesis. (e) Spermatids: relatively small rounded cells, with a thin ring of cytoplasm surrounding an elliptically-shaped nucleus. (f) Spermatozoa: developed from the spermatids, with an elongated compact nucleus and a cytoplasmic tail of approximately 32/z in length (varying between 27 and 36/~). Maturation of the fish testis does not always proceed simultaneously throughout the gonad, and spermatogenesis may be further advanced in the central than in the peripheral areas. This was the case in the guppy, and in order to obtain a representative assessment of the state of gonadal maturity, every twentieth section along the length of the testis was examined. The cell population of the lobules were classified as "ripe"--those containing spermatids and spermatozoa---or "unripe"--those with earlier stages of cell development. The findings were expressed as the percentage of ripe lobules in the testis. The amount of connective tissue and germinal tissue in the testis was assessed by projecting an image of a section on to a screen, upon which a vertical line had been drawn, and marking off the extent of each component. Measurements were made every 100/~ along the testis, and the results were expressed as the percentage of connective tissue in the testis. Measurements were made of the thickness of the testis wall, taking 25 measurements from sections near the centre of the testis. RESULTS
The testis in young fish The testis, covered by a thin layer of peritoneum, was seen to be subdivided into large numbers of lobules supported by connective tissue extensions of the tunica albuginea. Each lobule was composed of numerous cysts of germ cells, which apparently divide and develop through successive cell generations before producing mature spermatozoa (Fig. 1). In the fully ripe testis, the cyst walls have ruptured, and spermatozoa were seen free in the lumen of the lobule, which communicated with the efferent duct of the testis. In young males, ranging in age from 2½ to 8 months, the tunica albuginea was composed of one or two layers of connective tissue cells, and measured less than 5/z in thickness (except in one individual). The lobular walls were tightly packed with cysts which contained cells at all stages of spermatogenesis, the most frequently occurring cell stage in unripe lobules being the primary spermatocyte. Generally the lobules at the periphery of the testis were unripe, whilst towards the centre of the gonad the number of lobules containing spermatids and spermatozoa was increased. Cysts in different stages of development were found in some of the lobules, but in the majority of lobules the phases of development were synchronous. In the ripe unruptured cysts, the heads of the spermatozoa were oriented towards the cyst wall; after rupture, bundles of spermatozoa
FIc. 1. Photomicrograph of the testis of the guppy, showing various cell types : (a) Secondary spermatogonia; (b) Primary spermatocytes; (c) Secondary spermatocytes ; (d) Spermatids ; (e) Spermatozoa. Magnification × 469.
facing p. 18
b FIG.
5.
Photomicrograph
of the testis wall: (a) in a guppy 59 months. Magnification x 490.
aged 8 months;
(b) in a guppy
aged
ENDOCRINE ASPECTS OF AGEING IN THE GUPPY, LEBIS~ES RgTICUIMTU$ (PETERS)
19
were seen lying closely packed around the lobule walls. The central lumen of the lobules usually contained a fluid which was faintly eosinophilic. There was some variability in the percentage of ripe lobules in the testis of young males. Thus in two guppies aged 2½ months the proportion of ripe lobules was 34 per cent and 46 per cent, and in one of 3½ months it was 70 per cent.
Ageing changes in the testis The length of the testis in relation to body length. There was no evidence of any significant variation in the length of the testis in relation to body length during life; correlation between these two factors was significant only at the 50 per cent level. In nine fish less than 2 years old the testis extended over 20-30 per cent of the standard body length, and in four older fish it had increased only to 31-34 per cent. The width of the testis. There was no evidence of a change in testis width withincreasing age. A regression analysis of age against testis width, expressed as a percentage of the body width, showed significance at the 10 per cent level only. Changes in the numbers of ripe lobules with age. During the lifespan all the males showed an increase in the percentage of lobules containing spermatids and spermatozoa, which was significant at the 1 per cent level (Fig. 2). The relationship obtained was Y - ~ 0.63X + 45.45 where X = age in months and Y-~-percentage of ripe lobules. The correlation coefficient 'r' was 0.75. 100
.~-
75 • / I
i /
--
50
• I
i
I
t
t
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25
I
I
I
I
I
I
I
I
I
I
I
I
5
10
15
20
25
30
35
~0
~5
50
55
60
Age in months
Fzo. 2. The percentage of lobules containing spermatozoa or spermatids in the testis of the guppy with increasing age. The regression is shown by a broken line.
20
A. D. WOODHEAD AND SALLY ELLETT
In seven fish less than 1 year old, the proportion of ripe lobules (27-70 per cent) was more variable than in seven older fish (50-87 per cent). Unripe lobules were generally found at the periphery of the testis. In fish older than 30 months, the spermatozoa in some of the lobules appeared degenerate (these are discussed below), and the number of these lobules increased with advancing age. However, all cell stages of spermatogenesis were represented in the testis throughout life, and even in the testes of senile fish cysts of spermatogonia and spermatocytes were seen, although these were fewer in number. Changes in the amount of connective tissue in the testis with age. There was a pronounced increase in the amount of connective tissue in the testis with advancing age (Fig. 3). In fish of 2~L8 months the connective tissue between the lobules was sparse, and an average value of 10 per cent was obtained. The first indication of an increase in the amount of stroma was seen in a fish of 13½ months of age, where areas of thickened stroma were found at the periphery of the testis. When tested, this increase was found to be significant at the I per cent level. 5045 ~- 4O ._= 35 / ,,.e
/
3O
•
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~
t I /
25
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10
I 5
I 10
I 15
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I I 25 30 Age in months
I 35
1 40
I 45
i 50
I 55
i 60
FIc. 3. T h e percentage of connective tissue in the testis o f the g u p p y at various ages. regression is s h o w n b y a broken line.
The
Linear regression analysis gave a relationship: Y ~ 0.38X + 12.10 where X ~ age in months and Y ~ percentage connective tissue in the testis. The correlation coefficient 'r' was 0.76. Proliferation of the connective tissue was usually greater at the outer areas of the testis, where isolated cysts containing spermatids and spermatozoa lay embedded in the
ENDOCRINE ASPECTS OF AGEING IN THE GUPPY, LEBISTE8 RETICUL,4TU,,q (PETEI~S)
21
thickened stroma. In young guppies the centre of the testis remained relatively free from infiltration. Multiplication of connective tissue continued with age, gradually extending into large areas, particularly at the periphery of the testis. A comparison of the percentage of stroma at a point one-tenth of the way along the length of the testis (from the anterior end) gave values of 12 per cent in two fish less than 2 years old, and 52 per cent in two fish older than 2 years. However, at the centre of the testis (half-way along the length) the values were 13 and 14 per cent, respectively. Eventually, in senile fish, extensive thickening had taken place throughout the testis, until the stroma had gradually spread to occupy much of the space which had previously been filled with spermatogenic tissue. In association with this there was a decrease in the amount of germinal tissue in the testis with age; the testes of fish of less than 1 year old contained approximately 87 per cent germinal tissue, compared with 68 per cent in fish of 30 months and over. 15-
13 12 17 --I0 =c:l9 B
,....-. I
~7
1 t /
~5
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..... /
~5
I
4
3 2 I 0
I 5
I 10
I 15
i 20
I I I 25 30 35 Age in months
I 40
I L,5
I 50
55
I 60
FIG. 4. T h e increase in thickness of the testis wall with age. T h e regression is shown by a broken line.
Changes in thickness of the testis ~oall with age. The tunica albuginea thickened with age, until, in the older fish, it was frequently twice its original thickness; this increase was significant at the 5 per cent level (Fig. 4). A linear regression analysis of the thickness of the testis wall on age gave the relationship: Y = 0.08X + 3.51 where X = age in months and Y -k- thickness of tunica albuginea (~). The correlation coefficient 'r' was 0.51.
22
A. D. WOODHEAD AND SALLY ELLETT
In young fish the cells of the connective tissue sheath were elongated and thin, with a long narrow nucleus, and the covering was composed of one or two layers of cells. In older fish the walls had become thickened by proliferation of the connective tissue elements, and were composed of six or seven layers of cells. Some thickening of the tunica albuginea was undoubtedly due to contraction of the connective tissue cell fibres, which appeared shorter and thicker than in young fish, but the increase in the number of the cells accounted for the larger part of the recorded increase in thickness. The appearance of the testis wall in young and senile guppies is shown in Figs. 5(a) and
(b). Abnormalities of spermatogenesis with age The material available for this study was limited, so that it was not possible to follow in detail the chronological sequence of ageing events in the testis. However, several features were regularly recorded in the testes of old fish. Degeneration of the cells in the early phases of spermatogenesis was recorded very infrequently. In one guppy, nuclear fragmentation had taken place in the secondary spermatocytes, and, in the same individual, multinucleate cells, possibly also spermatocytes, were present; but both of these types were few in number. Generally cells in the earlier phases of spermatogenesis showed few morphological differences even in the most senile fish, compared with young individuals. Some abnormalities of the later stages of spermatogenesis were apparent in fish of 13½ and 16 months of age, where cysts with fragmented spermatozoa were sporadically recorded; their numbers increased with advancing age. In a few cysts only fragments of pycnotic nuclei remained. A further consistent change was one in which the stroma surrounding some of the cysts containing spermatozoa had proliferated and become arranged in closely packed concentric layers around the cyst. Another prominent change was one in which the cells of the cyst wall had become tall, in some cases almost obliterating the cyst cavity and the spermatozoa contained within. Other atypical cysts were seen far less frequently. In some of those which contained atypical spermatozoa, the fluid contained in the central cavity was basophilic or colourless, and was represented by a much shrivelled mass which appeared to have been formed by the solidification of the fluid and its gradual shrinkage. This change was not regarded as a fixation artefact. These features of the senile testis were usually found at its outer edges and in the proximal region. Whilst atretic cysts were common in senile guppies, complete degeneration of the testis was not encountered in any fish of this series, and often the centre of the testis of senile fish appeared normal and contained apparently normal spermatozoa. Pigmentation of the tunica albuginea of the testis was regularly seen in older fish, and accumulation of pigment also occurred in the stroma. Melanin deposits were generally localized in large cells with ragged, irregular outlines. Cells with a heavily staining nucleus and a yellow-brown pigmented cytoplasm were also found in young males, but occurred much more commonly in senile fishes; groups of these cells were sometimes associated with deposits of melanin. The yellow-brown cells were distinguishable from normal interstitial tissue and did not have the appearance of adipose cells. Adipose tissue was present in the testes of males of all ages, but in older fish there appeared to be more adipose cells associated with the sperm duct than were seen in young fish.
ENDOCRINE ASPECTS OF AGEING IN THE GUPPY, L E B I S T E S R E T I C U L A T U S (PETERS)
23
The interstitial tissue of the testis of the guppy Since the slides of the guppies had been stained with a general purpose stain, no detailed study of the interstitial cells of the testis could be made. These cells were seen embedded in the stroma, and usually abutting the walls of the lobules of all the testes examined. They were large and rounded, and had a central nucleus with a single prominent nucleolus; their cytoplasm was abundant and showed faintly eosinophilic granules. Their histological appearance in senile males was very similar to that in young fish. DISCUSSION The testes of Lebistes show well marked ageing changes, their gradual involution being characterized by a replacement of spermatogenic tissue with connective tissue, by a thickening of the tunica albuginea, and by an increasing incidence of atretic cysts. The first signs of ageing were seen in a fish of 13½ months, in which the testis wall had thickened and augmentation of connective tissue had occurred. Ageing changes were regularly established in fish over 30 months old, and progressively increased with advancing age, although the extent of these changes in the testis showed considerable individual variation. However, there seemed to be little change in the interstitial cells throughout life. We have only found one previous histological study of the senile testis in fish, made by Rasquin and Halter (1951) on characins, Astyanax mexicanus. These authors reported an increased formation of insoluble calcified "concentrations" in the testis of Astyanax, which they believed were similar to those found in the ageing mammalian prostate gland. The basophilic concentrations seen in some lobules of the testis of senile Lebistes appear to have arisen by solidification or condensation of sperm duct fluid. Since our material had already been fixed and stained, we could not employ the techniques used by Rasquin and Hafter to show the presence of calcium in these concentrations. No assumptions can therefore be made concerning the resemblances between these structures in the testes of Lebistes and Astyanax. However, the concentrations were not as numerous in senile Lebistes as in Astyanax. Rasquin and Hafter also described the infiltration of connective tissue into the gonad of senile Astyanax, although this did not occur throughout all the testis, a large part of which was still producing spermatozoa. Although apparently normal spermatozoa were present in the testis of aged guppies, it seems likely that the progressive increase in the number of atretic lobules with age might occasion a progressive decline in reproductive performance, and it is notable that Comfort (personal communication) finds an early and rapid decline in male fertility in Lebistes. Atretic lobules were not described in Astyanax, but Rasquin and Halter reported the presence of cells with hyperchromatic nuclei and yellow cytoplasm. A prominent component of the interlobular tissue of Astyanax was adipose cells; these were also found in all the guppies examined and became more abundant with age. Removal of the pituitary gland in bony fishes results in the gradual regression of the gonads, which become reduced in size (Atz, in Pickford and Atz, 1957). The later stages of spermatogenesis are arrested, and those present at the time of operation gradually disappear, until the reduced testis finally consists of cysts of spermatogonia. Barr (1963) found that the testes of hypophysectomized plaice, Pleuronectes platessa L., were eventually composed of a few isolated cysts of spermatogonia lying in a very thick connective tissue stroma. Whilst some of the ageing changes in the testis of Lebistes may be
24
A. D. WOODHEAD AND SALLY ELLETT
due to a decrease in the output of pituitary gonadotrophins, so that spermatogenic tissue was lost and progressively replaced by connective tissue, the resemblances between the aged testis in Lebistes and the atrophied testis of hypophysectomized teleosts were not great. T h e r e was no reduction in size of the testis with age in Lebistes, nor did spermatogenesis cease; even in senile individuals apparently normal spermatozoa were produced. Considerable differences in the extent of involution of the testis with age were seen in individual Lebistes, and in this respect the findings were closer to those obtained b y Rasquin and Rosenbloom (1954) who kept characins (.4styanax mexicanus) in darkness for prolonged periods. T h i s treatment, which was claimed to produce a state of "physiological" hypophysectomy, had variable effects on the testis, some fish having m u c h reduced testes and little spermatogenic activity, whilst others were but little affected. T h e present results appear to us to indicate endocrine disfunction with age, rather than a f o r m of "physiological" hypophysectomy.
,4cknowledgememts--We wish to thank Dr. ALEX COMFORT, University College, London, for generously allowing us to use his guppy material for this study. Dr. H. A. COLE, Director of the Fisheries Laboratory, Lowestoft, and Dr. I. W. ROWLANDSof the Wellcome Institute of Comparative Physiology, The Zoological Society of London, gave us considerable support and encouragement throughout the work. We are grateful to the Natural Environment Research Council and The Nuffield Foundation for grants for this study. This paper was written whilst one of us (A.D.W.) held a Research Fellowship of the University of Queensland at Heron Island Research Station, Australia.
REFERENCES
ATZ, J. W. (1957) In The Physiology of the Pituitary Gland of Fishes (Edited by PICKFORD,G. E. and ATZ, J. W.), p. 178-270. New York Zoological Society, New York. BARR, W. A. (1963) Gem. comp. Endocrinol. 3, 216-225. COMFORT, A. (1960) Gerontologia 4, 177-186. COMFORT, A. (1961) Gerontologia S, 209-222. RASQUIN, P. and HA~r~, E. (1951)ft. Morph. 89, 397-404. R_~SQUIN, P. and ROSENBLOOM,L. (1954) Bull..din. Mus. nat. Hist. 104, 359-426. WOODHEAD,A. D. and ELLETT, S. (1966) Exp. Geront. 1, 315-330. WOODHEAD,A. D. and ELLETT, S. (1967a) Exp. Geront. 2, 73-77. WOODHEAD, A. D. and ELLETT, S. (1967b) Exp. Geront. 2, 159-171. S u m m a r y - - A histological study was made of the testes of guppies ranging in age from 2½ to 59 months. The testis showed a gradual loss of spermatogenic tissue and a progressive infiltration of connective tissue with age. The connective tissue sheath of the testis had thickened considerably in old fish. Atrophic and degenerate cysts were also found in senile fish, but these usually occurred side by side with apparently normal cysts. Even in extreme old age apparently normal spermatozoa were produced by the testis. Some of the changes in the senile testis of Lebistes resembled those occurring in the testes of hypophysectomized fish, and it is possible that these may result in part from a decline in the output of pituitary gonadotrophins in old age. R t s u m G Les testicules de guppies (Lebistes du Vtntzuela) ~gts de deux mois et demi A 59 mois ont fait l'objet d'une 6tude histologique. On y a relevt, ~tmesure du vieillissement, une perte graduelle de tissu spermatog~ne et une infiltration progressive de tissu conjonctif. La gaine de tissu conjonctif du testicule s'~paississait
ENDOCRINE ASPECTS OF AGEING IN THE GUPPY, L E B I S T E S RETICUL.4TUS (PETERS)
consid~rablement chez les poissons ~g~s. Les poissons ~g~s pr~sentaient aussi des kystes atrophiques et d~g6n~r~s, mais ceux-ci voisinaient g~n~ralement avec des kystes apparemment norm_aux. Les testicules produisaient des spermatozoides apparemment normaux jusqu'~ u n {tgeextr~mement avanc6. Certaines modifications des testlcules s~niles de Leb/stes ressemblaient ~ celles qui se manifestent dans les tes= ticules de poissons ayant subi l'hypophysectomie et il est possible qu'elles soient, dans une certaine mesure, la consequence d'une diminution de la production de gonadotrophines bypophysaires au cours de la vieillesse.
Z u s a m m e n f a s s u n g - - E i n e histologische Untersuchung an Testes von Guppies im Alter yon 2½ bis 59 Monaten wurde durchgefdhrt. Die Testes zeigten einen aU= m~_lichen Rtickgang des sperrnatogenen Gewebes u n d fortschreitende Infiltration mit Bindegewebe mit dem Alter. Die bindegewebige Hiille der Testes war bei alten Fischen betr~chtlich verdickt. Bel senilen Fischen fanden sich auch atrophische u n d degenerative Zysten. Diese traten aber gew~hnlich gemeinsam n-tit offenbar normalen Zysten auf. Auch im extrem hohen Alter produzierten die Testes offenbar norrnale Spermatozoen. Einige der VerLnderungen in senilen Testes von Lebistes ~.nelten denen in Testes hypophysektomierter Fische. Es ist m6glich, daft diesc zum Tell durch Abfall der Gonadotropinproduktion der Hypophyse in h6hcren Alter entstehen.
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psi6 6sI.qH Taxze O6HapyxeH~ aTpocbHpovm~n.xe a nereHepRp3nonme traCTS, HO OH~ O6MKHOBeHHOBcTpe'laJmCl,pK/~OMC HHCTaMHHOpMaY~HOrOBK~a. ~ B CoBceM HpeK= aOHHOMcTapOMBoapacre CHepMaTO3OKn.h)HopMaJIbHOFO BK~aB~dpa6aTHBaymc],BceMeHHX= Kax. HeXOTOpHe~ H 3 M C H e ~ B ycTapeBm~c c e M e ~ a x Lebistes HanoMmtaymTagOBbIe B CeMet-mt4gaxpl~i6i i o ~ B e p ~ c ~ l rHIIO~H~KTOMHH,IIO~TOMy~I~TaeTCRBO3MOXH]dM,qTO OHHBSIZBaH~OTqaCTHCHHZeHXeMB~eJ~eim~ nn3TwrapHoro roHa~oTpomma B crapocTH.
25