- Email: [email protected]
The spermiation of goldfish (Carassius auratus) as a bioassay for salmon (Oncorhynchus tshawytscha) gonadotropin
GESERAL
AND
COMPARATIVE
ENDOCRINOLOGY
The Spermiation
383-391 (1968)
10,
of Goldfish
as a Bioassay
for Salmon
tshawytscha) FUMIO Fisheries
YAMAZAKI” Research Vancouver
(Carassius
auratus)
(Oncorhynchus
Gonadotropin1 EDWARD
AND
Board of 5, British
Received
M. DONALDSON
Canada, Vancouver Columbia,
October
Laboratory,
Canada
14, 1967
Spermiation in the goldfish (Carassius au7atu.s) is defined as the release of spermatozoa into the sperm duct by thinning of the semen. This phenomenon was completely inhibited by hypophysectomy. Intraperitoneal injection of 0.03 mg per 10 gm body weight of a salmon pituitary gonadotropic preparation caused spermiation in all mature male hypophysectomized goldfish. Human chorionic gonadotropin was also effective for spermiation. A linear relationship was observed between the logarithm of the dose of these two hormones and the spermiation response. Equine luteinieing hormone and ovine folhcle stimulating hormone did not cause spermiation. No cytological change was seen in the seminiferous lobules at spermiation. However, changes were noted in the interstitial eelIs and also in the epithelial cells of the sperm duct. The use of the spermiation response in hypophysectomizcd male goldfish as a bioassay for salmon gonadotropin is discussed.
To date no completely satisfactory biological assay technique for the detection and quantitat,ion of fish gonadotropin has been developed which uses a fish as the test animal. Consequently, animals belonging t.o other vertebrate classes such as the mouse (Otsuka, 1956)) weaver finch (Witschi, 1955) and frog (Fontaine and Chauvel, 1961; Stroganov and Alpatov, 1951) have been used as test animals for the bioassay of gonadotropic extracts of fish pit.uitary glands. However, fish pituitary gonadotropins seem to be different in chemical composition from mammalian pituitary gonadotropins (Pickford and Atz, 1957). Mammalian FSH has no observed effect on reproduction in female goldfish (Yamazaki, 1965) and also in male lake chub, Couesiss plumbeus (Ahsan, 1966). This ‘This work was carried out during the tenure of an F.R.B. Postdoctoral fellowship. ’ Regular address: Faculty of Fisheries, Hokkaido University, Hakodate, Japan. 383
phylogenetic, specificity of the pituitary hormones may put some limitation on the use of classes of vertebrates other than fish as test animals for the bioassay of fish gonadotropins. Therefore, during the extraction and purification of gonadotropins from fish for an investigation of their physiological act,ion it is very important to use fish as test animals. Robertson and Rinfret (1957) and Schmidt et al. (1965) assayed pituitary extracts using rainbow trout with infantile testes. However, these assays take several weeks to obtain results. Clemens and Grant (1964) demonstrated t,hat extracts of pituitary glands of carp and goldfish caused an increase in the water content of the testis, and t,hey pointed out that this gonadal hydration response, being relatively rapid and sensitive, provides an assay for gonadotropin. However, in this case the gonads are removed to check the effect of the injected hormone so that the same test animal cannot be used repeatedly.
384
YAMAZAKI
AND
In the present paper the spermiation response in goldfish is shown to be dependent on gonadotropin and a bioassay is described which utilizes this response for the repeated assay of salmon gonad@ropin. Histological changes occurring in the testis at spermiation are also described. MATERIALS
AND
METHODS
Goldfish (Carassius auratus) were obtained from the Goldfish Supply Company, Stouffville, Ontario. The fish were kept in an indoor stock aquarium at a temperature of lZ-14°C and were fed daily with dry pellets (J. R. Clarke Co., Salt Lake City, Utah). Male fish were chosen from the stock aquarium, their weights ranged from 7.8 gm to 28.5 gm.
The hypophysectomy and sham operations were carried out by the opercular approach (Yamazaki, 1961). The experimental fish received t,he same food as the stock fish. All the operated fish werr kept in 160-litre aquaria with aeration and filtration, which contained 0.25% NaCl solution at 20°C. At lower temperatures (below 14°C) spprmatogenesis occurs but not spermlation. The lattrr phenomenon is accelerated usually by higher water temperature (20°C) and occurs within 24 hours at the higher temperature (Yamamoto et al., 1966). In the first experiment the effect of hypophysectomy on spermiation was investigated. Twentyfour fish which did not show spermiation were selected from the stock tank. Twelve fish were hypophysectomized and the others were shamoperated by exposing the pituitary but not removing it. Spermiation was checked daily for 6 days. The occurrence of spermiation was determined by the application of gentle pressure to the abdomen in an anterior to posterior direction near the genital pore three times. In those fish where spermiation had occurred, this procedurr resulted in an effluence of semen at the urogenital pore. Fish which had been checked for spermiation were divided into three groups. The first group is that which shows no semen after being hand stripped three successive times. This group is scored as -. The second group is that in whirh semen appears at the first gentle stripping, but at, the third time shows none or only a very small amount. This is scored as +. The third one is that which shows a large amount of semen even at the third stripping. This is scored as ++. In the second experiment injections of HCG (Human chorionic gonadotropin, Sigma Chemical Company), LH (Luteinizing hormone equinr, Ma.nn Research Laboratories, Inc.), FSH (Follicle
DONALDSON
stimulating hormone, NIH-FSH-S-2 ovine) and preparations of salmon pituitary glands3 were made intraperiton~ally into hypophysectomized fish which did not show sprrmiation. The dosagcx per 10 gm body weight is presented in Table 2. The responses to the injection were checked 24 hours after the injection. The response of a group of fish was calculated as a percentage of the maximum response possible, a $- being given half the value of a ++. A third experiment was carried out to determine whether the same goldfish could be used for repeated bioassay of gonadotropin. In this experiment five male goldfish (mean BW 11.6 + 2.6 gm) were injected with 30 IU HCG/lO gm BW on day 6 after hypophysectomy. A similar group of control fish were injected with saline. All fish were checked for spermiation on days 7-11. The fish were reinjected on the day following the day on which all HCG-injected fish showed no spcrmiation response. Thus, the fish received further injections of HCG or saline on days 12, 19. 24, 30, and 36. On the intervening days all fish were checked for spermiation. To study the histological changes in the testis at spermiation two groups were examined. Each group contained four fish that had been hypophysectomized for 2 weeks. In one group, four fish were injected with saline solution (0.6% NaCl); in the other group, two fish were injected with 50 IU HCG and the other two fish with 0.1 mg of the salmon pituitary gland preparation. One day after injection, sprrmiation was checked by stripping and the testes were exci.qcd, fixed with Bouin solution, cut at 7p, and stained with Delafield’s hematosplin-eosin. To compare t.he size of the interstitial cells in both groups, the long axis (a) and minimum axis (b) of nuclei of 20 interstitial cells per fish were measured. Then the K value [K = (ab)“‘] was obtained for each cell. A total of 80 interstitial cplls in ca1.11 group were measured. All hypophysectomized fish were checked for completeness of hypophysectomy by dissecting the pituitary region. Results are only presented for those fish whirh were completely hypophysectomizrd. RESULTS Effects of Hypophysectomy on Spermiation. The data is summarized in Table 1.
In the sham-operated
control
group, four
3 The method of extraction and purification will be described in detail in another paper. One gram wet weight of pituitaries gave’ 2.6 mg gonadotropin powder.
EFFECT
OF HYPOPHYSECTOMY
Hypophysectomieed
B.L.a (cm)
B.W. (gm)
2 3 4 5
5.4 6.0 6.1 6.1 6.1
6 7 8 9 10 II 12 Mean
. 1
4
5
6
8.3 8.3 8.3 9.0 10.0
-
-
-
-
-
-
1
-
-
-
-
-
-
2 3 4 -
6.4 6.4 7.1 7.1 6.9 7.2 7.6
11.5 12.3 13.0 13.0 13.0 15.0 18.0
-
-
-
-
-
-
8
-
-
-
-
-
-
i
+ -
+ -
-
-
-
-
6.5
11.7
a Standard
4
0
0
0
0
B.L.a (cm)
B.W. km)
6.2 8.6 6.2 9.0 6.1 10.0 6811.0+++ 7.1 12.1
++ +
++ +
-
8 9 10 11 12
i.0 6.6 6.9 7.2 7.0 6.9 6.4
12.5 13.5 14.0 14.5 15.0 13.0 9.8
++ + + ++ +
Mean
6.i
11.9
Evaluation
(ye)
operativr
2
1
50
3
day 4
5
6
+ -
-
+ -
-
+c + -
++ + ++ +
++ ++ +
+ ++ +
+ ++ +
++ -
42
33
21
17
13
length.
SPERMIATION
TABLE 2 IN HYPOPHYSECTOMIZED MALE SALMON PITUFFARY PREPARATION,
RESPONSE OF
Mean body weight =t SD km)
Salmon pituitary preparation
fish
Post Fish No.
3
4
GOLDFISH
day
3
(%)
THE
Sham-operated
1
Evaluation
IN
fish
Post operative FOh
TABLE 1 ON SPERMIATION
No. of fish used
GOLDFISH 24 HOVRS HCG, LH, OR FSH
Dosage per 10 gm body weight
ReYnse
++
INJECTION
Evaluation (%)
20.0 18.0 19.0 15.0 22.0
+ + f * f
5.6 2.7 2.9 1.0 3.2
5 5 5 5 5
19.6 16.7 18.4 18.6 17.8 19.7
f + f f f _+
2.5 2.9 2.4 2.7 2.0 2.5
5 5 5 5 5 5
LH
11.0 11.0 12.0 11.0 12.0 12.0
+ f k f f +
2.9 2.9 2.4 2.9 2.4 2.4
4 4 4 4 4 4
0.025 mg 0.05 mg O.lmg 0.5 mg 1.0 mg 2.Omg
4 4 4 4 4 4
0 0 0 0 0 0
FSH
21.0 18.6 19.7 17.8 15.4
5 2 f f zk
2.5 2.7 2.5 2.0 1.9
5 5 5 5 5
0.01 mg O.lmg 0.3 mg l.Omg 3.0 mg
5 5 5 5 5
0 0 0 0 0
18.1
+ 3.3
30
30
0
HCG
Control NaCl)
(0.6%
0.0003 mg 0.001 mg 0.003 mg 0.01 mg 0.03 mg
-
AFTER
1 3 10 30 100 300
0.1
385
5 3
4 2 1
IU IU IU IU IU IU
cc/fish
2 5 3
1 3 3 1
2 5
0 20 50 70 100
1 4 5 5
10 30 50 90 100 100
3%
TAMAZAKI
AND
fish did not show any sign of sperm shedding during hand-stripping on any day. But the other eight fish showed spermiation, and they shed sperm by slight. hand-stripping for from l-6 days after the operation. On the other hand, in the hypophysectomixed group, only one fish showed slight semen effluence for 2 days, but the other eleven fish did not show any sign of sperm shedding during hand-stripping on any of the 6 days. Thus, the hypophysectomized group showed a 4% response on the first 2 days and zero response thereafter compared to a 50% response in the sham-operated group on day 1 which decreased gradually to 13% on day 6, indicating that hypophysectomy inhibits spermiation. Effects of Salmon Pituitary Gland Prepara tion ,and Mammalian Gonad0 tropins (HCG, LH and FSH) on Spermiation. The results are summarized in Table 2. Five different doses of salmon pituitary extract from 0.0003 mg to 0.03 mg were injected per 10 gm body weight. Of these dosages, 0.0003 mg/lO gm body weight gave no response, but above 0.001 mg/lO gm BW, the response increased in proportion to the dosage and a 100% response was obtained with the maximum dose of 0.03 mg/lO gm BW. Of the three mammalian gonadotropins tested, only HCG gave a positive response. The response to this hormone also increased in proportion to the dosage above 1 IU/lO BW. A 100% response was obtained after injection with more than 100 IU/gm BW. However, no spermiation rcsponse from the injection of LH or FSH was recorded even when 2.0 mg and 3.0 mg per 10 gm body weight was injected. Thirty hypophysectomized fish were injected with 0.1 cc of 0.6% NaCl solution. They did not show any sign of spermiation. Spermiation Response after Repeated Injection of HCG. The HCG-injected group of goldfish showed the spermiation response after the first and each successive injection of HCG. The mean response was 70 k 9% and there was no indication that the fish were becoming refractory to the HCG even after six injections. Changes in the Testes at Xpermiation. The experimental group in which spermia-
DONALDSON
tion was induced by the injection of HCG or salmon pituitary preparation did not show my cytological changes in the condition of the seminiferous tubules as compared with the control group which were injected with saline. The lobules were fillccl with spermatozoa and had sporadic openings making efferent ducts in both groups (Figs. 1, 2). However, clear changes were found in the sperm duct and in the interstitial cells. The sperm ducts of goldfish are not composed of a single duct but of several complicated ones mediating between the intricate mass of seminiferous lobules and t#he outside of the body (Fig. 3). The ducts are lined by a layer of columnar epithelial cells. With the approach of spermiation, these cells hypertrophy and possess protoplasmic processes on their free surface (Fig. 4). In the group in which spermiation was induced by injection of extract of salmon pituitary gland or HCG, most sperm were separated from the free surface of the sperm duct by a secretion of the sperm duct cells and only a few sperm were embedded in the epithelium of the duct. Cytoplasmic processes were found in some cells and in the free surfaces of the ducts had an irregular appearance (Fig. 6). On the other hand, in the group which were injected with saline and showed no spermiation, epithelial cells were greatly reduced in height and protoplasmic processes were only found rarely. The sperm heads were embedded in the epithelium and the free surfaces of the duct were filled wit,h sperm (Fig. 5). These findings indicate that the sperm duct cells produce a secretion, induced directly or indirectly by gonadotropin, which probably increases the fluidit’y of the semen. Interstitial cells of goldfish are distributed throughout the testes either singly or more generally in clumps surrounded by an interlobular septum or connective tissues. Sometimes they lie along the lobular septa and project, into the lobule (Fig. 7) or in mature fish they may be surrounded by spermatozoa (Fig. 8). They are large cells measuring lop-18 p in diameter. Their cyt,oplasmic borders are commonly indefinite or faint. The nuclei are oval or oblong,
SPERMlATION
BIOASSAY
FOR
GONADGTROPlh‘
387
FIG. 1. Portion of posterior part of testis of hypophysectomized fish injected with saline. x 120. FIG. 2. Portion of posterior part of testis of hypophysectomized fish in which sperm&ion was induced by injection of 50 IU HCG. X 120. FIG. 3. Portion of sperm ducts of intact goldfish. X 120. FIG. 4. Columnar epithelial cells of sperm duct of intact fish which had been transferred from 14°C to 20°C water temperature for 5 hours and then killed. x470. FIG. 5. Portion of sperm duct and columnar epithelial cells of sperm duct of the same fish shown in Fig. 1. The duct is filled with sperm and the epithelial cells are shrunken. Some sperm are embedded in the epithelium. x470. FIG. 6. Portion of sperm duct and sperm duct columnal epithelial cells of the fish shown in Fig. 2. The sperm are det,ached from the epithelium. X470.
388
YAMAZAKI
FIG. 7. Interstitial FIG. 8. Interstitial
50 IU
HCG.
AND DONALDSOS
cells of a hypophysectomized cells of a hypophysectomized
fish which was injected with saline. X690. fish in which spermiation was induced by injection
the long axis being 7 ,u--13p and the short axis being 4 p-8 ,LL.The nucleus has finely reticulated chromatin and a single nucleolus. The cytopIasm was vacuolated and not stained with hematoxylin-eosin. In Graph 1 the K values of the interstitial cells are shown by frequency curve for both the nonspermiation and spermiationinduced groups. In the nonspermiation
70
K
GRAPH 1. Comparison stion
of
x690.
had been induced
group, the K value ranges from 5.8 p-9.3P, the mean K value was 7.6,~ although the curve does not have a clear peak at that point. Some cells in this group had a hooked or polygonal shape or more complicated form, the mean cell size was 10.3~ in diameter (Fig. 7). ,On the other hand, in the group in which spermiation was induced by extract of salmon pituitary or HCG, the
6.0
value
of interstitial cell nucleus with those of hypophysectomized
of
the
K
9.0
10.0
(Id
nucleus
values of hypophysectomized goldfish in which spermigoldfish in which spermiation had not been induced.
SPERMIATION
BIOASSAY
K value ranged from 7.4 p-9.8 p*, the mean K value was 8.5~ and the frequency curve has its peak at 8.8~. The cytoplasm increased in volume, the mean cell size in this group was greater (13.2 p in diameter, Fig. 8). This fact shows that the interstitial cells hypertrophy at the time of spermiation. There were no differences between the interstit,ial cells of the salmon pituitary extract and HCG injected fish. DISCUSSION
In the chemical extraction and purification of fish pituitary gonadot.ropin, it is indispensable to find a biological reaction in fish which is very sensitive to fish pituitary gonadotropin. Roberston and Rinfret (1957) assayed pituitary extracts using rainbow trout with infantile testes under conditions of starvation which inhibits spontaneous testicular growth and development. Schmidt et al. (1965) showed that in immature male t’rout (Solmo gairdnerii) there is a linear relationship between the logarithm of the dose of salmon pituitary extract and response as measured by gonad weight, and suggested that the increase in testis weight of infantile trout may be useful for the bioassay of fish pituitary gonadotropin. However, in this bioassay, several injections are required over a period of a month for one bioassay as gonadal growth in fishes generally takes a long time. Thus this method is unsuitable when many assays are to be carried out. Clemens and Sneed (1962) evaluated the gonadot,ropic activities and relative potency of pituitary glands collected throughout the year from various species of fish by the ovulation test or by an increase in seminal plasma. Since that time Clemens and his co-workers have observed the seminal thinning response or gonadal hydration in carp, goldfish, and other fish and have shown that the response is a very sensitive and relatively quick method for detect,ing the active gonadotropic principle of the pit,uit,ary gland (Clemens and Grant, 1964, 1965; Clemens, Ciereszko, Shoemaker, and Grant, 1964; Clemens and Johnson, 1964). However, in this test., the testes of t,he fish must be removed to determine their water con-
FOR
GONADOTROPIN
389
tent. Thus, the fish are used only once and control animals have to be examined in each test owing to variations in water content that occur. Therefore, many fish are necessary for the bioassay of a large number of samples. The spermiation test using hypophyeectomized male goldfish is recommended for the bioassay of large numbers of samples for the following reasons: 1. The spermiation of hypophysect.omized male goldfish is very sensitive to salmon gonadotropin. 2. In this test the potency of the samples is evaluated by comparing the number of fish responding to the total number used and by the degree of response obtained in each fish. 3. The test requires only 1 day. 4. Spermiat,ion can be checked easily with the naked eye in a few minutes by stripping of the abdomen. 5. Hypophysectomy inhibits spermiation and saline injection to the hypophysectomized male fish never induces spermiation so that control fish are not necessary for the test. 6. One hypophysectomized fish is useful for at least six tests. 7. Suitable male fish can be obtained throughout the year if the fish are kept below 14°C (Yamamoto, et al., 1966), as goldfish are potentially functionally mature all through the year at this temperature. As stated above, the present. study clearly shows that hypophysectomy completely inhibits spermiation. This finding is in contradiction to the findings of Barr (1963) in plaice (Pleuronectes platesa), and Ahsan (1966) in lake chub, (Couesius plunzbeus) . They stated that spermiation in t.hese fish occurred normally even in the absence of the pituitary gland. However, this contradiction may be explained because we feel that they used fish which had already showed sperm oozing just before operation as test animals, and this sperm oozing continues for 3-5 days after hypophyscctomy (Yamazaki, 1962). In discussing spermiation further, it is necessary to suggest. that there is a difference between maturation and functional maturity (Henderson, 1962) in the male fish. The basis for mat,uration in males is spermatogenesis in the lobules and the basis for functional maturitv is
390
YAMAZARI
AND
spermiation, which is the release of spermat,ozoa into the sperm duct, by t.he thinning of semen to facilitatc~ t,ht ejaculation of sperm at the time of spawning. Therefore, after spermiation, white milt is easily shed when the fish is gently stripped. Replacement therapy of salmon pit,uitary extracts into hypophysectomized male goldfish reinitiated spermiation. Injection of 0.03 mg of extract. per 10 gm body weight induced 100% spermiation. HCG also has the ability to induce spermiation. With the latter hormone, 100% reaction was given by the injection of 100 IU/lO gm body weight,. The present. data indicate that there is a linear relationship between the logarithm of the dose of these hormones and the evaluation of the response as measured from the number of the fish responding and the strength of the response. In this test, however, mammalian FSH ancl LH gave no spermiation response. Spermatogenetic aspects of the testes at spermiation resembled those of fish which had not shown spermiation. The lumina and sperm ducts were packed with large masses of mature sperm and cysts of spermatocysts and spermatids could be seen in the peripheral region of the lobules in both groups. However, at spermiation clear changes were observed in the epithclial cells of the sperm duct and interstitial cells. The fact t,hat the epithelial cells of the sperm (1uct.s hypertrophiecl and the spermatozoa were detached from the ducts at spermiat.ion suggest t,hat the cells secrete some fluid into the duct and cause the thinning of the semen. This is supported by the observation of Henderson (1962) in brook t,rout. She described augumental and decreasing changes in the epithelial cells of the sperm ducts at the period of functional maturity, which was defined as the period during which viable spermatozoa are shed when the fish is stripped. Marshall and Lofts (1956) described the occurrence of Leydig cell homologues in the interlobular space and in t,he lining of the lobules of the testes in fish. They divided vertebrates into two categories according to the arrangement of the cells. The first. is the typical vertebrate inter-
DONALDSON
stitial cell (Leydig cell) pattern found in common teleoste such as the three-spined s:ticklc back, the sprat and Tilapia. The second is the so-called lobule boundary cell pattern found in pike ant1 Lnbeo. In goldfish, both int,erstitial cells and occasional lobule boundary cells were recognized as in the rainbow trout (Robertson, 1958), placing the goldfish in a category dist,inct from those which exhibit only one of these two ccl1 types. Craig-Bennett (1931) and others have correlated the seasonal cycle in the int,erstitial cells of the testis with the secondary sexual characterist.ics in Gasterosteus aculeatus. Sathyanesan (1959) observed interstit.ial cells in the cat-fish (Mystus seenghala) and found that the cells are identifiable throughout the year and exhibited increase in number and size which made the isolated groups of the cells prominent during stages C and D (corrcsponding to prespawning and spawning). Mathur (1962) stated that the interstitial cells of Rarbks stigma were seen most clearly in mature testes or in testes which had discharged their sperm. However, it. is still difficult to assign any functional correlation from the accounts in the literature, but the present observations have shown that the interstitial cells are hypertrophiecl at spermiation. This fact suggests that, the interstitial cells have some role at spermiation in the goldfish, but it is not possible to determine from the present experiment, whether spermiation is a direct effect of the pituitarv gonadotropin or whether it is mediated via the steroidogenic properties of the interstitial cells. Experiments are now being carried out to elucida.te this problem. ACKNOWLEDGMENT8 The authors wish to thank Dr. W. 6. Hoar. Department of Zoology, University of British Columbia, with whom we discussed the manuscript, Miss K. Forgeron for the histological, and Mrs. H. Dye for assistance in the purification of the salmon gonadotropin preparations. Purified follicle stimulating hormone (NIH.-FSH-S-2 ovine) was kindly donated by the Endocrinology Study Section of the National Institutes of Health. One of us (Fumio Yamaznki) is also very grateful to Professors Kiichiro Ynmxmoto and Hidejiro
SPERMIATION
BIOASSAY
Niiyama, Faculty of Fisheries, Hokkaido University, for their encouragement in the course of the present study, and to Dr. H. L. A. Tarr, Director Vancouver Laboratory, Fisheries Research Board, for giving him the opportunity to study in the Vancouver Laboratory. REFERENCES S. N. (1966). Effects of gonadotropic hormones on male hppophysectomized lake chub, Couesius plumbeus. Can. J. Zool. 44, 703717. BARR, W. A. (1963). The endocrine control of the sexual cycle in the plaice, Pleuronectes platessa (I,). III. The endocrine control of spermatogenesis. Gen. Camp. Endocrinoi. 3, 216225. CLEMENS, H. P., .~ND SNEED, K. E. (1962). Bioassay and use of pituitary materials to spawn warm-water fishes. U.S. Fish Wildlife Serv. Res. Rept. 61, l-30. CLEMENS, H. P., .~SD JOHNSON, W. W. (1964). Specificity of the gonadal hydration factor in the pituitary of some freshwater fishes. Copeia, No. 2, 389-398. CLEMENS, H. P., CIEHESZKO, L. S., SHOEMAKER, J. D., AND GRANT, F. B. (1964). Partial characterization of the gonadal hydration principle in the pituitaries of carp (Cyprinus carpio). Gen. Camp. Endocrit,ol. 4, 503-507. CLEMENS, H. P., ASJJ GRANT, F. B. (1964). Gonadal hydration of carp (C~prinus culti and goldfish (Carassius auratus) after injections of pituitary extracts. Zoologica 49, 193-210. CLEMENS, H. P., ASD GRANT, F. B. (1965). The seminal thinning response of carp (Cyprinus carpio) and rainbow trout (Salmo gairdnerii) after injections of pituitary extracts. Copeia NO. 2, 174-177. CRAIG-BENNETT, -1. (1931). The reproductive cycle of the three-spincd stickleback, Gasterosteus aculeatus Linn. Phil. Trans. Roy. Sot. London, B219, 197-279. FONTAINE, M., AND CHAUVEL, M. (1961). Evaluation of gonadotropic activity of the pituitary gland of teleost fish and in particular of Salmo salnr L. at diffrrent stages of their development and their migrations. Compt. Rend. Sot. Biol. 252, 822-824. HENDERSON, N. E. (1962). The annual cycle in the testis of the eastern brook trout, Salvelinzls fonlinalis (Mitchill). Can. J. Zool. 40, 631-640. M.~RSH.~I,L, A. J., .~SD LOFTS, B. (1956). The leydig-cell homologue in certain teleost fishes. AVature 177, 701-705. AHMN,
FOR
391
GONADOTROPIN
D. S. (1962). Seasonal variations in the and testes of Barbus stigma (Puntius sophore). Zool. Polon. 12, 131-144. OTSUKA, S. (1956). On the extraction and bioassay of the follicle stimulating and luteinizing substances of the salmon. Endocrinol. Japan. 3, 272-277. PICKFORD, G. E., AND ATZ, J. E. (1957). “The Physiology of the Pituitary Gland of Fishes.” N.Y. Zool. Sot. New York. ROBEIITSON, 0. H., AND RINFRET, A. P. (1957). Maturation of the infantile testes in rainbow trout (Salmo gairdnerii) produced by salmon pituitary gonadotropins administered in cholesterol pellets. Endocrinology 60, 559-561. ROBERTSON, 0. H. (1958). Accelerated development of testis after unilateral gonadectomy, with observations on normal testis of rainbow trout. U.S. Fish Wildlife Serv. Fishery Bull. 127, 9-30. SATHWNESAN, A. G. (1959). Seasonal histological changes in the testis of the catfish My&us seenghala (Sykes). J. Zool. Sot. India II, 52-59. SCHMIDT, P. J., MITCHELL, B. S., SMITH, M., AND TSUYUKI, H. (1965). Pituitary Hormones of the Pacific Salmon. 1. Response of Gonads in Immature Trout (Salrno gairdnerii) to Extracts of Pituitatry Glands from Adult Pacific Salmon (Oncorhynchus). Gen. Comp. Endocrinol. 5, 197-206. STROGANOV, N. S., AND ALPATOV, V. V. (1951). “A new unit for determining the activity of the hypophysis in fish.” Rybn Khozy. 27(g), 5660, cited from Pickford and Atz (1957). WITSCHI, E. (19.55). Vertebrate gonadotropins in comparative physiology of reproduction. Mem. Sot. Endocrinol. No. 4. 149-165. YAMAMOTO, K., NAGAHAMA, Y., AND YAMAZAKI, F. (1966). A method to induce artificial spawning of goldfish all through the year. Bull. Japan. Sot. Sci. Fisheries 32, 977-983. YAMAZ.~KI, F. (1961). The effects of hypophysectomy on the ovary of the goldfish, Carassius MATHUH,
ovary
artratus.
Bull.
Fat.
Fisheries
Hokkaido
ciniv.
12,
167-180. F. (1962). Effects of hypophysectomy on the ovulation, oviposition and sexual behavior in the goldfish, Carassira aura&. Bull. Fat. Fisheries Hokkaido Unit). 13, 39-46. YAMAZAKI, F. (1965). Endocrinological studies on the reproduction of the female goldfish, CarasSOILSartrufus L., with special reference to the function of the pituitary gland. Mem. Fat. Fisheries Hokknido L!niv. 13, l-64. YAMAZAKI,
AND
COMPARATIVE
ENDOCRINOLOGY
The Spermiation
383-391 (1968)
10,
of Goldfish
as a Bioassay
for Salmon
tshawytscha) FUMIO Fisheries
YAMAZAKI” Research Vancouver
(Carassius
auratus)
(Oncorhynchus
Gonadotropin1 EDWARD
AND
Board of 5, British
Received
M. DONALDSON
Canada, Vancouver Columbia,
October
Laboratory,
Canada
14, 1967
Spermiation in the goldfish (Carassius au7atu.s) is defined as the release of spermatozoa into the sperm duct by thinning of the semen. This phenomenon was completely inhibited by hypophysectomy. Intraperitoneal injection of 0.03 mg per 10 gm body weight of a salmon pituitary gonadotropic preparation caused spermiation in all mature male hypophysectomized goldfish. Human chorionic gonadotropin was also effective for spermiation. A linear relationship was observed between the logarithm of the dose of these two hormones and the spermiation response. Equine luteinieing hormone and ovine folhcle stimulating hormone did not cause spermiation. No cytological change was seen in the seminiferous lobules at spermiation. However, changes were noted in the interstitial eelIs and also in the epithelial cells of the sperm duct. The use of the spermiation response in hypophysectomizcd male goldfish as a bioassay for salmon gonadotropin is discussed.
To date no completely satisfactory biological assay technique for the detection and quantitat,ion of fish gonadotropin has been developed which uses a fish as the test animal. Consequently, animals belonging t.o other vertebrate classes such as the mouse (Otsuka, 1956)) weaver finch (Witschi, 1955) and frog (Fontaine and Chauvel, 1961; Stroganov and Alpatov, 1951) have been used as test animals for the bioassay of gonadotropic extracts of fish pit.uitary glands. However, fish pituitary gonadotropins seem to be different in chemical composition from mammalian pituitary gonadotropins (Pickford and Atz, 1957). Mammalian FSH has no observed effect on reproduction in female goldfish (Yamazaki, 1965) and also in male lake chub, Couesiss plumbeus (Ahsan, 1966). This ‘This work was carried out during the tenure of an F.R.B. Postdoctoral fellowship. ’ Regular address: Faculty of Fisheries, Hokkaido University, Hakodate, Japan. 383
phylogenetic, specificity of the pituitary hormones may put some limitation on the use of classes of vertebrates other than fish as test animals for the bioassay of fish gonadotropins. Therefore, during the extraction and purification of gonadotropins from fish for an investigation of their physiological act,ion it is very important to use fish as test animals. Robertson and Rinfret (1957) and Schmidt et al. (1965) assayed pituitary extracts using rainbow trout with infantile testes. However, these assays take several weeks to obtain results. Clemens and Grant (1964) demonstrated t,hat extracts of pituitary glands of carp and goldfish caused an increase in the water content of the testis, and t,hey pointed out that this gonadal hydration response, being relatively rapid and sensitive, provides an assay for gonadotropin. However, in this case the gonads are removed to check the effect of the injected hormone so that the same test animal cannot be used repeatedly.
384
YAMAZAKI
AND
In the present paper the spermiation response in goldfish is shown to be dependent on gonadotropin and a bioassay is described which utilizes this response for the repeated assay of salmon gonad@ropin. Histological changes occurring in the testis at spermiation are also described. MATERIALS
AND
METHODS
Goldfish (Carassius auratus) were obtained from the Goldfish Supply Company, Stouffville, Ontario. The fish were kept in an indoor stock aquarium at a temperature of lZ-14°C and were fed daily with dry pellets (J. R. Clarke Co., Salt Lake City, Utah). Male fish were chosen from the stock aquarium, their weights ranged from 7.8 gm to 28.5 gm.
The hypophysectomy and sham operations were carried out by the opercular approach (Yamazaki, 1961). The experimental fish received t,he same food as the stock fish. All the operated fish werr kept in 160-litre aquaria with aeration and filtration, which contained 0.25% NaCl solution at 20°C. At lower temperatures (below 14°C) spprmatogenesis occurs but not spermlation. The lattrr phenomenon is accelerated usually by higher water temperature (20°C) and occurs within 24 hours at the higher temperature (Yamamoto et al., 1966). In the first experiment the effect of hypophysectomy on spermiation was investigated. Twentyfour fish which did not show spermiation were selected from the stock tank. Twelve fish were hypophysectomized and the others were shamoperated by exposing the pituitary but not removing it. Spermiation was checked daily for 6 days. The occurrence of spermiation was determined by the application of gentle pressure to the abdomen in an anterior to posterior direction near the genital pore three times. In those fish where spermiation had occurred, this procedurr resulted in an effluence of semen at the urogenital pore. Fish which had been checked for spermiation were divided into three groups. The first group is that which shows no semen after being hand stripped three successive times. This group is scored as -. The second group is that in whirh semen appears at the first gentle stripping, but at, the third time shows none or only a very small amount. This is scored as +. The third one is that which shows a large amount of semen even at the third stripping. This is scored as ++. In the second experiment injections of HCG (Human chorionic gonadotropin, Sigma Chemical Company), LH (Luteinizing hormone equinr, Ma.nn Research Laboratories, Inc.), FSH (Follicle
DONALDSON
stimulating hormone, NIH-FSH-S-2 ovine) and preparations of salmon pituitary glands3 were made intraperiton~ally into hypophysectomized fish which did not show sprrmiation. The dosagcx per 10 gm body weight is presented in Table 2. The responses to the injection were checked 24 hours after the injection. The response of a group of fish was calculated as a percentage of the maximum response possible, a $- being given half the value of a ++. A third experiment was carried out to determine whether the same goldfish could be used for repeated bioassay of gonadotropin. In this experiment five male goldfish (mean BW 11.6 + 2.6 gm) were injected with 30 IU HCG/lO gm BW on day 6 after hypophysectomy. A similar group of control fish were injected with saline. All fish were checked for spermiation on days 7-11. The fish were reinjected on the day following the day on which all HCG-injected fish showed no spcrmiation response. Thus, the fish received further injections of HCG or saline on days 12, 19. 24, 30, and 36. On the intervening days all fish were checked for spermiation. To study the histological changes in the testis at spermiation two groups were examined. Each group contained four fish that had been hypophysectomized for 2 weeks. In one group, four fish were injected with saline solution (0.6% NaCl); in the other group, two fish were injected with 50 IU HCG and the other two fish with 0.1 mg of the salmon pituitary gland preparation. One day after injection, sprrmiation was checked by stripping and the testes were exci.qcd, fixed with Bouin solution, cut at 7p, and stained with Delafield’s hematosplin-eosin. To compare t.he size of the interstitial cells in both groups, the long axis (a) and minimum axis (b) of nuclei of 20 interstitial cells per fish were measured. Then the K value [K = (ab)“‘] was obtained for each cell. A total of 80 interstitial cplls in ca1.11 group were measured. All hypophysectomized fish were checked for completeness of hypophysectomy by dissecting the pituitary region. Results are only presented for those fish whirh were completely hypophysectomizrd. RESULTS Effects of Hypophysectomy on Spermiation. The data is summarized in Table 1.
In the sham-operated
control
group, four
3 The method of extraction and purification will be described in detail in another paper. One gram wet weight of pituitaries gave’ 2.6 mg gonadotropin powder.
EFFECT
OF HYPOPHYSECTOMY
Hypophysectomieed
B.L.a (cm)
B.W. (gm)
2 3 4 5
5.4 6.0 6.1 6.1 6.1
6 7 8 9 10 II 12 Mean
. 1
4
5
6
8.3 8.3 8.3 9.0 10.0
-
-
-
-
-
-
1
-
-
-
-
-
-
2 3 4 -
6.4 6.4 7.1 7.1 6.9 7.2 7.6
11.5 12.3 13.0 13.0 13.0 15.0 18.0
-
-
-
-
-
-
8
-
-
-
-
-
-
i
+ -
+ -
-
-
-
-
6.5
11.7
a Standard
4
0
0
0
0
B.L.a (cm)
B.W. km)
6.2 8.6 6.2 9.0 6.1 10.0 6811.0+++ 7.1 12.1
++ +
++ +
-
8 9 10 11 12
i.0 6.6 6.9 7.2 7.0 6.9 6.4
12.5 13.5 14.0 14.5 15.0 13.0 9.8
++ + + ++ +
Mean
6.i
11.9
Evaluation
(ye)
operativr
2
1
50
3
day 4
5
6
+ -
-
+ -
-
+c + -
++ + ++ +
++ ++ +
+ ++ +
+ ++ +
++ -
42
33
21
17
13
length.
SPERMIATION
TABLE 2 IN HYPOPHYSECTOMIZED MALE SALMON PITUFFARY PREPARATION,
RESPONSE OF
Mean body weight =t SD km)
Salmon pituitary preparation
fish
Post Fish No.
3
4
GOLDFISH
day
3
(%)
THE
Sham-operated
1
Evaluation
IN
fish
Post operative FOh
TABLE 1 ON SPERMIATION
No. of fish used
GOLDFISH 24 HOVRS HCG, LH, OR FSH
Dosage per 10 gm body weight
ReYnse
++
INJECTION
Evaluation (%)
20.0 18.0 19.0 15.0 22.0
+ + f * f
5.6 2.7 2.9 1.0 3.2
5 5 5 5 5
19.6 16.7 18.4 18.6 17.8 19.7
f + f f f _+
2.5 2.9 2.4 2.7 2.0 2.5
5 5 5 5 5 5
LH
11.0 11.0 12.0 11.0 12.0 12.0
+ f k f f +
2.9 2.9 2.4 2.9 2.4 2.4
4 4 4 4 4 4
0.025 mg 0.05 mg O.lmg 0.5 mg 1.0 mg 2.Omg
4 4 4 4 4 4
0 0 0 0 0 0
FSH
21.0 18.6 19.7 17.8 15.4
5 2 f f zk
2.5 2.7 2.5 2.0 1.9
5 5 5 5 5
0.01 mg O.lmg 0.3 mg l.Omg 3.0 mg
5 5 5 5 5
0 0 0 0 0
18.1
+ 3.3
30
30
0
HCG
Control NaCl)
(0.6%
0.0003 mg 0.001 mg 0.003 mg 0.01 mg 0.03 mg
-
AFTER
1 3 10 30 100 300
0.1
385
5 3
4 2 1
IU IU IU IU IU IU
cc/fish
2 5 3
1 3 3 1
2 5
0 20 50 70 100
1 4 5 5
10 30 50 90 100 100
3%
TAMAZAKI
AND
fish did not show any sign of sperm shedding during hand-stripping on any day. But the other eight fish showed spermiation, and they shed sperm by slight. hand-stripping for from l-6 days after the operation. On the other hand, in the hypophysectomixed group, only one fish showed slight semen effluence for 2 days, but the other eleven fish did not show any sign of sperm shedding during hand-stripping on any of the 6 days. Thus, the hypophysectomized group showed a 4% response on the first 2 days and zero response thereafter compared to a 50% response in the sham-operated group on day 1 which decreased gradually to 13% on day 6, indicating that hypophysectomy inhibits spermiation. Effects of Salmon Pituitary Gland Prepara tion ,and Mammalian Gonad0 tropins (HCG, LH and FSH) on Spermiation. The results are summarized in Table 2. Five different doses of salmon pituitary extract from 0.0003 mg to 0.03 mg were injected per 10 gm body weight. Of these dosages, 0.0003 mg/lO gm body weight gave no response, but above 0.001 mg/lO gm BW, the response increased in proportion to the dosage and a 100% response was obtained with the maximum dose of 0.03 mg/lO gm BW. Of the three mammalian gonadotropins tested, only HCG gave a positive response. The response to this hormone also increased in proportion to the dosage above 1 IU/lO BW. A 100% response was obtained after injection with more than 100 IU/gm BW. However, no spermiation rcsponse from the injection of LH or FSH was recorded even when 2.0 mg and 3.0 mg per 10 gm body weight was injected. Thirty hypophysectomized fish were injected with 0.1 cc of 0.6% NaCl solution. They did not show any sign of spermiation. Spermiation Response after Repeated Injection of HCG. The HCG-injected group of goldfish showed the spermiation response after the first and each successive injection of HCG. The mean response was 70 k 9% and there was no indication that the fish were becoming refractory to the HCG even after six injections. Changes in the Testes at Xpermiation. The experimental group in which spermia-
DONALDSON
tion was induced by the injection of HCG or salmon pituitary preparation did not show my cytological changes in the condition of the seminiferous tubules as compared with the control group which were injected with saline. The lobules were fillccl with spermatozoa and had sporadic openings making efferent ducts in both groups (Figs. 1, 2). However, clear changes were found in the sperm duct and in the interstitial cells. The sperm ducts of goldfish are not composed of a single duct but of several complicated ones mediating between the intricate mass of seminiferous lobules and t#he outside of the body (Fig. 3). The ducts are lined by a layer of columnar epithelial cells. With the approach of spermiation, these cells hypertrophy and possess protoplasmic processes on their free surface (Fig. 4). In the group in which spermiation was induced by injection of extract of salmon pituitary gland or HCG, most sperm were separated from the free surface of the sperm duct by a secretion of the sperm duct cells and only a few sperm were embedded in the epithelium of the duct. Cytoplasmic processes were found in some cells and in the free surfaces of the ducts had an irregular appearance (Fig. 6). On the other hand, in the group which were injected with saline and showed no spermiation, epithelial cells were greatly reduced in height and protoplasmic processes were only found rarely. The sperm heads were embedded in the epithelium and the free surfaces of the duct were filled wit,h sperm (Fig. 5). These findings indicate that the sperm duct cells produce a secretion, induced directly or indirectly by gonadotropin, which probably increases the fluidit’y of the semen. Interstitial cells of goldfish are distributed throughout the testes either singly or more generally in clumps surrounded by an interlobular septum or connective tissues. Sometimes they lie along the lobular septa and project, into the lobule (Fig. 7) or in mature fish they may be surrounded by spermatozoa (Fig. 8). They are large cells measuring lop-18 p in diameter. Their cyt,oplasmic borders are commonly indefinite or faint. The nuclei are oval or oblong,
SPERMlATION
BIOASSAY
FOR
GONADGTROPlh‘
387
FIG. 1. Portion of posterior part of testis of hypophysectomized fish injected with saline. x 120. FIG. 2. Portion of posterior part of testis of hypophysectomized fish in which sperm&ion was induced by injection of 50 IU HCG. X 120. FIG. 3. Portion of sperm ducts of intact goldfish. X 120. FIG. 4. Columnar epithelial cells of sperm duct of intact fish which had been transferred from 14°C to 20°C water temperature for 5 hours and then killed. x470. FIG. 5. Portion of sperm duct and columnar epithelial cells of sperm duct of the same fish shown in Fig. 1. The duct is filled with sperm and the epithelial cells are shrunken. Some sperm are embedded in the epithelium. x470. FIG. 6. Portion of sperm duct and sperm duct columnal epithelial cells of the fish shown in Fig. 2. The sperm are det,ached from the epithelium. X470.
388
YAMAZAKI
FIG. 7. Interstitial FIG. 8. Interstitial
50 IU
HCG.
AND DONALDSOS
cells of a hypophysectomized cells of a hypophysectomized
fish which was injected with saline. X690. fish in which spermiation was induced by injection
the long axis being 7 ,u--13p and the short axis being 4 p-8 ,LL.The nucleus has finely reticulated chromatin and a single nucleolus. The cytopIasm was vacuolated and not stained with hematoxylin-eosin. In Graph 1 the K values of the interstitial cells are shown by frequency curve for both the nonspermiation and spermiationinduced groups. In the nonspermiation
70
K
GRAPH 1. Comparison stion
of
x690.
had been induced
group, the K value ranges from 5.8 p-9.3P, the mean K value was 7.6,~ although the curve does not have a clear peak at that point. Some cells in this group had a hooked or polygonal shape or more complicated form, the mean cell size was 10.3~ in diameter (Fig. 7). ,On the other hand, in the group in which spermiation was induced by extract of salmon pituitary or HCG, the
6.0
value
of interstitial cell nucleus with those of hypophysectomized
of
the
K
9.0
10.0
(Id
nucleus
values of hypophysectomized goldfish in which spermigoldfish in which spermiation had not been induced.
SPERMIATION
BIOASSAY
K value ranged from 7.4 p-9.8 p*, the mean K value was 8.5~ and the frequency curve has its peak at 8.8~. The cytoplasm increased in volume, the mean cell size in this group was greater (13.2 p in diameter, Fig. 8). This fact shows that the interstitial cells hypertrophy at the time of spermiation. There were no differences between the interstit,ial cells of the salmon pituitary extract and HCG injected fish. DISCUSSION
In the chemical extraction and purification of fish pituitary gonadot.ropin, it is indispensable to find a biological reaction in fish which is very sensitive to fish pituitary gonadotropin. Roberston and Rinfret (1957) assayed pituitary extracts using rainbow trout with infantile testes under conditions of starvation which inhibits spontaneous testicular growth and development. Schmidt et al. (1965) showed that in immature male t’rout (Solmo gairdnerii) there is a linear relationship between the logarithm of the dose of salmon pituitary extract and response as measured by gonad weight, and suggested that the increase in testis weight of infantile trout may be useful for the bioassay of fish pituitary gonadotropin. However, in this bioassay, several injections are required over a period of a month for one bioassay as gonadal growth in fishes generally takes a long time. Thus this method is unsuitable when many assays are to be carried out. Clemens and Sneed (1962) evaluated the gonadot,ropic activities and relative potency of pituitary glands collected throughout the year from various species of fish by the ovulation test or by an increase in seminal plasma. Since that time Clemens and his co-workers have observed the seminal thinning response or gonadal hydration in carp, goldfish, and other fish and have shown that the response is a very sensitive and relatively quick method for detect,ing the active gonadotropic principle of the pit,uit,ary gland (Clemens and Grant, 1964, 1965; Clemens, Ciereszko, Shoemaker, and Grant, 1964; Clemens and Johnson, 1964). However, in this test., the testes of t,he fish must be removed to determine their water con-
FOR
GONADOTROPIN
389
tent. Thus, the fish are used only once and control animals have to be examined in each test owing to variations in water content that occur. Therefore, many fish are necessary for the bioassay of a large number of samples. The spermiation test using hypophyeectomized male goldfish is recommended for the bioassay of large numbers of samples for the following reasons: 1. The spermiation of hypophysect.omized male goldfish is very sensitive to salmon gonadotropin. 2. In this test the potency of the samples is evaluated by comparing the number of fish responding to the total number used and by the degree of response obtained in each fish. 3. The test requires only 1 day. 4. Spermiat,ion can be checked easily with the naked eye in a few minutes by stripping of the abdomen. 5. Hypophysectomy inhibits spermiation and saline injection to the hypophysectomized male fish never induces spermiation so that control fish are not necessary for the test. 6. One hypophysectomized fish is useful for at least six tests. 7. Suitable male fish can be obtained throughout the year if the fish are kept below 14°C (Yamamoto, et al., 1966), as goldfish are potentially functionally mature all through the year at this temperature. As stated above, the present. study clearly shows that hypophysectomy completely inhibits spermiation. This finding is in contradiction to the findings of Barr (1963) in plaice (Pleuronectes platesa), and Ahsan (1966) in lake chub, (Couesius plunzbeus) . They stated that spermiation in t.hese fish occurred normally even in the absence of the pituitary gland. However, this contradiction may be explained because we feel that they used fish which had already showed sperm oozing just before operation as test animals, and this sperm oozing continues for 3-5 days after hypophyscctomy (Yamazaki, 1962). In discussing spermiation further, it is necessary to suggest. that there is a difference between maturation and functional maturity (Henderson, 1962) in the male fish. The basis for mat,uration in males is spermatogenesis in the lobules and the basis for functional maturitv is
390
YAMAZARI
AND
spermiation, which is the release of spermat,ozoa into the sperm duct, by t.he thinning of semen to facilitatc~ t,ht ejaculation of sperm at the time of spawning. Therefore, after spermiation, white milt is easily shed when the fish is gently stripped. Replacement therapy of salmon pit,uitary extracts into hypophysectomized male goldfish reinitiated spermiation. Injection of 0.03 mg of extract. per 10 gm body weight induced 100% spermiation. HCG also has the ability to induce spermiation. With the latter hormone, 100% reaction was given by the injection of 100 IU/lO gm body weight,. The present. data indicate that there is a linear relationship between the logarithm of the dose of these hormones and the evaluation of the response as measured from the number of the fish responding and the strength of the response. In this test, however, mammalian FSH ancl LH gave no spermiation response. Spermatogenetic aspects of the testes at spermiation resembled those of fish which had not shown spermiation. The lumina and sperm ducts were packed with large masses of mature sperm and cysts of spermatocysts and spermatids could be seen in the peripheral region of the lobules in both groups. However, at spermiation clear changes were observed in the epithclial cells of the sperm duct and interstitial cells. The fact t,hat the epithelial cells of the sperm (1uct.s hypertrophiecl and the spermatozoa were detached from the ducts at spermiat.ion suggest t,hat the cells secrete some fluid into the duct and cause the thinning of the semen. This is supported by the observation of Henderson (1962) in brook t,rout. She described augumental and decreasing changes in the epithelial cells of the sperm ducts at the period of functional maturity, which was defined as the period during which viable spermatozoa are shed when the fish is stripped. Marshall and Lofts (1956) described the occurrence of Leydig cell homologues in the interlobular space and in t,he lining of the lobules of the testes in fish. They divided vertebrates into two categories according to the arrangement of the cells. The first. is the typical vertebrate inter-
DONALDSON
stitial cell (Leydig cell) pattern found in common teleoste such as the three-spined s:ticklc back, the sprat and Tilapia. The second is the so-called lobule boundary cell pattern found in pike ant1 Lnbeo. In goldfish, both int,erstitial cells and occasional lobule boundary cells were recognized as in the rainbow trout (Robertson, 1958), placing the goldfish in a category dist,inct from those which exhibit only one of these two ccl1 types. Craig-Bennett (1931) and others have correlated the seasonal cycle in the int,erstitial cells of the testis with the secondary sexual characterist.ics in Gasterosteus aculeatus. Sathyanesan (1959) observed interstit.ial cells in the cat-fish (Mystus seenghala) and found that the cells are identifiable throughout the year and exhibited increase in number and size which made the isolated groups of the cells prominent during stages C and D (corrcsponding to prespawning and spawning). Mathur (1962) stated that the interstitial cells of Rarbks stigma were seen most clearly in mature testes or in testes which had discharged their sperm. However, it. is still difficult to assign any functional correlation from the accounts in the literature, but the present observations have shown that the interstitial cells are hypertrophiecl at spermiation. This fact suggests that, the interstitial cells have some role at spermiation in the goldfish, but it is not possible to determine from the present experiment, whether spermiation is a direct effect of the pituitarv gonadotropin or whether it is mediated via the steroidogenic properties of the interstitial cells. Experiments are now being carried out to elucida.te this problem. ACKNOWLEDGMENT8 The authors wish to thank Dr. W. 6. Hoar. Department of Zoology, University of British Columbia, with whom we discussed the manuscript, Miss K. Forgeron for the histological, and Mrs. H. Dye for assistance in the purification of the salmon gonadotropin preparations. Purified follicle stimulating hormone (NIH.-FSH-S-2 ovine) was kindly donated by the Endocrinology Study Section of the National Institutes of Health. One of us (Fumio Yamaznki) is also very grateful to Professors Kiichiro Ynmxmoto and Hidejiro
SPERMIATION
BIOASSAY
Niiyama, Faculty of Fisheries, Hokkaido University, for their encouragement in the course of the present study, and to Dr. H. L. A. Tarr, Director Vancouver Laboratory, Fisheries Research Board, for giving him the opportunity to study in the Vancouver Laboratory. REFERENCES S. N. (1966). Effects of gonadotropic hormones on male hppophysectomized lake chub, Couesius plumbeus. Can. J. Zool. 44, 703717. BARR, W. A. (1963). The endocrine control of the sexual cycle in the plaice, Pleuronectes platessa (I,). III. The endocrine control of spermatogenesis. Gen. Camp. Endocrinoi. 3, 216225. CLEMENS, H. P., .~ND SNEED, K. E. (1962). Bioassay and use of pituitary materials to spawn warm-water fishes. U.S. Fish Wildlife Serv. Res. Rept. 61, l-30. CLEMENS, H. P., .~SD JOHNSON, W. W. (1964). Specificity of the gonadal hydration factor in the pituitary of some freshwater fishes. Copeia, No. 2, 389-398. CLEMENS, H. P., CIEHESZKO, L. S., SHOEMAKER, J. D., AND GRANT, F. B. (1964). Partial characterization of the gonadal hydration principle in the pituitaries of carp (Cyprinus carpio). Gen. Camp. Endocrit,ol. 4, 503-507. CLEMENS, H. P., ASJJ GRANT, F. B. (1964). Gonadal hydration of carp (C~prinus culti and goldfish (Carassius auratus) after injections of pituitary extracts. Zoologica 49, 193-210. CLEMENS, H. P., ASD GRANT, F. B. (1965). The seminal thinning response of carp (Cyprinus carpio) and rainbow trout (Salmo gairdnerii) after injections of pituitary extracts. Copeia NO. 2, 174-177. CRAIG-BENNETT, -1. (1931). The reproductive cycle of the three-spincd stickleback, Gasterosteus aculeatus Linn. Phil. Trans. Roy. Sot. London, B219, 197-279. FONTAINE, M., AND CHAUVEL, M. (1961). Evaluation of gonadotropic activity of the pituitary gland of teleost fish and in particular of Salmo salnr L. at diffrrent stages of their development and their migrations. Compt. Rend. Sot. Biol. 252, 822-824. HENDERSON, N. E. (1962). The annual cycle in the testis of the eastern brook trout, Salvelinzls fonlinalis (Mitchill). Can. J. Zool. 40, 631-640. M.~RSH.~I,L, A. J., .~SD LOFTS, B. (1956). The leydig-cell homologue in certain teleost fishes. AVature 177, 701-705. AHMN,
FOR
391
GONADOTROPIN
D. S. (1962). Seasonal variations in the and testes of Barbus stigma (Puntius sophore). Zool. Polon. 12, 131-144. OTSUKA, S. (1956). On the extraction and bioassay of the follicle stimulating and luteinizing substances of the salmon. Endocrinol. Japan. 3, 272-277. PICKFORD, G. E., AND ATZ, J. E. (1957). “The Physiology of the Pituitary Gland of Fishes.” N.Y. Zool. Sot. New York. ROBEIITSON, 0. H., AND RINFRET, A. P. (1957). Maturation of the infantile testes in rainbow trout (Salmo gairdnerii) produced by salmon pituitary gonadotropins administered in cholesterol pellets. Endocrinology 60, 559-561. ROBERTSON, 0. H. (1958). Accelerated development of testis after unilateral gonadectomy, with observations on normal testis of rainbow trout. U.S. Fish Wildlife Serv. Fishery Bull. 127, 9-30. SATHWNESAN, A. G. (1959). Seasonal histological changes in the testis of the catfish My&us seenghala (Sykes). J. Zool. Sot. India II, 52-59. SCHMIDT, P. J., MITCHELL, B. S., SMITH, M., AND TSUYUKI, H. (1965). Pituitary Hormones of the Pacific Salmon. 1. Response of Gonads in Immature Trout (Salrno gairdnerii) to Extracts of Pituitatry Glands from Adult Pacific Salmon (Oncorhynchus). Gen. Comp. Endocrinol. 5, 197-206. STROGANOV, N. S., AND ALPATOV, V. V. (1951). “A new unit for determining the activity of the hypophysis in fish.” Rybn Khozy. 27(g), 5660, cited from Pickford and Atz (1957). WITSCHI, E. (19.55). Vertebrate gonadotropins in comparative physiology of reproduction. Mem. Sot. Endocrinol. No. 4. 149-165. YAMAMOTO, K., NAGAHAMA, Y., AND YAMAZAKI, F. (1966). A method to induce artificial spawning of goldfish all through the year. Bull. Japan. Sot. Sci. Fisheries 32, 977-983. YAMAZ.~KI, F. (1961). The effects of hypophysectomy on the ovary of the goldfish, Carassius MATHUH,
ovary
artratus.
Bull.
Fat.
Fisheries
Hokkaido
ciniv.
12,
167-180. F. (1962). Effects of hypophysectomy on the ovulation, oviposition and sexual behavior in the goldfish, Carassira aura&. Bull. Fat. Fisheries Hokkaido Unit). 13, 39-46. YAMAZAKI, F. (1965). Endocrinological studies on the reproduction of the female goldfish, CarasSOILSartrufus L., with special reference to the function of the pituitary gland. Mem. Fat. Fisheries Hokknido L!niv. 13, l-64. YAMAZAKI,