Table Of ContentZOOLOGICAL SCIENCE 10: 337-351 (1993) © 1993 Zoological SocietyofJapan
Annual Patterns of Testicular Development and Activity in the
Chinese Bullfrog (Rana rugulosa Wiegmann)
Yung-Hsi Kao1'2 Paul S. Alexander2 Vivian V. Cheng Yang2
, ,
and John Yuh-Lin Yu1-3
1Endocrinology Laboratory, Institute ofZoology, Academia Sinica, Nankang,
Taipei, Taiwan 115, R.O.C. and 1Department ofBiology,
Tunghai University, Taichung, Taiwan 407, R.O.C.
—
ABSTRACT Annual patternsoftesticulardevelopment andoftesticularandplasma androgen levels
in the Chinese bullfrog, Rana rugulosa Wiegmann, are reported. The animals were collected from a
frog culture farm from November of 1986 through November of 1987, at monthly intervals or more
frequently during the breeding season. Proliferation of primary spermatogonia was observed in
November-December. In January-February, the number of primary spermatogonia decreased while
secondary spermatogonia increased. Primaryand secondaryspermatocytesshowed littlechanges until
February when theirnumber rose sharply. Althougheverystage ofspermatogenesiswaspresent year
around, spermatozoa were massively formed in April-June. Thus, the spermatogenetic cycle of R.
rugulosa, inhabitingsubtropicalTaiwan, isofcontinuoustype. Leydigcellsbegantoproliferate inlate
MarchandwereabundantfromAprilthroughJuly. Testicularandrogencontentshowedbasalvaluesin
January-February and August-December, began to increase in late March, peaked in April, and
dropped markedly in May. The pattern of plasma androgen levels was similar to that of testicular
androgencontent, except peakinginmiddle April-earlyMayanddroppedin lateJune. The unimodal
patternofseasonalchangesinplasmaandrogenlevelsparallelsLeydigcellproliferation,spermatogene-
sis, testicular androgen content, and reproductive activities. In addition, the testicular weight was
negativelycorrelatedwithliverweight,butnotwithfatbodyweightduringanannualreproductivecycle
ofR. rugulosa.
INTRODUCTION ly continuous spermatogenesis; and R. hexa-
dactyla, R. cyanophlyctis, and R. catesbeiana, the
The patterns of reproductive cycle have been continuous spermatogenesis [6, 22, 43, 44, 58].
investigated in many species of anurans. The The secretion patterns of the pituitary gonado-
annual spermatogenetic pattern in anurans varies tropins in relation to testicular development and
from discontinuous spermatogenesis in the activity during a reproductive cycle of anurans
temperate zone to continuous spermatogenesis in have been relatively less studied [13, 19]. It was
tropical species [22], Earlier studies have shown demonstrated that the circulating luteinizing hor-
that ranids, one of the major groups of anurans, mone (LH) and follicle-stimulating hormone
display a diverse type ofspermatogenesis: such as (FSH) were highly correlated, respectively, with
Rana temporaria, R. arvalis, and R. dalmatina the changes in plasma androgen levels and in
exhibiting the discontinuous spermatogenesis; R. testicular weight during an annual cycle of male
tigerina, R. esculenta, and R. perezi, thepotencial- toad, Bufo japonicus [13]. On the other hand,
seasonal changes in testicular and/or circulating
Accepted December 3, 1992 androgen levels in relation to spermatogenetic
ERR„neepdcjroeicinrvtiendroeAliqouuggeyutssttsLuas1b8h,ooru1al9td9o2rbye,seItnntstitot:uteDr.oftJ.YZt.oLo.lioYguy,, ia.nnds/evoerrarle,parnoudruacntsi,vesuacct,hivaistyR_.haevsecubl,eeennt4aesLr7nt,ab3~l8i,sh4.3e„,d
Academia Sinica, Nankang, Taipei, Taiwan 115, 56L R- Perezi [6J< R- catesbeiana [19, 58], B.
R.O.C. japonicus [13], B. mauritanicus [48], Pachymedusa
338 Y. Kao, P. S. Alexander, et al.
dacnicolor [41], R. ridibunda [55], and Rana occi- Young tadpoles with external gills were first
pitalis [17]. However, the annual patterns of observed in late May, and metamorphosing frog-
circulating androgen levels in anurans exhibit a lets were first observed in early June.
great variation of species specificity. Data on rainfall, temperature, and photoperiod
The Indian bullfrog (R. tigerina Daudin) was atTungshih, are summarized in Fig. 1. Theperiod
reported to show a potentiallycontinuous sperma- between MarchandSeptember, 1987wasthe rainy
togenetic cycle [1, 45]. Correlations of the season. The lowest monthly mean airtemperature
androgen levelsin testis andin bloodwith sperma- was 15.4°C in January and the highest was 28.5°C
togenesis and reproductive activity during an in August. Monthly minimum air temperature
annual cycle of this species were, however, not averaged about 5°Cin January andFebruary. The
investigated. photoperiodwasshortest in Decemberandlongest
The Chinese bullfrog, R. rugulosa, is phylogeni- inJune. Shortest andlongestdayshadaboutthree
cally closely related to the Indian bullfrog, R. hours difference in photoperiod.
tigerina [9, 20, 39]. Little information has been
available in male R. rugulosa, inhabiting subtropi- Collection ofblood and tissues
cal Taiwan, and central and southern China, with Monthly samples of 12-25 sexually mature male
respect to its patterns of reproductive cycle: sper- frogs (hatched in April, 1986) were collected from
matogenetic cycle, androgen levels, breeding November, 1986 through November, 1987, except
activity, secondary sexual characters, and changes forsamplingatweeklyorbiweeklyintervalsduring
in various organs in associationwith reproduction, the breeding season from March through early
such as liver and fat body. We have conducted a July. Frogs, after collection, were imediately
series ofstudiesinvestigatingthe annual reproduc- placedintoawetfishingnet andwere anesthetized
tive patterns of R. rugulosa in both males and with ether. Blood (0.8-2.0ml) was drawn from
females. We report here the annual patterns of the conus arteriosus with a 2-ml heparinized
male R. rugulosawith respect to: 1) the changesin syringe, and centrifuged at 1000G for 10min at
the sizes ofbody, testis, liver, and fat body; 2) the 8°C; and plasma (0.4-1.0 ml) was collected and
—
spermatogenetic cycle, and the changes in the stored at 20°C until androgen assay. Both right
proliferationofLeydigcells;3)theandrogenlevels andlefttestesofeach frogwereremovedandtheir
in testis and plasma; and 4) the correlation of lengths and weights were measured. One testis
plasmaandtesticularandrogenlevelswithsperma- was fixed in Bouin's fluid for histological observa-
—
togenesis and other reproductive activity. tions, and the other was stored at 20°C for
androgen assay. Liver and fat bodies were re-
moved and weighed as well.
MATERIALS AND METHODS
Histological examination
Study site Bouin's-fixed testes were wax-embedded, sec-
Ranarugulosawerecollectedfrom afrogculture tioned at 7/im, and stained with Harris' hemato-
farm located in Tungshih (23°40'N, 120°15'E), xylin and eosin. We determined spermatogenetic
Yunlin County, Central Taiwan. Briefly, the frogs cycle by two phases: spermatogenesis, and sper-
were raisedin anopenpondwhichwassurrounded miogenesis. The stages of spermatogenesis were
with fishing nets. They were fed with "frog food" identified accordingtothe method used by Rastogi
manufactured by Fu-Shou Food Co., Taichung, etal. [43]: stage 1, primaryspermatogonia (ISPG);
and the natural preys as well. During hibernation, stage 2, secondary spermatogonia (IISPG); stage
the frogs were dormant under digged earth caves 3, primary spermatocytes (ISPC); stage 4, secon-
or the hayricks of rice and woody box. dary spermatocytes (IISPC); and stage 5, sperma-
Chorussing ofmale frogs was heard from March tids (SPT). Spermiogenesis was recorded by the
through July at the frog culture farm, and was extent ofspermatozoa present in the seminiferous
occasionally heard in August and September. tubules. Both spermatogenesis and sper-
Annual Testicular Activity in Bullfrog 339
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1986 1987
Months
Fig. 1. Monthlycahgnesintherainfall, photoperiod (daylength), andairtemperatureinTungshih, YunlinCounty,
Taiwan.
miogenesis of each animal were determined from androgens, in relative to testosterone (100%),
twentycross-sectioned seminiferoustubules repre- were: 5a-dihydrotestosterone (74%), andro-
senting four different cross-sections ofeach testis. stenedione (1.23%), and androstenediol (0.59%)
The proliferation of Leydig cells in the interstitial [59]. Thus, the data are expressed as "androgen"
tissues was recorded. which mainly representing testosterone and dihy-
drotestosterone.
Radioimmunoassay of androgen in plasma and The coefficient ofvariation (CV) ofthe interas-
testis say was 11.0% (N=5) and that of intraassay was
Androgen concentrations in blood plasma and 6% (N=6) for plasma androgen. The CV of
testis were measured by the radioimmunoassay interassay was 9.2% (N=8) and that ofintraassay
described previously by Chen et al. [4]. The was 2.4% (N=6) for testicular androgen.
steroids in samples were extracted with diethyl
ether. The average recoveries of the added ster- Statistical analysis
oids were 70.2% and 83.3% for plasma and tes- Analysis of variance (ANOVA) and Duncan's
ticular androgen, respectively, and individuals re- multiple range tests were used to examine differ-
coveries were run with each sample. The cross encesamongthenumerousmeans. Linearcorrela-
reactivities of the antiserum with various tionswere performedforallvariables. Probability
340 Y. Kao, P. S. Alexander, et al.
levels of 0.05 and 0.01 were used to indicate than those duringDecember-April. Theweightof
significance in comparison of means and correla- paired testes increased during Dcember-Febru-
tions [49]. ary, peaked in March, sharply declined in April-
May, and remained at low levels in June-October
(Table 1). Differential changes in the weights and
RESULTS
lengths of left and right testes were observed (Fig.
2). The mean weight of the left testis was 20%
Annualchangesinthesizesofbody, testis, liverand higherthan that ofthe right testis;while, themean
fat body length ofthe lefttestiswas40% higherthanthatof
Changes in body weight and body length are the right testis. The shape of left testis was less
shown inTable 1. During the annual reproductive uniform than that of right testis. In addition, the
cycle of R. rugulosa, body weights ranged from annual changes in lengths of both testes were not
30-82g; and body lengths from 70-92mm. The as great as those oftesticular weight although the
bodyweights were greater duringMay-November lengths of both testes rose in February-April.
Table 1. Changes in the sizes of body, testis, liver, and fat body during an annual reproductive
cycle of R. rugulosa
Body3 Organ weights3
Mont)lis1 N2 length weight testes liver fat bodies
(cm) (g) (mg) (g) (g)
1986
Nov 21 21 78.5+1.0 46.9+1.4 86.4+5.9 1.33+0.06 2.40+0.20
Dec 28 12 80.1+1.4 40.9+2.0 88.5+8.7 0.89+0.05 1.63+0.18
1987
Jan 23 20 78.8+0.9 42.5+1.3 117.4+9.2 1.05+0.06 1.38+0.15
Feb 26 20 80.4+0.7 43.0+1.3 132.8+10 0.97+0.05 1.52+0.14
Mar 12 16 80.2+0.8 42.7+1.0 133.2+9.7 0.78+0.03 1.10+0.17
26 16 82.8+0.6 47.9+1.2 131.3+4.8 0.85+0.03 1.28+0.16
Apr 2 15 79.2+0.6 38.5+0.9 115.7+8.2 0.66±0.02 0.82+0.11
9 19 79.2+0.6 39.2+0.9 107.6+4.8 0.59+0.03 0.61+0.09
16 14 79.4+0.7 39.4+1.2 122.1+10 0.62±0.03 0.51+0.01
23 17 77.6+0.7 37.7+3.9 102.5+5.3 0.62+0.03 0.34±0.10
30 16 77.5+0.7 36.2+1.0 110.6+5.2 0.58+0.03 0.29+0.06
May 7 11 78.3+1.0 39.1+1.5 109.8+6.5 0.65+0.04 0.29+0.06
21 17 80.5+1.3 44.8+2.0 85.0+4.6 0.88+0.06 0.14+0.07
Jun 4 19 79.9+0.6 49.2+1.5 71.5+5.7 1.36+0.09 0.14+0.03
28 21 81.3+0.7 55.1+1.9 72.6+4.2 2.07+0.14 0.19+0.03
.
Jul 29 25 81.3+0.8 43.8+1.2 79.4+7.1 1.28+0.07 1.07+0.22
Aug 29 22 84.8+0.7 56.6+2.5 60.8+6.3 3.14+0.28 1.35+0.17
Sep 24 17 81.6+0.9 52.8+1.2 65.0+7.4 3.40+0.28 2.96+0.25
Oct 30 12 80.6+0.6 51.1+1.9 67.3±9.4 3.00+0.27 2.42+0.14
Nov 23 17 81.3+0.7 50.7+2.0 99.0+6.4 1.68+0.11 2.39+0.26
1 The frogs were collected monthly, or more frequently during the active breeding season from march
through June.
2 Number of animals
3 The data are expressed as the means+SE.
Annual Testicular Activity in Bullfrog 341
A A Left a Right
14-
12-
3
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1986 1987
Months
Fig. 2. Changes in the weights and lengths of the left and right testes during an annual reproductive cycle ofR.
rugulosa. The data are expressed as the means+SE, and the number ofanimlas as indicated in Table 1.
Annual pattern in the weights of liver and fat
body are shown inTable 1. The liverandfat body Testicular development andspermatogenesis
weights decreased in November-February, Proliferation of Leydig cells changed with sea-
reached the minimal sizes in early May and late son (Fig. 4). In November-December and
June, repsectively, and increase in early June and January-February, Leydig cells were sparse and
late July, repsectively. In September, both organs difficultto distinguishfromotherconnective tissue
attained the maximal sizes, and then began to (Fig. 4-A). The Leydig cells began to proliferate
decrease in late October. Highly significant cor- in late March and were abundant in the broad
relation was observed between liver and fat body intertubule space in April-July (Fig. 4-B) after
weights (r=0.60, p<0.01). Asshown inFig. 3, an which they rapidly decreasd between the decreas-
inverse relationship existed between the hepato- ing intertubular space (Fig. 4-C).
somatic index and the gonadosomatic index (r'= Seasonal changes in histological observations of
-0.828, p<0.01). The correlation between fat the spermatogenetic cycle in the testis ofR. tiger-
body-somatic index and the gonadosomatic index ina rugulosa are also representatively shown in
was, however, not significant. Fig. 4. The spermatogenetic cycle showed every
I
342 Y. Kao, P. S. Alexander, et al.
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1986 1987
Months
Fig. 3. Therelationsofliverandfatbodyweightstothetesticularweightsduringanannual reproductivecycleofR.
rugulosa. The data are expressed asmean±SE, and the numberofanimals as indicatedinTable 1. GSI. testes
weight/body weight; HSI, liverweight/body weight; FBSI, fat body weight/body weight.
Fig. 4. SeasonalchangesinthespermatogeneticcycleandtheproliferationofLeydigcellsofR. rugulosa. a, thenest
ofprimary spermatogonia; b, the nest ofsecondaryspermatogonia; c, the nest ofprimary spermatocytes; d. the
nest of secondary spermatocytes; e, the nest of spermatids; f. spermatozoa; g. Leydig cells. A. Hibernation
(December). Primary and secondary spermatogonia were the most numerous spermatogenetic cells in this
period. A few clustersofspermatozoa were produced. Leydigcells existed in sparse between the seminiferous
tubules. B. Breeding Season (May). Massive spermatozoa were formed in this period. Leydig cells were
abundantin the intertubularspace. C. PostbrccdingSeason (August). Cellnestsofallstagesofspermatogenetic
cyclewereexistent. Fewspermatozoaoccurredinthisperiod. Leydigcellsweregreatlyreducedinnumberinthe
intcrtubules.
Annual Testicular Activity in Bullfrog 343
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344 Y. Kao, P. S. Alexander, et al.
stage ofspermatogenesis yeararound. Only a few ing in August-October (Fig. 4-C).
clusters of spermatozoa were found in the semi- Significant annual changes occured in the sper-
niferous tubules during hibernation (November- matogenetic cycle (ANOVA, p<0.01) based on
February) (Fig. 4-A). Massive formation of sper- theestimateofthe numberofvariousstagesofcell
matozoa occurred during the active breeding sea- nests from primary spermatogonia to spermatids
son mainlyinApril-June (Fig. 4-B). Spermatozoa (Fig. 5). In November-December of 1986, ISPG
were greatly reducedin numberduringpostbreed- were most numerous, accounting for 48% of the
k
(N 3-
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1986 1987
Months
Fig. 5. Quantitative changes in different stages of spermatogenesis during an annual reproductive cycle of R.
rugulosa. Values are expressed as the average number of cell nests of various spermatogenetic stages per
seminiferous tubule. ISPG and IISPG, primary and secondary spermatogonia; ISPC and IISPC, primary and
secondary spermatocytes; SPT, spermatids.
1
Annual Testicular Activity in Bullfrog 345
MIIIIIIIIIHIIIIIINj
IIIIIIIIIIIIII|IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIII1III1 IIII
hibernation breeding
11:1: early breeding post-breeding
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Months
Fig. 6. The unimodal patterns of changes in testicular androgen content and plasma androgen levels during an
annual reproductive cycle of R. rugulosa. The annual reproductive cycle was divided into four periods:
hibernation, early breeding, breeding, and post-breeding. The data are expressed as the means±SE, and the
number ofanimlas as indicated with Arabic numerals.
346 Y. Kao, P. S. Alexander, et al.
total germinal cell nests; otherstages ofgerm cells (<lng/ml) during hibernation (November-
began to multiply in December. In January and February), increased in late March, peaked in
February, the number of ISPG and SPT declined, early May (14.50+1.05 ng/ml), decreased sharply
but those of IISPG, ISPC, and IISPC increased in late May and June, and reached basal levels
with the rise in ISPC and IISPC lagging behind afterwards (from July through October). Testicu-
that of IISPG. In March there were increases in lar androgen content was also low during hiberna-
the ISPG and IISPG followed by sharp decreases tion period (<1 ng/testis), increased rapidly in
in April. ISPG then exhibited a slight increase early April (3.28+0.28ng/testis) and peaked in
with some fluctuations in May-August; it de- middle (3.88+0.45 ng/testis) and late April (3.90
creased again in September, followed by sharp +0.44ng/testis); the androgen levels were de-
increases in October and November. IISPG re- creased markedly in May, followed by a gradual
mained virtually constant from May through drop afterwards, and reached basal values in
September, and then increased in October. ISPC September (0.7+0.07 ng/testis). During the
decreased in March-May, and increased again in annual reproductive cycle of R. rugulosa, the
June-July; by contrast, a reverse pattern existed difference between maximal and minimalvaluesin
for IISPC in March-July. At this period (March- testicular androgen contentwas 10fold, while that
July), the cell nests of SPC (the sum of ISPC and in plasma androgen was 50 fold.
IISPC) became the most numerous. During the Correlations of plasma androgen concentration
breeding season (March-July), spermatozoa were with testicular development, liver, and fat body
massively formed from SPT. ISPC, IISPC, SPT, are summarized in Table 2. Highly significant
and spermatozoa all reduced in August, and in- correlations were observed between plasma
creased in September-October. androgen levels and testicular androgen content.
There were significant negative correlations of
Plasma and testicular androgen levels plasma and testicular androgen levels with liver
Plasma and testicular androgen levels showed weight and fat body weight respectively, as well as
significant seasonal changes (ANOVA, p<0.05, with two stages of spermatogonia (the number of
Fig. 6). Plasma androgen levels were very low nests of primary spermatogonia, ISPG, and of
Table2. Correlation coefficients (r) ofplasma and testicular androgen levels with sperma-
togenetic cell types, and liver andfat bodyweightsofmale R. rugulosa during an annual
reproductive cycle
Plasma androgen Testicular androgen
Testicular androgen 0.734" 1.0
Testicular weight 0.087 0.188
GSI 0.288 0.426
Liver weight -0.438+ -0.460*
HSI -0.448f -0.466*
Fat body weight -0.702" -0.696**
FBSI -0.719" -0.707**
Primary spermatogonia -0.515+ -0.628*
Secondary spermatogonia -0.499* -0.403
Primary spermatocytes 0.025 0.139
Secondary spermatocytes 0.389 0.141
Spermatids -0.074 -0.019
Correlation coefficients (r) were analyzed from the monthly means of those parameters. GSI,
gonadosomatic index=testis weight/body weight; FBSI, fat body weight/body weight; HSI. liver
weight/body weight; \ p<0.10; *, p<0.05; **, p<0.01.
