Vol. 8, pp. 75-80

Asiatic Herpetological Research

1999

Seasonal Variations of Testicular and Epididymal Structure and Plasma Levelsof Testosterone in the Soft-shelled Turtle (Pelodiscus sinensis)
Wei-ping Mao and Zhao-xian Wang
Department of Biology, Nanjing Normal University; 122 Ninghai Road, Nanjing 210097, China.
Abstract.- For purpose to consider the annual cycle of testis in soft-shelled turtle, Pelodiscus sinensis, the testes

and epididymides were examined histologically, and the plasma levels of testosterone was measured byradioimmunoassay (RIA) though the year. Gradual increase in testicular weight from May to August was
followed by a decrease in degrees. Spermatocytogenesis was first observed from late April to early May which
remained active to August. Spermatogonial division became to decline in September and stopped by the end of

November. The epididymal weights rose from August to November and remained heavy during hibernation witha rapid decline of about 53.76% in the mating period of next year. From July to September, the epithelial cells of

epididymal ductus grew and increased in height especially. There were a great number of secretory granules in
the cytoplasm with active synthesis. The plasma concentration of testosterone started to rise in April, which thenfell in

May and June. In July, it rose rapidly to peak levels and then declined to minimum. We suggested that it's

more importantthat

the peripheral testosterone promotes reproductive behaviour and stimulates secretion

synthesis in epithelial cells of epididymis.

Ke\ words.- Pelodiscus sinensis, testosterone.

Introduction
Only scattered and equivocal literature can be foundconcerning the reptilian gametogenetic cycle, theendocrinological function of testis and pituitary gonadinterrelationships.

sively derived

The information is almost exclu-

didymal canals from October until their discharge inthe following April. Seasonal changes in size and histology of the testis and the accessory sexual organs(epididymis) are well known in a lot of reptile species(Lofts, 1969; Courty, 1979), and correlated variations
infrom investigations on squamate spe-

androgen levels are documented but still poorly

detailed.

cies (Bartholomew, 1953; Lofts, 1971; Courty, 1980).
It

One does find, however, a few studies on such reptiles
as turtles,

for example: Chrysemys picta (Callard,1976); Pelodiscus sinensis (Lofts and Tsui, 1977);

Chrysemys dorbigni(Silva,1984).

The authors of

those studies hold identical views that the testicular
structureand function of reptiles changeinannualcycle, and the epithelium of seminiferous tubule has

spermatogenetic ability in several months while inother months it is in a static condition (Lofts, 1987).

some reptiles as in mammals (Callard,erature on its reproduction relates only to its gonads.

Hu Zeng-Gao (1988) reported that the testis of Pelodiscus

the results of their studies on the histological and his-tochemical changes in the testis. They were of theopinion that Pelodiscus sinensis has a postnuptial patternwhich the spermatozoa are produced soon aftermating period. The spermatozoa are stored in the epi-

Hews

reported in a few species of reptiles but data tend to bediffer with different species (Bourne and Seamark.

1975; Callard, 1976; Silva, 1984). Some researchers

suggested that the spermatogenesis correlated withplasma testosterone levels

in turtles(Kuchling.

1981; Silva, 1984). In other references we found thatthe highest plasma testosterone levels did not appear
in spermatogenesis period, but in mating period (Callard, 1976). Lofts and Tsui (1977) considered that thesinensisproduced sperms in reproductive
period, which were used for copulation in same
period. Lofts and Tsui ( 1977) in Hong Kong reported

1967;

and Kime, 1978; Courty, 1979). Seasonal changes in
the level of circulating testosterone have beenthe

The aquatic soft-shelled turtle, Pelodiscus sinensis, lives in rivers, lakes and ponds. The available lit-

has been shown by metabolic conversion of pre-cursors that testosterone is the principal androgen ininterstitial tissue

was active in steroids synthesis dur-

ing mating period, but inactive during spermatogenesis period. The purpose of our study, therefore, was todetermine if the variations in the plasma testosterone

levels during the annual cycle in Pelodiscus sinensis

are correlated with histological variations of the ger-minal epithelium and epididymal canals.

Vol. 8, p. 76

Asiatic Herpeiological Research

1999

Material and Methods

twice with 5 ml of anhydrous ether, with an average

Animals and samples collection

recovery of 96%. The extract was dried at 40° by constant temperature bath, and dissolved in 2 ml phos-

The samples used in our experiments were provided
by Chang Zhou Breeding Center of Soft-Shelled Tur-phate-buffered saline containing gelatin (GPBS). weplaced 0.5 ml of this solution in duplicate assay tubes(near Nangjing in Jiangsu Province in east China).

and incubated with radio labeled testosterone (H-T

The breeding environment was similar to their naturalliving condition. Except December and January every
month we obtained blood plasma, testes and epid-

for 18-24 h at 4°C. After removing the unbound frac-tle

idymides that those were from six adult male

soft-

Pelodiscus

bodyshelledturtles,sinensis(there10.000 cpm) and anti-testosterone antiserum (0.1 ml)tion with dextran-coated chorale, theweights were about 650-1000g) in one year. The
blood plasma samples were stored at -24° until thegram

testosterone analysis was performed. The tissue sam-analysis

were weighted and±SEM.

ples of testes and epididymides

samples wereplaced in scintillation fluid (TP-POPOP-toluene) andthe radioactivity (cpm) was detected on a liquid scin-

RIA data were analyzed with a pro-tillation counter.

autilizingweightedlogic-log

regression

IBM PC. All data are as the mean

on anfixed in Bouin fluid.

Results
Histological procedures
For the histological examination, a piece of testis anda piece of epididymis (about

25mm"

)

Weight and histology
The seasonal weight

were fixed invariations in testes

and epid-

Bouin fluid for 24 hours. After discolored, the sam-idymides of Pelodiscus sinensis are shown as unit

ples were embedded in paraffin, cut in 5um sections,and then stained with haematoxylin and eosin.

body weight in Figure 1. From March (shortly after
emergence from hibernation) to April, testicular

Testosterone assay

weights remained low (particularly in April). The
seminiferous tubules remained atrophied and sper-

We determined the plasma concentration of testosterone by radioimmunoassay (RIA) using a technique provided by WHO. The kits used in our

experiments were from Hua Mei Biological Engineering Co. (a Sino-US joint venture). Briefly, the hor-mone was extracted from plasma samples (250-7501)

matogenetically inactive. The germinal epithelium
contained Sertoli cells and spermatogonia only (Fig.

2A). In April there was a significant reduction (byabout 53.76%) in the epididymal weights as spermatozoa were evacuated from the epididymal canals.

During

May

and

June

declined continuously
35

(by

the

epididymal weights
about 19.65%, Fig. 1).
16

r

14

30
12

1

«

25
10
20

1
M
— "
m

15

6

J
10

8-

%

3
(

s.

8

O

month )

Figure 1 Seasonal variations in testis and epididymis weight in the soft-shelled turtle, Pelodiscus sinensis. Each point repre.

sents mean of six values.

1999

Asiatic Herpetological ResearchVol. 8, p. 77

Figure 2. Microscopic figures of testis in the soft-shelled turtle, Pelodiscus sinensis. A. The seminiferous tubules
contracted and contained Sertoli cells (St) and spermatogonia (Sg) in April 200. B.The germinal epithelium contained dividing spermatogonia, spermatocytes and spermatids in May 200. C. Spermatozoa (S) were released

from germinal epithelium in June 132. D. The thickness of spermatogenetic epithelium increased but spermatozoa

decreased in July 132. E: Spermatozoa occluded the lumen of the seminiferous tubules in August132. F: The germinal epithelium atrophied in February 200.

Spermatocytogenesis started from late April to early
May, and the spermatocyte layers increased progressively in germinal epithelium. The testicular weights

began to increase in May. By the end of the month,thegerminal

epithelium

contained dividing

sper-matogonia, spermatocytes and a few spermatides (Fig.

2B). In June, spermatozoa were released from germinal

epithelium

(Fig.

2C)

without

accompanying

apparent increase in epididymal weights. The active
spermatogonia division progressed until July. The
thickness of spermatogenetic epithelium increased
and the diameter of seminiferous tubules reachedVol. 8, p. 78

Asiatic Herpetological Research

T^mm

2500

2000

1999

-

•

1000

500

Figure 4. Seasonal variations in plasma testosterone
concentration in soft-shelled turtle, Pelodiscus sinensis.

Each point represents mean SEM of six values
In the ductus of the epididymis, spermatozoa
could be recognized in great number in most months.

Although in March soon after emergence from hibernation epididymal weights were high and their ductus
were full of numerous spermatozoa (Figs. 1, 3A).

Only in June (after mating period) were there markedly less spermatozoa present in the ductus of the epididymis. At this time the simple columnar epitheliumof epididymis became thin and the nuclei moved
toward the basal region of the cells. From the end ofJuly,

we could see that the spermatozoa were trans-

ported form the testis into the ductus of epididymis,
Figure 3. Microscopic figures of epididymis in the softshelled turtle, Pelodiscus sinensis. A. There were great

and their epithelial cells increased their heights andamount of spermatozoa (S) in the ductus, The simple
columna was thin in March 200. B. The heights of epithelial cells were at maximum, great amount of spermatozoa could be seen in the ductus in August 200.

heights reached the maximum (Fig. 3B).

maximum (Fig. 2D). By the end of August, when thewere at their top values, a large
amount of spermatozoa gathered in the lumen of thetesticular weights

distended seminiferous tubules (Fig. 2E) and passed
into

the

epididymal

canalswiththeepididymal

In September, spermatogonia! division started to

decline with less frequent mitotic figures. The germi-

epithelium

contained

mainlyspermatides andspermatozoa. At that time, testicular weights began todecrease. By the end of November, testis atrophiedhighly.

Andremainingwere only a few spermatozoa
seminiferous tubules. During hiber-there

in thenation, the testis remained atrophied, and the germinal

epithelium was composed of Sertoli cells as well as

spermatogonia only and was heavily lipoidal (Fig.
2F). Often, there were necrotic cells in the lumen of
the seminiferous tubules.

granulesincytoplasm.

In

August, their

Plasma testosterone concentration
The variations of the plasma testosterone concentrations are shown in Figure 4. In March and April(spring), when the testis still remained atrophied, themean values of plasma testosterone concentrationfrom 160.87 pg/ml to 613.79 pg/ml.
After that, there was a fall in plasma testosterone constarted to risecentrations which coincided with the progressive atro-

weights increasing rapidly.

nalsecretory

phy of the epididymides. The plasma content
remained relatively low in June, but rapidly increased
to the highest levels (2104.97 pg/ml) in July. It did not
until September. After that, thevaluesfell
plasmasharply to their baseline levels in
October. It could be seen that in an annual cycle of thedrop significantly

plasma testosterone concentration had two peaks. The
lower peak occurred during the spring and the higher
one was in summer. The higher peak coincided withthe maximal testicular weight and the hypertrophy of
the germinal epithelial cells as well as the full growthof epithelial cells at the epididymal ductus.

1999

Asiatic Herpetological Research

Discussion
The present study has demonstrated

the sper-thatmatogenetic activity of germinal epithelium changes
with season in Pelodiscus sinensis. Resemhling that ofmost turtles reported (Lofts, 1987), its spermatogenesis resumes relatively soon after themating period, sothat advanced germinal stage is completed before theonset of the colder winter months. Spermatozoa are

stored in the epididymal canals until theson of next year. This result

breeding sea-Vol. 8, p. 79

the plasma testosterone. Therefore, there is adiscrepancy about the annual cycle of the secretory activityof interstitial cells between the histochemical

study
reported by Lofts and Tsui (1977) with the direct
determination of plasma testosterone levels in ourstudy. We also found that the seminiferous tubules
have the ability to secrete testosterone (unpublishedresult).
In the turtle
Chrysemys dorbigni and the tortoise
Testudo hermanni, the maximum value of
test-

that

plasmaon the secondary sexual organs inautumn, preparing them for the storage of spermatozoa which occurs in winter (Kuchling, 1981; Silva,

We suggest that this discrepancy might be due to the1984). This situation which can also be observed inour study. The decline of plasma testosterone levels inisconsistent with thatreported by Lofts an Tsui (1977) in Pelodiscus sinen-

We have to pointed out that spermatogenetic peaksis.

we observed is in July and August, which issomewhat later than that observed by Lofts and Tsui.
different temperaturesand photoperiods

atdifferent

latitudes.

The measurement of plasma testosterone in themale Pelodiscus sinensis indicatesthatan annualincrease in peripheral levels begins in spring after
emergence from hibernation period and is interrupted

from the end ofMaythrough June. In July andAugust, the plasma testosterone rises to its maximum
level. This finding is identical to that in tortoise,
Testudo h. hermanni, reported by Kuchling
fresh water turtle, Chrysemys picta, the(1981 ). In

highest levels

were in spring and the lowest were in summer, while

osteroneacts

Pelodiscus sinensis in

May and June is associatedwith the decrease in epididymal weights and the atrophy of the epididymal epithelium. The epididymal
epithelial cells enlarge in July and August, when theplasmalevels arehighest.

They increase

secretary

granules progressively. During this period, the epididymal weights increase significantly accompanying
with the spermatogenesis. The spermatozoa are stored

and matured

inthe epididymis until the following

mating period.
In our study the seminiferous tubules are
spermatogenetically inactive when the plasma testoster-

the testosterone levels increased
significantly (Callard, 1976). Although in Chrysemys dor-

one increases in the mating period. However, whenplasma levels are low in May, the spermatogenesis

higni there was only one peak of plasma testosterone

begins. It implied that the germinal epithelium is sen-inautumnlevels,theincreasereached thestartedinmating period andmaximum value in autumn during theprincipal period of spermatogenesis {Silva, 1984). It issimilar to that in Pelodiscus sinensis.

Lofts (1968) described lipid accumulation in theinterstitialcellsof thetestisinsitive to testosterone when environmental
rises.

temperature

Our conclusion is that the peripheral testoster-

one in Pelodiscus sinensis is more important to support reproductive behavior and stimulate the actionsof epididymis.

Pelodiscus sinensis

which occurred prior to regression of the cells themselves, this indicated the cessation ofsteroidogenesis.
Lofts and Tsui (1977) reported that they detected 3-

HSD (-3-hydroxy steroid deydrogenase) activity in
Pelodiscus sinensis. It showed that the interstitial cells
had a positive reaction for 3-HSD and were considerably depleted of lipoidal droplets in the mating period

Acknowledgments
We wish to express our deep gratitude to Mr. Chang
Hue for assistance in our experiment. We are gratefulto Professor Ning-Zheng Sun and Mr. Shu-Yu

Gu forgraphics technical assistance. This work was supported by a grant of Education Commission of Jingsu
Province, China.

(March- April) and in the period of enhanced epididymal weight (August-October), but in active
spermatogenesis period the interstitial cells were negative tothe tests. And they said that the seminiferous tubules

gave same histochemical feature of high testosterone
secretory activity at the onset of spermatogenesis. As
well known, the secretory activity of interstitial cells

coincides with the variations of plasma testosterone
levels, because the Leydig cells are the main origin of

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