photothermal effects on ovarian growth and function in the soft-shelled turtlelissemys punctata...

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 274~41-55 (1996)

Photothermal Effects on Ovarian Growth and Function in the Soft-Shelled Turtle Lissemys punctata punctata

SUPRITI SARKAR, N.K. SARKAR, €? DAS, AND B.R. MAITI Histophysiology Laboratory, Department of Zoology, University of Calcutta, Calcutta-700 019 (S.S., B.R.M.), Department of Zoology, Presidency College, Calcutta-700 073 (N.K.S.), and Department of Parasitology, National Institute of Cholera a.nd Enteric Diseases, Calcutta-700 010 (PD.), India

ABSTRACT Investigations were carried out to find out the role of higher and lower ambient temperatures together with short and long photoperiods on ovarian growth and secretion of steroids (estradiol-17p and progesterone) in the adult female soft-shelled turtle Lissemys punctata punctata during the preparatory, recrudescent, and quiescent phases of its seasonal reproductive cycle. In each phase, females were treated with 3°C higher and 3°C lower than the mean highest and lowest ambient temperatures in association with short and long photoperiods for short-term (4 week) and long-term (8 weeks or more) durations. Short-term high-temperature treatment significantly stimulated ovarian growth and secretion of estradiol-17p during the preparatory phase. Eight-week treatment failed to exercise further stimulation over 4-week treatment, whereas 10- week treatment caused regressive changes of ovary (atresia of follicles). Both short- and long-term high-temperature treatments caused degeneration of ovarian follicles during the recrudescent phase. Low-temperature treatment slowed down ovarian growth and secretion in both the preparatory and recrudescent phases. In the quiescent phase, low- as well as high-temperature treatments exercised little influence over the refractory ovarian growth and secretion. Photoperiod appeared to have no effect on female reproduction in any phase. Thus, high temperature is suggested to have a triggering role on ovarian growth and secretion of estrogen at the early preparatory phase, but once the ovarian function sets in, high temperature seems to have a regressive rather than stimulatory effect on ovarian growth and function. o 1996 Wiley-Liss, Inc.

Temperature and photoperiod are two important environmental factors which have been suggested to exercise various degrees of influence on reptilian reproduction (Licht, '84; Owens and Morris, '85; Whittier and Crews, '87). Ambient temperatures higher than the normal have been reported to stimulate spermatogenesis and testosterone secretion in the males of a number of turtle species, viz., IIFionyx sinensis (Lofts and Tsui, '77; Lofts, '781, Sternotherus odoratus (Graham and Hutchin- son, '79; Mendonca and Licht, '86a), Lepidochelys kempi (Morris, '82), Testudo harmanni harmanni (Kuchling, '82a,b), Chrysemys picta (Ganzhorn and Licht, '83), Caretta caretta (Owens et al., '84), and Chelydra serpentina (Mahmoud, '92). While an earlier report has suggested that photoperiod plays a stimulatory role in testicular recrudes- cence in Pseudemys elegans (Burger, '37), recent workers have reported little influence of photope- nod on male reproduction in Testudo hermanni hermanni (Kuchling, '82a,b), Lepidochelys kempi 0 1996 WILEY-LISS, INC.

(Morris, '82), Chrysemys picta (Ganzhorn and Licht, '83), Sternotherus odoratus (Mendonca and Licht, '86a), and nrrapene carolina (Vivien-Roels et al., '87).

In female turtles, studies have been carried out in a relatively fewer number of species, and diverse results have been reported. In the painted turtle, Chrysemys picta, warm temperature (28OC) inhibits follicular growth and causes regression of ovaries, whereas constant low temperature (17°C) maintains maximum ovarian growth and function; follicles develop but ovulation does not occur at a lower temperature (13°C) (Ganzhorn and Licht, '83). A similar inhibitory effect of prolonged exposure to warm temperature (28°C) on ovarian activity has been reported in the musk

Received May 1, 1995; revision accepted October 12, 1995. Address reprint requests to Dr. Supriti Sarkar, Department of Para-

sitolom, Room No. 207, National Institute of Cholera and Enteric Diseases, P-33 C.I.T. Road, Scheme XM, Beliaghata, Calcutta-700 010, West Bengal, India.

42 S. SARKAR ETAL.

turtle, Sternotherus odoratus; the ovarian function is highest at a lower temperature (18OC) in this turtle (Mendonca, '87). On the contrary, high temperature has been reported to promote ovarian growth and function in a few species. In Chrysemys scripta elegans, the populations living in the lake waters being heated by industrial ef- fluent show a larger clutch-size, indicative of a higher reproductive potential, than those inhab- iting the lake away from industries (Thornhill, '82). In the blandingi turtle Emydoidea blandingi, a few warm days are found to be essential for the females to complete their vitellogenesis during the recrudescent phase of their reproductive cycle (Congdon et al., '83). The females of two species of sea turtles, Lepidochelys kempi and Chelonia mydas, show complete ovarian growth and function at a high temperature (25°C); a significant decline of their reproductive function at cold (11°C) is indicated by decreased plasma levels of vitel- logenic indices (calcium and phosphorus) and estrogen (Moon et al., '90). Controversies also exist with regard t o the effect of photoperiod on female reproduction in turtles. In the soft- shelled turtle (Lissemys punctata granosa, the longest day length during July-August coincides with the peak breeding activity, whereas the short-day length period extending between November and February coincides with the refractory phase (Singh, '77). In Chrysemys picta, long day length in the spring has been suggested t o be important for the final growth of ovarian follicles (Ganzhorn and Licht, '83). On the contrary, photoperiod has been reported to bear no influence on the ovarian activity in any season in the musk turtle, Sternotherus odoratus (Mendonca, '87).

The pattern of female reproductive cycle in the soft-shelled turtle Lissemys punctata punctata is very interesting and distinct from that of the temperate turtles in two important respects, 1) whereas the temperate turtles are either multiclutched, with several peaks of ovarian growth in a year (e.g., Sternotherus odoratus, McPherson et al., '82; Mendonca and Licht, '86b), or single- clutched, with two peaks of ovarian growth, a spring peak followed by ovulation and a fall peak without ovulation (e.g., Chrysemys picta, Callard et al., '78), Lissemys punctata punctata is single- clutched, with a single annual peak of ovarian growth occurring in the rainy season (July-Au- gust) in India (Sarkar et al., '95; in press) and 2) the natural ability of Lissemys to carry on its reproductive functions at warm temperature (28°C

and above) is quite higher than the temperate species. The mean lowest and highest daily temperatures during the recrudescent phase of its reproductive cycle are 26.2 f 0.89"C and 33.65 f 0.94"C, respectively (Sarkar, '93). The temperatures affecting ovarian function in temperate species are naturally not likely to affect sub- tropical turtles. At present, little information is available on the dual role of temperature and photoperiod on the ovarian activity of subtropi- cal turtles. Against this background, we have at- tempted to study the effects of lower and higher ambient temperatures with short and long photoperiods on female reproductive functions in adult Lissemys punctata punctata.

MATERIALS AND METHODS Animals and experimental schedule

Adult soft-shelled turtles, Lissernys punctata punctata Bonnaterre, were collected from different natural populations and reared during the last few years (1988-1995) in a dug-out tank (20' x 15' x 5') under natural environment with food and water available ad libitum and natural shore around the tank, where turtles could nest as well as hibernate according to their necessity The seasonal reproductive cycle of the female turtle has been found to consist of five phases, viz., preparatory phase (March-June), recrudescent phase (July), breeding phase (August-September), regressive phase (October-November), and quiescent phase (December-February) (Sarkar, '93; Sarkar et al., '95; in press). Adult females (body weight: 1-1.25 kg; plastron length > 14 cm) were collected from the rearing tank and neighbouring hibernation field during the preparatory, recrudescent, and quiescent phases of the reproductive cycle. The experimental turtles of each phase were divided into four groups which were treated with different photothermal regimes. Two of the four groups in each phase were exposed to 3°C lower of mean lowest ambient temperature, and the other two were exposed to 3°C higher of mean highest ambient temperature of the respective phase. The mean lowest and the mean highest ambient temperatures for each phase were calcu- lated based on the temperature records of the last 4 years. Either lower or higher thermal treatment was done in association with short and long photoperiod, respectively. Each group was again divided into two or more subgroups for short-term (4 weeks) and long-term (8 weeks or more) treatments.

PHOTOTHERMAI, EFFECTS ON OVARY IN TURTLE 43

TABLE 1. Schematic presentation of different photothermal treatments of adult female Lissemys punctata punctata'

Duration of Reproductive Experiment Group and Mode of Water treatment (week) for phase of turtle started on subgroup treatment temperature Photoperiod different subgroups

Preparatory March 1 1 (A,B,C) LTSP 21°C lOL, 14D 1A4,1B:8, 1C:lO 2 (A,B,C) LTLP 21°C 14L, 10D 2A:4, 2B:8,2C:10 3 (A,B,C) HTSP 38°C lOL, 14D 3A:4, 3B:8, 3C:lO 4 (A,B,C) HTLP 38°C 14L, 10D 4A4,4B:8,4C:lO

Recrudescent July 1 5 (A,B) LTSP 23°C lOL, 14D 5A4,5B:8 6 (A,B) LTLP 23°C 14L, 10D 6A4,6B:8 7 (A,B) HTSP 36.5"C lOL, 14D 7A4, 7B:8 8 (A,B) HTLP 36.5"C 14L, 10D 8A.4, 8B:8

Quiescent December 15 9 (A,B) LTSP 13°C lOL, 14D 9A:4, 9B:8 10 (A,B) LTLP 13°C 14L, 10D 10A:4,10B:8 11 (A,B) HTSP 29°C lOL, 14D llA:4, llB:8 12 (A,B) HTLP 29°C 14L, 10D 12A:4,12B:8

'LT, low temperature; HT, high temperature; SP, short photophase; LP, long photophase; L, light; D, dark.

The schedule of different photothermal treatments has been summarised in Table 1.

A sample of five animals was autopsied on the day of collection of turtles for different experiments in each phase. These samples served as the initial control, whereas the experimental control turtles were maintained in the rearing tank and were autopsied on the day of termination of each set of experiment. All experiments were conducted in thermostatically controlled aquaria adjusted with desired photoperiods. Each aquarium was provided with wooden platforms which allowed the animals to leave the water as needed. Turtles were supplied with small fishes, shrimps, chopped fishes, and earthworms ad libitum twice a day throughout the experiments. The aquarium water was let out twice daily, and the temperature of the freshly added water was approximately matched to that of the previous water. Water temperature and day length were recorded throughout the year.

Autopsy and morphological study Turtles were autopsied by decapitation on the

day of the experiment, for the initial controls and on the day of termination of different experiments for experimental controls together with the treated groups. Following laparatomy, both ovaries of each turtle were dissected out, made free of extrane- ous tissues, and weighed to the nearest gram; the weight was expressed as gonadosomatic index (GSI: g/lOO g body weight). All the macroscopic yolky follicles from each ovary were counted, mea- sured by a slide caliper, and divided into different class sizes according t o the diameter: class I (small: 5-10 mm), class I1 (medium: 10.1-15 mm),

class I11 (large: 15.1-20 mm) and class Tv (preovulatory/ovulable > 20 mmj. Gross morphology of the ovary with its atretic follicles was also studied.

Hormone assay Blood (7-10 ml) was collected in heparinized

tubes from each turtle by cardiac puncture and then centrifuged at 2,000 rpm a t 4°C for 1/2 hr. The plasma was removed and stored at -20°C un- til analysed.

Plasma levels of estradiol-17P (E-17Pj and progesterone (PRO j were determined by radioim- munoassay (Abraham et al., '72). Antisera for both E-17P and PRO and radiolabelled steroids were purchased from Biotecx Laboratories, Inc (Texas). Known amounts of radioinert E-17P and PRO were added to turtle plasma, and the recovery of plasma E-17P and PRO were found to be 86.2% and 84.8%, respectively. The antisera for E-17P and PRO had crossreactivity of 98.2% and 98.8%, respectively. The minimum detectable levels of E- 17p and PRO that could be distinguished from the zero standards were 10 pg/ml and 0.60 ng/ml, respectively The intra-assay and inter-assay coeffi- cients of variation for E-17P were 3.3% and 3.8%, respectively, whereas those for PRO were 5.6% and 9.2%, respectively All plasma samples (200 pyas- say) were analysed in duplicate, and the radioac- tivity was counted in a gamma scintillation counter for 1 min.

Statistics 'Jh determine the differences between the data

on GSI, follicular population, and hormone titers of control and treated groups, Fisher's t-test was performed (Snedecor and Cochran, '71).

44 S. SARKAR. ETAL.

RESULTS Experiments during the preparatory phase

The GSI, number of ovarian follicles, and plasma levels of E-17P and PRO were found to be very low in the initial control turtles at the beginning of the preparatory phase of the reproductive cycle. The values were higher except for the plasma PRO in the experimental controls after 4 weeks (Table 2). In the turtles exposed to low temperature for 4 weeks under both photophases (subgroups 1A and 2A: LTSP and LTLP), ovarian growth, GSI, and plasma E-17P hardly increased beyond the initial control levels, and the values were significantly lower (P < .005) than in the experimental controls; plasma PRO, however, remained unchanged compared to both initial and experimental controls (Table 2). On the contrary, the turtles exposed to high temperature under both photophases (subgroups 3A and 4A: HTSP and HTLP) showed significant increase (P < .OOl) in GSI over that of the initial as well as experimental controls due to an increase in the number of small and medium yolky follicles and also due to the development of some large yolky follicles which were absent in the controls (Fig. 1). Plasma E-17P was also higher than in the initial and experimental controls (P < .001), but PRO remained unchanged (Table 2; Fig. 1).

After 8 weeks, the experimental control turtles showed significant increase (P < .001) in GSI, number of yolky follicles in the ovaries, and in plasma E-17P, compared to the initial controls (Table 3; Figs. 1 ,2) , and the ovaries contained not only many small and medium follicles but a few large follicles too; plasma PRO, however, remained unchanged. In the turtles exposed to low temperature under both photophases (subgroups 1B and 2B: LTSP and LTLP), ovarian growth, GSI and plasma E-17P increased slightly (P < .05 to P < .Ol) beyond the initial control levels but the values were much lower (P < . O O l ) than in the experimental controls (Table 3; Figs. 1, 2). In the turtles exposed to high temperature under both photophases (subgroups 3B and 4B: HTSP and HTLP), the GSI, number of yolky follicles, and plasma E-17p were significantly higher (P < .OOl) than in the experimental controls; plasma PRO showed no discernible change (Table 3; Figs. 1,2).

After 10 weeks, a high GSI along with large number of yolky follicles in the ovaries and high plasma E-17P were found in the experimental control turtles compared to the initial controls (Table 4; Figs. 1, 2). The values observed in the turtles

exposed to low temperature under both photophases (subgroups 1C and 2C: LTSP and LTLP) were slightly higher than the initial control values but were significantly lower (P < .001) than that observed in the experimental controls (Table 4; Figs. 1, 2). Plasma PRO remained unchanged in both initial and experimental controls as well as in treated turtles. In the high-temperature-treated turtles irrespective of short and long photoperiods (subgroups 3C and 4C), the GSI as well as the number of ovarian follicles significantly increased ( P < . O O l ) over the initial control values, but the values were unchanged from those of the experimental controls (Fig. 1). In addition, many atretic follicles of different sizes appeared in the ovaries of the high-temperature-treated turtles. Plasma E- 17p and PRO levels remained unchanged in the latter groups compared to the experimental controls (Table 4; Fig. 2).

Experiments during the recrudescent phase At the arrival of the recrudescent phase, the

initial control females showed much higher values of GSI, number of ovarian follicles, and plasma E-17P than in the initial controls of the preparatory phase (Table 5; Figs. 3,4). After 4 weeks, the experimental control turtles developed a few preovulatory follicles in addition to a large number of other types of follicles in the ovaries (Table 5 ) . As a result, the GSI along with the plasma E-17p level were also high in these turtles (Figs. 3, 4). On the contrary, the GSI and the number of ovarian follicles were significantly lower (P < . O O l ) in the turtles exposed to low temperature under short and long photophases (subgroups 5A and 6A: LTSP and LTLP) in comparison to that of the experimental controls, and the values were similar to that of the initial controls (Table 5; Fig. 3). Like the ovarian morphology, plasma levels of hormones also failed to show any change from the initial control levels (Table 5; Fig. 4). Following same duration (4 weeks) under high temperature regimes with both short and long photoperiods, the treated females (subgroups 7A and 8A: HTSP and HTLP) exhibited a regressed ovarian morphology compared to the experimental control turtles. The GSI was slightly higher than the initial GSI but was lower (P < . O O l ) than the experimental controls (Table 5 ; Fig. 3). Follicular development could not proceed beyond the initial stages as evidenced from atresia of many larger (class I1 and 111) follicles. Plasma E-17P was much lower (P < .001) than in the experimental controls, whereas plasma

TAB

LE 2

. E

ffect

s of

4-w

eek

phot

othe

rmal

trea

tmen

t on

ovar

ian

grow

th a

nd p

lasm

a st

eroi

d le

vels

du

ring

the

prep

arat

ory

phas

e in

Lis

sem

ys tu

rtle

’

Num

ber

of v

olkv

ova

rian

folli

cles

Ani

mal

GSI

body

wt.

) (g

/100

g

Cla

ss I

(sm

all:

5-10

mm

)

Cla

ss I1

(m

ediu

m:

10.1

-15

mm

)

Cla

ss I1

1 (l

arge

: 15

.1-2

0 m

m)

Cla

ss IV

(pre

ovul

ator

y :

>20

mm

)

Init

ial

cont

rol (

5)

Exp

erim

enta

l co

ntro

l LT

SP (1

A)

(8)

LTLP

(2A

) (8

)

HT

SP (3

A)

(8)

HT

LP

(4A

) (8

)

0.35

2 0

.07

0.68

2 0

.06

P <

.Ol*

0.

41 2 0

.03

N.S

.‘”’

P <

.005

**

0.40

2 0

.04

N. S .

(a’

P <

.005

**

2.02

f 0

.19

P <

.001

* P

< .0

01**

2.

42 f 0

.41

P <

.001

* P

< .0

01**

1.90

f 0

.06

2.88

0.

16

P <

.05*

2.

01

0.14

N

. S. ‘a

)

P <

.005

**

2.00

f 0

.12

N.S

.‘“’

P <

.001

**

8.12

T 1

.28

P <

.001

**

P <

.001

**

9.93

f 1

.73

P <

.001

* P

< .O

Ol*

*

0

1.10

2 0

.13

0 0

7.28

2 1

.03

P <

.001

**

8.42

? 1

.93

P <

.005

**

0

3.79

f 0

.99

4.01

? 1

.03

0 0 0

Shel

led

E-1

7P

egg

(pg/

ml)

0 17

.00

0.94

0 24

.83f

1.6

3

0 18

.09 2 0

.90

P <

.01”

N. S .(”’

Pi

.05*

*

N. S

.(a’

P <

.001

**

P <

.001

* P

< .0

01**

P <

.001

* P

< .0

01**

0 18

.60 ”f 0

.83

0 88

.65

k 3

.77

0 87

.95

2.33

PRO

cp

dml)

78.2

6 f 1

.03

80.1

9 f 2

.15

N.S

ta’

79.1

3 2 2

.91

N.S

.‘“’

N.S

.@’

80.4

6 f 2

.92

N.S

.‘“’

N.S

.lb’

79.3

7 2.

80

N . S.‘

”’

N . S

.‘b’

80.2

0 f 2

.83

N.S

.‘”’

N.S

.‘b’

‘Fig

ures

in

pare

nthe

ses

indi

cate

the

No.

of t

urtl

es in

the

resp

ectiv

e gr

oups

. E-l

7P, e

stra

diol

-17P

; PR

O, p

roge

ster

one.

*S

igni

fica

nce o

f dif

fere

nce

from

initi

al c

ontr

ol.

‘*Si

gnif

ican

ce o

f dif

fere

nce

from

exp

erim

enta

l co

ntro

l. ‘“

’N.S

., not

sig

nifi

cant

ly d

iffe

rent

fro

m in

itial

con

trol

. ‘b

’N.S

., not

sig

nifi

cant

ly d

iffe

rent

fro

m e

xper

imen

tal c

ontr

ol.

S. SARKAR ET AL.

4 8

I 1 I. CONTROL

10

Duration of treatment (week)

Fig. 1. Effects of 4-, 8-, and 10-week photothermal treatments on GSI during the preparatory phase of the reproductive cycle in Lissemys. I. Control, initial control; E. Control, experimental control; LTSP, low temperature and short photophase; LTLP, low temperature and long photophase; HTSP, high temperature and short photophase; HTLP, high temperature and long photophase.

PRO remained similar in all groups of turtles (Table 5; Fig. 4).

After 8 weeks, the GSI as well as ovarian growth increased maximally in the naturally reared experimental control turtles (Fig. 3). All types of yolky follicles increased in number with the development of many preovulatory follicles (Table 6). Moreover, the presence of a few shelled eggs in their uteri and a corresponding number of corpora lutea in the ovaries indicated the oc- currence of ovulation in these turtles. Both plasma E-17P and PRO showed a sharp rise over the initial controls (Table 6; Fig. 3). In the turtles exposed to lower and higher temperatures with both photophases (LTSP, LTLP, HTSP, and HTLP), the GSI and plasma levels of E-17P and PRO were found to be significantly lower than that of the experimental controls (Table 6; Figs. 3, 4). At low temperature (subgroups 5B and 6B: LTSP and LTLP), the number of ovarian follicles remained similar to that in the initial controls, since the follicular development slowed down; however, at high temperature (subgroups 7B and 8B: HTSP and HTLP) small yolky follicles were higher in

number from the initial controls, but other follicles underwent atresia and decreased in number both from the initial as well as experimental controls (Table 6).

Experiments during the quiescent phase The quiescent females at the beginning of the

experiments had least GSI (Table 7), and their ovaries appeared as leaf-like structures contain- ing only non-yolky follicles. The plasma level of E-17P was almost undetectable, and PRO was low (Table 7). The GSI, ovarian morphology, and plasma levels of hormones did not show any perceptible changes in the experimental controls and in the treated turtles following either 4 or 8 weeks of different photothermal regimes (subgroups 9A, 10A, 11A, 12A, 9B, 10B, 11B, and 12B) (Tables 7, 8).

DISCUSSION The obtained results clearly indicate that tem-

perature had a pronounced effect on ovarian growth and consequent secretion of E-17P in the soft-shelled turtle Lissemys punctata punctata. However, the effect was not similar in different phases of the seasonal reproductive cycle.

During the preparatory phase, short-term (4- week) treatment of the turtle at high temperature (38°C) caused significant increase of GSI due to stimulation of follicular growth and secretion of E-17P7 whilst long-term treatment (%week) at the same thermal regime failed to induce further stimulation of those functions over that obtained from 4-week treatment (P < .05). However, the GSI did not increase up to what had been noted to be the maximum GSI (8.16 * 1.03) during the natural breeding phase of the turtles by one of the present authors (Sarkar, '93). Finally, high-temperature treatment, when continued for 10 weeks, had a distinct regressive effect on ovarian function as evidenced from the atresia of ovarian follicles of all sizes in large number. In the preparatory phase, exposure of turtles to lower ambient temperature (21°C) retarded normal ovarian activities irrespective of the duration of treatment; follicular development could not proceed beyond the initial stage in the case of 4-week7 %week, and 10-week treatments.

During the recrudescent phase, the turtles were quite sensitive to higher ambient temperature (365°C). Only the small yolky follicles appeared normal; while many of the larger follicles became atretic, the rate of atresia was higher in the case of prolonged treatment (8 weeks). Ovarian activ-

TAB

LE 3

. Ef

fect

s of

&

wee

k ph

otot

herm

al tr

eatm

ent o

n ov

aria

n gr

owth

and

pla

sma

ster

oid

leve

ls

duri

ng th

e pre

para

tory

pha

se in

Lis

sem

ys t

urtle

'

Num

ber

of y

olky

ova

rian

folli

cles

GSI

C

lass

I

Cla

ss I1

C

lass

111

Cla

ss IV

(g/lO

O g

(s

mal

l: (m

ediu

m:

(lar

ge:

(pre

ovul

ator

y:

Shel

led

E-1

7P

PRO

A

nim

al

body

wt.

) 5-

10 m

m)

10.1

-15

mm

j 15

.1-2

0 m

m)

120

mm

j eg

g (P

&a

(pdr

nlj

Init

ial

cont

rol

(5)

Exp

erim

enta

l co

ntro

l (6

) LT

SP (1

B)

(8)

LTLP

(2B

) (8

)

HT

SP

(3B

) (8

)

HT

LP

(4B

) (8

)

0.35

2 0

.07

1.59

2 0

.23

P <

.OO

l*

0.52

2 0

.09

P <

.05*

P

< .0

01**

0.

53 2 0

.09

P <

.05*

P

< .0

01**

2.

92 2

0.7

7 P

< .0

01*

P <

.001

**

2.88

f 0

.92

P <

.001

* P

< .0

01**

1.90

0.

06

5.82

2 0

.82

P <

.001

* 2.

84 2 0

.42

P< .05*

P <

.001

**

2.97

f 0

.38

P <

-025

" p

< .0

01**

10

.24 2 1

.07

P <

.001

**

P c

.005

**

11.4

9 2 1

.01

P i .0

01*

P <

.OO

l**

0

3.70

2 0

.31

0 0

9.10

2 0

.88

P <

.001

**

8.90

2 1

.03

P <

.001

**

0

1.70

2 0

.05

0 0

4.00

2 0

.43

Pi

.001

**

5.92

2 0

.93

P <

.001

**

0 0

17.0

0 2 0

.94

0 0

48.6

0 2 1

.12

0 0

20.3

9 f 0

.36

P <

.001

*

P <

.01*

P

< .0

01**

Pi .

01*

P <

.001

**

P <

.001

" P

< .0

01*"

P <

.001*

P <

.001

**

0 0

20.6

8 2 0

.37

0 0

90.6

0 2 3

.81

0 0

92.8

12 2

.36

78.2

8 2 1

.03

83.2

9 2.

96

N.S

.'"'

78.2

3 2 1

.83

N.S

.'"'

N.S

.'b'

80.1

9 2 2

.33

N.S

.'"'

N . S

.'b'

80.2

6 2 1

.67

N.S

.'"'

N.S

kb'

77.2

6 f 2

.48

N.S

N

.S.'b

'

'Fig

ures

in

pare

nthe

ses

indi

cate

the

No.

of t

urtl

es in

the

resp

ectiv

e gr

oups

. E-1

7P, e

stra

diol

-17P

; PR

O, p

roge

ster

one.

*S

igni

fica

nce o

f dif

fere

nce f

rom

init

ial c

ontr

ol.

**Si

gnif

ican

ce of

dif

fere

nce f

rom

exp

erim

enta

l con

trol

. (a

'N.S

., not

sig

nifi

cant

ly d

iffe

rent

from

init

ial c

ontr

ol.

(b!N

.S., n

ot s

igni

fica

ntly

dif

fere

nt fr

om e

xper

imen

tal

cont

rol.

48 S. SARKAR ETAL.

loor

0 I . CONTROL

E. CONTROL

L T S P

L T L P

H T S P

H T L P

4 8 10

Duration of treatment ( w e e k 1

Fig. 2. Effects of 4-, 8-, and 10-week photothermal treatments on plasma level of estradiol-17P (E-17P) during the preparatory phase of the reproductive cycle in Lissemys (for abbreviations, see Fig. 1).

ity was significantly retarded by low-temperature (23°C) treatment of the turtles of the recrudescent phase. The quiescent turtles were sensitive neither to higher nor to lower ambient temperature treatments of any duration (4 or 8 weeks). In the quiescent phase, both ovarian growth and steroidogenesis remained negligible in the treated turtles as in the hibernating experimental controls.

The effects of higher ambient temperature treatments on ovarian growth and secretion in Lis- semys punctata punctata at the preparatory and recrudescent phases appear to resemble what have been described earlier by others in two species of temperate turtles. In Chrysemys picta, warm temperature (28°C) inhibited follicular growth both in the fall and spring females (Ganz- horn and Licht, '83). Similarly, in the multiclutched musk turtle Sternotherus odoratus, in the spring, high temperature (28OC) completely inhibited ovarian growth, while in the fall, high temperature was initially stimulatory but caused atresia of follicles after 10 weeks (Mendonca, '87). However, the effect of temperature in the quiescent phase has not been described in these temperate turtles.

The differential effect of temperature on ovarian growth and steroidogenesis in different

phases of the seasonal reproductive cycle of Lissemys turtle may be explained as follows. Our previous study has revealed that in Lissemys vitellogenesis starts in the early preparatory phase (March), which results in perceptible ovarian growth during the late preparatory phase (May- June) (Sarkar et al., '95; in press). The mean highest daily temperature in Calcutta during March has been found to be 34.97"C from the temperature records of the last four years. A high temperature treatment (38°C) for 4 weeks in this phase considerably stimulate ovarian activity, while the prolongation of the treatment caused an initial stimulation of ovarian growth (up to 8 weeks), which was finally followed by follicular atresia after 10 weeks. The observations clearly indicate that high temperature plays an instru- mental role only in triggering the ovarian activity at the beginning of the preparatory phase; once the ovarian growth sets in, high temperature can- not exercise a sustained stimulatory effect and, rather, causes degeneration of ovarian follicles in the long m. This inference is reinforced from the observation on the behaviour of the experimental control turtles maintained under natural environment. These turtles are sometimes found to retreat to summer aestivation during May when the highest daily temperature becomes too high (around 40°C). The ovarian follicles are prevented from atresia owing to this habit of summer aestivation.

In the recrudescent phase, when ovarian growth is already in progress, high temperature (36.5"C) causes atresia of larger follicles, irrespective of the duration of treatment, indicat- ing that high temperature at this phase of the reproductive cycle is not conducive to ovarian growth and activity. In nature, the recrudescent females do not experience prolonged exposure to high temperature; the mean highest daily temperature in the month of July in Calcutta is found to be 33.65%. This is why the experimental control turtles maintained under natural environment gradually proceeded toward the breeding phase while the high-temperature- treated recrudescent turtles failed to do so.

During the quiescent phase, neither ovarian growth nor steroidogenesis was at all influenced by different photothermal treatments, which in- dicates a total unresponsiveness of the females in respect to their ovarian function to photother- ma1 changes in the quiescent phase. A complete shutdown of reproductive responsiveness to environmental stimuli has also been demonstrated in many squamate reptiles during the refractory or

TABL

E' 4

. E

ffec

ts o

f I0

-wee

k ph

otot

herm

al tr

eatm

ent o

n ov

aria

n gr

owth

and

pla

sma

ster

oid

leve

ls

duri

ng th

e pr

epar

ator

y ph

ase

in L

isse

mys

turt

le'

Ani

mal

GSI

bodv

wt.)

(d

l00

g

Num

ber

of y

olky

ova

rian

folli

cles

C

lass

I

Cla

ss I1

C

lass

I11

Cla

ss lV

(sm

all:

(med

ium

: (l

arge

: (p

reov

ulat

ory:

5-

10 m

m)

10.1

-15

mm

) 15

.1-2

0 m

m)

>20

mm

)

Init

ial

cont

rol (

5)

Exp

erim

enta

l co

ntro

l (6)

L

TSP

(1C

) (8

)

LTLP

(2C

) (8)

HT

SP (

3C)

(8)

HT

LP

(4C

) (8

)

0.35

2 0

.07

2.10

2 0

.17

P <

,001

" 0.

62 2

0.0

7 P

< .0

5*

P <

.001

**

0.58

0.

06

P <

.05*

P

< .0

01""

2.

32 -+

0.6

2 P

< .0

01*

N.S

.'h'

2.47

2 0

.59

P <

.001

* N

.S.'b

'

1.90

+ 0.

06

6.90

2 0

.73

P <

.001

*:

3.07

2 0

.38

P<

.05*

P

< .0

01""

2.

99 f 0

.42

Pi .

05*

P <

.001

**

0.46

2 0

.87

P <

.001

**

N.S

.'b)

5.89

f 0

.47

P <

.001

* N

. S .rb

)

0

4.18

-+ 0

.30

0 0

3.80

2 0

.16

N. S

. ib)

4.10

2 0

.22

N.S

.'b'

0 0

1.79

2 0

.23

0

0 0

0 0

2.03

2 0

.33

0

N .S

.'b'

1.99

2 0

.48

0

N.S

.'b'

Shel

led

E-1

7P

egg

(Pdm

v

0 17

.00 f 0

.94

0 68

.79

.t 2

.22

0 20

.26 f 0

.52

P <

.001

*

P <

.05*

P

< .0

01"*

P< .0

5*

P <

.001

**

P <

.001

* N

.S.'b

'

P <

.001

*

0 21

.19

$0.6

1

0 72

.36 t 1

.68

0 69

.44

& 2

.03

N.

PRO

(p

drnl

)

78.2

6 f 1

.03

81.2

9 f 2

.19

N.S

.'"'

79.1

3 2 2

.52

N.S

.'"'

N.S

.'b'

80.4

9 3.

01

N.S

.'"'

N.S

.ib'

81.9

3 -t 2

.01

N.S

.'"'

N.S

.'"

80.0

0 f 1

.88

N.S

.'"'

N.S

.'b'

'Fig

ures

in

pare

nthe

ses

indi

cate

the

No.

of t

urtl

es in

the

resp

ectiv

e gr

oups

. E-l

7P, e

stra

diol

-171

J; PR

O, p

roge

ster

one.

*S

igni

fica

nce o

f dif

fere

nce

from

initi

al c

ontr

ol.

""Si

gnif

ican

ce of

dif

fere

nce

from

exp

erim

enta

l con

trol

. ia

)N.S

., not

sig

nifi

cant

ly d

iffe

rent

from

ini

tial

con

trol

. 'b'

N.S

., no

t sig

nifi

cant

ly d

iffe

rent

from

exp

erim

enta

l con

trol

.

TAB

LE 5

. E

ffect

s of

4-w

eek

phot

othe

rmal

tre

atm

ent o

n ov

aria

n gr

owth

and

pla

sma

ster

oid

leve

ls

duri

ng t

he r

ecru

desc

ent p

hase

in

Liss

emys

turt

le'

Num

ber

of v

olkv

ova

rian

folli

cles

G

SI

Cla

ss I

Cla

ss I1

C

lass

I11

Cla

ss IV

(gI1

00 g

(sm

all:

(med

ium

: (l

arge

: (p

reov

ulat

ory:

Sh

elle

d E

-17P

PR

O

Ani

mal

bo

dy w

t.)

5-10

mm

) 10

.1-1

5 m

m)

15.1

-20

mm

) >2

0 m

m)

egg

(pg

hl)

(p

dml)

Init

ial

2.97

2 0

.31

10.2

0 2 0

.01

5.38

f 0

.81

1.86

f 0

.21

0 0

70.4

8 f 2

.01

87.1

6 2 3

.19

cont

rol (

5)

Exp

erim

enta

l 5.

88 f 0

.47

18.1

3 -c

0.89

8.

78 -c

0.4

3 4.

42 2

0.3

9 1.

50 2

0.22

0

197.

68 2 4

.01

82.5

8 3.

01

cont

rol (

6)

P <

.001

* P

< .0

01*

P <

.005

* P

< .0

01*

N.S

.'"'

LT

SP (5

A)

2.81

5 0

.23

9.33

2 0

.76

5.03

2 0

.64

2.01

5 0

.33

0 0

79.2

9 2 1

.81

80.4

6 f 4

.56

(8)

N.S

.'"'

N. S

.("I

N.S

.'"'

N. S

.'"I N

. S.(d

' N

.S.'"

' P

< .0

01**

P

< .0

01**

P

< .0

01**

P

< .0

01**

P

< .0

01**

N

.S.'b

' L

TL

P (6

A)

3.00

0.

21

11.1

0 2 1

.07

5.00

2 0

.20

1.50

5 0

.17

0 0

72,4

15 1

.73

85.5

35 3

.33

(8)

N.S

.'"'

N.S

.'"'

N.S

.'"'

N.S

.'"'

N.S

.'"'

N.S

.'"'

P <

.001

**

P <

.001

**

P <

.001

**

P <

.001

**

P <

.001

**

N.S

.'b'

(8)

P <

.025

* P

< .0

1*

N.S

.'"'

N.S

.'"'

N . S

.'")

N.S

.'"'

P< .0

05**

P

< .0

25**

P

< .0

01**

P

< .0

05**

P

< .0

01**

N

.S.'b

' H

TL

P (8

A)

3.99

2 0

.28

15.0

1 -c

0.78

3.

78 -c

0.5

2 1.

98 +.

0.3

8 0

0 82

.12

+. 1

.89

82.9

8 2 2

.67

(8)

P <

.05*

P

< .0

05*

N.S

.'"'

N.S

.'"'

N.S

.'"'

N. S

.'"I

P <

.005

**

P <

.025

**

P <

.001

**

P <

.001

* P

< .0

01*

N.S

.@'

HT

SP (7

A)

3.87

2 0

.21

14.5

6 2 0

.96

4.30

2 0

.67

2.08

2 0

.44

0 o

84.2

3 -c

1.02

84

.34

5 2

.89

'Fig

ures

in

pare

nthe

ses

indi

cate

the

No.

of t

urtl

es in

the

resp

ecti

ve g

roup

s. E

-17a

, est

radi

ol-1

7P; P

RO

, pro

gest

eron

e.

*Sig

nifi

canc

e of

dif

fere

nce f

rom

init

ial c

ontr

ol.

**Si

gnif

ican

ce o

f dif

fere

nce f

rom

exp

erim

enta

l con

trol

. '@

N.S

., no

t sig

nifi

cant

ly d

iffe

rent

fro

m in

itia

l con

trol

. 'h

lN.S

., not

sig

nifi

cant

ly d

iffe

rent

from

exp

erim

enta

l con

trol

.

PHOTOTHERMAL EFFECTS ON OVARY IN TURTLE 51

200

- - E 160-

m a

9

\

- - h -

I 120- W

E - 0 I

a 60-

F 0

6 -

E m

2 E 4 - m

m - 0

0 -

-. Y -

2 -

0

-

T

0 I CONTROL

E CONTROL

L T S P

L T L P

H T S P

4 8

Duration of treatment (week)

Fig. 3. Effects of 4- and 8-week photothermal treatments on GSI during the recrudescent phase of the reproductive cycle in Lissemys (for abbreviations, see Fig. 1).

4

l!l T-T

1 I CONTROL

Q E CONTROL

R L T S P

69 L T L P

H T S P

H T L P

I .

6

Duration of treatment (week 1

Fig. 4. Effects of 4- and 8-week photothermal treatments on plasma level of estradiol-17P (E-17P) during the recrudescent phase of the reproductive cycle in Lissemys (for abbreviations, see Fig. 1).

quiescent phase of their seasonal reproductive cycle (Duvall et al., '82; Whittier and Crews, '87). While the unresponsive nature of the quiescent ovaries of those reptiles has been attributed to the presence of large corpora atretica (Crews and Licht, '74), nutritional factors are supposed to be the main reasons for the unresponsiveness of the quiescent ovaries of Lissemys turtle, which contained no trace of corpora atretica at all. An earlier study by one of the present authors revealed that vitellogenesis and ovarian growth in Lissemys were significantly dependent on food intake and the nature of the food (Sarkar, '92). In the present study it was found that neither the experimental controls nor the treated turtles consumed any food during the quiescent phase, and this lack of food intake, in turn, might have been responsible for suppression of ovarian activity in either group.

Finally, photoperiod appeared to have exerted little influence over ovarian growth and function not only in the quiescent phase but also in the preparatory and recrudescent phases of the seasonal reproductive cycle of Lissemys turtle. The effect of HTSP did not differ from that of HTLP on the one hand, and the effect of LTSP did not differ from that of LTLP on the other, at any phase of the reproductive cycle. The ab- sence of any marked effect of photoperiod on ovarian activity in the present turtle species re- sembles tha t observed in the musk turtle, Sternotherus odoratus (Mendonca, '871, whereas, photoperiod has been claimed to exert con- siderable influence on ovarian activity in Lissemys punctata granosa (Singh, '77) and Chrysemys picta (Ganzhorn and Licht, '83). Such variations in the effect of photoperiod on ovarian activity in different turtles are difficult t o explain. In fact, not only the turtles but also many other reptiles have shown diverse kinds reproductive responsiveness t o photoperiods (Bartholomew, '50, '53; Mayhew, '61, '64; Licht, '73, '84; Duvall e t al., '82; Whittier and Crews, '87). Finally it seems logical t o conclude that temperature rather than photoperiod plays a regulatory role on the female reproductive function in the turtle species studied.

ACKNOWLEDGMENTS We gratefully acknowledge financial support and

Junior and Senior Research Fellowship awards (38/669/87)/EMR-II and 9/28 (349) 92-EMR-I to the first author from the Council of Scientific and Industrial Research, Government of India. Kind acknowledgment is also due to the Director, Na-

TAB

LE 6

. E

ffec

ts of

&w

eek

phot

othe

rmal

trea

tmen

t on

ovar

ian

grow

th a

nd p

lasm

a st

eroi

d le

vels

du

ring

the

recr

udes

cent

pha

se i

n L

isse

mys

turt

le'

Num

ber o

f yol

ky o

vari

an fo

llicl

es

GSI

C

lass

I

Cla

ss I1

C

lass

I11

Cla

ss IV

(d

l00

g (s

mal

l: (m

ediu

m:

(lar

ge:

(pre

ovul

ator

y:

Shel

led

E-1

7P

PRO

A

nim

al

body

wt.

) 5-

10 m

m)

10.1

-15

mm

) 15

.1-2

0 m

mj

>20

mm

) eg

g (p

g/m

l)

(pg/

mlj

Init

ial

2.97

+. 0

.31

10.2

0 2 1

.01

5.38

-t 0

.81

1.86

2 0.

21

0 0

70.4

8 2 2

.01

87.1

6 f 3

.19

cont

rol (

5)

Exp

erim

enta

l 8.

08 2

1.0

0 32

.00

2 1

.41

12.5

2 2 0

.87

9.36

ir 0

.89

12.3

5 2 1

.12

3.89

2 0

.67

440.

38 2 1

2.26

510

.18

2 1

8.29

co

ntro

l (6)

P

< .0

01*

Pi

.001

* P

< .0

01*

P< .0

01*

P <

.001

* P

< .0

01*

LT

SP (5

B)

2.88

2 0

.31

11.3

6 & 0

.88

6.00

2 0

.77

1.89

2 0

.47

0 0

72.6

8 &

1.5

6 82

.36

2 2

.81

(8)

N. S

.(a'

N.S

.'"'

N.S

.'"'

N.S

.'"'

N.S

.'"'

N. S

.(a)

LT

LP

(6B

) 3.

08 2

0.6

1 10

.93 2 1

.21

6.68

2 0

.93

1.52

2 0

.53

0 0

70.8

3 2 1

.33

80.8

6 2 3

.29

(8)

N.S

.'"'

N.S

.'"'

N.S

.'"'

N.S

.'"'

N.S

."'

N. S

.("I

HT

SP (7

B)

3.68

2 0

.73

14.2

8 2 1

.12

4.08

f 0

.77

1.50

+. 0

.20

0 0

77.8

2 2 3

.36

82.9

32 3

.12

(8)

N. S

.("I P

< .0

25*

N.S

.'"'

N.S

.'"'

N.S

.'"'

N.S

.'"'

HT

LP

(8B

) 3.

32 2

0.6

9 15

.31

+. 1.

92

3.00

2 0

.55

1.98

? 0

.20

0 0

75.2

9 2 2

.86

84.3

8 2 2

.89

(8)

N . S

.'"'

P <

.05*

P

< -0

5"

P< .0

25*

N.S

.'"'

N.S

.'"'

P <

.001

**

P <

.001

**

P <

.001

**

P <

.OOl

**

P <

.001

**

Pi .

001*

*

P <

.001

**

P <

.001

**

P <

.001

**

P <

.OOl

**

P <

.001

**

P< .0

01*"

P< .0

01**

P

< .001**

P <

.001

**

P <

.001

**

P <

.001

**

P< .0

01**

P <

.006

**

P <

.001

**

P <

.001

**

P <

.001

**

P <

.001

**

P <

.001

**

'Fig

ures

in

pare

nthe

ses

indi

cate

the

No.

of t

urtl

es in

the

resp

ectiv

e gr

oups

. E-1

7fi,

estr

adio

l-17

P; PR

O, p

roge

ster

one.

*S

igni

fica

nce o

f dif

fere

nce

from

init

ial c

ontr

ol.

**Si

gnif

ican

ce of

dif

fere

nce

from

exp

erim

enta

l con

trol

. (a

)N.S

., not

sig

nifi

cant

ly d

iffe

rent

from

init

ial

cont

rol.

%T.

S.,

not s

igni

fica

ntly

dif

fere

nt fr

om e

xper

imen

tal

cont

rol.

TAB

LE 7.

Eff

ects

of 4

-wee

k pho

toth

erm

al t

reat

men

t on

ova

rian

gro

wth

and

pla

sma

ster

oid

leve

ls

duri

ng th

e qu

iesc

ent p

hase

in

Lis

sem

ys tu

rtle

'

Num

ber o

f yol

ky o

vari

an fo

llicl

es

GSI

C

lass

I C

lass

I1

Cla

ss 1

11

Cla

ss IV

(g

/100

g

(sm

all:

(med

ium

: (l

arge

: (p

reov

ulat

ory:

Sh

elle

d E

-17P

PR

O

Ani

mal

bo

dy w

t.)

5-10

mm

) 10

.1-1

5 m

m)

15.1

-20

mm

) 22

0 m

m)

egg

(pdm

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PHOTOTHERMAL EFFECTS ON OVARY IN TURTLE 55

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photothermal effects on ovarian growth and function in the soft-shelled turtlelissemys punctata...

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