Comp. Biochenl. Physiol.. 1975. Ibl. 52A. I'P. 519 to 525. Pergamon Press. Printed in Great Britai,

STUDIES ON THE ANNUAL REPRODUCTIVE cYCLE OF THE FEMALE COBRA, NAJA NAJA--I. SEASONAL VARIATION IN PLASMA CHOLESTEROL VALENTINE LANCE

Biological Science Center, Boston University, Boston, MA 02215. U.S.A.

(Received 14 September 1974) Abstract--l. Total plasma cholesterol was measured in monthly samples of female cobras over a period

of 3 years. 2. Cholesterol levels ranged between 155 and 550 mg~ but were generally between 200 and 300 mg~o. 3. Significant seasonal differences were noted with a sharp drop in plasma cholesterol in May, an increase in September, and a second decrease in November. 4. The decrease in plasma cholesterol in May was strongly correlated with maximum ovarian development, and the decrease in November appeared to be associated with fat body deposition. INTRODUCTION

IN TELEOSTSplasma cholesterol increases significantly at the onset of spawning (Idler & Tsuyuki, 1958; Lewander et al., 1974). There is evidence that a similar rise in plasma cholesterol occurs in amphibia prior to ovulation (Follett & Redshaw, 1968). In the domestic fowl (Romanoff & Romanoff, 1949) and the domestic turkey (Mukherjee et al., 1969) egg laying is associated with an increase in plasma cholesterol. Increases in plasma cholesterol are also seen in pigeons (Hoffman, 1960) and white-crowned sparrows (Kern et al., 1972) during the breeding season. There is no information on the association between reproduction and plasma cholesterol levels in reptiles, though Chaikoff & Entenman (1946) did report that whole blood cholesterol levels in the turtle Chrysemys picta belli were higher in females with active ovaries or eggs in the oviduct than in males or females with undeveloped ovaries. Other studies on plasma cholesterol in reptiles have failed to detect these differences. Jackson & Legendre (1967) and Stenroos & Bowman (1968) noted considerable variation both within species and between species in turtles, but did not detect any sex difference. However, the number of animals examined was small and the reproductive state was not indicated. Differences in diet or phylogentic separation were given as possible reasons for the observed variation (Jackson & Legendre, 1967; Jackson et al., 1971). In Pseydemys concinna suwanniensis there appeared to be a correlation between an increase in blood serum cholesterol with an increase in age (size) of the turtle (Jackson et al., 1970). Increase in temperature has been shown to cause significant changes in plasma cholesterol in Chrysemys picta. When turtles maintained at 22°C were subjected to a temperature of 32°C for 24 hr plasma cholesterol increased from a mean of 200 to 238 mg~. However, if the turtles were exposed to 38°C for 24 hr plasma levels dropped to 129mg~ (Wilber & Lieb, 1950). The control of cholesterol metabolism is extremely complex; age, sex, diet, genetic constitution and a 519

number of hormones have been implicated in its regulation (Ho & Taylor, 1970). The little experimental work done on the control of plasma cholesterol levels in reptiles indicate that it differs in many ways from what is known in mammals. Hypophysectomy or thyroidectomy result in an increase in plasma cholesterol in the rat (Byers et al., 1970), whereas hypophysectomy (Rangneker & Padaonkar, 1972) or thyroidectomy (Pandha et al., 1968; Thapliyal et al., 1974) in the snake Natrix piscator cause a decrease in plasma cholesterol. Injection of insulin in the alligator causes a marked decrease in plasma cholesterol but has little effect in the rat (Coulson & Hernandez, 1964). As can be seen (Table 1) most of our information on blood cholesterol levels in reptiles comes from observations made on a limited number of specimens, and in virtually every case neither is the time of year at which the sample was collected nor the sex of the animal given. In view of the paucity of information on plasma cholesterol in reptiles, especially with regards to reproduction and season, it was decided to measure this important parameter as part of a study on the annual reproductive cycle in the female cobra, Naja naja. As no information exists on the cholesterol content of snake eggs the yolk cholesterol content of eggs taken from the oviducts of gravid snakes was also measured. MATERIALS AND M E T H O D S

Monthly samples of between 5 and 15 adult female cobras ranging in size from 850 and ll50mm in length, and weighing between 200 and 650 g were obtained from a Hong Kong snake dealer throughout 1969, 1970 and part of 1971. The snakes, all of which came from Kwangtung Province, China, were deranged immediately before leaving the shop and were killed without anaesthesia by decapitation within 24hr of arriving in the laboratory. Blood was collected directly into heparinized centrifuge tubes by exposing the heart and incizing the ventricle. After

520

VALENTINE LANCE

A

o~

,o~.~

~I~ =~-~-~.~.~ ~ ~ ~.~ ~

o

o

p.o_ o

6

o

o

o

o

~

~

o

o

~

6

I~

--~+1~÷1~+1~+1~

"6 0 0

I-

0

"6

+1+1+1

Annual reproductive cycle of the female cobra

MDr~

O~ o~ (3'~

O~

ox

o.o . . . .

-

F~

~,~

~

~o~oo o-~ +I +I ~ +I r--

.3=

~ +I +I

. ~ . ~ . ~

~

oo

_

~-

_

~ +I+I+I

.~

-

~

~ - -~ ----+I

. ~ g ~ o ~ ~ ~ ~ ~

~.~.~:~ ~ e~J

"El

-

2= E

o~ .m

521

522

VALENTINE LANCE

centrifugation the plasma was separated and stored at - 15°C until analyzed. Body weights, body lengths and the weights of the various organs were recorded, and ovarian, oviduct, adrenal and throid tissues were fixed in Bouin's solution for subsequent histological examination. Total plasma cholesterol was determined using the method of Mann (1961) with the minor modification that the FeCI3 stock solution was dissolved in phosphoric acid rather than glacial acetic acid (Rosenthall et al., 1957). Duplicate determinations were made on each plasma sample. All reagents were of Analar grade (E. Merck) and the cholesterol standard (lkapharm) was checked for purity by thin layer chromatography. The method was checked against a commercial serum standard (Versatol A, Warner Lambert Co.). With minor modifications the method was adapted for the measurement of yolk cholesterol content of recently ovulated eggs. The shelled eggs were removed from the oviducts of gravid snakes and fixed in I0~o formalin until analyzed. Statistical analysis of the data was performed on an Olivetti desk top computer. RESULTS

Plasma cholesterol values in the female cobra showed considerable individual variation ranging from a low of 155mg~o (May 1970) to a high of 550mg~ (October 1971). The majority of values, however, fell between 200 and 300 mg~. When the month to month values were analyzed it was seen that there was a highly significant decrease in plasma cholesterol in the month of May when compared to all other months except November and December (P < 0.1301); an increase in September, significantly higher than all other months except April, June, July, August and October (P < 0"05); and a second decrease in November which was significantly lower than all other months except May, June, December, January and February (P < 0.001). These differences were similar in each of the 3 years studied. The pooled data is shown graphically in Fig. 1. To show the relationship with seasonal plasma cholesterol variation the seasonal changes in fat body and ovarian weight are shown in Fig. 2 and the seasonal changes in liver weight in Fig. 3. The increase in plasma cholesterol in September shows a highly significant correlation ( r = - 0 " 6 5 , P < 0'001)with the decrease in liver weight. The low

r"--o Ovary weight g ,.-e Fat body, g/IOOG body weight

- 15

o

.E "

(221 (261~:r1~21 , , J

F

M

, M

A

, J

L.

(21) J

A

S

- 5

,2o,

22

(221(341 12810

N

O

Fig. 2. Seasonal variation in ovary and fat body weight in the female cobra, Naja naja. Symbols as in Fig. 1. cholesterol levels in November appear to be correlated with the sudden increase in fat body weight, but since all snakes examined in this month had fully developed fat bodies no direct correlation could be calculated. The highly significant decrease in plasma cholesterol in May occurs when the ovary is at its maximum development. When the cholesterol values from individual snakes in May are paired with the ovarian weight it was found that there was a high degree of correlation between these two parameters (r = -0'62, P < 0.001). No correlation between body weight or body length and plasma cholesterol was noted, though in the month of October when the total body weight of the snakes is lowest the plasma cholesterol is significantly higher than in November or December. No relationship between the seasonal variations in adrenal or thyroid weights and plasma cholesterol could be detected. The cholesterol content of the yolks of 11 oviducal eggs is given in Table 2. DISCUSSION

The plasma cholesterol levels in female Naja naja show considerable individual variation but are within the range of values reported for other snakes (see Table l). One uncontrollable variable in this study

T |

325 -- Plasma cholesterol in mg/100 mt

221 ~ .

/

iver absolute weight, g 15 -

B~ 275

14

22) (~26) (231

r ~

~

12 i

50

O

(

II IO

225

2oo

I

F

I

M

L

A

I

M

I

d

]

d

I

A

I

S

I

O

I •

N

D

Fig. I. Seasonal variation in total plasma cholesterol in

the female cobra, Naja naja. Each point represents the mean___S.E.M. for each month. The numbers in parentheses indicate the number of animals examined each month.

7

(221

I

I

I

L

I

I

I

I

I

I

I

I

d

F

M

A

M

J

J

A

S

O

N

O

Fig. 3. Seasonal variation in liver weight in the female cobra, Naja ozuja. Symbols as in Fig. I.

Annual reproductive cycle of the female cobra

523

Table 2. Cholesterol content of Naja naja eggs No. 1 2 3 4 5 6 7 8 9 10 11

Size in mm 40 45 48 58 53 52 49 44 54 43 53

x x x x x × x x x × x

25 23 24 27 26 26 27 27 25 24 26

Weight (g)

Weight of yolk Cholesterol content (g) (rag/100 mg wet wt of yolk)

8'9 9"1 9'1 14'4 12'8 11"9 12"9 10"2 13"5 8-7 11"1

was the time between capture in the field and arrival in the laboratory. All of the snakes examined had empty stomachs but were generally in good condition. Since fasting for up to 20 weeks has little effect on plasma cholesterol levels in Crotalus atrox (Martin & Bagby, 1973), whereas feeding can raise the level by 65~ in Crotalus horridus (Carmichael & Petcher, 1945) it is assumed that the levels observed in Naja naja are normal fasted values, and that the seasonal variation described probably reflects the annual cycle in nature. The fact that the same pattern was observed in 3 years in succession strengthens this view. From a base mean of about 260 m g ~ there is a single peak of about 320 m g ~ in September, a sharp drop to around 210 m g ~ in May just prior to ovulation, and a second decrease in November to 230 mgTo. The increase in plasma cholesterol in September is strongly correlated with a decrease in liver weight, and the drop in plasma cholesterol in November appears to be associated with the onset of fat body development. Afroz et al. (1971) reported that the fat body of the lizard Uromastix hardwickii contains an unusually high proportion of cholesterol as compared to mammalian fat, and that this cholesterol content is highest shortly after the fat body is laid down in November. When the fat body regresses after hibernation the following April the cholesterol content declines. If a similar phenomenon is occurring in Naja the drop in plasma cholesterol in November is probably due to uptake by the fat body, and the increase in plasma levels in September could be due to synthesis and release by the liver. Afroz et al. (1971) noted that the cholesterol content of the liver in Uromastix was at its highest immediately prior to the deposition of the fat body. Dessauer (1955) made a detailed study on the seasonal changes in distribution of protein, carbohydrate and lipid in the various tissues of the iguanid lizard, Anolis carolinensis. He showed that there is a marked turnover and exchange of lipid between the liver and the fat body. It is likely that a similar movement of cholesterol occurs in Naja. No correlation between plasma cholesterol and body weight or length was apparent as was reported by Jackson et al. (1970) in the turtle; and none of the other parameters measured (adrenal weight, thyroid weight or plasma ions) showed any relationship to the annual cholesterol pattern. The mean plasma cholesterol levels in the female cobra appear to be close to those seen in the male

6"0 6"3 6-5 11"5 10"9 10"0 11'2 8'7 11"0 7-4 10'2

0"316 0'248 0"220 0"357 0"212 0'217 0'248 0"184 0'210 0'211 0"194

(Wong & Chiu, 1974), whereas in the turtle Chrysemys picta there is a pronounced sex difference (Chaikoff & Entenman, 1947; Salhanick & Callard, personal communication). The drop in plasma cholesterol in May appears to be directly correlated with ovarian follicular maturation. A strong negative corelation between ovarian weight and plasma cholesterol was observed in this month in each of the three years of this study. In all cases snakes with low plasma cholesterol were seen to have large yolked follicles, but not all snakes with large follicles had low plasma cholesterol levels. In some instances snakes examined in May with large yolked follicles had high plasma cholesterol levels. Many of these follicles were seen to bc atretic on subsequent histological examination. Betz (1963) noted similar cases of atresia in otherwise healthy looking preovulatory follicles in Natrix rhombifera. This drop in plasma cholesterol corresponding to the late stage of vitellogenesis in the cobra is contrary to what is known in birds where blood cholesterol increases during vitellogenesis and egg laying (Romanoff & Romanoff, 1949; Mukherjee et al., 1969). Izard et al. (1961) were unable to detect any seasonal variation in serum cholesterol in Vipera berus. However, their sample was probably too small to detect such a transitory change as is seen in the cobra. Only a single paper on cholesterol levels in vitellogenic reptiles has appeared (Chaikoff & Entenman, 1947), and in that study it was noted that the whole blood cholesterol was higher in turtles with yolked follicles and oviducal eggs than in males or immature females. Since cholesterol measurements were not made at other times of the year it is not certain that the higher levels in females is directly related to egg production. Although there was no marked increase in plasma cholesterol in the cobra before vitellogenesis began in April, there did appear to be a gradual increase from the low winter levels to a peak in April. However, the differences were not statistically significant, and were nowhere near the magnitude of the changes seen in birds. The decrease in plasma cholesterol observed in Naja naja with mature vitellogenic follicles appears to last only a very short time. It is possible that previous studies on blood cholesterol in reptiles failed to detect any short term preovulatory decline as sample sizes were too small. Despite the fact that cholesterol content of the yolk of developing follicles was not measured, the considerable drop in plasma cholesterol levels at

524

VALENTINE LANCE

the period of maximum follicular development in Nc~ja would seem to indicate that the cholesterol is being transported into the yolk. Several lines of evidence suggest that this is the case. Brenner (1970) noted that the lipid content of the ovarian follicles of the turtle Clemmys insculpta increased as the follicles increased in size, suggesting that lipid is being selectively accumulated by the developing ova. A similar accumulation of lipid and cholesterol by the developing ovary has been shown to occur in teleosts (Idler & Bitners, 1960; Lewander et al., 1974). There is evidence that circulating cholesterol is transported into the yolk of ovarian follicles in the Cyclostomata. Fernholm (1972) injected C l,Llabelle d cholesterol into mature Myxine glutinosa and found significant amounts of radioactivity in the yolk and follicular epithelium after l l hr. If, as has been suggested by Tienhoven (1968), gonadotropins increase the permeability of the egg membrane, the drop in plasma cholesterol in the female cobra prior to ovulation could be due to an increase in circulating gonadotropin levels at this time. Neaves (1972) has indicated that FSH injections in Anolis carolinensis increase the non-specific uptake of large proteins by mature oocytes; and the work of Follet et al. (1968) on the frog Xenopus laevis has shown that gonadotropin causes an uptake of radioactively-labelled vitellogenic protein and circulating esterified cholesterol by the ovarian follicles. It is possible therefore, that the transitory drop in plasma cholesterol in Naja at the peak of follicular development is due to a preovulatory surge of gonadotropin release from the anterior pituitary which results in a rapid uptake of cholesterol by the ovum. Whether this is a non-specific effect simply due to an increased permeability of the egg membrane or due to some selective energy-linked uptake remain to be determined. The uptake of vitellogenic protein by Xenopus oocytes is highly specific (Wallace & Dumaont, 1969). Acknowledoements--This research was supported by the Department of Zoology, University of Hong Kong. REFERENCES

AFROZH., ISHAQ M. t~¢ ALl S. S. (1971) Seasonal changes in the lipids of adipose tissue in a hibernating lizard (Uromastix hardwickii). Proc. Soc. exp. Biol. Med. 136, 894-898. BE'rZ T. W. (1963) The ovarian histology of the diamondbacked water snake, Nao'ix rhomhifera during the reproductive cycle. J. Morph. 113, 245-260. BRENNER F. J. (1970) The influence of light and temperature on fat utilization in female Clemmys insculpta. Ohio J. Sci. 70(4), 233-237. BYERSS. O., FRmDMANM. & ROSENMANR. H. (1970) Prevention of hypercholesteraemia in thyroidectomized rats by growth hormone. Nature, Lond. 288, 464-465. CARMICrt~EL E. B. & PETCHER P. W. (1945) Constituents of the blood of the hibernating and normal rattlesnake, Crotalus horridus. J. biol. Chem. 161,693-696. CHAmOFF I. L. & EN~NMAN C. (1946)The lipides of blood, liver and egg yolk of the turtle. J. biol. Chem. 166, 683689. COULSON R. A. & HERNANDEZT. (1964) Biochemistry of the Alligator. A Study of Metabolism in Slow Motion. Louisiana State University Press, Baton Rouge.

DESSAUERH. C. (1955) Seasonal changes in the gross organ composition of the lizard. Anolis carolinensis. J. exp. Zool. 128, 1-12. FERNHOLM B. (1972) Is there any steroid hormone formation in the ovary of the hagfish, Myxine ghltinosa? Acta zool., Stockh. 53, 235-242. FOLLE'rr B. K. & REDSHAW M. R. (1968) The effects of oestrogen and gonadotrophins on lipid and protein metabolism in Xenopus laevis (Daudin). J. Endocr. 40, 439-456. FOeLE'rr B. K., NJCHOLLST. J. & REDSHAW M. R. (1968) The vitellogenic response in the South African clawed toad (Xenopus laevis Daudin). J. Cell. Physiol. 72(2), Suppl. 1, 90-102. Ho K. J. • TAYLOR C. B. (1970) Control mechanisms of cholesterol biosynthesis. Arehs Path. 90, 83-92. HOFFMAN R. A. (1960) Observations on the serum and gonad cholesterol in pigeons. Endocrinology 67, 311-318. HOLCOMBC. M., JACKSONC. G. & JACKSONM. M. (1972) Serum cholesterol values in three species of turtles. J. Wildl. Dis. g, 181-182. IDLER D. R. & BRINERS I. (1960) Biochemical studies on sockeye salmon during spawing migration--IX. Fat, protein, and water in the major internal organs and cholesterol in the liver and gonads of standard fish. J. Fish. Res. Bd Can. 17. 113-122. IDLER D. R. & TSUYUKI H. 0958) Biochemical studies on sockeye salmon during spawning migration--I. Physical measurements, plasma cholesterol and electrolyte levels. Can. J. Bioehem. 36, 783-791. IZARD Y., DETRAITJ. & BOWUETP. (1961) Variations saisonnieres de la composition du sand de Vipera aspis. Ann. Inst. Pasteur 100, 539-545. JACKSONC. G. JR. &. LEGENDRER. C. (1967) Blood serum cholesterol levels in turtles. Comp. Biochem. Physiol. 20, 331-312. JACKSON C. G., JR,, HOLCOMB C. M. & JACKSON M. M. (1970) The relationship between age, blood serum cholesterol level and aortic calcification in the turtle. Comp. Biochem. Physiol, 35, 491-494. JACKSON C. G., JR., HOLCOMB C. M. & JACKSON M. M. (1971) Blood serum cholesterol levels in two congeneric species of molluscivorous turtles. Comp. Biochem. Physiol. 38B, 459-461. JoY M. & DASTAGUEG. (1944) Nouvelles recherches sur la composition du sang chez Vipera aspis: II Les constituents chimiques. Arch. phys. Biol. 17 suppl. 67, p. 61. KERN M. D., DEGRAWW. A. & KING J. A. (1972) Effects of gonadal hormones on the blood composition of white-crowned sparrows. Gen. & conlpar. Endocr. 18, 43-53. LEWANDER K., DAVE G., JOHANSSONM.-L., LARSSON A. & LIDMAN U. (1974) Metabolic and hematological studies on the yellow and silver phases of the European eel, Anguilla anguilla--I. Carbohydrate, lipid, protein and inorganic ion metabolism. Comp. Biochem. Physiol. 47B, 571 581. LUCK J. M. t~£ KEELER L. (1929) The blood chemistry of two species of rattlesnake, Crotalus atrox and Crotalus oregonus. J. biol. Chem. 82, 703-707. MANN G. V. (1961) A method for the measurement of cholesterol in blood serum. Clin. Chem. 7, 275-284, MARTIN J. H. & BAGBY R. M. (1973) Effects of fasting on the blood chemistry of the rattlesnake, Crotalus atrox. Comp. Biochem. Physiol. 44A, 813-820. MUKHERJEET. K., FRIARSG. W. 8/. SUMMERSJ. D. (1969) Estimated of change in plasma cholesterol and protein in relation to certain reproductive traits in female breeder turkeys. Poult. Sci. 48, 2081-2086. NEAVES W. B. (1972) The passage of extracellular tracers through the follicular epithelium of lizard ovaries. J. exp. Zool. 179, 339-364.

Annual reproductive cycle of the female cobra PANDHA S. K., CHANDOLA A. & THAPLIYAL J. P. (1968) Thyroid in the chequered water snake, Natrix piscator (Abstract). In Symposium on Comparative Endocrinology, Banaras University, Varanasi, India, Oct. 2-6, 1968. RANGNEKER P. V. & SURYAVANSHIS. A. (1969) Studies on the pancreatic islets of the lizard Uromastix hardwickii (Gray). J. Univ. Bombay 38, 56--68. RANGNEKER P. V. d~ PADGAONKAR A. S. (1972) Effects of total hypophysectomy on the glycemic and plasma cholesterol levels in the snake, Natrix piscator (Russell). Acta zool., Stockh. 53, 1-7. ROMA~OFF A. L. & ROMANOFFA. J. (1949) The Avian Egg. John Wiley & Sons, New York. ROSENTHAL H. L., PFLUKE M. L. & BUSCAGLtA S. (1957) A stable iron reagent for determination of cholesterol. J. Lab. clin. Med. 50, 318-322. STENROOS O. O. & BOWMANW. A. (1968)Turtle blood--I. Concentrations of various constituents. Comp. Biochem. Physiol. 25, 219-222. THAVLXYALJ. P., GUPTA S. C. & GARG R. K. (1974) Effects of thyroidectomy on the chequered water snake, Natrix piscator. J. Endocr. 60, 517-524.

525

T|ENHOVEN A. VAN. (1968) Vertebrate Reproduction. Academic Press, New York. VASTESAEGERM. M., DELCOURT R. & GILLET P. H. (1965) Spontaneous atherosclerosis in fishes .and reptiles. In Comparative Atherosclerosis (Edited by ROBERTS C. J., JR. & STRAUS, R.) pp. 129-149. Harper & Row, New York. WALLACE R. A. & DUMONT J. N. (1968) The induced synthesis and transport of yolk proteins and their accumulation in the oocyte in Xenopus laevis. J. Cell. Physiol. 72. 73-89. WILBER C. G. & LIEB J. (1950) The effects of increased environmental temperatures on the lipids in the blood of the turtle, Chrysemys picta. St. Louis Univ. Studies 1 (3), 1-16. WONG K. L. & CHXU K. W. (1974) The snake thyroid gland--I. Seasonal variation of thyroidal and serum iodoamino acids. Gen. & compar. Endocr. 23, 63-70,

Key Word Index--Cholesterol; cobra; Naja naja.