Camp. Bioehem. Physiol.. 1976. Vol. 53A. pp. 285 to 289. Pergamon Press. Printed in Great Britain

STUDIES ON THE ANNUAL REPRODUCTIVE CYCLE OF THE FEMALE COBRA, NAJA NAJA. SEASONAL

VARIATION IN PLASMA INORGANIC IONS

VALENTINE LANCE

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

(Received 2 April 1975)

Abstract--l. Measurements of plasma calcium, magnesium, phosphate, potassium, sodium and chloride were made on monthly samples of plasma from female cobras, Naja naja from south China throughout the year.

2. Plasma calcium and plasma inorganic phosphate increased significantly in April, May and June, and showed a high correlation with ovarian follicular development.

3. Plasma sodium rose significantly only in April and May and did not appear to be related to ovarian development. It is suggested that plasma sodium levels may be related to seasonal adrenal gland activity. None of the other ions measured showed any significant seasonal variation.

4. Injection of estradiol-benzoate at a dose of 50/~g/100g body wt every 2 days for 26 days into sexually quiescent snakes in December caused a highly significant increase (P 0.001) in plasma calcium, but had no effect on plasma magnesium or plasma cholesterol. Injection of progesterone or bovine prolactin had no effect on plasma calcium, magnesium or cholesterol.

I N T R O D U C T I O N

DURING the process of vitellogenesis in egg laying ver- tebrates there are marked elevations in plasma cal- cium and protein. Such changes have been described in teleosts (Fleming et al., 1964), amphibians (Shapiro & Zwarenstein, 1933), reptiles (Dessauer & Fox, 1959) and birds (McDonald & Riddle, 1945). Similar changes can be evoked in males or ovariectomized females by injections of estrogen (see Review by Ches- ter Jones et al., 1972). Other steroid hormones have no effect, though in reptiles growth hormone may be necessary for the full vitellogenic effect of estrogen (Callard et al., 1972; Gerstle & Callard, 1972). A great deal of work on the amphibian, Xenopus laevis (see reviews by Redshaw, 1972; Wallace & Bergink, 1974) and the domestic fowl (Bergink et al., 1974) has estab- lished that the changes in plasma constituents during vitellogenesis are directly related to gonadotropin- induced estrogen secretion by the ovary. The estrogen then acts on the liver to stimulate the synthesis of a specific protein or lipoprotein complex known as vitellin. This yolk precursor, vitellin, is released into the circulation and taken up by the developing oocyte in response to a gonadotropic hormone from the anterior pituitary gland. It is believed that a basically similar mechanism exists in all oviparous vertebrates with the possible exception of the elasmobranchs (Urist & Schjeide, 1961).

The literature on seasonal changes in blood plasma chemistry of reptiles has been reviewed exhaustively in three recent publications (Dessauer, 1970; Duguy, 1970; Gilles-Ballien, 1974). However, despite the large number of studies made on reptiles there have been very few reports that relate seasonal changes in plasma constituents to reproduction.

This work was supported by the Department of Zoo- logy, University of Hang Kong.

Increases in serum calcitim prior to ovulation have been found in turtles (Clark, 1967) snakes (Dessauer & Fox, 1959) and lizards (Gersfle & Callard, 1972).

In studies on the snakes Natrix and Thmnnophis it was noted that as plasma calcium, protein and phosphate increased during vitellogenesis plasma magnesium also increased (Dessauer et al., 1956; Des- sauer & Fox, 1959). Estrogen injections in male Thamnophis caused a rise in plasma calcium, protein and phosphate, but the plasma magnesium values were not reported (Dessauer & Fox, 1959). Since that time there have been no further published reports that relate changes in plasma magnesium concentrations to yolk deposition in reptiles.

In view of the paucity of such studies, especially in seasonally breeding tropical reptiles, measurements of some of the plasma constituents of the female cobra, Naja naja from south China were made throughout the year during a study of the reproduc- tive cycle. The effects of injections of various hor- mones on plasma chemistry in reproductively quies- cent snakes was also examined.

MATERIALS AND METHODS

Mature female cobras, Naja naja body wt 250--4508 from Kwangtung Province, China were purchased at monthly intervals from a Hang Kong snake dealer throughout 1969, 1970 and part of 1971. All snakes, except where indicated, were killed by decapitation, without anes- thesia within 24 hr of arriving in the laboratory. Blood was collected from the ventricle into heparinized tubes, centrifuged, and the plasma stored at -20"C until ana- lyzed. Plasma cations were determined by atomic absorp- tion spectrophotometry (Perkin-Elmer, Model 290) in 0"25 ml samples of well mixed plasma diluted to 10 ml with 0.5% lanthanum chloride. Plasma Ca 2+ was measured in every sample taken whereas plasma Na +, K + and Mg 2+ were measured in the monthly samples from September

285

2 8 6 VALENTINE LANCE

1969 to November 1970. Plasma chloride was determined by an electro--titration method using the Amico-Cotlove Chloride Meter (American Instruments). In a limited number of samples plasma inorganic phosphate was measured by the mahod of Fisk and Subbarow {Bauer et al., 1968).

In December :1970 a number of snakes were tested for the effects of injections, of various hormones on some of the plasma constituents. After being weighed the cobras were placed in individual wooden cages, provided with drinking water, but not fed, and after 2 days were injected according to the following scheme:

Group 1. Eight snakes, each of which received 50/.tg,/100 g body wt of estradiol benzoate intramuscularly every second day for 26 days. The steroid was dissolved in arachis oil (1 mg/ml).

Group 2. Eight snakes, each of which received progester- one at a dose of 50/zg/100 g body wt. Vehicle and injection regimen as in Group 1.

Group 3. Eight snakes each of which received bovine prolactin (NIH-P-B-2) (I mg/ml in 0.65% saline) at a dose of 50/zg/100g body wt per injection every 2 days for 26 days.

Group 4. Eight snakes, which received injections of arachis oil only.

In addition to plasma calcium and magnesium, plasma cholesterol was also measured in the experimental animals using the method of Mann 0961).

RESULTS

Plasma calcium Except for the months when the cobra is under-

going vitellogenesis (April, May, June) the plasma total calcium showed very little variation with the values falling between a very narrow range of 2.44-6.00mM/1. During April, May and June the mean total plasma calcium values showed a highly significant increase (P < 0.001) despite considerable individual variation, and in May when ovarian devel- opment was maximal a mean of 8-0mM/l was

tl

S

t

( i ~ (12} t~l IIW .... ¢ ~ .... (1}) {S)

J f ~ A N J -~ A S O N 0

Fig. 1. Seasonal variation in plasma calcium in the female cobra, Naja naja. The figures in parentheses indicate the number of samples measured each month, and the bars

about the means are equal to the S.E.M.

observed. The highest individual recorded value was 34-08 mM/1 in a late stage preovulatory snake in June. The combined data from all plasma samples measured is shown in Fig. 1. There is a highly signifi- cant correlation between plasma calcium levels and ovarian weight for the months of April, May and June (r = 0"314, P < 0"01).

Plasma sodium, potassium, maynesium and chloride

The results of the analyses of plasma samples for sodium potassium, chloride, and magnesium are given in Table 1. No significant seasonal variation in plasma potassium, magnesium or chloride were apparent. Plasma sodium however, did show a signifi- cant increase in April and May, but could not be correlated with any change in ovarian function.

Table 1. Monthly plasma inorganic ion values in the female cobra

~al.or£de Sodi~ Potalut£~a Hagnelt£tm ~ t h d l L -WL - * I L .H/L

Sept 1969 12(~_2 (12)* 143~$ (12) 6.38~0.43 (12) 2.1_9-~-0.15 (12)

Oct 1969 128~-2 (12) 141_+3 (12) 6.10~-O.18 (12) 1.64-/-0.07 (12)

Nov 1969 128~ (12) 165+2 (12) 7.4?._+0.26 (12) 1.$_tr~.03 (12)

Dec 1969 120~3 (12) 146+_17 (12) 6.17. .+0.21 (12) 1.61+0.0A (12)

Jan 1970 11.~3 (12) 164+3 (12) 5.99~-O.23 (12) 1.62_+0.07 (12)

Feb 1970 120~-_3 (10) 165+2 (10) 5.82_+0.18 (10) 1.4_5-!-O.07 (10)

Hat 1970 117+2 (10) 164_+3 (10) 5.7_]-±0.22 (10) 1.4_6-~0.08 (10)

~ 1970 119~_1 (10) 180~2 (10) 6.1_6±-O.27 (10) 1.25-±O.06 (10)

May 1970 120~2 (10) 172+_2 (10) 5.96-!-0.29 (10) 1.41--+0.01 (10)

June 1970 122~-3 (10) 164+.2 (10) $.9~-0.19 (10) 1.5_~-0.04 (10)

July 1970 117+_1 (10) 162_+2 (10) 5.42._+0.25 (10) 1.3_5~-0.04 (10)

Aug 1970 116~1 (10) 156!-2 (10) 5.08~0.10 (10) 1.42._+0.01 (10)

Sept 1970 11~-2 (10) 1.41+0.03 (10)

OCt 1970 116~-3 (10) 1.2_0~-O.05 (10)

~lov 1970 119~-A (10) 1.1~-0.04 (10)

The filturee in imrantlmeee IndXcate the nulber of emtplee measured In *inch month

Annual reproductive cycle of the female cobra 287

Table 2. Plasma inorganic phosphate in the female cobra

Heath n Mean 4. S.ILN. Mange

Jan 1970 6 3 .58 + 0 . 2 9 *

h b 1970 6 3,S8 4. 0 .15

Mar 1970 8 4 .02 ~ 0 .48

Apt 1970 6 5 .35 ~ 0 .36

May 1970 6 4 .62 + 0 .59

June 1970 12 3 .58 + 0.49

July 1970 6 3 .70 ~ 0 .48

An8 2970 7 3.28 4. 0 .20

Sept 1970 8 3.99 _+ 0 .12

O~t 1970 9 3 .50 ~ 0 .25

2 .67 - A.67

3 .30 - 4 .30

2.83 - 3 .70

4 .33 - 6 .50

2.85 - 6 .70

2.95 - 7 .60

2 .67 - 5 .25

2.67 - 3.95

3.65 - 4 .67

2 .67 - 4 ,84

Apr 1971 14 4.65 ~ 0 .45

May 1971 12 4 .90 ~ 0 .36

June 1971 12 3 .29 + 0 .47

July 1971 18 4.35 ~ 0 , 2 0

2.05 - 8 .20

3.25 - 7 ,70

3.95 - %94

2.95 - 6 .40

* q/100~. 2.5 P L A S M A M A G N E S I U M

Plasma inorganic phosphate A complete set of data for the annual cycle was

not obtained for plasma inorganic phosphate. The results are given in Table 2. There was a significant increase in plasma phosphate in the months April, May and June in 1970 and 1971. ( P < 0.05 when com- pared to all other months using the Student t-test).

The effects of estradiol benzoate, progesterone and pro- lactin on plasma constituents.

The results of the experiment in which hormones were injected for 26 days are shown in Fig. 2. The only parameter measured which showed any signifi- cant change was plasma calcium. The plasma calcium levels in the snakes receiving 50 #g of estradiol ben- zoate -17/~/100g body wt every 2 days were signifi- cantly higher than the control and both of the other groups when tested by the Student t-test (P < 0.001). None of the other constituents measured showed any change when compared to controls.

D I S C U S S I O N

Total plasma calcium in the female cobra showed very little variation outside the breeding season. Dur- ing the months July-March there were no significant differences in the monthly means. However, at the onset of vitellogenesis in April plasma calcium levels were observed to rise significantly, and by July, after ovulation was completed, the levels rapidly dropped to non-breeding levels. There was a highly significant correlation between plasma calcium and ovarian weight during the 3 month breeding period.

Very few studies on the female reproductive cycle of reptiles have reported changes in plasma calcium Dessauer et al. (1956) and Dessauer & Fox (1959) measured calcium, magnesium, phosphate and pro- tein in the plasma of two genera of ovoviviparous snakes, Thamnophis and Natrix during the reproduc- tive cycle and showed that all of these components

_J

E

2 .0

1.5

'1

Control estrogen progest, prolactin (5) (6) (6) (7)

P L A S M A C A L C I U M

.J

:E 5 E

8

control estrogen progest, prolactin (5) (6) (6) (7)

3 0 0 P L A S M A C H O L E S T E R O L

150 control estrogen progest, prolact|n (5} {6) (S) (7)

Fig. 2. The effects of injections of various hormones on plasma magnesium, calcium and cholesterol in the female cobra, Naja maja. The figures in parentheses represent the number of animals in each group,' and the bars about the

means represent the S.E.M.

'~88 VALENTINE E.ANCE

rose dramatically during viteUogenesis. The highest recorded individual plasma calcium value was 90 mM/l in Thamnophis sauritis near ovulation. The highest recorded individual value in the female cobra was 34.08 raM/1 in a snake with fully developed ovar- ian follicles. The values reported by Dessauer & Fox (1959) for anestrus snakes and snakes with hydration stage follicles are within the range obtained in Naja naja, but the values reported in Thamnophis near ovu- lation are far in excess of those found in this study. It is likely that there are species differences with re- spect to plasma calcium levels in reproducing reptiles, since in a study on Dipsosaurus dorsalis values of 3.73 and 1.84mM/l were obtained in preovulatory and postovulatory lizards respectively (Gerstle & Callard, 1972). Turtles likewise show less dramatic changes in plasma calcium with a preovulatory level of only 4"07 mM/l and a postovulatory level of 2.74 mM/l (Rubin & Callard, in press) though they do respond to injections of estrogen by showing supranormal levels of calcium (Clark, 1967).

Plasma magnesium values in the cobra were in the range 1.10-5.08 mM/1 and did not show any seasonal variation. Dessauer & Fox (1959) however, reported highly significant increases in plasma magnesium cor- responding to the increase in plasma calcium during vitellogenesis in Thamnophis. The plasma magnesium values in the cobra are similar to those found in Thamnophis and Natrix in the non-breeding condi- tion. The plasma magnesium values in a vitellogenic Natrix however, were only slightly eleyated (Dessauer et al., 1956). Since both calcium and magnesium were measured in the same plasma sample from the cobra it would seem that a rise in plasma magnesium coinci- dent with a rise in calcium does not occur in this species.

Plasma inorganic phosphate showed a significant increase during vitellogenesis in Naja, but the indivi- dual values varied considerably throughout the year. Dessauer & Fox (1959) noted similar increases, but with less variability in plasma inorganic phosphate during vitellogenesis in Thamnophis.

These changes in plasma components during vitel- logenesis are now known to be the result of increased levels of vitellin, a unique plasma protein synthesized by the liver in response to estrogen (see review by Chester-Jones et al., 1972). All of the above changes can be induced by injections of estrogens into non- breeding, immature or male reptiles. No other ovar- ian or adrenal steroid can produce similar changes. The rise in plasma calcium and phosphate in the female cobra during vitellogenesis would seem to in- dicate that estrogen is being produced by the ovary at this time. The increase in liver weight (Lance, 1975) prior to the increase in plasma calcium in April and the heightened steroid dehydrogenase activity in the ovary at this time (unpublished observations) tend to support this view. Although plasma calcium increases and plasma cholesterol decreases (Lance, 1975) during the final stages of vitellogenesis, and both parameters show a strong correlation with ovarian weight, this doe not .necessarily imply that the tw O events are cau- sally rf, lated. When plasma calcium values and plasma:cholesterol values from individual snakes are compared the correlation between the two variables though signitic~nt, is fairly weak. In many cases

snakes with low cholesterol levels also had low cal- cium levels, and vice versa. This would seem to indi- cate that the plasma calcium and plasma cholesterol titers vary independently, and that the hormone or hormones controlling their metabolism are different. The fact that estrogen caused a highly significant in- crease in plasma calcium in sexually quiescent Naja but did not appear to affect cholesterol levels strengthens this suggestion.

Since plasma sodium was measured for only one seasonal cycle the increases in the summer months are difficult to interpret. It is possible that this in- crease is related to the increased activity of the adrenal gland during these months. Considerable variation both within species and between species in seasonal plasma sodium levels have been reported, and as yet no clear cut picture has emerged (see Gil- les-Baillien, 1974, for discussion).

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