THE CONTROL OF VITELLOGENESIS BY OVARIAN HORMONES IN THE LIZARD

XANTUSIA VIGILIS

Z. YARON and LIDIA WIDZER

Department of Zoology, The George S. Wise Center for Life Sciences. Tel-Aviv University, Tel-Aviv. Israel

(Rrcricrd 29 July 1977)

Abstract-l. The plasma vitelloprotein concentration (VP) of the lizards was measured by densitometry of electrophoretically fractionated plasma proteins.

2. VP increased in ovariectomized lizards treated with estradiol alone or combined with progesterone. 3. No increase of VP was found in ovariectomized lizards treated with progesterone. 4. Progesterone inhibited the PMS-induced ovarian growth of intact lizards. 5. Ovarian weight decreased and VP increased in intact lizards treated with progesterone. 6. The inhibitory effect of progesterone on ovarian growth is attributed to the reduction of VP

incorporation in the oocytes. 7. The clutch size in lizards is controlled, presumably, by an intraovarian mechanism in which

progesterone is involved

INTRODUCTION

The reptilian ovary is known to synthesize and secrete

progesterone and estrogens (Callard & Leathem,

1965; Lupo di Prisco et al., 1968; Callard et al., 1972c,d; Klicka & Mahmoud, 1972, 1973; Chan et al., 1973; Chan & Callard, 1974; Veith, 1974; Col- ombo et al., 1974; Highfill & Mead, 1975; Colombo & Yaron, 1976; Callard et al., 1976; Licht & Crews, 1976; Judd et al., 1976). One of the functions attri- buted to estrogens in reptiles is the induction of the synthesis and the secretion of vitelloprotein by the liver (reviewed by Dessauer, 1974). However, the func- tion of progesterone is not that clear. The fact that this steroid is found in both viviparous and oviparous reptiles (Browning, 1973) emphasizes that in reptiles this steroid may have physiological functions which are not related directly to gestation (Yaron, 1972b; Veith, 1974; Highfill & Mead, 1975).

The administration of progesterone to intact lizards has resulted in the regression of yolk in the ovaries of Chamaeleo pumilus (Veith, 1974). This steroid also inhibited the PMS-induced ovarian growth in Scelo- porus cyanogenys and in Dipsosaurus dorsalis (Callard et al., 1972c,d). Progesterone implanted into the hypothalamus of Sceloporus cyanogenys had no effect on either ovarian growth or on ovulation. However, the subcutaneous administration of this steroid inhi- bited both processes (Callard et al., 1972b. d).

Since there is no conclusive evidence in reptiles for an “antigonadal” effect of progesterone at the hypo- thalamo-hypophyseal level, it seemed necessary to search for the mechanism of ovarian inhibition at another level. The present work describes the effect of the ovarian hormones, estradiol-178 and progester- one on vitellogenesis in the viviparous lizard Xantusia

vigilis, and the attempt to localize the site where pro- gesterone may exert its inhibitory effect on ovarian growth.

MATERIALS AND METHODS

Animals and treatment

Lizards were purchased from a dealer in Calimesa, Cali- fornia. Groups of 10 lizards (about 2g body wt) were housed in a 5-l. polypropylene box. Lizards were offered daily water and housefly larvae grown on meat and fish. Temperature, illumination and surgical procedures were as described earlier (Yaron, 1972a).

Aqueous suspensions of the steroids were prepared with gum arabic (1.2 mg/ml, 0.9://, NaCl).

Experimenf I. Intact males were injected i.p. with 0.6 fig estradiol every other day for 6, 14 and 20 days.

Exprrirrlenr II. Ovariectomized females were treated with estradiol (total dose 6.3 pg) or 5OOpg progesterone or both, divided into ten equal i.p. injections given on alternate days.

Exp~rimenr‘III. Intact females were injected i.p. on alter- nate days for 16 days with either 4i.u. PMS (Gestyl. Organon) dissolved in 0.9Y, NaCl, or with the saline only.

Experiment IV. Intact females were injected daily for 21 days with either PMS (’ i.u./lizard), progesterone (80 lg/ lizard). with both or witl. bovine albumin (0.14 mg in 50 ~1 0.9% NaCl).

Vitelloprotein determination

The lizards were killed by decapitation I day after the last injection of the hormones. The blood from the wound was collected into heparinized capillaries. Plasma was separated after centrifugation and the proteins were separ- ated by electrophoresis on 7% polyacrylamide gel columns (Ornstein. 1964). The gels were stained with 0.29:. amido black, and after ele&ophoretic destaining they were scanned with a Gilford Spectrophotometer 2400. The plasma level of the vitelloprotein was expressed as the ratio between the area under the vitelloprotein peak and that of the plasma albumin. Livers were removed from the decapitated lizards, and weighed on a torsion balance to the nearest 0.1 mg. Liver weight is expressed as “hepato- somatic index” (mg liver/g body wt). The significance of the differences between group means was evaluated using the Student t-test.

279

The clectrophoretic pattern of plasma proteins from intact females of Xantusia vigilis was character- ized by a prominent protein band at R, = 0.15 -0.18. This band was not found in the plasma of males (Fig. I ). The intensity of this band in the plasma electro- phoresis decreased one month or more following ovariectomy. The concentration of this protein, rela- tive to that of plasma albumin on the same column. was found to correlate to the accumulated dose of estradiol injected into male lizards (r = 0.978. Fig. 2). It is suggested. therefore. that this band represented the plasma vitelloprotein of X~~~u.sia riclilis.

The separate effects of exogenous estrogen or pro- gesterone on the concentration of plasma vitellopro- tein. in the absence of the ovaries. was studied in ovaricctomized lizards (Exp. II). The administration of progesterone alone to these lizards did not alter the electrophoretic pattern of the plasma. and no in- crease in the plasma vitelloprotein;!albumin ratio could be detected. However. treatment with estradiol resulted in a five-fold increase of the plasma vitrllo- protein,‘albumin ratio. A somewhat higher increase in this ratio was noted in lizards treated simultaneously with both steroids (Fig. 3). The hepato-somatic index in estradiol-treated lizards was higher than that of the controls (33 k 1.3 vs 25.8 ) 0.2) or progesterone- treated. ovariectomized lirards (27 f 0.9, P < 0.001). The hepato-somatic index of lizards treated with both

steroids (39 & ?.I) did not differ from that of the

estradiol group (P = 0.053).

Ftg. I. Electrophorcsis of plasma proteins from intact male and female Xn,~r~r~iu rGgi/is. 7”,, polyacrylamide gel stained with amido black. Arrow designates vitelloprotein band.

l

0 2 4 6 8

pgESTRADIOL

Fig. 2. Vitelloprotem/albumin ratio in plasma of male Xun- tusiu t3rgili.s injected with estradiol-17/I for 6, 14 and 20

days (total dose: 1.8. 4.2 and 6.0 pg, respectively).

The effect of endogenous ovarian hormones on plasma vitelloprotein was investigated by stimulating their secretion with an exogenous gonadotrophin in intact lizards (Exp. III). A ten-fold increase in the vitelloprotein/albumin ratio occurred in the plasma of female lizards following the administration of 32 i.u. PMS (2 i.u./lizard every 2 days). The increase in the weight of the ovaries in these lizards was about five-fold (Table I). In the following experiment (Exp. IV), in which intact lizards were treated more inten- sively with PMS (2 i.u./lizard, daily for 3 weeks), the weight of the ovaries in the treated lizards was 30 times higher than that of the controls, whereas the vitelloprotein/albumin ratio did not change at all (Table 2).

The administration of progesterone to the intact lizards (Exp. IV) resulted in a significant decrease in the weight of the ovaries (P < 0.01). However, in these lizards the vitelloprotein/albumin ratio in- creased three-fold (P < 0.001). Addition of progester- one to the PMS treatment resulted in a diminished growth of the ovary as compared with the PMS group (V < 0.002, Table 2).

DISCUSSION

Treatment of Xantusiu vigilis males with estra- dial- I7p resulted in the appearance of a new protein in the plasma (Figs I and 2). The concentration of a protein with the same electrophoretic mobility in- creased in the plasma of ovariectomized Xantusia fol- lowing the injection of the same steroid (Fig. 3). The clectrophoretic mobility of this protein resembled that of the protein which appeared or increased in the plasma of other lacertilians following a treatment with estradiol. This protein has been designated as “plasma vitellin” in Utu .stan.shurinna (Hahn, 1967), “y-globulin” in Scrloporus cyunogenys (Suzuki & Prosser. 1968) and in Churnuelro pumilus (Veith. 1974t and. probably, “fraction 4, b-globulin” in Dip- ,smauru.s dorsu[is (Gerstle & Callard, 1972). It is assumed, therefore, that the protein band at R, = 0.15~0.18 in the plasma electrophoresis also represents the vitelloprotein of Xantusia uigilis.

Treatment of Xantusia females with estradiol resulted also in an increased hepatic weight, due to the stimulated synthesis of vitelloprotein in this gland (Hahn, 1967). Hepatic growth during the vitellogenic phase of reproduction or in response to treatment

Vitellogenesis in a lizard 281

E

EP

Fig. 3. Gel electrophoreses and electropherogrammes %f plasma taken from ovariectomized Xantusia vigilis treated with ovarian steroids. Lizards were injected i.p. on alternate days for 3 weeks with either the vehicle, progesterone alone (total 500 &izard), estradioL17fi alone (total 6.3 pg/lizard) or both steroids combined. Gel columns were stained with amido black and were scanned with a Gilford spectrophotometer. V-vehicle injected; P-progesterone treated; Em+stradiol treated; EP- combined treatment; X-vitelloprotein. The mean vitelloprotein/albumin ratios of the above groups

were: V = 0.044; P = 0.041; E = 0.250; EP = 0.370.

with estradiol has been reported in other reptiles (Cal- n-rents in Xantusia were performed on ovariectomized lard et al., 1972a, b; Gerstle & Callard, 1972; Lance, lizards with intact pituitaries. Although our results 1975). In Dipsosaurus dorsalis and in Sceloporus can neither support nor negate the idea about the cyanogenys, estrogen-induced vitellogenesis and the requirement of GH in the vitellogenic response, they concomitant hepatic growth required the presence of do indicate that the absence of the ovaries did not the intact pituitary or the administration of Growth interfere with the formation of plasma vitelloprotein Hormone (GH) (Callard et al., 1972a, d). Our experi- in lizards subjected to an exogenous estrogen.

2x2 %. YARO~ and LIIIIA WII)/I K

Table I. Effect of PMS treatment on vitellogemc parameters an .t. ~rrr,r\r,i : /I/I/~\ ! t \!I III)

____--- Treatment II Body wt (g) Ovary (mg) ,z Vitelloprote~n .~lhumiii

mean f S.E.M. mean + S.E.M. mean i s F M __.~

Saline 6 1.x2 + 0.07 4.55 f 0.86 i 0, I20 3 il.0~5 PMS 7 I .94 * 0. I3 20.64 * 5.14* 6 I.iXO 4 02X1+

* Sigmficantly different from control (P < 0.02). I Significantly different from control (P < 0.01). PMS (Gestyl, Organon) was injected (4 i.u..ilirard) X times on alternate day” rhc

vitellourotein/albumin ratio was calculated from plasma electropherogrammec (<et’ Fig. 3;.

The administration of progesterone alone to ovar- iectomized lizards was not followed by a considerable increase in the concentration of plasma vitelloprotein (Fig. 3). The addition of progesterone to the estradiol treatment did not reduce the increase of plasma vitel- loprotein in response to the estrogen (Fig. 3). It may be concluded that progesterone did not interfere with the vitellogenic response of the liver to estrogen in Xantusia.

In Exps III and IV we examined the effect of endo- genous estrogens, the secretion of which had been stimulated by the administration of an exogenous gonadotrophin. In Exp. III the ratio of vitelloprotein/ albumin in the plasma increased from 0.120 f 0.035 in the controls to 1.180 & 0.286 in the PMS-treated lizards. Surprisingly, in Exp. IV the ratio was similar in the control and PMS-treated lizards. The discre- pancy between the results of PMS treatments in these experiments can be explained by the difference in the growth of the ovaries. In Exp. III ovarian weight in- creased only 5-fold, whereas in Exp. IV the weight of ovaries increased about thirty-fold.

The inverse relation between plasma concentration of vitelloprotein and ovarian growth illustrates two of the processes by which gonadotrophins exert their effect on the growth of the ovary: (I ) the stimulation of estrogen synthesis and secretion by the ovary, and the consequent production and release of vitellopro- tein by the liver: and (2) the uptake and incorporation

of vitelloprotein into the growing follicles. Such pro- cesses controlled by gonadotrophins were described in amphibians (Redshaw, 1972). The lower dose of PMS in Exp. III was presumably sufficient to pro- mote estrogen secretion by the ovary and, in turn. the vitelloprotein production by the liver. Probably, the level of the gonadotrophin was not high enough to stimulate also the incorporation of the vitellopro- tein into the oocyte. The net result was, therefore. an accumulation of the vitelloprotein in the plasma. and a moderate increase in ovarian weight. The higher dose of PMS in Exp. IV stimulated both pro- cesses. This was reflected by an enormous increase in the weight of the ovaries and by the low plasma concentration of the vitelloprotein.

An inverse relation between ovarian weight and plasma cholesterol (indicating yolk constituents) was described in Naja nuja. In this snake, the main in- crease of ovarian weight occurs in May, together with a significant decrease in plasma cholesterol. When cholesterol values from individual snakes were paired with ovarian weights. a high degree of inverse correla- tion was found between the two parameters (Lance.

1975).

The role of progesterone as an “antigonadal” hor- mone was suggested in a review on the endocrine control of the reptilian gonad (Callard et al., 1972~). The reviewers suggest that “the hormone might exert an inhibitory action on the biosynthesis of estrogen.

Table 2. Etrects of PMS and progesterone on vitellogenesis in Xnnfusitr rigilrs” (Exp. IV)

Treatment )1 Body wt

(S)

Ovary

(mg)

Diameter of largest follicle Vitelloprotem

(mm) II albumln”

Albumin‘ Progesterone” PMY PMS + progesterone

12 1.97 * 0.08 4.58 k 0.75 1.56 + 0.15 7 0.17 * 0.04 IO 2.1 I k 0.09 2.13 i 0.19’ 0.76 + 0.08 6 0.56 f 0.08’ 8 2.34 + 0.07 148.00 + 6.74’ 6.22 + 0.22’ 6 0. 16 * 0.04

6 1.99 f 0.10 80.10 + 10.50” 4.70 rf: 0.32h 5 0. 15 f 0.02

“The experiment was conducted on adult lizards with initial ovarian weight of less than 5 mg. Results are expressed as group means (+SEM).

‘The ratio was calculated from plasma electropherogrammes. ‘ Bovine albumin (0.14 mg) was injected daily as a control. ’ Progesterone (80 /cg.!lizard per day) was injected i.p. for 21 days. ’ Different from control (P i 0.01). ‘Different from control (P < 0.001). r PMS (Gestyl. Organon) 2 i.u./lizard per day in 50111 0.9”,, NaCl was mlectcd 1.p. for 21

days. h Different from PMS-injected (P < 0.002).

Vitellogenesis in a lizard 283

thus reducing vitellogenesis, or it could possibly block other reactions essential to vitellogenesis, which occur either in the liver or fat body, or both”.

Our results in Xantusia vigilis cannot corroborate these assumptions. Although progesterone alone did not induce vitellogenesis in ovariectomized lizards, it did not reduce the effect of estrogen on the liver in a combined treatment (Fig. 3). Although progesterone treatment of intact lizards resulted in a significant reduction of ovarian weight and of follicular diameter, the plasma vitelloprotein level was increased in the same lizards (Table 2). We suggest, therefore, that the antigonadal effect of progesterone in Xantusia was exerted at the follicular level. Progesterone, probably, inhibited the incorporation of vitelloprotein into the oocytes. Such inhibition resulted in a decrease of ovarian weight together with the accumulation of vitelloprotein in the plasma. This inhibitory effect of progesterone antagonized the stimulatory effect of the gonadotrophin on the same process.

In a preliminary study at our laboratory, silicone implants containing progesterone were attached uni- laterally to ovaries of Lacerta sicula. After treatment with PMS the growth of progesterone-implanted ovaries was significantly retarded, as compared with the contralateral ovaries implanted with silicone only (unpublished results).

If such inhibition by progesterone operates during the normal ovarian cycle of reptiles it may, indeed, serve as a mechanism controlling the size of the clutch, i.e. the number of ripe follicles produced by the ovary in one cycle. In fact, the preovulatory ovary of Xanrusia vigilis is capable of progesterone synthesis (Colombo et al., 1974). Moreover, progesterone levels in the plasma of several reptiles, like Natrix sipedon pictiuentris begin to rise in animals with large preovu- latory follicles (Chan et al., 1973). These data indicate that in reptiles large follicles may secrete progesterone before ovulation.

Removal of the largest follicles from the largest ovary or both ovaries in Anolis carolinensis resulted in a “greater than normal growth rate of smaller follicles”. The explanation of this phenom- enon was that “larger follicles may secrete a sub- stance(s) which acts locally, desensitizing smaller folli- cles to gonadotropic stimulation” (Jones et al., 1973). We suggest that progesterone secreted by the larger follicles in Xantusia inhibits the growth of the adja- cent smaller follicles by reducing their rate of vitello- protein uptake and incorporation.

Acknowledgements---We would like to thank Dr B. Moav and Mr Z. Ilan, Department of Zoology, Tel-Aviv University, for their valuable criticism of the manuscript.

REFERENCES

BROWNING H. (1973) The evolutionary history of the corpus luteum. Biol. Reprod. 8, 128-1.57.

CALLARD I. P. & LEATHEM J. H. (1965) In uitro steroid synthesis by the ovaries of elasmobranchs and snakes. Archs Anat. microsc. Morph. exp. 54, 3548.

CALLARD I. P., BANKS S. H. & BANKS W. L. (1972a) Hepa- tic protein and nucleic acid content in Dipsosaurus dor- salis following hypophysectomy and treatment with estradiol-17/I and growth hormone. Comp. Biochem. Phy- siol. 41B. 5033510.

CALLARD I. P., BAYNE C. G. & MCCONNELL W. F. (1972b) Hormones and reproduction in the female lizard Scelo- porus cyanogenys. Gen. camp. Endocr. 18, 175-194.

CALLARD I. P., CHAN S. W. C. & POTTX M. A. (1972~) The control of the reptilian gonad. Am. 2001. 12, 273-287.

CALLARD I. P., D~~LITTLE J., BANKS W. L. JR. & CHAN S. W. C. (1972d) Recent studies on the control of the reptilian ovarian cycle. Gm. camp. Endocr. Suppl. 3, 65-75.

CALLARD I. P., MCCHESNEY I., SCANES C. & CALLARD G. V. (1976) The influence of mammalian and avian gonado- tropins on in vitro ovarian steroid synthesis in the turtle (Chrysemys picta). Gen. camp. Endocr. 28, 2-9.

CHAN S. W. C. & CALLARD I. P. (1974) Reptilian ovarian steroidogenesis and the influence of mammalian gonado- trophins (FSH and LH) in oifro. J. Endocr. 62, 2677275.

CHAN S. W. C., ZIEGEL S. & CALLARD I. P. (1973) Plasma progesterone in snakes. Camp. Biochem. Physiol. 44A, 63 l-637.

COLOMBO L. & YARON Z. (1976) Steroid 21-hydroxylase activity in the ovary of the snake Storeria dekayi during pregnancy. Gen. camp. Endocr. 28, 403-412.

COLOMBO L., YARON Z., DANIELS E. & BELVEDERE D. (1974) Biosynthesis of 11-deoxycorticosterone by the ovary of the Yucca Night Lizard, Xantusia uigilis. Gen. camp. Endocr. 24, 331-337.

DESSAUER H. C. (1974) Plasma proteins in Reptilia. In Chemical Zoology (Edited by FLORKIN M. & SCHEER B. T.), Vol. 9, pp. 1877216. Academic Press, New York.

GERSTLE J. F. & CALLARD 1. P. (1972) Reproduction and estrogen-induced vitellogenesis in Dipsosaurus dorsalis. Comp. Biochem. Physiol. 42A, 791-801.

HAHN W. E. (1967) Estradiol-induced vitellinogenesis and concomitant fat mobilization in the lizard Uta stansbur- iana. Camp. Biochem. Physiol. 23, 83-93.

HIGHFILL D. R. & MEAD R. A. (1975) Sources and levels of progesterone during pregnancy in the garter snake, Thamnophis elegans. Gen. camp. Endocr. 27, 389400.

JONES R. E., GERRARD A. M. & ROTH J. J. (1973) Endocrine control of clutch size in reptiles. II. Compensatory fol- licular hypertrophy following partial ovariectomy in Anolis carolinensis. Gen. camp. Endocr. 20. 55&555.

JUDD H. L., LAUGHLIN G. A., BACON J. P. & BENIRSCHKE

K. (1976) Circulating androgen and estrogen concen- tration in lizards (Iguana iguana). Gen. camp. Endocr. 30, 391-395.

KLICKA J. & MAHMOUD I. Y. (1972) Conversion of preg- nenolone-4i4C to progesterone-4i4C by turtle corpus luteum. Gen. camp. Endocr. 19, 367-369.

KLICKA J. & MAHMOUD I. Y. (1973) Conversion of choles- terol to progesterone by turtle corpus luteum. Steroids 21, 483-493.

LANCE V. (1975) Studies on the annual reproductive cycle of the female cobra, Naja naja-I. Seasonal variation in plasma cholesterol. Comp. Biochem. Physiol. 52A, 519-525.

LIGHT P. & CREWS D. (1976) Gonadotropin stimulation of in vitro progesterone production in reptilian and amphibian ovaries. Gen. camp. Endocr. 29, 141-151.

LUPO DI PRISCO C., DELRIO G. & CHIEFFI G. (1968) Sex hormones in the ovaries of the lizard Lacerta sicula. Gen. camp. Endocr. 10, 292-295.

ORNSTEIN L. (1964) Disc electrophoresis. I. Background and theory. Ann. N.Y. Acad. Sci. 121, 321-349.

REDSHAW M. R. (1972) The hormonal control of the amphibian ovary. Am. 2001. 12. 289-306.

SUZUKI H. K. & PROOFER R. L. (1968) The effects of estra- dio1 valerate upon the serum and bone of the lizard Sceloporus cyanogenys. Proc. Sot. exp. Biol. Med. 127, 47.

284 %. YAKON ;Illd LII)IA Wll)/l I<