Plasma levels of estradiol during vitellogenesis and early gestation in
oviparous and viviparous Lacerta (Zootoca) vivipara
Benoît Heulin1,*, Danielle Garnier2, Yann Surget-Groba3, Jean Deunff3
Abstract. The evolution of viviparity in lizards and snakes is always associated with a reduction of the eggshell membrane. There is strong evidence indicating that estradiol is the primary factor involved in seasonal development of the uterine glands in preparation for eggshelling. However, the hypothesis that the thinner eggshells of viviparous species could be the consequence of lower pre-ovulatory levels of circulating estradiol has not been tested. In a previous histological study we showed that the pre-ovulatory growth of the uterine shell glands is significantly more pronounced in oviparous than in viviparous females of the lizard Lacerta (Zootoca) vivipara. During the current study we assayed plasma levels of estradiol before and during vitellogenesis and during early gestation. We did not find any significant difference of estradiol concentrations between oviparous and viviparous females. In both reproductive forms the plasma estradiol concentration was significantly higher during late vitellogenesis than during early gestation. Future research should address whether variation in the growth of the uterine shell glands could be predominantly mediated by modification affecting estrogen receptors of the uterus rather than by concentration of the circulating hormone. Keywords: estradiol, live-bearing, lizards, oviparity, reproductive mode.
The parchment-like eggshell observed in most
of the uterine shell glands, which occurs during
oviparous species of squamates is composed
the period of vitellogenesis preceding ovulation,
of a thick layer of proteinaceaous fibers, the
appears to be regulated by estradiol secreted
eggshell membrane, overlain by a thin (some-
by the ovary (Girling, 2002). Reduced develop-
times absent) calcite crust (Schleich and Kas-
ment of uterine shell glands in viviparous forms
tle, 1988). Although some viviparous species
of squamates is thought to be the consequence
of squamates still have an eggshell membrane
either of lower pre-ovulatory levels of circulat-
enveloping the embryo during development,
ing estradiol and/or of a lower sensitivity (lesser
this structure is always much thinner than the
number or blocking of the estrogen receptors)
eggshell membrane of oviparous species (Stew-
of the uterine target tissue (Guillette, 1993).
art, 1985; Heulin, 1990; Guillette, 1993; Qualls,
Testing this hypothesis requires comparison
1996). Understanding the evolution of vivipar-
of closely related oviparous and viviparous taxa
ity in squamates requires study of the factors
in order to minimize the confounding effect
that influence eggshell thickness. The eggshell
of phylogenetic differences. The lizard Lacerta
membrane of oviparous and viviparous species
(Zootoca) vivipara, which is one of the rare
is secreted by uterine glands immediately after
species of squamates that are reproductively bi-
ovulation (Guillette, 1993; Palmer et al., 1993;
modal (i.e. species with allopatric oviparous
Heulin et al., 2005). Differentiation and growth
and viviparous populations), is an ideal modelfor such a comparative study. Viviparous fe-
1 - CNRS UMR 6553, Station Biologique de Paimpont,
males of L. (Z.) vivipara give birth to fully
formed offspring (stage 40 of Dufaure and Hu-
2 - Inserm, U625; GERHM; IFR140; University of Rennes
I, Campus de Beaulieu, Rennes, F-35042, France
bert, 1961) enveloped in a thin (6-10 µm)
3 - CNRS UMR 6553, Laboratoire de Parasitologie Phar-
eggshell membrane, whereas oviparous females
maceutique, Avenue du Professeur Léon Bernard,
oviposit eggs containing embryos of stage 30
to 35 encased in a thicker (40-65 µm) eggshell
membrane (Panigel, 1956; Heulin, 1990; Heulin
Koninklijke Brill NV, Leiden, 2008.
Also available online - www.brill.nl/amre
et al., 1991, 2002; Stewart et al., 2004). A re-
N.H., USA) dissolved in redistilled methanol. Radioac-
cent histological investigation revealed that the
tive estradiol-17β were purchased from Amersham-France(Les Ulis, France) ([2,4,6,7,16,17-3H]Œstradiol; sp act 5.14
uterine shell glands are significantly thicker
TBq/mmol, 139 Ci/mmol). Estradiol-17β, was extracted
in oviparous than in viviparous females dur-
from plasma (0.03 to 0.1 ml) with 2 ml dichloromethane
ing vitellogenesis and that, in both forms, the
(Recovery > 80%). Steroid concentrations in the bloodplasma were measured using radioimmunoassay according
secretion of the eggshell membrane and the
to Terqui et al. (1973). Bound and free fractions separa-
regression of uterine shell glands occur very
tions were made by the dextran-charcoal method. The an-tiserum, anti-estradiol-6-O-carboxymethoxyme-BSA (final
rapidly after ovulation (Heulin et al., 2005).
dilution 1/240 000) cross-reacted with 6-keto-estradiol 17β
In the present paper we report plasma estra-
(12.3%), but with neither estradiol-17α nor estrone (<1%)
diol levels measured on the blood samples col-
(Dray et al., 1971). Oviparous and viviparous plasma sam-ples were assayed in a random order in 3 assays. Each sam-
lected from females autopsied in our histologi-
ple was assayed in duplicate. We also ran quality control
cal study (i.e. Heulin et al., 2005) to test whether
standards of known concentration in each assay. Sensitiv-
the lesser pre-ovulatory development of uterine
ity was 0.5 pg for the standard curve and <0.1 ng/ml forplasma. The inter-assay and intra-assay coefficients of vari-
shell glands in viviparous females is correlated
ation were 15% and 13%, respectively.
with a lower level of circulating estradiol.
All averages are given ± standard deviation. We ana-
lyzed the data using a two-factors analysis of variance (re-
The data presented here were obtained from lizards that
productive mode X stages of reproductive cycle), followed
were caught in September 1998 in the oviparous popula-
by pairwise (Student-t tests) comparisons of means. The
tion of Louvie (43◦06 N, 0◦23 W, Alt. 370 m) in south-
Minitab 11.11 program was used for all statistics.
western France and in the viviparous populations of Paim-
A detailed account of reproductive condition, including
pont (48◦N, 2◦W, Alt 150 m) in north-western France. The
embryonic stage, eggshell thickness and the histology and
lizards hibernated (4 months at 4◦C) in our lab. The females
morphometrics of the uterus of the females used in the cur-
were allowed to copulate with males for 2 or 3 days during
rent study has already been presented in Heulin et al. (2005). The categories of females used in our table 1 correspond to
the third week following hibernation. During the activity pe-
those described in Heulin et al. (2005): NV, non-vitellogenic
riod (before and after hibernation) the lizards were reared
with translucent follicles of less than 4 mm3; VT1, vitel-
separately in plastic terraria., in a room where large win-
logenic with yellow follicles of 5 to 65 mm3; VT2, vitel-
dows provided natural photoperiod. Each terrarium (30 ×
logenic with follicles of 65 to 125 mm3; OE, with oviductal
20 × 20 cm) was equipped with a shelter, dishes of food and
eggs containing stage 3 or 4 embryos of Dufaure and Hubert
water, and a 40W bulb that provided heat for 6 h/day. The
rearing conditions described above allow normal vitellogen-esis and the onset of ovulation occurs about one month after
The oviparous and viviparous data sets are
the end of hibernation (Gavaud, 1983; Heulin, unpubl. obs.).
presented in table 1. A two-way analysis of
In our study, we sacrificed some females during the two days
variance revealed significant variation of the
following their removal from hibernation (N = 6 oviparousand N = 6 viviparous). The other females (27 oviparous,
plasma estradiol concentration between repro-
29 viviparous) were sacrificed 20 to 30 days after the end
ductive stages (F = 4.96, P < 0.01), no sig-
of hibernation during the period of peak development of the
nificant variation between reproductive modes
uterine shell glands (in pre-ovulatory vitellogenic females)
= 3.06, P = 0.09), and no interac-
and the subsequent decrease in size of these glands follow-ing eggshell formation (in females with recently ovulated
tion between reproductive mode and stages
eggs). This timing allowed us to get a majority of samples
(F = 0.28, P = 0.84). Pairwise compar-
(tissues and blood) for the period during which there are the
isons (Student-t tests) of reproductive stages re-
most pronounced differences in growth and activity of shell
vealed that the plasma estradiol concentration
glands between oviparous and viviparous females (Heulinet al., 2005).
was higher during late vitellogenesis (category
Each female was sampled once (when sacrificed).
VT2) than during early embryonic development
The females were chilled to 3◦C for 20 min before
(category OE), both in oviparous (significant at
decapitation. Blood samples were collected from thecarotid arteries into heparinised tubes immediately af-
P = 0.0005) and in viviparous (significant at
ter decapitation. The blood sample was centrifuged at
P = 0.0001) females. In viviparous females
1200 tpm for 10 min and the resultant plasma was
the plasma estradiol concentration was signif-
stored at −70◦C until assayed for estradiol. Standard
icantly higher during, than before vitellogene-
solutions of non-radioactive estradiol-17β were preparedin phosphate buffer from 10 µg/ml stock solutions re-
sis (significant at P = 0.03 for the compari-
constituted from powdered steroids (Steraloid, Wilton,
son of categories VT1 versus NV; significant at
Table 1. Plasma estradiol levels, thickness of the eggshell membrane and thickness of the uterine shell glands of oviparous and viviparous Lacerta (Zootoca) vivipara during the pre-ovulatory and post-ovulatory periods. All data except estradiol levels are from Heulin et al., 2005.
Categories of females: NV, non-vitellogenic; VT1, vitellogenic with follicles of 5 to 65 mm3; VT2, vitellogenic with folliclesof 65 to 125 mm3; OE, with oviductal eggs containing stage 3 or 4 embryos of Dufaure and Hubert (1961). Days post-emergence: days after removal from hibernation. *, ** respectively indicate significant differences at P < 0.01 or P < 0.001 between oviparous and viviparous values,Student t-test. P = 0.04 for the comparison of VT2 versus
ley and Moore, 2002). In viviparous species,
NV). Other pairwise comparisons of reproduc-
this peak of estradiol concentration generally
tive stages were not significant. Whatever the
ranges from 0.07 ng/ml to 6 ng/ml, (Gorman
reproductive stage (NV, VT1, VT2 or OE) the
et al., 1981; Kleis-San Francisco and Callard,
mean values of plasma estradiol concentrations
1986; Ghiara et al., 1987; Whittier et al., 1987;
were always slightly elevated in oviparous fe-
Bonnet et al., 1994; Jones and Swain, 1996;
males compared to viviparous females (table 1),
Woodley and Moore, 1999; Edwards and Jones,
but these differences were not statistically sig-
2001; Girling et al., 2002). In the absence of
nificant (Student-t tests, P > 0.05 in all cases).
a phylogenetically based comparative analysis,
The highest plasma concentrations of estra-
a possible relationship between reproductive
diol in squamate reptiles occur during the pe-
mode and concentration of estradiol is obscured
riod of follicular growth preceding ovulation
by the high level of interspecific variation.
(Girling, 2002). Estradiol, which is secreted
The sole previous study of lacertid lizards re-
by the follicles of the ovaries, induces vitel-
ported a concentration of pre-ovulatory plasma
logenin synthesis by the liver and simultane-
estradiol (4 ng/ml) in a wild population of
ously stimulates the development of the uterus
the oviparous Podarcis sicula (Carnevali et al.,
in preparation for eggshelling and/or for gravid-
1991) that is very close to the value we obtained
ity. There is considerable interspecific variation
for oviparous Lacerta (Zootoca) vivipara. Al-
in pre-ovulatory levels of circulating estradiolamong squamates and the range for oviparous
though our investigations show that the mean
species overlaps that for viviparous species. In
values of plasma estradiol concentrations were
oviparous species, the peak of plasma concen-
slightly elevated in oviparous females compared
tration of estradiol measured during the pre-
to viviparous females for all three of our pre-
ovulatory period ranges from 0.12 ng/ml to
ovulatory categories, this tendency was not sta-
4 ng/ml (Bonna-Gallo et al., 1980; Gorman
tistically significant. Although the lack of sig-
et al., 1981; Joss, 1985; Moore and Crews,
nificant differences in circulating levels of estra-
1986; Carnevali et al., 1991; Diaz et al., 1994;
diol might be due to the small samples, it strik-
Phillips and Millar, 1998; Rhen et al., 2000;
ingly contrasts with the significant difference in
Radder et al., 2001; Weiss et al., 2002; Wood-
pre-ovulatory growth of the uterine shell glands
in oviparous females compared to viviparous fe-
experimental protocols are approved (authorization number
males (Heulin et al., 2005, and table 1).
DSV35-38) by the animal protection and health committeeof the French veterinary services.
There is strong experimental evidence indi-
cating that estradiol, secreted by the ovariesduring follicular growth, is the main hormone
triggering the pre-ovulatory development ofuterine shell glands in Squamates (Panigel,
Bonna-Gallo, A., Licht, P., MacKenzie, D.S., Lofts, B.
(1980): Annual cycles and levels of pituitary and plasma
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THE MYSTERY OF SOMA In the realm of Hinduism, Soma can be recognized as, and is considered to be, a god ( deva ), a magical and hallucinogenic plant, and the juice of that plant. Soma is extraordinary in that it is recognized as one of only a few plants humans have ever deified (see Wasson 3). There are a large variety of other sacred plants (even within India), but none of which have had q