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 - 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 References
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Received: August 6, 2007. Accepted: September 22, 2007.


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