Leptodactylus ocellatus is a well-known neotropical anuran in Argentina and Brazil, typically inhabiting marshes and streams, from sea level to 1000-1200 m in the Pampean mountains.
Leptodactylus ocellatus is sympatric with L. chaquensis in Paraguay and on the Parana borders. Previous work (Cei, 1948,1949,1950,1980; Cei et al., 1955) with these two sibling species, L. ocellatus and L. chaquensis, revealed diversity in their reproductive cycles. Leptodactylus ocellatus exhibits less gametogenetic discontinuity and variety of secondary sex characters; the annual ovarian cycle of L. chaquensis, involves a fallwinter period of atresia for the residual mature oocytes from the former cycle, and at the same time a progressive auxocytic activity. The spring-summer period of mating and egg-laying of L. chaquensis is followed by resorption of retained oocytes. Moreover, in both sexes, a striking seasonal rhythm in the development of secondary sex characters has been found, together with dramatic growth of skeletal humeri and significant variation in serum calcium values (Cohen 1962a, b, 1963).Material digitalizado en SEDICI gracias a la colaboración del Dr. Jorge Williams (FCNM-UNLP).Facultad de Ciencias Naturales y Muse

Battisti, A.

Carnevali, O.

Cei, José Miguel Alfredo María

Ibañez, N.

Mosconi, G.

Polzonetti-Magni, A. M.

Álvarez, B B.

Journal of Herpetology

English

El Servicio de Difusión de la Creación Intelectual

Journal of Herpetology, Vol. 30, No. 1, pp. 68-70, 1996
Copyright 1996 Society for the Study of Amphibians and Reptiles
Changes in Plasma Sex Steroids in 
Females of Two Sympatric 
Leptodactylus from Subtropical 
South America
G. Mosconi,1 J. M. Cei,2 N. Ibanez,2 B. B. Alvarez,3 
O. Carnevali,1 A. Battisti,1 and A. M. Polzonetti- 
Magni.1 'Dipartimento di Biologia MCA, Università di 
Camerino, Italy, and 2Universidad National de Rio Cuarto 
(Dep. Ciencias Naturales y Fac. Veterinaria) Rio Cuarto, 
Cordoba, Argentina, and 3Universidad National del Nord- 
este (Fac. Ciencias Exactas, Naturales y Agrim.), Corrientes, 
Argentina.
Leptodactylus ocellatus is a well-known neotropical 
anuran in Argentina and Brazil, typically inhabiting 
marshes and streams, from sea level to 1000-1200 m 
in the Pampean mountains.
Leptodactylus ocellatus is sympatric with L. chaquensis 
in Paraguay and on the Parana borders. Previous work 
(Cei, 1948,1949,1950,1980; Cei et al., 1955) with these 
two sibling species, L. ocellatus and L. chaquensis, re­
vealed diversity in their reproductive cycles. Lepto­
dactylus ocellatus exhibits less gametogenetic discon­
tinuity and variety of secondary sex characters; the 
annual ovarian cycle of L. chaquensis, involves a fall­
winter period of atresia for the residual mature oo­
cytes from the former cycle, and at the same time a 
progressive auxocytic activity. The spring-summer 
period of mating and egg-laying of L. chaquensis is 
followed by resorption of retained oocytes. Moreover, 
in both sexes, a striking seasonal rhythm in the de­
velopment of secondary sex characters has been found, 
together with dramatic growth of skeletal humeri and 
significant variation in serum calcium values (Cohen 
1962a, b, 1963).
Endocrine regulatory mechanisms involved in re­
production in these species are unknown. We as­
sessed the plasma sex steroid changes during the pre- 
reproductive and reproductive periods. In females of 
both species living in sympatry, plasma androgens, 
estradiol-17/?, and progesterone were assessed in re­
lation with the ovarian changes, evaluated through 
the gonadosomatic index (GSI).
Wild populations of L. chaquensis and L. ocellatus 
were studied alongside the Parana river in the Laguna 
Brava swamps of Corrientes, Argentina. In this zone 
and in other areas of sympatry, the mean monthly 
temperature in winter does not fall below 16 C, while 
in summer months it is about 26 C. The mean rainfall 
is 80-100 mm in the driest months, and 150-210 mm 
in the wettest months (September-October and April- 
May, respectively). The climate in the riparian Parana 
environment is mild and relatively moist.
Ten females of both species were captured at each 
sampling at intervals over two years (1989-1990 and 
1991-1992) during their pre-reproductive and repro­
ductive periods. Each animal was anesthetized with 
3-amino benzoic acid ethyl ester (Sigma, St. Louis, 
MO; lOg/L tap water) within 5 min after capture, and 
blood was immediately collected in a heparinized sy­
ringe by cardiac puncture. Blood samples were stored 
in ice until processed; after centrifugation, plasma 
was frozen on dry ice and stored at — 70 C until assay. 
Each animal was weighed, and ovaries were removed 
and weighed. The gonadosomatic indexes (GSI) were 
calculated as a ratio of gonadal weight to body weight.
Plasma Hormone Determinations.—Plasma samples 
from 3 or 4 individuals randomly assigned to each 
batch of assays, were extracted with ether. Subse­
quently, radioimmunological analyses (RIA) of an­
drogens, estradiol- 17/3, and progesterone, were car­
ried out in duplicate as described by Polzonetti-Mag- 
ni et al. (1984).
The following sensitivities were observed: testos­
terone 5 pg (intra- and interassay coefficients of vari­
ation were 5.5% and 10%, respectively); estradiol-17/3, 
7 pg (intra- and interassay coefficients of variation 
were 4.5% and 7.5%, respectively); and progesterone, 
7 pg (intra- and interassay coefficients of variation 
were 4% and 8%, respectively). Steroid antisera were 
provided by Dr. G. Bolelli (Physiopathology of Re­
production Service, University of Bologna, Italy); tri­
tium-labelled steroids were purchased from Amer- 
sham International (Buckinghamshire, England) and 
authentic steroids were obtained from Sigma. The 
testosterone antibody was cross-reacted (> 80%) with 
5a-dihydrotestosterone and since the two steroids were 
not separated data are expressed as "androgens."
Statistical Analysis.—Plasma hormone data were an­
alyzed by one-way analysis of variance (ANOVA) us­
ing Stat View 512+® (Brain Power Inc., USA). A prob­
ability level of 0.05 was taken to indicate a statistically 
significant difference between means. Results are ex­
pressed as mean ± SD of data from ten different fe­
male samples.
Baseline androgen levels were found over the sum­
mer and fall-winter months in L. chaquensis, and then 
peaked (Fllg = 466.78; P < 0.05) at about 9 ng/ml in 
November, during the spring spawning season (Fig. 
1). In L. ocellatus, the androgen trend was similar to 
that of L. chaquensis, but the androgen peak (Fu8 = 
275.31; P < 0.05) values (8.5 ng/ml) occurred in early 
spring (August). In L. chaquensis estradiol-17/3 levels 
of the three samples in the spring were significantly 
higher (P < 0.05) than those in fall-winter and the 
summer (Fig. 2). The GSI behaved similarly to the 
estradiol-17/3, showing the highest values in Novem­
ber (Fu8 = 37.09; P < 0.05). In L. ocellatus, the lowest 
levels of estradiol-17/3 were found only in November 
and significantly higher (F118 = 32.40; P < 0.05) peak 
values of about 1000 pg/ml were found in August at 
the beginning of spring. Again, in L. ocellatus the GSI 
paralleled estradiol-17/3, reaching its zenith in Au­
gust. In L. chaquensis, increasing plasma titers of pro­
gesterone were found in the fall-winter months 
reaching peak (F1>18 = 12.64; P < 0.05) values (1800 
pg/ml, Fig. 3) at the beginning of the spring spawn­
ing season. On the contrary, no significant changes 
of plasma progesterone were observed in L. ocellatus.
Ecologically, L. ocellatus and L. chaquensis are active 
and vigorous frogs, feeding on a number of small 
vertebrates and arthropods. Remarkable develop-
SHORTER COMMUNICATIONS 69
Fig. 1. Seasonal variations of plasma androgens 
levels in female Leptodactylus chaquensis and Leptodac- 
tilus ocellatus. Each point is the mean of 10 determi­
nations ± SD.
ment of sex characters of males in both species has 
been reported (Cei, 1980). During the breeding season 
eggs are laid in foam nests on the surface of the water, 
usually in shallow ponds or lagoons. Given the shel­
tering effects of the foam nest, eggs and developing 
larvae are suitably protected from desiccation, and 
likely from many predators, such as water insects, 
carnivorous tadpoles, or snakes. Tadpoles of both spe­
cies are gregarious and peculiar parental care was 
reported (Cei, 1980). Some differences exist in their 
larval morphology: a larger tail in L. ocellatus, a more 
rounded snout in L. chaquensis, and different location 
of the sinistral spiracle.
Plasma sex steroids showed the highest values dur­
ing the spring spawning season (August-November). 
However, in L. ocellatus androgen peak values oc­
curred in early August, while in L. chaquensis they 
occurred in November. Moreover, in L. chaquensis, 
estradiol-17/3 was lowest in summer-winter months, 
and was highest in the spring spawning season (Au­
gust-November). Such a striking difference was not 
recorded for L. ocellatus, in which a slight but signif­
icant fluctuation in estradiol-17/3 plasma levels was 
found between spring and winter months. The clear­
cut seasonal pattern of the estradiol-17/3 plasma level 
in L. chaquensis may physiologically support the out­
standing seasonal ovarian changes (GSI) found for 
these frogs. Similarly the GSI for L. ocellatus is in 
agreement with the above-reported annual trend of 
the estradiol-17/3 plasma level. Finally, throughout 
the whole year the progesterone levels in L. ocellatus 
fluctuated between 850 and 1500 pg/ml, while those 
in L. chaquensis peaked in August, the other seasonal 
values being low.
These findings suggest a difference in reproductive 
physiology that both species maintain under similar 
environmental conditions. Even though now sym­
patric on the Parana borders, L. chaquensis and L. ocel­
latus may have evolved from allopatric ancestors in 
distinctly different ecological settings. Extreme con­
tinental thermic conditions led in L. chaquensis to an 
autonomous or internal rhythm. Instead, more equa­
ble climatic parameters in the wider continental dis-
Fig. 2. Seasonal variations of gonadosomatic in­
dex (GSI) and plasma estradiol-17/3 levels in female 
Leptodactylus chaquensis and Leptodactylus ocellatus. Each 
point is the mean of 10 determinations ± SD.
tribution of L. ocellatus (from 10° to 40° South Lat.) 
seem to have been effective in determining a more 
irregular and extensive mating period (Cei, 1980).
The hypothalamus-hypophysial-gonadal axis rela­
tionship, well ascertained in other amphibians living 
both in temperate and tropical areas (Chieffi and Pier-
Fig. 3. Seasonal variations of plasma progesterone 
levels in female Leptodactylus chaquensis and Leptodac­
tylus ocellatus. Each point is the mean of 10 determi­
nations ± SD.
70 SHORTER COMMUNICATIONS
antoni, 1987; Rastogi and lela, 1994), presumably reg­
ulates sex steroids in both Leptodactylus. In fact, an­
drogens in female anurans seem to play a role, as in 
males, in inducing reproductive behavior, and are a 
source of estradiol-170 through aromatization (Chieffi 
and Pierantoni, 1987; Dubowsky and Smalley, 1993). 
The estradiol-170 increase, related to ovarian devel­
opment and growth, seems to be involved in inducing 
liver vitellogenin synthesis. Vitellogenin synthesis is 
a hormonally controlled process, and vitellogenin 
synthesized by the liver is a precursor molecule of 
the yolk proteins, lipovitellin and phosvitin in all 
oviparous vertebrates so far studied (Wallace, 1985; 
Carnevali et al., 1994). Moreover, the increasing val­
ues of progesterone found in L. chaquensis during the 
spawning season may play a role in ovulation, as in 
other amphibians (Jones, 1987).
Acknowledgments.—This study was supported by 
grants from MURST and CNR (Progetto Bilaterale 
Italia-Argentina).
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Accepted: 28 July 1995.


Changes in Plasma Sex Steroids in Females of Two Sympatric Leptodactylus from Subtropical South America

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Changes in Plasma Sex Steroids in Females of Two Sympatric Leptodactylus from Subtropical South America

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