Reproductive maturation of the tropical clawed frog: Xenopus tropicalis (2023)

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General and Comparative Endocrinology

Volume 160, Issue 2,

15 January 2009

, Pages 117-123

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The tropical clawed frog, Xenopus tropicalis, is a relatively new model species being used in developmental biology and amphibian toxicology studies. In order to increase our understanding of reproductive maturation and the role of steroid hormones in X. tropicalis, we collected baseline reproductive data in this species from metamorphosis to adulthood. One cohort of frogs was maintained for 42 weeks post-metamorphosis (PM) with endpoints representative of important reproductive parameters collected at 1- or 2-week intervals. These endpoints were then correlated to titers of either estradiol or testosterone. Male frogs exhibited nuptial pads, starting at 8 weeks (PM) when measureable concentrations of circulating testosterone (5.3ng/mL plasma) first appeared. Testosterone concentrations remained above this level at all later time points, but were highly variable among individuals. Testes sizes in males reached their peak at 22 weeks PM (21mg) with sperm counts peaking at the same time (25 million sperm/male). In females, estradiol becomes elevated in the blood at 16 weeks PM (1.5ng/mL plasma) which corresponds with the presences of vitellogenin (4.4mg/mL plasma), vitellogenic oocytes in the ovary, ovarian growth, and oviduct growth. Vitellogenic oocytes increased in number up to 15,000 per female at 30 weeks PM and accounted for 75% of the total number of oocytes present in the ovary. The ovary and oviducts continued to grow in mass until 30 weeks PM at which point they had reached sizes of 3.6g and 0.8g, respectively. These data indicate that male and female X. tropicalis reach reproductive maturation at 22 and 30 weeks PM, respectively. Results from this study are valuable for the design of amphibian toxicology assays and increase our understanding of the reproductive biology of this relatively new model species.


The African clawed frog, Xenopus laevis, is an important historical and current model species in developmental biology; recently, interest in the related X. tropicalis has been increasing as an alternative to X. laevis due to its faster generation times, diploid and sequenced genome, and smaller body size (Hirsch et al., 2002). In aquatic toxicology, X. tropicalis is being utilized as a model species for reproductive toxicity studies (Fort et al., 2004, Takase et al., 2007, Knechtges et al., 2007, Pettersson and Berg, 2007, Olmstead et al., in press). While the larval development of these amphibians has been extensively characterized, less is known about the details of their reproductive maturation after metamorphosis.

In X. laevis, the differentiation of the gonad occurs during larval development with ovaries being distinguishable from the testes before metamorphosis is complete (Kelley, 1996). After metamorphosis these immature gonads grow and develop into full functionality as adults. During this period secondary sex characteristics develop in males and females and are controlled by circulating steroid hormones produced by the gonads (Kelley, 1996). Secondary sex characteristics in Xenopus include oviducts in females and nuptial pads on the forelimbs of males.

Oviducts are paired convoluted tubules derived from Mullerian ducts (Wake and Dickie, 1998). These oviducts function in the maturation of oocytes once they have been released from the ovaries (Wake and Dickie, 1998). At the completion of metamorphosis, these Mullerian ducts are present on either side of the kidneys in both male and female Xenopus. In mature females, oocytes that are released from the ovaries pass through the oviducts before being released for fertilization during mating. During the passage through the oviducts, these oocytes acquire several protective jelly coats. Oviducts begin to become thickened and convoluted in X. laevis around 7 months PM (Witschi, 1971). Ovariectomy in adult female X. laevis results in the regression of oviducts suggesting that these structures are maintained by circulating estrogens (Kelley, 1996). In X. laevis the males retain their Mullerian ducts until 7 months post-metamorphosis (PM). Castration prior to this period results in the maintenance of these immature oviducts in males. The regression of these ducts in males has been hypothesized to be due to the testicular secretion of an anti-Mullerian hormone in a similar fashion to the loss of Mullerian ducts in male mammals (Kelley, 1996).

Nuptial pads form on the forelimbs or chest in some male anurans that are characterized by keratinized epidermal spines and modified mucus glands that are thought to aid the male in maintaining amplexus during mating (Wells, 2007). In male X. laevis, nuptial pads form on the undersides of the forelimbs beginning between 6 and 10 months PM (Kelley, 1996). These nuptial pads are characterized by black, thickened epidermal spines and the presence of breeding glands on the undersides of the forelimbs and digits (Thomas and Licht, 1993, Kelley, 1996). Breeding glands are only found in the dermis of males where the epidermal spines develop and are not present at all in the female (Fujikura et al., 1988). These nuptial pads regress in adult male X. laevis that have been castrated, while exposure of castrated males or females to androgens results in nuptial pad development (Kelley and Pfaff, 1976).

Vitellogenin is a female-specific protein produced by the liver of non-mammalian vertebrates and incorporated into oocytes by the ovary where it forms the basis for egg yolk (Palmer et al., 1998). Production of vitellogenin is induced by circulating estradiol; it is not normally found in males. It is induced by estrogenic chemicals and has been widely used to study endocrine disruption in fish (Hutchinson et al., 2006) and amphibians (Palmer et al., 1998). In X. laevis, the liver develops the ability to become stimulated by estrogens to produce vitellogenin during larval development (May and Knowland, 1980); however, evidence of yolk deposition in the oocytes does not occur until 5–6 months PM (Kelley, 1996).

A mature Xenopus ovary contains an asynchronous population of oocytes at every stage of development. A classification scheme for the development of these oocytes has been developed that includes six stages based on size and pigmentation (Dumont, 1972). Progression of oocytes through these stages requires 8 months in X. laevis (Smith et al., 1991). Female X. laevis have been reported to become sexually mature from 10 to 24 months PM (Kelley, 1996), while some X. tropicalis females have been reported to reach maturity at the earliest by 6 months PM (Hirsch et al., 2002). In males the presence of viable spermatozoa have been reported in X. laevis testes by 6 months PM, although complete sexual maturity of the males is considered to occur later (Mikamo and Witschi, 1963, Kelley, 1996). In X. tropicalis, males have been reported to become reproductively viable as early as four months PM (Hirsch et al., 2002).

(Video) The Xenopus laevis (African Clawed Frog) | A Valuable Partner in Biomedical Research (2020)

The goal of this research is to characterize the reproductive maturation of X. tropicalis from metamorphosis to adulthood. We hypothesize that the development of secondary sex characteristics and maturation of the gonads will correlate to circulating sex steroid concentrations in the blood of maturing juvenile X. tropicalis. The results obtained from this study will increase our knowledge of anuran reproductive biology and be useful in the design and data analysis of amphibian reproductive toxicity assays.

Section snippets

Animal care and culture

Xenopus tropicalis, “golden strain”, were originally obtained from the University of California, Berkeley lab of Richard Harland. The breeding pair used to generate animals for this study has undergone at least three generations of inbreeding. Frogs were maintained at 25°C in flow-through 7L glass aquaria at densities of 30 tadpoles or 10 frogs per tank. Water was from nearby Lake Superior and was ozone treated and filtered prior to use. Characterization of this water is conducted on a regular

Sex steroid profiles

In females estradiol was first detected around 16 weeks PM, peaking around 24 weeks PM (Fig. 1). Levels of testosterone, which can act as a precursor for estradiol synthesis in females (Norris, 1997), have the same overall profile as that of estradiol. On average these testosterone levels are lower than estradiol levels until the estradiol peak at 24 weeks, after which testosterone levels are on average greater than estradiol levels. This peak at 24 weeks PM and concomitant change in


While levels of circulating sex steroids in adult Xenopus tropicalis have not been reported in the literature, several studies have measured plasma concentrations of testosterone and estradiol in adult X. laevis. Circulating testosterone concentrations in adult male X. laevis have been reported in the range of 2–26ng/mL plasma (Kang et al., 1995, Hayes et al., 2002, Hecker et al., 2004, Hecker et al., 2005, Matsumura et al., 2005, Urbatzka et al., 2007). These levels are consistent with those


The authors would like to recognize Annelie Lindberg-Livingston and Ivy Leland for their technical support. The suggestions and comments of Mike Hornung and Cynthia Rider are also appreciated for their improvement of this article. This paper has been reviewed according to ORD guidelines; however, the statements made do not represent the views of the US Environmental Protection Agency, nor does mention of trade names indicate endorsement by the Federal government.

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      Citation Excerpt :

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      Transcriptomic profiling in Silurana tropicalis testes exposed to finasteride

      General and Comparative Endocrinology, Volume 203, 2014, pp. 137-145

      Investigations of endocrine disrupting chemicals found in aquatic ecosystems with estrogenic and androgenic modes of action have increased over the past two decades due to a surge of evidence of adverse effects in wildlife. Chemicals that disrupt androgen signalling and steroidogenesis can result in an imbalanced conversion of testosterone (T) into 17β-estradiol (E2) and other androgens such as 5α-dihydrotestosterone (5α-DHT). Therefore, a better understanding of how chemicals perturb these pathways is warranted. In this study, the brain, liver, and testes of Silurana tropicalis were exposed ex vivo to the human drug finasteride, a potent steroid 5α-reductase inhibitor and a model compound to study the inhibition of the conversion of T into 5α-DHT. These experiments were conducted (1) to determine organ specific changes in sex steroid production after treatment, and (2) to elucidate the transcriptomic response to finasteride in testicular tissue. Enzyme-linked immunosorbent assays were used to measure hormone levels in media following finasteride incubation for 6h. Finasteride significantly increased T levels in the media of liver and testis tissue, but did not induce any changes in E2 and 5α-DHT production. Gene expression analysis was performed in frog testes and data revealed that finasteride treatment significantly altered 1,434 gene probes. Gene networks associated with male reproduction such as meiosis, hormone biosynthesis, sperm entry, gonadotropin releasing hormone were affected by finasteride exposure as well as other pathways such as oxysterol synthesis, apoptosis, and epigenetic regulation. For example, this study suggests that the mode of action by which finasteride induces cellular damage in testicular tissue as reported by others, is via oxidative stress in testes. This data also suggests that 5-reductase inhibition disrupts the expression of genes related to reproduction. It is proposed that androgen–disrupting chemicals may mediate their action via 5-reductases and that the effects of environmental pollutants are not limited to the androgen receptor signalling.

      (Video) How to sex African Clawed Frogs (Xenopus laevis) || Xenopus sexing

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    Copyright © 2008 Elsevier Inc. Published by Elsevier Inc. All rights reserved.


    Reproductive maturation of the tropical clawed frog: Xenopus tropicalis? ›

    tropicalis would reach reproductive maturation by 22 weeks (5.5 mo) for males and 30 weeks (7.5 mo) for females based on sperm counts and ovarian mass, respectively.

    What is the life cycle of a Xenopus? ›

    The life cycle of Xenopus, as in most other frogs, is made up of three main stages: fertilized egg, tadpole, and adult frog. Mating occurs in fresh water with the male clinging to the back of the larger female.

    How long does it take for an African Clawed Frog to grow? ›

    The total change from egg to small frog takes about six to eight weeks. Adults exhibit no parental care. African clawed frogs can live up to 15 years.

    Can African clawed frogs change gender? ›

    Frogs can change their sex even in pristine, pollution free settings. Past research suggested that male-to-female sex changes happening in frogs in suburban ponds may be caused by increased levels of estrogen released into the water. They found more female frogs than males in suburban areas.

    How do African clawed frogs mate? ›

    Mating in clawed frogs involves pelvic amplexus. The smaller male grasps the larger female from behind, in front of her hind legs. The female sheds eggs (small black dots visible in the photo), which are fertilized by the male as they are released.

    What are the stages of Xenopus tropicalis development? ›

    Individual NF stages have been grouped into six superstages: cleavage, blastula, gastrula, neurula, tailbud and tadpole.

    What are Xenopus gastrulation movements? ›

    The process of gastrulation allows for the formation of the germ layers in metazoan embryos, and is generally achieved through a series of complex and coordinated cellular movements. The process of gastrulation can be either diploblastic or triploblastic.

    How often do African clawed frogs lay eggs? ›

    African clawed frogs can breed up to 4 times each year. Mating can take place during any time of the year, but is most common in the spring.

    Do African clawed frogs start as tadpoles? ›

    African clawed frogs range from 2 to over 5 inches in body length. The tadpoles are fairly transparent, tend to swim “upside down,” and can be distinguished by the presence of long, catfish-like barbels (whiskers).

    Can African clawed frogs live in a 5 gallon tank? ›

    A small Aquarium Tank 21 W 5240 (2–10 gallons) is sufficient in housing a few frogs. It is recommended that you house no more than five frogs in a 5-gallon tank. The Dwarf African Clawed Frogs are completely aquatic and will live their whole lives underwa- ter, swimming to the surface for air.

    Can you keep two African clawed frogs together? ›

    You may also maintain one in such a tank, but they behave better and look better when others of their species accompany them. They may also be maintained in a group with one male and many females, but because of the possibility of competition, you should avoid keeping male species in the same tank.

    How many babies do African clawed frogs have? ›

    Females deposit 1-5 eggs at a time, which are fertilized by the male upon release. When egg-laying has been completed, remove the adults as they will be hungry after their efforts and very willing to consume their progeny.

    Will African clawed frogs breed in captivity? ›

    It is an atypical frog, with claws on its toes, and spends most of its time in water – unlike other frogs. This fascinated many researchers. They also realised these frogs are easy to maintain in captivity, and even breed under these conditions.

    Can female frogs reproduce without a male? ›

    Female frogs can produce thousands of eggs per year! They retain them in their bodies until it's time to mate. If she doesn't mate with a male and her eggs don't get fertilized, they die off inside her body.

    Why are my African clawed frogs hugging? ›

    African Dwarf Frog Behavior

    Many people will express worry for their frogs when they see them hugging each other and not moving for up to a day and a half. This is a normal behavior for the frogs and indicates that they are mating. Female African Dwarf Frogs are wider, whereas the males are skinnier.

    What states are African clawed frogs illegal? ›

    Due to incidents in which these frogs were released and allowed to escape into the wild, African clawed frogs are illegal to own, transport or sell without a permit in the following US states: Arizona, California, Kentucky, Louisiana, New Jersey, North Carolina, Oregon, Vermont, Virginia, Hawaii, Nevada, and Washington ...

    What are the four stages of development of a frog embryo? ›

    Stage 1: Egg. Stage 2: Tadpole. Stage 3: Froglet (young frog) Stage 4: Adult frog.

    What is the developmental stage of frog embryology? ›

    A female frog lays eggs in the water, which are fertilized by sperm from a male frog. The resulting zygote goes through embryonic development to become a free-living tadpole, which then metamorphoses into an adult frog—for instance, by losing its tail through programmed cell death, or apoptosis.

    At what stage of frog development does the germ layers develop? ›

    At the end of gastrulation, we end up with a three-layered frog embryo. These three layers are the germ layers, which are the basis for all of the organs that will form later on. The outermost layer of the embryo is the ectoderm.

    What are the stages of embryonic development gastrulation? ›

    Gastrulation occurs during week 3 of human development. The process of gastrulation generates the three primary germ layers ectoderm, endoderm, and mesoderm. Gastrulation primes the system for organogenesis and is one of the most critical steps of development.

    What are the steps of gastrulation embryology? ›

    In amniotes, gastrulation occurs in the following sequence: (1) the embryo becomes asymmetric; (2) the primitive streak forms; (3) cells from the epiblast at the primitive streak undergo an epithelial to mesenchymal transition and ingress at the primitive streak to form the germ layers.

    How long is mating season for tree frogs? ›

    Most reproduction takes place early on, although the calling season lasts from late April to early August. Individuals may mate up to three times in a season.

    What season do female frogs lay eggs? ›

    Frogs lay eggs during the mating season between March and July in most of the Northern Hemisphere. Frogs reproduce sexually by amplexus and lay eggs in water among vegetation so they do not float away.

    What season do frogs lay eggs? ›

    While most frogs breed in late Spring and early Summer, you'll still get a few doing their thing in March.

    Why does my African clawed frog keep floating? ›

    This action is called "burbling," when your frog spreads its limbs and floats on the water. They usually do this when they want a few breaths of air, as African Dwarf Frogs have a full set of lungs instead of gills.

    Can African clawed frogs live in tap water? ›

    Description: African clawed frogs do very well in a 10 or 20 gallon aquarium. Fresh spring water or dechlorinated tap water (not distilled water) is recommended. The water should be filtered.

    How long can a African clawed frog live out of water? ›

    These frogs cannot survive out of water for longer than 20 minutes in low humidity, as they dry out. Because they are fragile animals, care should be taken when handling African dwarf frogs, especially when considering them as a pet for young children.

    Do clawed frogs need a heater? ›

    Heat. A heat source usually isn't required for African clawed frogs, as they do well at room temperature. Use an aquarium thermometer to make sure the tank stays at around 60 to 80 degrees Fahrenheit (16 to 27 degrees Celsius).

    What is the best substrate for African clawed frogs? ›

    Appropriate tank housing

    In the wild these frogs usually live in murky water with a substrate of thick mud.

    What is the best feeder fish for African clawed frogs? ›

    Adult African Clawed Frogs can have a variety of feeds. A mixed diet of Comets, Rosies, feeder Goldfish, earthworms, crickets, and bloodworms help your frog thrive. With larger food like; Comets, Rosies, feeder goldfish, and large earthworms, I would feed 3-4 items twice a week.

    Can you touch African clawed frogs? ›

    Handling Your Frog: Don't. African Dwarf Frogs are “look at me! (but don't touch me)” pets. They can be injured during handling and do not tolerate being out of water.!

    Can African clawed frogs and Axolotls live together? ›

    I don't recommend putting an axolotl with a clawed frog. They can transmit disease to one another, plus I think it would stress them both.

    Can African clawed frogs survive freezing temperatures? ›

    "They can even survive severe drought and freezing conditions!" In fact, they can even survive in outer space.

    What pH level do African clawed frogs like? ›

    They require water temperatures between 72 and 80 degrees Fahrenheit with pH levels between 7.0 and 7.8.

    How many times should I feed my African clawed frog? ›

    African clawed frogs have no teeth and no tongue. However, they are carnivorous frogs with a healthy appetite. Feed appropriately sized food such as earthworms, wax worms, small guppies, bloodworms and small crickets 3–4 times per week.

    How many gallons does an African clawed frog need? ›

    Housing. African Clawed Frogs are small at up to 3” and fully aquatic which makes their housing fairly simple. For other species of African Clawed Frog, an Aqueon 10 Gallon Aquarium is enough room for one frog throughout it's life. If you decide to keep more than one frog, add 10 gallons to the tank size per frog.

    Can frogs inbreed? ›

    These results suggest that, for wood frogs, inbreeding has a bigger effect on fitness in the wild than in captivity and that measurements of survival are more sensitive than measures of growth or development.

    How do you encourage African dwarf frogs to mate? ›

    Dwarf African Frogs can be brought into breeding condition by a rapid or gradual lowering of the water level (to ½ of its former depth). After 2 weeks or so, refill the tank with de-chlorinated water that is approximately 5 degrees cooler than that already in the tank.

    What percentage of tadpoles make it to adulthood? ›

    “Most studies have found that only one to five percent of frogs in the wild make it to adulthood, so this is not a given,” said Katy Delaney, wildlife ecologist for the National Park Service.

    What is the life cycle of a forest frog? ›

    "The life cycle of a frog consists of three stages: egg, larva, and adult. As the frog grows it moves through these stages in a process known as metamorphosis.

    What is the lifecycle stage of a frog? ›

    Stage 1: Egg. Stage 2: Tadpole. Stage 3: Froglet (young frog) Stage 4: Adult frog.

    What is the life cycle of a frog stages? ›

    They have four stages in their life cycle: egg - tadpole - metamorph - adult.

    What is the growth and development of a frog? ›

    A female frog lays eggs in the water, which are fertilized by sperm from a male frog. The resulting zygote goes through embryonic development to become a free-living tadpole, which then metamorphoses into an adult frog—for instance, by losing its tail through programmed cell death, or apoptosis.

    Why is the froglet stage very important in a frog's life cycle? ›

    Answer. When the tadpole reaches the froglet stage, it is almost a full adult. At this point, the tadpole's gills have disappeared, and its lungs have enlarged. This means it is ready to leave the water and live on land.

    How do frogs reproduce? ›

    In most species of frogs, fertilization is external. The male frog grabs the female's back and fertilizes the eggs as the female frog releases them (Figure 2.2B). Rana pipiens usually lays around 2500 eggs, while the bullfrog, Rana catesbiana, can lay as many as 20,000.

    What are the 5 stage of a frog? ›

    The 5 life stages comprise egg (frog spawn), tadpole, tadpole without gills, tadpole with legs, and frog.

    How long are frogs froglets? ›

    Once hatched, tadpoles take about 14 weeks to transform into tiny frogs. Toad tadpoles take a little bit longer, becoming toadlets after about four months. They develop back legs first, then front legs, while the tadpole's tail shrinks and its body becomes less rounded. They also develop lungs and eardrums.

    What is the first stage of their life cycle? ›

    The life cycle starts as a fertilized egg. Then after roughly 40 weeks in utero an infant is born. Infancy is considered from birth until approximately one year of age. After one year a human enters the next stage of its life cycle, childhood.

    How long is a frog pregnant for? ›

    3. Eggs generally hatch in 2-3 weeks, but the time varies based on water temperature.

    What is the final change in the life cycle of a frog? ›

    Stage 4: Adult Frog

    As the frog develops into an adult, it will begin to eat insects rather than vegetation. It can take up to four years before the frog becomes fully mature. Once it is, the frog can then lay eggs or fertilize them, and the life cycle of a frog can start all over again!

    Which type of fertilization and development is found in frogs? ›

    The process of external fertilization takes place in frogs. It is a type of fertilization in which the fusion of male and female gametes occurs outside of the female's body. In the water, the female lays hundreds of eggs.

    How do frogs reproduce asexually? ›

    All frogs reproduce sexually–there are no known species of asexual frogs. However, whether they use internal or external fertilization to fertilize their eggs depends on their species. Most frogs use external fertilization, in which a female and male release their eggs and sperm around the same time.


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