Effects of age, weight, hormones, and hibernation on breeding success in boreal toads (Bufo boreas boreas)

The goals of this study were to test the effects of exogenous hormones and hibernation on breeding behavior and gamete release by boreal toads (Bufo boreas boreas). Each year, a subset of 77 toads was hibernated and then paired with hibernated or nonhibernated mates and treated with luteinizing hormone releasing hormone analogue (LHRHa), human chorionic gonadotropin (hCG), or left untreated. Amplexus and egg and sperm production were recorded. At 1 yr of age, only 19% of pairs exhibited amplexus, and no sperm or eggs were produced. At 2 and 3 yr of age, most male toads treated with LHRHa exhibited amplexus (56.9% and 100%, respectively). Among 2-yr-old males, amplexus was more prevalent (P < 0.05) in those that were hibernated than in those that were nonhibernated (54.0% and 33.3%, respectively), but most males in each group (93.3% and 75%, respectively) produced sperm in response to LHRHa treatment. Only one 2-yr-old and two 3-yr-old females produced eggs. At 4 yr of age, eight females produced eggs, but two died from egg retention. More nonhibernated than hibernated females developed eggs (7 of 10 vs. 1 of 10, P < 0.05). Mean (±SD) weight of female toads producing eggs (58.9 ± 11.9 g) was greater (P < 0.05) than that of nonproducing females (43.6 ± 7.0 g). Similarly, four of seven nonhibernated females (58.8 ± 8.3 g) produced eggs at 5 yr of age. All eggs were produced by females treated once with LHRHa. Number of eggs per female varied (141 to 3307), and development to tadpoles was low (0 to 36.5%), although tadpoles did become toadlets. In conclusion, male and female boreal toads matured at 2 and 4 yr of age, respectively, and heavier females were more likely to produce eggs. To enhance breeding success, males should be hibernated and treated with LHRHa. In contrast, female productivity was enhanced by improving their body condition instead of subjecting them to hibernation prior to LHRHa treatment.     

Progesterone improves the number and quality of hormone induced Fowler toad (Bufo fowleri) oocytes

Combinations of progesterone, lutenizing hormone releasing hormone analogue (LHRHa), human chorionic gonadotrophin (hCG), and the dopamine-2 (DA2) receptor antagonist 1-[1-[4,4-bis(4-Fluorophenyl)butyl]-4-piperidinyl]-1,3-dihydro-2H-benzimidazol-2-one (Pimozide; Orap) were tested for improvement of spawning rates, oocyte numbers, fertilization and neurulation rates of the Fowler toad (Bufo fowleri). Only treatments combined with progesterone produced large numbers of oocytes. The best treatment on oocyte numbers, neurulation rates, and the number of neurulas was with 5 mg progesterone, 20 mic.g LHRHa, and 0.25 mg Pimozide. Progesterone (5 mg) with 60 mic.g LHRHa gave high spawning rates, oocyte numbers, and fertilization rates but neurulation rates were low. Progesterone alone in high repeated doses did not result in ovulation. High doses of LHRHa (60 mic.g) with hCG, progesterone, and Pimozide gave the greatest number of toads spawning, however, they resulted in low oocyte numbers, fertilization and neurulation rates. A low dose of LHRHa (4 mic.g) with hCG, or hCG alone as a second administration, and progesterone with Pimozide produced few good quality oocytes. Toads were given normal ovulatory doses of hormones 24 or 48 hrs after their initial dose, but these resulted in low oocyte numbers followed by poor fertilization. Overall, these results suggest that progesterone with a dose between 20 mic.g and 60 mic.g of LHRHa may be optimal for the induction of ovulation in these toads. Moreover, Pimozide can supplement low doses of LHRHa but not replace it.     

A comparison of human chorionic gonadotropin and luteinizing hormone releasing hormone on the induction of spermiation and amplexus in the American toad (Anaxyrus americanus)

ABSTRACT: BACKGROUND: Captive breeding programs for endangered amphibian species often utilize exogenous hormones for species that are difficult to breed. The purpose of our study was to compare the efficacy of two different hormones at various concentrations on sperm production, quantity and quality over time in order to optimize assisted breeding. METHODS: Male American toads (Anaxyrus americanus) were divided into three separate treatment groups, with animals in each group rotated through different concentrations of luteinizing hormone releasing hormone analog (LHRH; 0.1, 1.0, 4.0 and 32 micrograms/toad), human chorionic gonadotropin (hCG; 50, 100, 200, and 300 IU), or the control over 24 hours. We evaluated the number of males that respond by producing spermic urine, the sperm concentration, percent motility, and quality of forward progression. We also evaluated the effects of hCG and LHRH on reproductive behavior as assessed by amplexus. Data were analyzed using the Generalized Estimating Equations incorporating repeated measures over time and including the main effects of treatment and time, and the treatment by time interaction. RESULTS: The hormone hCG was significantly more effective at stimulating spermiation in male Anaxyrus americanus than LHRH and showed a dose-dependent response in the number of animals producing sperm. At the most effective hCG dose (300 IU), 100 % of the male toads produced sperm, compared to only 35 % for the best LHRH dose tested (4.0 micrograms). In addition to having a greater number of responders (P < 0.05), the 300 IU hCG treatment group had a much higher average sperm concentration (P < 0.05) than the treatment group receiving 4.0 micrograms LHRH. In contrast, these two treatments did not result in significant differences in sperm motility or quality of forward progressive motility. However, more males went into amplexus when treated with LHRH vs. hCG (90 % vs. 75 %) by nine hours post-administration. CONCLUSION: There is a clear dichotomy between the two hormones' physiological responses on gamete production and stimulation of amplexus. Understanding how these two hormones influence physiology and reproductive behaviors in amphibians will have direct bearing on establishing similar breeding protocols for endangered species.     

A DNA-based assay identifies Batrachochytrium dendrobatidis in amphibians

Chytridiomycosis caused by Batrachochytrium dendrobatidis (Chytridiomycota) has been implicated in declines of amphibian populations on four continents. We have developed a sensitive and specific polymerase chain reaction-based assay to detect this pathogen. We isolated B. dendrobatidis from captive and wild amphibians collected across North America and sequenced the internal transcribed spacer regions of the rDNA cassette of multiple isolates. We identified two primers (Bd1a and Bd2a) that are specific to B. dendrobatidis under amplification conditions described in this study. DNA amplification with Bd1a/Bd2a primers produced a fragment of approximately 300 bp from B. dendrobatidis DNA but not from DNA of other species of chytrids or common soil fungi. The assay detected 10 zoospores or 10 pg of DNA from B. dendrobatidis and detected infections in skin samples from a tiger salamander (Ambystoma tigrinum), boreal toads (Bufo boreas), Wyoming toads (Bufo baxteri), and smooth-sided toads (Bufo guttatus). This assay required only small samples of skin and can be used to process a large number of samples.     

Mucormycotic dermatitis in captive adult Wyoming toads

During late May 1995, 50 adult captive endangered Wyoming toads (Bufo baxteri) were brought out of hibernation. Approximately 3 to 10 days after hibernation emergence, all toads were hormonally induced to breed, and paired. Each pair was placed in their own breeding tank. Four toads developed clinical signs of disease which included lethargy and multiple (4 to 12) small (2 mm) raised hyperemic nodules with white fuzzy caps on the ventral skin. The condition progressively worsened until death occurred, within 3 to 6 days. Mycotic dermatitis caused by Mucor sp. was diagnosed in the four toads through histology and isolation of the organism. This is the first case report of a Mucor sp. causing a fatal dermatitis in an amphibian without significant inflammatory response and without systemic involvement.     

Rapid visual detection of sperm-egg fusion using the DNA-specific fluorochrome Hoechst 33342

When unfertilized sea urchin eggs are pretreated with the bisbenzimide DNA-specific fluorochrome Hoechst 33342, then washed and fertilized, a single sperm bound to the egg surface becomes intensely fluorescent. The location of the fluorescent sperm on the egg surface coincides exactly with the epicenter of the cortical reaction and the site at which the insemination cone subsequently appears. These observations, coupled with studies of eggs treated with quercetin to prevent fusion, as well as eggs made polyspermic by halothane exposure, indicate that the sperm acquires fluorescence as a consequence of fusion with the fluorochrome preloaded egg. Using a modification of this technique, we have found that cytoplasmic continuity between the sperm and egg is established at 4-8 sec after the onset of the sperm-induced conductance increase in the egg.     

Dermatitis in captive Wyoming toads (Bufo baxteri) associated with Fusarium spp

From May 2007 to June 2008, 30 of 49 Wyoming toads (Bufo baxteri) kept at Omaha's Henry Doorly Zoo (Nebraska, USA) died showing clinical signs of ventral erythema, inappetance, lethargy, and delayed righting reflex. Treatment with antifungals and antibiotics was unsuccessful in all cases. Histopathologic analyses revealed dermatitis as the primary problem in 20 of 21 toads in which skin was examined. Fungal dermatitis was present in 17 toads, with hyphae approximately 1-3 μm in diameter, and parallel cell walls and frequent septations. In 14 animals, the fungal dermatitis was the main pathologic lesion. Several species of bacteria were associated with all cases. A few animals tested positive for Ranavirus using polymerase chain reaction. Fusarium sp. was consistently cultured from skin, feces, kidneys, and from powdered food provided to crickets. Four isolates were identified as Fusarium proliferatum, Fusarium oxysporum, Fusarium solani, and Fusarium verticillioides, which suggested a secondary role of fungi. A specific underlying cause of disease could not be found, although the roles of humidity and Ranavirus infection are discussed, along with the well-known susceptibility of bufonids to fungal dermatitis.     

Advances in transgenic rat production

Predictable and reproducible production of transgenic rats from a standardized input of egg donors and egg recipients is essential for routine rat model production. In the course of establishing a transgenic rat service, transgenic founders were produced from three transgenes in outbred Sprague-Dawley (SD) rats and four transgenes in inbred Fischer 344 (F344) rats. Key parameters that affect transgenesis efficiency were assessed, including superovulation treatments, methods to prepare pseudopregnant recipients, and microinjection technique. Five superovulation regimens were compared and treatment with 20 IU PMSG and 30 IU HCG was selected for routine use. Four methods to prepare pseudopregnant egg recipients were compared and estrus synchronization with LHRHa and mating to vasectomized males was selected as most effective. More than 80% of eggs survived microinjection when modified pronuclear microinjection needles and DNA buffers were used. The efficiencies of transgenic production in rats and C57BL/6J (B6J) mice were compared to provide a context for assessing the difficulty of transgenic rat production. Compared to B6J mice, SD rat transgenesis required fewer egg donors per founder, fewer pseudopregnant egg recipients per founder, and produced more founders per eggs microinjected. Similar numbers of injection days were required to produce founders. These results suggest that SD rat transgenesis can be more efficient than B6J mouse transgenesis with the appropriate technical refinements. Advances in transgenic rat production have the potential to increase access to rat models.     

A comparison of the fecundities of two species of toad (Bufo bufo and B. calamita) from different habitat types in Britain

The fecundities of Natterjack toads, Bufo calamita , on a Cumbrian dune system and a relict heathland site in Hampshire were compared and contrasted with the fecundity of Common toads, B. bufo , on the Cumbrian dunes. For both species, fecundity was positively correlated with length, while in B. calamita length was also correlated with size of eggs produced, and age of the female. Bufo calamita was found to invest more energy exponentially in egg production with increasing length, and therefore age. This species also laid more than twice as many eggs as comparably-sized female B. bufo , and some probably produced two clutches of spawn per year in southern England. The enhanced fecundity of the species was believed to be an adaptation to living in areas with unpredictable spawning sites. Small female B. bufo on the dunes appeared to put more energy into egg production when small than individuals of the same species on other habitat types in Britain, possibly as a result of reduced life expectancy on the dune habitat. There was a link between old age and the production of non-viable spawn in B. calamita , and males of this species which spawned at the end of the prolonged breeding season were found to have reduced fertilization efficiency when spawning on consecutive evenings.     

Fertilization and preimplantation development in vitro of mouse eggs obtained following stimulation with different doses of pregnant mare serum: a comparison of the responses in two strains

Unfertilized eggs were recovered from TO and (C57BL/10 X CBA)F1 females, induced to ovulate with 0.75--10.0 IU PMS and 5.0 IU HCG/mouse, and mixed with TO sperm in vitro. Among the groups of F1 eggs, there were no significant differences in either fertilization or development to the blastocyst stage in vitro. With TO eggs, however, there was significantly lower fertility in the 0.74 and 1.5 IU PMS groups than in the higher PMS dose groups. Conversely, preimplantation development was significantly better withe low doses. To determine whether polyploidy migh affect viability in vitro, digynic polyploidy was experimentally induced in both F1 and TO eggs with cytochalasin B. Development of polyploid F1 X TO embryos to the blastocyst stage did not differ significantly form that of untreated embryos. Development of polyploid TO X TO embryos obtained from eggs stimulated with 1.5 IU PMS was still higher than that for eggs stimulated with 7.5 IU PMS. This suggests that cytoplasmic inadequacies, rather than genome imbalance, are responsible for the reduced ability of the eggs stimulated with high doses of PMS to reach the blastocyst stage in vitro.     

Optimal dose of human chorionic gonadotropin for inducing ovulation in the ferret

The optimal dose of human chorionic gonadotropin (hCG) for induction of ovulation was determined by comparing the ovulatory response of 119 mated ferrets (controls) with that of estrous females induced to ovulate with five different dosages of hCG. Copulation induced formation of 12.7 ± 4.5 corpora lutea (CL) in all 119 females and resulted in a 90.7% conception rate as evidenced by finding approximately eight blastocysts/female in the uteri of 108 ferrets. All doses of hCG tested induced ovulation; however, the lower doses (50 and 75 IU) resulted in a lesser percentage of females ovulating. The highest doses of hCG (150 and 300 IU) resulted in fewer CL/female being formed. The optimal dose of hCG for simulating copulation induced ovulation was 100 IU. Tubal transport of unfertilized oocytes in pseudopregnant females was found to be significantly retarded when compared to the rate of transport of embryos in the control group.     

Sperm binding and penetration of the zona pellucida in vitro but not sperm-egg fusion in an Australian marsupial, the brushtail possum (Trichosurus vulpecula)

Sperm capacitation and in vitro fertilisation (IVF) have been achieved in most eutherian mammals and American marsupials under relatively simple culture conditions. In contrast sperm capacitation in Australian marsupials has not been achieved in vitro and attempts at IVF have previously been characterised by a complete lack of sperm-zona pellucida (ZP) binding. Recently, co-culture of sperm with oviduct epithelial cell monolayers or with oviductal explant conditioned media has been shown to prolong the viability and motility of brushtail possum spermatozoa, as well as to induce capacitation-associated changes such as transformation of sperm to the T-shape orientation. In this study we report that these in vitro produced T-shaped sperm, and in vivo derived T-shaped sperm flushed from the oviduct of artificially inseminated possums as a control, are able to bind to and penetrate the ZP of approximately 25% of eggs recovered from PMSG/LH-superovulated possums in vitro. Development of ZP receptivity and penetrability towards sperm was also identified as a major factor affecting the outcome of IVF. Neither in vivo nor in vitro derived T-shaped sperm were able to bind to or penetrate the ZP if eggs were obtained from animals that were treated with pLH less than 76 h after PMSG. Thus this study provides preliminary evidence for the necessity of sperm-oviduct epithelial cell interactions for capacitation in Australian species and lends further support to the suggestion that the T-shape head orientation is indicative of sperm capacitation. Despite the occurrence of sperm-ZP binding and penetration, sperm-egg membrane fusion and egg activation were not observed. Although the factor(s) responsible for the lack of sperm-egg membrane fusion in the possum have not been identified it is possible that egg capacity for membrane fusion develops independently of zona receptivity and is defective in these eggs, or alternatively that membrane fusion requires strictly defined ionic conditions which are not provided by the IVF media used in this study.     

Superovulation in immature and mature Chinese hamsters

Mature female Chinese hamsters ovulate an average of 8.8 ± 1.0 (mean ± SD) eggs per female in each estrous cycle. Superovulation can be induced in both immature and mature females by subcutaneous or intraperitoneal injections of pregnant mare serum gonadotropin (PMSG) and either human chorionic gonadotropin (hCG) or pituitary luteinizing hormone (PLH). The best superovulation in immature females was induced by the administration of 15 IU of PMSG followed 72 hr later by injection of 15 IU of hCG (about 25 eggs per female) or 0.2 mg (200 IU) PLH (about 46 eggs per female). Ovulation started about 13–15 hr after administration of hCG (or PLH) and was completed during the next 5–6 hr. Superovulation in mature females could be induced by injecting PMSG any day of the estrous cycle, but the best superovulation (about 39 eggs per female) was induced by injecting 15 IU of PMSG on day 1 (day of ovulation) followed by the injection of 0.4 mg of PLH 72 hr later. When immature females treated with the best superovulatory protocol were mated on the evening of PLH injection, only 5% of the eggs were found fertilized 50 hr after PLH administration. On the other hand, about 60% of the eggs were found fertilized in mature females mated following treatment with the best superovulatory protocol. The majority (83–85%) of superovulated eggs obtained from both immature and mature females were normally fertilized in vitro.     

Optimisation of an oviposition protocol employing human chorionic and pregnant mare serum gonadotropins in the Barred Frog Mixophyes fasciolatus (Myobatrachidae)

ABSTRACT: BACKGROUND: Protocols for the hormonal induction of ovulation and oviposition are essential tools for managing threatened amphibians with assisted reproduction, but responses vary greatly between species and even broad taxon groups. Consequently, it is necessary to assess effectiveness of such protocols in representative species when new taxa become targets for induction. The threatened genus Mixophyes (family Myobatrachidae) has amongst the highest proportion of endangered species of all the Australian amphibians. This study developed and optimised the induction of oviposition in a non-threatened member of this taxon, the great barred frog (Mixophyes fasciolatus). METHODS: Gravid female M. fasciolatus were induced to oviposit on one or more occasions by administration of human chorionic gonadotropin (hCG) with or without priming with pregnant mare serum gonadotropin (PMSG). Treatments involved variations in hormone doses and combinations (administered via injection into the dorsal lymph sacs), and timing of administration. Pituitary homogenates from an unrelated bufonid species (Rhinella marina) were also examined with hCG. RESULTS: When injected alone, hCG (900 to 1400 IU) induced oviposition. However, priming with two time dependent doses of PMSG (50 IU, 25 IU) increased responses, with lower doses of hCG (200 IU). Priming increased response rates in females from around 30% (hCG alone) to more than 50% (p = 0.035), and up to 67%. Increasing the interval between the first PMSG dose and first hCG dose from 3 to 6 days also produced significant improvement (p<0.001). Heterologous pituitary extracts administered with hCG were no more effective than hCG alone (p = 0.628). CONCLUSIONS: This study found that M. fasciolatus is amongst the few amphibian species (including Xenopus (Silurana) and some bufonids) that respond well to the induction of ovulation utilising mammalian gonadotropins (hCG). The optimal protocol for M. fasciolatus involved two priming doses of PMSG (50 IU and 25 IU) administered at 6 and 4 days respectively, prior to two doses of hCG (100 IU), 24 hours apart. This study is also the first to demonstrate in an amphibian species that responds to mammalian gonadotropins that an increase in the ovulation rate occurs after priming with a gonadotropin (PMSG) with FSH activity.     

A consistently successful procedure for in vitro fertilization of golden hamster eggs

Complete details are described for the first time of the procedures used in the author's laboratory for obtaining in vitro fertilization (IVF) of golden hamster eggs leading to the first cleavage division. These IVF procedures have been developed during the past 20 years and are very reproducible: IVF of at least 75% of eggs is routinely achieved, and on average 65% of inseminated eggs undergo the first cleavage division in vitro. These results can easily be obtained by inexperienced investigators. The ease and reproducibility of the hamster IVF procedures make them very suitable for studies of sperm:egg interaction and associated events. Studies in the author's laboratory have included analysis of sperm fertilizing ability under chemically defined conditions, the presence of sperm acrosome reaction stimulating factors in the egg investments, maturation of oocytes in vitro, the block to polyspermy, and the contribution of egg aging to fertilization anomalies. In addition, the motility of hamster sperm under chemically defined conditions is used in a routine screening protocol for detecting contaminants in the culture milieu. Golden hamster gametes of Ter several distinct advantages for IVF studies, including the large size of the sperm acrosome, the persistence of the very large sperm tail in the ooplasm for many hours following fertilization, and the translucence of the ooplasm, which facilitates observation of the sperm tail and pronuclei. The female golden hamster exhibits a regular 4 day estrous cycle, with distinctive indications of estrus and postestrus phases. Because of the advantages of using the golden hamster, the procedures described in this report may be useful to other investigators wishing to conduct research using IVF. Essentially the same IVF procedures can be used with monkey and bovine gametes.     

Causes of mortality of the Wyoming toad

Wyoming toads (Bufo baxteri) that died from January 1989 to June 1996 were submitted to the Wyoming State Veterinary Laboratory (Laramie, Wyoming, USA) for postmortem evaluation. These consisted of 108 free-ranging toads and 170 animals from six captive populations. Ninety-seven (90%) of 108 free-ranging toad carcasses were submitted during September and October. From 1989 to 1992, 27 (77%) of 35 mortalities in the captive populations occurred in October, November, and December. From 1993 to 1996, mortality in captive toads occurred without a seasonal pattern and coincided with changes in hibernation protocols that no longer mimicked natural cycles. Cause of mortality was determined in 147 (53%) of the 278 cases. Mycotic dermatitis with secondary bacterial septicemia was the most frequent diagnosis in 104 (71%) of 147 toads. Basidiobolus ranarum was found by microscopic examination of skin sections in 100 (96%) of 104 of these mortalities. This fungus was isolated from 30 (56%) of 54 free-ranging and 24 (48%) of 50 captive toads. This research documents the causes of mortality for both free-ranging and captive endangered Wyoming toads over a 7 yr period.     

Induced ovulation in Atlantic croaker (Sciaenidae) using hCG and an LHRH analog: A preliminary study

A single dose of LHRHa (D-Ala(6), des Gly(10)- LHRH-ethylamide; 100 mug/kg body weight); administered by intramuscular injection, effectively induced ovulation in the Atlantic croaker (Micropogonias undulatus ); 37 of 40 (92%) females were successfully hand-stripped and there was no mortality. A single dose of human chorionic gonadotropin (hCG; 500 IU/kg body weight) was successful in inducing oocyte hydration. However, only 16 of 30 (53%) females ovulated successfully and could be hand-stripped. Another 8 females became extremely bloated and died. Thus LHRHa was shown to be the more reliable method of inducing ovulation in the Atlantic croaker. The response interval between hormone treatment and ovulation at different water temperatures was also determined.     

Spawning response of mature female sea bass,Lates calcarifer (Bloch), to a single injection of luteinizing hormone-releasing hormone analogue: effect of dose and initial oocyte size1

The effect of various doses of luteinizing hormone-releasing hormone analogue (LHRHa) ranging from 1 to 100 μg/kg body weight on the spawning response of mature female sea bass, Lates calcarifer (Bloch) was tested. A single intramuscular injection of LHRHa resulted in a dose-related increase in the spawning rate (number of spawnings of each fish over four consecutive days) of mature fish. An LHRHa dose of 5 μg/kg and less induced low spawning rates of 16.7% to 37.5% or at least one spawning every four days. However, mature sea bass spawned more than once (43.8–58.3%) in four days at dose levels of 10 μg/kg and above. Hormone treatment within the dose range tested did not influence the number, fertilization and hatching rates of spawned eggs. The influence of initial oocyte size on the LHRHa-induced spawning response of mature sea bass was also examined. Sea bass with an initial oocyte diameter of 0.30–0.39 mm did not respond to the single injection of 100 μg LHRHa/kg. In contrast, LHRHa induced spawning among sea bass with an initial egg size of 0.40–0.49 mm, although two of four sea bass of the same stage of ovarian maturity spawned spontaneously. Fish having an initial oocyte size of 0.50–0.55 mm spawned with and without LHRHa treatment. Spontaneous spawning among saline-injected sea bass occurred at a later time (24–58 h post-injection) compared to fish induced to spawn by a single injection of LHRHa (8–36 h post-injection). The initial spawning response time interval for fish with an initial egg size of 0.50 mm or greater was further reduced to 8–9 h by LHRHa. These results indicate that LHRHa can successfully induce spawning in mature female sea bass which have attained a critical oocyte diameter and that the spawning response interval is reduced with a further increase in egg size beyond the critical oocyte diameter limit.     

Characterization of the ooplasmic factor inducing decondensation of and protamine removal from toad sperm nuclei: involvement of nucleoplasmin

Immunohistochemical studies with antiserum against the protamines of the toad, Bufo japonicus, revealed that the sperm nucleus loses protamines within 5 min after entry into the egg. Likewise, lysolecithin-permeabilized sperm incubated with the egg extract lose the protamines within 1 min, accompanied by nuclear decondensation. The activities that induce both protamine removal and decondensation in sperm nuclei were found in extracts from growing and mature oocytes and pregastrula embryos, but not in postneurula embryos or adult tissues. SDS-PAGE analyses revealed that the egg extract removed not only protamines from the Bufo sperm, but also selectively the sperm-specific basic proteins from sperm nuclei of Xenopus laevis. The protamine-removing activity (PRA) was partially purified from egg extracts as negatively charged macromolecules by anion-exchange chromatography and gel filtration. The PRA was heat-stable (100 degrees C, 10 min) and sensitive to proteinase K, but not to RNase A and DNase I. Immunoblot analysis of the supernatant after incubation of Bufo sperm in the fraction with the PRA revealed that protamines derived from sperm nuclei were associated with a major protein of the fraction. This protein exhibited mobilities of 140 and 36 kDa on native- and SDS-PAGE, respectively, with the isoelectric points in the range 4.2 to 4.5 and possessed an amino acid composition quite similar to that reported for Xenopus nucleoplasmin. We propose that in fertilized eggs the protamines are removed from sperm nuclei by nucleoplasmin by binding to but not by enzymatic degradation of the protamine.     

Life history consequences of feeding versus non-feeding in a facultatively non-feeding toad larva

Summary Bufo periglenes, a toad endemic to montane Costa Rica, produces an unusually small clutch of large, yolk-rich eggs. The toads breed in small ephemeral pools that are unpredictable in duration and may be low in food availability. Two congeners, Bufo coniferus and Bufo marinus, occur nearby, breed in more permanent bodies of water that offer more food, and exhibit the typical toad pattern of large clutches of small eggs. Tadpoles of all three species feed on detritus and suspended organic material. By raising tadpoles of the three species individually with and without food I investigated the relationship between egg size (yolk provision) and tadpole survival. All of the unfed B. coniferus and B. marinus tadpoles grew little and died soon after developing to the hindlimb bud stage. On the other hand, all of the unfed B. periglenes tadpoles metamorphosed successfully, demonstrating that the tadpoles are facultatively non-feeding; developmental time from hatching to metamorphosis was significantly shorter for unfed tadpoles than for fed tadpoles, but fed individuals were significantly larger at transformation. Faster developmental rate and larger body size at transformation are both advantageous for frogs and toads, but cannot be attained simultaneously. Large egg size may afford flexibility in unpredictable environments. In pools where food is available, tadpoles presumably eat, take longer to metamorphose, but are larger at transformation than tadpoles developing in nutrient-poor sites. Small body size at transformation (a consequence of not eating) has potential costs, but the large quantity of yolk provided by a large egg enhances the probability of metamorphosis in food-limited environments.