Newly identified Mycobacterium species in a Xenopus laevis colony

The University of Massachusetts Medical School maintains 3 separate research colonies of Xenopus laevis, with each colony located in a separate building on campus. After a 5-wk in-house quarantine period, 34 wild-caught X. laevis were transferred into one of the existing colonies. As a result, this colony grew from 51 to 85 frogs. All animals were housed in a recirculating frog housing system. During the first 2 mo, 6 frogs died suddenly, and health reports were generated for another 10 frogs in this colony. The majority of health reports were written in response to acute coelomic distention. These patterns continued until, after 1 y, only 25 of the original 85 animals remained. Necropsies performed showed large accumulations of serosanguinous fluid in the subcutaneous space or body cavity. Granulomatous inflammatory lesions with acid-fast bacilli were generally present in the liver, lung, or spleen. Culture of affected tissues grew Mycobacterium sp. within 40 d. Polymerase chain reaction analysis confirmed the isolated organism to be the same species of Mycobacterium (provisionally named M. liflandii) recently reported by 2 other groups. However, previous clinical publications suggested that this bacterium originated only from X. tropicalis. The cases we present highlight the rapidly lethal effects of M. liflandii in a colony of wild-caught X. laevis and illustrate the need to dedicate further attention to this emerging Xenopus disease.     

Cryopreservation of sperm of Xenopus laevis and Xenopus tropicalis

Now that transgenic strains of Xenopus laevis and X. tropicalis can be generated efficiently and with genomic sequence resources available for X. tropicalis, early amphibian development can be studied using integrated biochemical and genetic approaches. However, housing large numbers of animals generated during genetic screens or produced as novel transgenic lines presents a considerable challenge. We describe a method for cryopreserving Xenopus sperm that should facilitate low maintenance, long-term storage of male gametes. By optimising the cryoprotectant, the rates of cooling and thawing, and conditions for fertilisation, sperm from the equivalent of one-eighth of a X. laevis testis or of two X. tropicalis testes have been cryopreserved and used to fertilise eggs of both species after thawing. Sperm undergo a substantial loss of viability during a freeze-thaw cycle, but sufficient survive to fertilise eggs. Gametes of mutagenised frogs are being stored in connection with a screen for developmental mutations.     

Skeletal development in Xenopus laevis (Anura: Pipidae).

Postembryonic skeletal development of the pipid frog Xenopus laevis is described from cleared-and-stained whole-mount specimens and sectioned material representing Nieuwkoop and Faber developmental Stages 46-65, plus postmetamorphic individuals up to 6 months old. An assessment of variation of skeletogenesis within a single population of larvae and comparison with earlier studies revealed that the timing, but not the sequence, of skeletal development in X. laevis is more variable than previously reported and poorly correlated with the development of external morphology. Examination of chondrocranial development indicates that the rostral cartilages of X. laevis are homologous with the suprarostral cartilages of non-pipoid anurans, and suggests that the peculiar chondrocranium of this taxon is derived from a more generalized pattern typical of non-pipoid frogs. Derived features of skeletal development not previously reported for X. laevis include 1) bipartite formation of the palatoquadrate; 2) precocious formation of the adult mandible; 3) origin of the angulosplenial from two centers of ossification; 4) complete erosion of the orbital cartilage during the later stages of metamorphosis; 5) development of the sphenethmoid as a membrane, rather than an endochondral bone; and 6) a pattern of timing of ossification that more closely coincides with that of the pelobatid frog Spea than that recorded for neobatrachian species.     

Circadian Locomotor Activity Rhythms in the African Clawed Frog, Xenopus laevis: The Role of the Eye and the Hypothalamus

The effects of hypothalamic lesioning and removal of the eyes on locomotor activity rhythms of African clawed frog, Xenopus laevis were examined under light-dark cycles (LD12:12) and constant conditions. Frogs were kept individually and the activity rhythms at the bottom layer of water tank were recorded by means of the infrared photocells. Intact frogs displayed clear entrained nocturnal activity and expressed freerunning activity rhythms in constant darkness (DD), while some frogs did not freerun under co nstant dim light (dimLL) and constant light (LL). Freerunning periods in intact frogs were significantly shorter in dimLL than in DD. Although freerunning periods were shortened after blinding in same individuals, no significant changes in the freerunning periods were observed after blinding under dimLL and LL. When electrolytic lesions to the hypothalamus were performed, all frogs with more than 70% damage of the SCN abolished freerunning rhythms and in frogs with less than 70% damage, 57% of the animals became arrhythmic. In conclusion, (1) There is a circadian pacemaker somewhere outside the eyes, and it is probably situated in the hypothalamusincluding the SCN. (2) Both the eyes and the SCN are involved in the circadian system of the frogs.     

Circadian Locomotor Activity Rhythms in the African Clawed Frog,Xenopus laevis: The Role of the Eye and the Hypothalamus

The effects of hypothalamic lesioning and removal of the eyes on locomotor activity rhythms of African clawed frog, Xenopus laevis were examined under light-dark cycles (LD12:12) and constant conditions. Frogs were kept individually and the activity rhythms at the bottom layer of water tank were recorded by means of the infrared photocells. Intact frogs displayed clear entrained nocturnal activity and expressed freerunning activity rhythms in constant darkness (DD), while some frogs did not freerun under co nstant dim light (dimLL) and constant light (LL). Freerunning periods in intact frogs were significantly shorter in dimLL than in DD. Although freerunning periods were shortened after blinding in same individuals, no significant changes in the freerunning periods were observed after blinding under dimLL and LL. When electrolytic lesions to the hypothalamus were performed, all frogs with more than 70% damage of the SCN abolished freerunning rhythms and in frogs with less than 70% damage, 57% of the animals became arrhythmic. In conclusion, (1) There is a circadian pacemaker somewhere outside the eyes, and it is probably situated in the hypothalamusincluding the SCN. (2) Both the eyes and the SCN are involved in the circadian system of the frogs.     

Cutaneous acariasis in the African clawed frog (Xenopus laevis)

Increased mortality was observed in a single colony of 50 Xenopus laevis. The frogs were used as oocyte donors in developmental biology studies. Necropsy findings included dermal erythema and petechiation consistent with red leg syndrome; dermal ulcerations and white, filamentous growths on the skin were consistent with Saprolegnia sp. Microscopic evaluation of the skin and fungus revealed an astigmatid mite similar to those of the genus Rhizoglyphus. The mite was also found in the water and the biological filter of the tanks housing the frogs. This mite is considered not to be a parasite of X. laevis; instead, it feeds off moss, fungi, and detritus. Subsequent evaluation of the sphagnum moss used for shipping the frogs from the supplier revealed the same mite in the moss. Our hypothesis is that the mite was introduced into the tank with the shipment of new frogs in sphagnum moss. The mites lived within the biological filter, and were only found after the growth of Saprolegnia sp. attracted the mites to the frogs. Laboratory animal care and veterinary personnel should consider non-pathogenic species of mites in the differential diagnosis of acariasis in Xenopus frogs.     

Effects of available cover and feeding schedule on the behavior and growth of the juvenile African clawed frog (Xenopus laevis)

Though the African clawed frog (Xenopus laevis) is the most commonly used amphibian in biological research, there are no standard protocols for the husbandry of this species. With the goal of developing optimal conditions for raising these frogs, the authors assessed the effects of available cover and feeding schedule on post-metamorphic growth and behavior of juvenile X. laevis. Frogs, which were housed four per tank, had access to varying numbers of shelters (zero, two or four) and were fed either daily or episodically (three times per week) over a period of 6 months. Though X. laevis growth was not influenced by the availability of cover, frogs that were fed daily grew larger than frogs that were fed episodically. Additionally, frogs that were fed daily and had no access to cover were most likely to exhibit foraging behavior when disturbed. These results suggest that feeding frogs daily will promote growth and fecundity. Furthermore, although labs often provide frogs with hiding places, such cover does not enhance X. laevis growth.     

Cement gland as the adhesion organ in Xenopus laevis embryos

The cement gland in batrachians is a temporal ectodermic organ which is necessary for an embryo's attachment to the substrate. In this review, some notions about the origin of the cement gland of Xenopus laevis frogs, its functioning, genes being expressed in it, and regulation of its formation and development are provided. The role of some homologies of agrgenes of the cement gland in Xenopus laevis is noted at different conditions of other animals and man.     

Replacement of protamine by F1 histone during reactivation of fused human sperm nuclei

Summary Rabbit antisera, specific for the histones F1, F2a2, F2b, F3 and for protamine were used to monitor a possible transition from protamine towards somatic-type histones during sperm nucleus reactivation, following human sperm fusion with mouse fibroblasts.Mature human sperm nuclei were shown to contain the histones F2a2, F2b, F3 and protamine, but were missing F1 histone by immuno cytochemistry using the indirect fluorescence method. However, a gradual disappearance of protamine from fused sperm nuclei, could be observed during the first 24 h of reactivation. Subsequently, F1 histone could be detected in increasing concentrations in 60% of reactivated sperm nuclei, during the next four days.The shift from protamine towards F1 histone could also be visualized cytochemically via staining with brilliant sulphaflavine, which appears to discriminate between sperm nuclei on the basis of their F1 histone content.     

Replacement of protamine by F1 histone during reactivation of fused human sperm nuclei.

Rabbit antisera, specific for the histones F1, F2a2, F2b, F3 and for protamine were used to monitor a possible transition from protamine towards somatic-type histones during sperm nucleus reactivation, following human sperm fusion with mouse fibroblasts. Mature human sperm nuclei were shown to contain the histones F2a2, F2b, F3 and protamine, but were missing F1 histone by immuno cytochemistry using the indirect fluorescence method. However, a gradual disappearance of protamine from fused sperm nuclei, could be observed during the first 24 h of reactivation. Subsequently, F1 histone could be detected in increasing concentrations in 60% of reactivated sperm nuclei, during the next four days. The shift from protamine towards F1 histone could also be visualized cytochemically via staining with brilliant sulphaflavine, which appears to discriminate between sperm nuclei on the basis of their F1 histone content.     

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.     

Nuclear Basic Proteins of Xenopus laevis Sperm: Their Characterization and Synthesis during Spermatogenesis

Nuclear basic proteins from morphologically and functionally mature sperm of Xenopus laevis were analyzed by acid/urea/Triton X-100 polyacrylamide gel electrophoresis (AUT-PAGE). Six sperm-specific proteins (SP1-6) were identified in addition to somatic histones H3, H4 and smaller amount of H2A and H2B, but not H1. Of these, SP3–6 were unique in containing 33–41% arginine and having very low lysine/arginine ratios, while SP2 was more similar to H3 and H4 in having a lower arginine and higher lysine content. Fractionations of testicular cells at different spermatogenic stages by unit gravity sedimentation showed that primary spermatocytes and acrosomal vesicle spermatids possess typical somatic type histones but no SPs. Injection of [¹⁴C]-arginine into the testis and its tracing by fluorography on AUT-PAGE gels indicated that all somatic histones are synthesized during the stages between spermatogonia and primary spermatocytes, whereas SPs are synthesized at differentially regulated rates during the stages after acrosomal vesicle formation. In indirect immunofluorescence studies with anti-SP3-5 rabbit antiserum, a positive reaction was observed in the last step of spermiogenesis after the commencement of nuclear coiling.     

Histone- and Protamine-DNA Association: Conservation of Different Patterns within the β-Globin Domain in Human Sperm

Most DNA in human sperm is bound to highly basic proteins called protamines, but a small proportion is complexed with histones similar to those found in active chromatin. This raises the intriguing possibility that histones in sperm are marking sets of genes that will be preferentially activated during early development. We have examined the chromatin structure of members of the β-globin gene family, which are expressed at different times in development, and the protamine 2 gene, which is expressed in spermatids prior to the widespread displacement of histones by transition proteins. The genes coding for and γ globin, which are active in the embryonic yolk sac, contain regions which are histone associated in the sperm. No histone-associated regions are present at the sites tested within the β- and δ-globin genes which are silent in the embryonic yolk sac. The trends of histone or protamine association are consistent for samples from the same person, and no significant between-subject variations in these trends are found for 13 of the 15 fragments analyzed in the two donors. The results suggest that sperm chromatin structures are generally similar in different men but that the length of the histone-associated regions can vary. The association of sperm DNA with histones or protamines sometimes changes within as little as 400 bp of DNA, suggesting that there is fine control over the retention of histones.     

Androgen-induced myogenesis and chondrogenesis in the larynx of Xenopus laevis

We investigated a possible role for testosterone-induced cell proliferation in the development of sexual dimorphism in the larynx of South African clawed frogs, Xenopus laevis. Androgen-induced cell proliferation was studied using [3H]thymidine autoradiography. Nuclei of cartilage, perichondrium, and muscle were labeled in the larynx of sexually immature frogs of both sexes but not in adults. Cell proliferation did not occur with estradiol treatment nor was it seen in nonlaryngeal muscle or cartilage. Electron microscopic/autoradiographic studies of laryngeal muscle indicate that testosterone stimulates satellite cell division which later results in formation of myonuclei. We conclude that testosterone induces both chondrogenesis and myogenesis in juvenile larynx and that this process may contribute to the pronounced sexual dimorphism of the adult vocal organ.     

Pattern regulation in defect embryos of Xenopus laevis

Defect embryos of 24 series were prepared by removing increasing numbers of blastomeres from an 8-cell embryo of Xenopus laevis. They were cultured and their development was examined macroscopically when controls reached a tailbud stage or later. Results show that most of defect embryos of 12 series develop normally, and some of them become normal frogs. Each of these defect embryos contain at least two animal blastomeres, one dorsal, and one ventral blastomere of the vegetal hemisphere. This suggests that a set of these four blastomeres of the three types is essential for complete pattern regulation.     

Absence of paternal accessory sex gland secretions disturbs epigenetic reprogramming and expression of Igf2 and Dlk1 in golden hamster embryos

Accessory sex gland (ASG) secretion is known to exert an effect on sperm that is heritable in hamster embryos. We hypothesized that ASG secretion changes the sperm epigenome, which in turn is propagated in sired embryos. To test our hypothesis, we produced male hamsters that were devoid of either all ASG (TX) or only the ventral lobe of the prostate gland (VPX). A sham-operated control group (SH) was also established. These males were mated with normal females; uterine sperm, fertilized oocytes, and pre-implantation embryos were harvested from the females after mating. Epididymal sperm were collected at the end of experiments. Immunofluorescent staining was performed on these harvested specimens using antibodies against 5-methylcytosine, Dnmt1, Dnmt3a, Dnmt3b, protamine 1, protamine 2, and aectyl-H4K5. Expression of Igf2 and Dlk1 were analyzed by real-time RT PCR and in situ hybridization. We demonstrated that the DNA methylation pattern changed dynamically in SH, TX, and VPX fertilized oocytes. In VPX and TX embryos, DNA demethylation was slower and remethylation was delayed when compared with SH embryos. In addition, Dnmt3b expression was also abnormal. When sperm from VPX and TX males were exposed to whole ASG secretion in vivo, the resulting embryos all methylated normally. Immunofluorescent staining revealed that there was no difference in protamine packaging of uterine sperm from VPX and TX males. The staining also showed a lower level of acetyl-H4K5 expression in the male pronuclei of TX produced embryos. Furthermore, the VPX and TX embryos also expressed higher levels Igf2, and Dlk1. We concluded that interactions between ASG and sperm affected: (1) histone acetylation in male pronuclei; (2) DNA methylation in fertilized oocytes; and (3) Igf2 and Dlk1 expression embryos.     

The natural stimulus for spawning in Xenopus laevis (Amphibia)

I have already reported (Savage, 1965), preliminary experiments in which "natural spawning", that is, spawning by frogs kept under as natural conditions as aquaria allow, and by frogs not injected or manipulated in any other way, had been induced by a variety of external stimuli, including unialgal cultures of various kinds.
These experiments were planned and executed using as a guide, at every possible point, the statistical investigations of Rana temporaria obtained entirely from field data (Savage, 1935, 1961). The reasons for using Xenopus laevis and not R. temporaria were purely practical. R. temporaria breeds but once a year, and has other disadvantages, but X. laevis is a good laboratory animal.
Since conclusions drawn from mathematical relations in one species have led without important deviation to the expected experimental results in another, it is unlikely that what I now have to report is peculiar to these two species, selected because of availability in one case and ease of working in another. The conclusions are probably quite general. The earlier conclusions have been extended, active materials have been isolated, and other factors relevant to reproduction have been found.