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Article

Chronic Feeding of a Low Boron Diet Adversely Affects Reproduction and Development in Xenopus laevis^{1 ,2}

Douglas J. Fort³, Enos L. Stover, Philip L. Strong^*, F. Jay Murray and Carl L. Keen^**

The Stover Group, R & D Laboratory Division, Stillwater, OK 74074; Murray & Associates, San Jose, CA 95138; ^* U.S. Borax, Valencia, CA 91355-1847; and ^** Department of Nutrition, University of California, Davis, CA 95616

³To whom correspondence should be addressed.

	ABSTRACT

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The aims of this work were as follows: 1) to determine whether a purified diet currently used for studies with rats was acceptable for reproductive studies in frogs; and 2) to determine whether frogs are sensitive to a deficit of boron (B) in the diet. Adult Xenopus laevis were fed a nonpurified beef liver and lung (BLL) diet (310 µg B/kg), a purified diet supplemented with boron (+B; 1850 µg B/kg), or a purified diet low in boron (-B; 45 µg B/kg) for 120 d. Frogs fed the BLL and +B diets produced 11.3 and 12.2% necrotic eggs, respectively. Abnormal gastrulation occurred in <4% of the fertilized eggs in both groups, and 96-h larval survival exceeded 75% in both groups. In contrast, frogs fed the -B diet for 120 d produced a high proportion of necrotic eggs (54%). Fertilized embryos from the -B diet–fed frogs showed a high frequency of abnormal gastrulation (26.8%), and >80% of the embryos died before 96 h of development. Mean embryo cell counts at X. laevis developmental stage 7.5 (mid-blastula) were significantly lower in the -B embryos than in the BLL or +B embryos. BLL and -B embryos grown in low boron culture media had a high frequency of malformations compared with embryos grown in boron-supplemented media. These studies show that a purified diet that has been used in rodent studies was acceptable for reproduction studies in X. laevis. This work also demonstrates that a diet low in boron markedly impairs normal reproductive function in adult X. laevis, and that administration of the low boron diet results in an increase in both incidence and severity of adverse effects. In addition, these studies demonstrate the usefulness of the X. laevis model in nutrition studies.

KEY WORDS: • development • reproduction • nutritional essentiality • Xenopus laevis

	INTRODUCTION

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Boron in the form of inorganic borates has been recognized as an essential micronutrient for the growth and viability of plants for >75 y (Blevins and Lukaszewksi 1994, Loomis and Durst 1992 , Warington 1923 ). With a few geographical exceptions, natural bioavailable levels of boron in soils usually fall within a range that is necessary to support growth and development of plant species. There is considerable support for an essential role for boron in human health (Hunt 1994 and 1996b , Nielsen 1994 and 1996 ); on the basis of these data, WHO (1996) has recently listed boron as a "probable essential trace element." Positive health benefits that have been associated with the intake of supplemental boron include improved bone health, reduction in the severity of some forms of arthritis and improved cognitive function (Fracp et al. 1990 , Hunt and Nielson 1984, Penland 1998 , Shah and Vohora 1990 ). Although these observations are intriguing, some have argued that positive health effects of boron in higher life forms are due to pharmacologic responses and that boron is not essential. For this reason, a series of studies, specifically designed to evaluate the essentiality of boron during early development, have been initiated. It has been shown recently that boron may be essential for early development in trout (Eckhert 1998 ) and zebrafish (Rowe et al. 1998 ). Boron deficiency in zebrafish has been reported to result in delayed growth and embryo mortality (Rowe et al. 1998 ).

The frog, particularly the South African clawed frog Xenopus laevis, has been used as a model of embryonic development for >100 y (Loomis 1986 , Hausen and Riebesell 1991 ). The utility of X. laevis as a model for evaluating abnormal development has also been reported (Dumont et al. 1983a ); however, the X. laevis model has not been used extensively in nutrition studies, possibly due to the lack of a purified diet. The X. laevis model has been used recently to evaluate the effects of zinc and copper deficiency on early developmental processes (Fort et al. 1999). The specific objectives of this study were as follows: 1) to determine whether a purified diet currently used for studies in rats would support normal reproduction in frogs; 2) to confirm that frogs are sensitive to a deficit of boron during reproduction and early development; and 3) to corroborate and extend our preliminary studies, indicating that boron deprivation for 28 d impaired reproduction and increased the incidence of developmental abnormalities in Xenopus (Fort et al. 1998 ). Previous studies with frogs (Fort et al. 1998 ), which evaluated the effect of a low boron diet during organogenesis, indicated that boron (B) concentrations 3 µg B/L resulted in abnormal development in X. laevis. Similarly, in our previous 28-d boron depletion studies, we observed that adult frogs fed a low boron diet had a higher incidence of necrotic eggs, embryos that gastrulated abnormally at a greater rate and larvae that were less viable at 96 h than embryos from adult frogs fed a ground beef lung and liver (BLL)⁴ diet or a +B diet [American Society for Testing and Materials (ASTM) 1991 ].

To accomplish the above, we characterized the effects of a low boron diet on reproduction in X. laevis after a 120-d feeding trial. In addition, the deleterious effect of low boron on X. laevis embryo-larval development was evaluated using a standardized embryo-larval development model, the frog embryo teratogenesis assay–Xenopus (FETAX), a 4-d whole-embryo bioassay that evaluates organogenesis in X. laevis (Dawson and Bantle 1987a, Dawson et al. 1988 , Dumont et al. 1983b , Fort et al. 1988 and 1989 , Fort and Bantle 1990 ).

	MATERIALS AND METHODS

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Reagents and culture water.

Boric acid (premium grade, >99% purity) was obtained from U.S. Borax (Valencia, CA). To reduce boron levels in the culture water used for both the embryos and adults, FETAX solution⁵ was prepared with ACS-grade FETAX salts (>99.5% pure) (Sigma Chemical, St. Louis, MO) and deionized water (<0.1 ± 0 µg B/kg) and stored in a polyethylene plastic vessel. Low boron FETAX solution contained ~0.6 ± 0.1 µg B/L. Dechlorinated tap water, in which the frogs were maintained under standard conditions (ASTM 1991 ), contained ~116 ± 15.2 µg B/L. FETAX solution (ASTM 1991 ), which is prepared with standard-grade salts (>95% pure) and deionized water in borosilicate glass, contained 5.6 ± 0.3 µg B/L and was used as the normal control. Because X. laevis are a purely aquatic species regardless of life stage, both young and adults are continually maintained in a low boron environment.

Diets.

The -B (45 ± 6 µg B/kg) and +B (1850 ± 42 µg B/kg) purified diets (Hunt 1996a ) were obtained from the USDA Human Nutrition Research Center (Grand Forks, ND). These diets were developed for studying the effects of a low boron diet in rats. The compositions of the diets used in these studies are provided in Table 1 . The composition of the -B and +B diets was identical, with the exception of the boron content. In an effort to demonstrate that the +B diet supported growth, reproduction and development of X. laevis consistent with the ASTM diet (ASTM 1991 ) of BLL, a nonpurified BLL dietary treatment was used in this study. BLL was obtained from a local meat packaging facility, ground and frozen as patties until used. The BLL diet contained liver and lung in the ratio 10:1 (wt/wt) and was supplemented with the following nutrients: 15.0 IU vitamin A palmitate; 4.0 IU vitamin D-3; 0.05 IU vitamin E succinate; 0.35 mg vitamin C; 0.005 mg thiamin; 0.006 mg riboflavin; 0.08 mg niacin; 0.004 mg vitamin B-6; and 0.02 µg vitamin B-12 per gram of BLL. The level of boron in the BLL diet was ~310 ± 10.9 µg B/kg.

Adult frogs purchased from Xenopus I (Ann Arbor, MI) specifically for this phase of experiments were cultured in the low boron FETAX solution for 120 d. Each of the three diets was fed throughout the 120-d depletion period. Laboratory equipment that minimized contamination (i.e., polyethylene plastic vessels instead of glass) was used throughout these studies. The frogs were isolated from atmospheric boron. The adult frogs for each diet were housed in separate polyethylene plastic tanks and separated by sex. Research was conducted in compliance with the Animal Welfare Act and other Federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the NIH guidelines.

	Test methods

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Design.

In the first trial of this study, four adult female and four adult male frogs were used per diet. In the second trial, 10 newly acquired male and female X. laevis were used per dietary regimen. Consistent culture conditions and experimental design were maintained for both trials 1 and 2. The frogs were fed the -B, +B and BLL diets for 120 d, at which time each of the 14 adult frogs was bred and developmental effects tests performed as described in the following.

Breeding and embryo collection.

To induce mating, the male and female frogs received 500 and 750 IU, respectively, of human chorionic gonadotropin (Sigma Chemical, St. Louis, MO) via injection into the dorsal lymph sac (ASTM 1991 , Fort and Bantle 1990 ). Amplexus normally ensued within 2–6 h, and deposition of eggs occurred from 9 to 12 h after injection. Embryo collection and reproductive evaluations were performed as close to the time of egg deposition as practicable. Oocytes and embryos were collected from each dietary group for boron analysis.

The jelly coating surrounding the embryos was removed at stage 7 (large cell blastula) by gentle swirling for 3–4 min in a low boron (~0.5 µg B/L) 2% (wt/v) cysteine solution, prepared in the low boron FETAX solution (~0.4 µg B/L) (ASTM 1991 , Fort et al. 1991 and 1992 ). The pH of the cysteine solution was adjusted to 8.1 with low boron (<0.1 µg B/L) NaOH. After removal of the jelly coat, embryos were rinsed in the low boron FETAX solution and then used as described. The embryos used in these studies were sorted and selected such that only blastula-stage embryos of normal appearance were used. Test vessels used for each experiment were organized in a randomized design to minimize potential testing bias.

The pH of the FETAX solution was maintained at 8.0 with bicarbonate buffering (ASTM 1991 ). Each treatment contained a total of 8 mL of solution. Embryos were cultured in an insulated incubator at 23 ± 1.0°C throughout the test. Solutions were changed every 24 h of the 4-d test; dead embryos were counted, recorded and removed at 24-h intervals. After the 96-h culture period, surviving embryos were fixed in 3% formalin, pH 7.0. The number of live, dead and malformed live embryos, and the stage of development was recorded (ASTM 1991 , Fort et al. 1993 and 1996 ).

Reproductive assessment.

Reproductive data (including the number of eggs or embryos undergoing necrosis [stage 1–2], fertilization [stage 2], and the frequency of abnormal gastrulation [stage 12]), embryo cell number (stage 7.5) and samples for boron concentrations were collected quantitatively as soon as practical after egg deposition. Stage 1 X. laevis are newly fertilized one-celled embryos (zygotes). Stage 2 represents the two-celled embryo, stage 5 the 16-celled embryo, stage 7.5 the mid-blastula and stage 12 the advanced gastrula (mid-yolk plug). The time elapsed between stages 0 and 12 is ~13 h. Necrotic eggs were identified by the presence of discoloration, mottled pigmentation, lack of development, abnormal shape and morphology, and a white appearance, with no discernible development in the advanced stages. Total egg counts were performed by counting the entire mass of eggs harvested from the breeding chamber (Fort et al. 1998 ). Quantitation of the data was obtained by three successive counts. Similar procedures were involved in determining the number of unfertilized eggs, the number of necrotic eggs and the number of embryos demonstrating abnormal gastrulation. Embryo cell number was determined by manually counting the animal pole cells of 150 developmental stage 6.5, 7 and 7.5 embryos that were randomly selected from the pool of developing embryos, in accordance with standardized embryo-larval staging information compiled by Nieuwkoop and Faber (1975) . These stages represent early and middle blastula development in X. laevis; at that time, the embryo is a single cell layer thick. Only animal pole cells were considered in this assessment.

Developmental effects assessment.

Before initiating the developmental effects studies, the embryos were dejellied as described previously (ASTM 1991 , Fort et al. 1991 and 1992 ). An assessment of embryonic viability was determined using normally cleaving blastula-stage embryos by monitoring developmental progress through 96 h, when the major stages of organogenesis are complete. Developmental effects data were recorded at 24-h intervals throughout the evaluation of embryonic viability. Randomly selected embryos were collected for boron and copper analysis before the initiation of the viability studies. Developmental effects and viability frequency were evaluated after each of the seven individual treatments of the culture media as follows: 1) low boron FETAX solution (for each diet administered to the adult frogs); 2) low boron FETAX Solution supplemented with 10 µg B/L; and 3) low copper FETAX solution (positive control). For boron and copper analyses, 5 replicates of 20 embryos each were added, for a total of 100 embryos. These five replicates were combined at the end of the 96-h test and analyzed for boron and copper concentrations.

Positive control.

We described previously the teratogenic nature of low copper FETAX solution (<0.2 µg Cu/L) (Fort et al. 1999). Embryos collected from the adult frogs fed the BLL diet were cultured for 96 h in the low copper culture water. The low copper FETAX solution was prepared using deionized water and highly purified (>99.5%), low copper salts. Developmental effects were monitored as described in the preceding sections. FETAX solution typically contains ~2.0 µg Cu/L.

Boron and copper analysis.

Boron and copper concentrations were measured in water and biological samples with the use of inductively coupled plasma-mass spectrometry (ICP-MS) analysis according to Environmental Protection Agency (EPA) method 200.8 (Long and Martin 1991 ). Method detection limits for boron and copper were 0.1 and 0.2 µg/L for water samples, and 10.0 and 1.0 µg/L for biological samples, respectively.

Data analysis.

Reproductive performance, including frequencies of necrosis, fertilization, abnormal gastrulation and viability, were determined for each breeding pair. At the conclusion of each developmental effects study, mortality and malformation frequencies were determined. Comparisons of reproductive fitness evaluations and developmental effects treatments were performed using ANOVA and Dunnett's test (Dawson and Bantle 1987b , Fort and Bantle 1990 ). Differences were considered significant at P 0.05. Initially, the two trials were analyzed separately; however, because the two data sets were not significantly different (P = 0.05), the results were pooled into one set for the purpose of presentation (n = 14 adult frogs per diet).

	RESULTS

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Adults.

The overall appearance of female and male frogs was similar among each of the three diet groups throughout the 120-d depletion period. There were no overt differences in health or in the presence of disease in adults fed the different diets. Furthermore, the weight gain obtained in both the female and male frogs during the 120 d was similar among each of the three diets (Dunnett's test, P = 0.05). The weight gain in females fed the BLL, +B or -B diets was 61.2 ± 5.3, 59.3 ± 6.2 and 58.2 ± 7.3 g, respectively. The weight gain in males fed the three diets was 42.6 ± 3.8, 45.8 ± 6.3 and 40.9 ± 8.2 g, respectively. These gains in weight, including the difference in weight gain between female and male frogs, are consistent with normal weight gain in Xenopus (ASTM 1991 , Fort et al. 1999).

Reproductive outcome.

Boron concentrations in embryos from adult frogs fed the BLL diet (890 ± 54 µg B/kg) were similar to those of the +B embryos. Boron concentrations in embryos from adult frogs fed the +B diet for 120 d were markedly higher than those in embryos from frogs fed the -B diet (1120 ± 84 vs. 10 ± 2 µg B/kg). These results show that the +B diet was capable of providing a significantly greater amount of boron to the adult frogs and, subsequently, to the embryos. These results also show that maternal and paternal consumption of the -B diet resulted in the production of embryos with very low boron concentrations relative to control values.

The effect of the BLL, +B, and -B diets on X. laevis reproductive performance after the 120-d dietary treatment is depicted in Table 2 . Egg mass size, rates of fertilization, oocyte necrosis, abnormal gastrulation, embryonic viability and total embryo cell counts were similar in the +B and BLL diet groups. On the basis of these results, it appears that the purified rat diet used in this study will support normal reproductive performance in X. laevis. The egg mass size from the frogs fed the -B diet for 120 d did not differ from (P > 0.05) the egg mass collected from the frogs fed the +B diet. Fertilization rates did not differ (P > 0.05) in embryos from frogs fed the +B and -B diets.

Developmental effects.

The influence of low boron and boron-supplemented culture media on the frequency of lethality in 4-d embryos collected from frogs fed the +B and -B diets in each respective trial is presented in Table 3 . Overall, the frequency of mortality in each of the treatments was significantly greater (P < 0.05) in embryos derived from the adults fed the -B diet than in embryos from frogs fed the +B diet. In these 120-d depletion studies, 10 µg B/L supplementation to the embryos collected from adults fed the -B diet did not reduce the rate of mortality, an observation consistent with a previous study in which a 28-d boron depletion period was used (Fort et al. 1998 ).

View larger version (103K): [in this window] [in a new window]   Figure 1. Effect of B deficiency on X. laevis development. Panel A: 4-d larvae from ground beef liver and lung (BLL) diet-fed frogs cultured in 10 µg B/L frog embryo teratogenesis assay–Xenopus (FETAX) solution (adequate); panel B: 4-d larvae from the +B diet-fed adults cultured in 10 µg B/L FETAX solution (adequate); panel C: 4-d larvae from -B diet-fed adults cultured in low boron FETAX solution (~0.6 µg B/L).

	DISCUSSION

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Importantly, the developmental abnormalities noted in the -B group were distinct from those previously reported for copper or zinc (Fort et al. 1999). In this study, boron deficiency induced maldevelopment of the craniofacial region, mouth, and pigmented retina, and abnormal myotome assembly. This syndrome was similar to the terata observed in the preliminary 28-d depletion study; however, the frequency and severity of the abnormalities were appreciably greater in this study. The addition of boron (10 µg B/L) to the low boron culture water did not decrease the adverse effects noted in embryos collected from adult frogs fed the -B diet.

Copper deficiency in X. laevis induced incomplete notochord development, abnormal lens and optic nerve development, hydro- and microencephaly, hind limb maldevelopment, including reduction deficits distal to the femur, and incomplete heart formation, including atria and the dorsal aorta. Zinc deficiency caused pericardial and ophthalmicedema, axial flexure of the notochord, and maldevelopment of the mouth similar to cleft palate. Although some general similarities exist between these syndromes, the terata associated with each of these deficiencies were visually distinct from each other. Furthermore, the malformation syndrome noted in the -B embryos is distinct from that observed for embryos exposed to developmental toxicants evaluated with the X. laevis model system, including notable teratogens such as ethanol, thalidomide and diphenylhydantoin (Bantle et al. 1998 , Fort and Bantle 1990 ). We would suggest that the specificity of the syndrome provides additional support for the concept that boron is essential for normal development.

Results from the 28-d boron depletion studies indicated that the adverse effects of low boron consumption during organogenesis in embryos collected from adults fed the -B diet could be partially reversed by the addition of boron (10 µg B/L) into the culture water (Fort et al. 1998 ). Results of these studies, after 120 d of low boron administration to the parental frogs, indicated that the adverse effects on embryo-larval development could not be completely reversed by the addition of boron (10 µg B/L). Because boron repletion was noted in the previous 28-d depletion studies but not in the 120-d depletion studies, it is likely that the severe effect of boron deficiency in frogs disrupted boron repletion processes during the longer deficiency period. This finding suggests that the physiologic state of the organism as the result of the extent and duration of boron deficiency may determine repletion capacity. Furthermore, these results may be the effect of a greater extent of boron depletion in the embryos from frogs fed the -B diet with the longer depletion program or the effect of a longer duration of the boron-deficient state in the adults. In either case, the longer boron depletion period damaged the developing embryos such that they were incapable of developing normally. Thus, the embryonic damage induced after the 120-d boron depletion period was apparently irreversible. Tissue boron levels in embryos/larvae from the adults fed the -B diet for 120 d (<0 µg B/g) were appreciably lower than those in their 28-d depletion counterparts. On the basis of these findings, it is interesting to speculate that boron may play a role in the structural integrity and physiologic function of biological membranes.

In conclusion, the results from these studies show that exposure of adult X. laevis and embryos to a low boron environment is capable of inducing impaired reproductive performance, abnormal organogenesis and increased embryo lethality. These results are consistent with findings in other species, including trout (Eckhert 1998 ), zebrafish (Rowe et al. 1998 ) and the preimplantation mouse embryo (Lanoue et al. 1999 ), concerning the essential role of boron. In addition, these studies demonstrate the usefulness of the X. laevis frog model in nutrition studies.

	ACKNOWLEDGMENTS

 
The authors thank Rachel Felter for her assistance in the preparation of this manuscript. The authors would also like to acknowledge the assistance of Curtis Eckhert for his critical review of this research program. We also thank Curtiss Hunt, Forrest Nielsen and the U.S. Department of Agriculture Human Nutrition Research Center, Grand Forks, ND, for their generous supply of the low boron and boron-supplemented diets, which were critical to the success of this study.

	FOOTNOTES

 
¹ Presented at the 22nd Annual Teratology Society Meeting, San Diego, CA, June 1998.

² Funding for this research was provided by U.S. Borax Inc.

⁴ Abbreviations used: ASTM, American Society for Testing and Materials; BLL, beef liver and lung; EPA, Environmental Protection Agency; FETAX, frog embryo teratogenesis assay–Xenopus.

⁵ FETAX solution (amphibian culture media) consisted of 625 mg NaCl, 96 mg NaHCO₃, 30 mg KCl, 15 mg CaCl₂, 60 mg CaSO₄·2H₂O, and 75 mg MgSO₄ per liter of boron-free (<0.1 µg B/L) deionized water (Dawson and Bantle 1987b ).

Manuscript received May 28, 1999. Initial review completed July 8, 1999. Revision accepted July 23, 1999.

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