Assessing the effects of low boron diets on embryonic and fetal development in rodents using in vitro and in vivo model systems

To date, boron (B) essentiality has not been conclusively shown in mammals. This article summarizes the results of a series of in vitro and in vivo experiments designed to investigate the role of B in mammalian reproduction. In the first study, rat dams were fed either a low (0.04 μg B/g) or an adequate (2.00 μg B/g) B diet for 6 wk before breeding and through pregnancy; reproductive outcome was monitored on gestation day 20. Although low dietary B significantly lowered maternal blood, liver, and bone B concentrations, it had no marked effects on fetal growth or development. The goal of the second study was to assess the effects of B on the in vitro development of rat postimplantation embryos. Day 10 embryos collected from dams fed either the low or adequate B diets for at least 12 wk were cultured in serum collected from male rats exposed to one of the two dietary B treatments. Dams fed the low B diet had a significantly reduced number of implantation sites compared to dams fed the B-adequate diet. However, embryonic growth in vitro was not affected by B treatment. The aim of study 3 was to define the limits of boric acid (BA) toxicity on mouse preimplantation development in vitro. Two-cell mouse embryos were cultured in media containing graded levels of BA (from 6 to 10,000 μM). Impaired embryonic differentiation and proliferation were observed only when embryos were exposed to high levels of BA (>2000 μM), reflecting a very low level of toxicity of BA on early mouse embryonic development. Study 4 tested the effects of low (0.04 μg B/g) and adequate (2.00 μg B/g) dietary B on the in vitro development of mouse preimplantation embryos. Two-cell embryos obtained from the dams were cultured in vitro for 72 h. Maternal exposure to the low B diet for 10, 12, and 16 wk was associated with a reduction in blastocyst formation, a reduction in blastocyst cell number, and an increased number of degenerates. Collectively, these studies support the concept that B deficiency impairs early embryonic development in rodents.     

Somatic embryogenesis in mature zygotic embryo culture of Glehnia littoralis

In vitro somatic embryogenesis of Glehnia littoralis Fr. schm. was observed when zygotic embryos were cultured on a medium containing 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid (0.01-10 μM), with 1 μM being the optimum. Microscopic observations revealed globular, heart-shaped and torpedo-shaped embryo formations and plantlet regeneration. These somatic embryos seemed to be produced directly from cells of the zygotic embryos used as explants. Of seven types of media tested, Nitsch's medium showed the highest rate of somatic embryogenesis. Somatic embryos developed into normal plantlets and were able to be potted. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sequential culture medium promotes the in vitro development of Macaca fascicularis embryos to blastocysts

In vitro production of blastocyst stage embryos from Macaca fascicularis (Mf) has not previously been demonstrated without cell support. Historical data indicates that a large proportion of Mf embryos arrest at the morula stage in nonsequential culture medium (NSM) lacking serum supplementation and/or cell support. Here we report the application of a sequential culture system supporting in vitro production of Mf blastocysts. Mf embryos produced by in vitro fertilization (IVF; n = 69) were subjected to in vitro culture without cell support in either a commercial sequential embryo culture medium (SM) or an NSM. At 24 hr post-insemination (PI) embryos generated from in vivo and in vitro matured oocytes and cultured in the NSM cleaved to two or more cells in significantly greater proportions (15/23; 65%) compared to embryos cultured in SM (14/46; 30%). However, by day 3 PI embryo development beyond eight cells was not different in NSM (9/23; 39%) compared to SM (25/46; 54%). At day 5 PI embryo development to the morula stage was slightly lower in NSM (8/23, 35%) compared to SM (21/46, 45%), and embryo degeneration was slightly higher in NSM (9/23, 39%) compared to SM (9/46, 20%). After 7-9 days of in vitro culture, embryo development to the blastocyst stage and embryo degeneration were significantly lower and higher, respectively, in NSM (0/23, 0%; and 23/23, 100%) compared to SM (9/46, 20%; and 26/46, 56%). In this study the sequential culture system was better able to support in vitro development of Mf embryos compared to nonsequential culture systems.     

Effects of temperature, sorbitol, alanine and diapause hormone on the embryonic development in Bombyx mori: in vitro tests of old hypotheses

Abstract.  An in vitro culture method is described in which embryonic development in Bombyx mori is traced at various temperatures and treatments. The results show that the induction, intensification and termination of diapause are distinct processes. Prediapause embryos, explanted from 40-h-old diapause-destined eggs and cultured in Grace's medium, continue to develop to the appendage-formation stage without arrest, which indicates that the isolated embryos have not entered diapause, whereas the development of embryos from diapausing eggs (15 days after being laid) is significantly slower. The rate of development of embryos dissected from diapause eggs increases during chilling (5 °C) and incubation (at 25 °C) gradually during chilling and dramatically at 25 °C. The in vitro experiments also reveal that sorbitol directly inhibits the development of embryos explanted from diapausing eggs but has no affect on the development of embryos from prediapause eggs. Neither alanine nor diapause hormone prevent isolated embryos from developing.     

Chick embryo retina development in vitro: The effect of insulin

In this paper we study the development of chick embryo retina culturedin vitro and the effects exerted by insulin. Retinas were removed from 7-day embryos and cultured in serum-and hormone-free medium for 7 additional days. Under these conditions retinal cells survived and underwent cholinergic differentiation, as previously ascertained by Hausman et al. (Dev. Brain Res., 1991, 59: 31–37). However, a great retardation of development was noted compared to uncultured control, 14-day retina. In fact both wet weight and DNA and protein content increased much slower than in ovo and the tubulin content decreased below even the starting value. In addition, although after 7 days in culture retinal cells were organized in identifiable layers, nevertheless the typical organization equivalent to 14-day in ovo retina was absent. The addition of insulin in the medium markedly increased the wet weight of cultured retinas, their protein content and the level of tubulin pools, particularly that of non-assembled fraction. Nevertheless insulin did not modify DNA synthesis and did not induce the increment of both neuron specific enolase and actin. Morphological observations show that insulin markedly increased the number and the thickening of the fiber layers. These results, together with the facts that retina synthesizes and secretes insulin and possesses specific insulin receptors suggest that insulin can have autocrine or paracrine regulatory functions in retinal development by exerting a general effect on retinal growth and a more specific one on tubulin production.     

Somatic embryogenesis in cell suspension cultures of pigeonpea (Cajanus cajan)

Suspension cultures of calli derived from seedling leaf explants of Cajanus cajan L. var. Vamban-1 produced somatic embryos. The highest embryogenic frequency was induced on semisolid MS (Murashige and Skoog, 1962) medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The maximum frequency of somatic embryogenesis was observed when this callus was transferred to MS liquid medium supplemented with 4.52 μM 2,4-D. Further studies on ontogeny of somatic embryos showed that the cells destined to become somatic embryos divided into spherical proembryos. Subsequent divisions in the proembryo led to globular, heart and torpedo-shaped somatic embryos. The germination of somatic embryos occurred on auxin-free MS basal medium. Effects of various auxins, cytokinins and carbohydrates on induction and frequency of somatic embryogenesis were studied. A medium supplemented with 4.52 μM of 2,4-D and 87.64 mM sucrose was effective in inducing a higher frequency of somatic embryos, whereas cytokinin had no effect and led to recallusing of embryos. About 5–6% of embryos converted into plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mineral metabolism in the developing turkey embryo--I. The effects of developmental age and shell-less culture on trace element contents of selected tissues.

1. Turkey embryos were incubated in ovo or in long-term shell-less culture (ex ovo) for 14, 18, 22 or 26 days. The embryos incubated ex ovo exhibited a progressive decline in the rate of growth and were hypocalcemic and hypoproteinemic compared to their in ovo counterparts from day 18 to day 26 of incubation. 2. The ratio of the concentrations of alpha-fetoprotein and albumin (AFP/A) in serum was determined for both groups of embryos. The AFP/A ratio may be useful as a biochemical index to stage avian embryonic development. Using this index it was concluded that ex ovo embryos exhibited a progressive developmental retardation compared to in ovo embryos. 3. Significant differences were observed in serum trace element concentrations for embryos incubated in ovo vs ex ovo. Most notably, serum copper concentration was significantly lower in ex ovo embryos on days 18 and 22 of incubation and significantly higher on day 26 of incubation compared to serum from embryos incubated in ovo. 4. Livers from embryos incubated ex ovo exhibited significant differences trace element levels compared to those incubated in ovo. By day 26 of incubation the concentration and total amount of zinc and iron were markedly elevated, whereas copper was greatly reduced in the livers of embryos incubated ex ovo compared to the corresponding in ovo levels. 5. Hearts from embryos incubated ex ovo contained less zinc and copper and more iron by day 26 of incubation than those from embryos incubated in ovo.(ABSTRACT TRUNCATED AT 250 WORDS)     

The postnatal age of rat lung fibroblasts influences G1/S phase transition in vitro

Summary In the neonatal rat lung, alveolar development occurs from postnatal Days 4–13, during which time there is a fourfold increase in interstitial fibroblasts. Factors influencing emergence of new septa and cell proliferation associated with septal elongation have yet to be identified, in part because of difficulties inherent in studying this process in vivo. Using flow cytometric analysis of the DNA content of freshly isolated lung fibroblasts, we found that proliferation, as indicated by the percentage of cells in S plus G₂/M phases, peaked on postnatal Day 4 (P<0.04). By Days 9–10 the proliferation rate was lower than on Days 3, 4, 5, or 6 (P<0.005). We then evaluated rates of in vitro proliferation as a function of postnatal age in first passage fibroblasts and found that the proliferative phenotype expressed in vivo persists in vitro. Fibroblasts from 4–5-d-old pups increased in number and incorporated ³H-thymidine at a faster rate than did fibroblasts obtained from pups at other postnatal ages (P<0.0001). Age-dependent differences in cell cycle transit time were compared in fibroblasts synchronized by serum starvation and analyzed by flow cytometry at 2-h intervals from 13–21 h after release from serum starvation. A greater percentage of cells from 5-d-old pups entered S phase during this period than was seen for cells obtained from 2-, 9-, 13-, or 23-d-old rat pups (P=0.0001). Cells from 5-, 9-, and 13-d-old pups reentered G₀/G₁ by 21 h after release from serum starvation, in contrast to fibroblasts from 2- and 23-d-old rats which did not. Throughout the 15-h period after release from serum starvation, levels of cyclin E, which peaks at the G₁/S border, were highest in the 5-d-old cells (P<0.025). Synchronization with 2.5 mM hydroxyurea which inhibits DNA synthesis completely abolished age-related differences in cell cycle transit time, implying that age-dependent differences in lung fibroblast proliferation rates are the result of events occurring before S-phase entry.     

Development of early bovine embryos to the blastocyst stage in serum-free conditioned medium from bovine granulosa cells

Summary Bovine granulosa cell — conditioned medium (BGC-CM) was prepared in a serum-free medium consisting of TCM 199, 5μg/ml insulin, and 0.5μg/ml aprotinin (TCM 199 IAP). Granulosa cells surrounded with embryos were denuded 24 to 30 h after in vitro fertilization. The proportion of denuded granulosa cell-free embryos that developed to the blastocyst stage in BGC-CM (43/219; 20%) as well as in the co-culture system (43/178; 24%) was significantly greater (P<0.001) than in fresh TCM 199 IAP medium (FM: 10/191; 5%), whereas the proportion of embryos that developed to the eight-cell stage was similar (P>0.05) in all three culture systems (95/178; 53% in co-culture, 111/219; 51% in BGC-CM, and 86/191; 45% in FM, respectively). Higher rates of hatching and hatched blastocysts 8.5 days after in vitro fertilization were observed in co-culture (13/44; 29.5%) and in conditioned medium (8/39; 20.5%). On the other hand, no hatching or hatched blastocysts were obtained in the fresh medium (0.7; 0%). Cell numbers per blastocyst in BGC-CM (178.3 cells/blastocyst) were approximately two-fold higher than those in FM (97.1 cells/blastocyst). However, higher cell numbers (249.3 cells/blastocyst) were observed in co-culture with BGC than in BGC-CM. The embryotrophic activity in BGC-CM was stable upon freezing and thawing, lyophilization, and heating at 56° C whereas activity was reduced by dilution in fresh medium, dialysis, pronase digestion, and heating at 80° C. These results suggest that BGC cultured in a serum-free medium can synthesize and secrete an embryotrophic factor(s) that supports blastocyst formation in vitro beyond the 8- to 16-cell stage.     

Somatic Embryogenesis and in vitro Flowering in <Emphasis Type="Italic">Saposhnikovia divaricata</Emphasis>

Efficient somatic embryogenesis (SE) and in vitro flowering and fruiting were achieved in Saposhnikovia divaricata (Turcz.) Schischk. Friable embryogenic callus developed from the root, internode, and leaf explants on Murashige and Skoog medium (MS) with 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D), and subsequently developed into somatic embryos on MS medium containing 4–5% sucrose, 1.74 μM naphthaleneacetic acid (NAA), 4.44 μM 6-benzylaminopurine (BA), and 1.90 μM abscisic acid (ABA). Then the mature embryos were separated and transferred onto MS with 3% sucrose and 0.6% agar for further development and conversion to plantlets. In vitro flowering and fruiting were obtained when the subcultures were carried out for over 15 months. Paclobutrazol (PP333) or ethephon (ETH) at low levels promoted flowering significantly. Also, abnormal rootless somatic embryos of S. divaricata could form flowers and fruits in vitro.     

Two-phase chemically defined culture system for preimplantation rat embryos

A limiting factor in the development of new technologies and transport of rats worldwide has been the inability to robustly culture preimplantation embryos. Previously, culture in vitro to the blastocyst stage from one-cell embryos was successful only if the one-cell embryos were isolated near the time of the first cleavage and from only a few strains. Here we report the use of commonly available, chemically defined culture media to overcome these limitations. In vitro culture of young one-cell embryos using common embryo media (KSOM, BMOC, or HTF) for 18-22 h followed by culture in mR1ECM medium allows the successful in vitro development of blastocysts from one-cell embryos after 5 days from both outbred (SD) and inbred strains of rat (WF, LEW, F344, and PVG). This system allows the parthenogenetic development of chemically activated, unfertilized oocytes to the blastocyst stage. Embryos cultured in this system develop to term and are live-born following transfer to surrogate mothers.     

Organotypic slice culture based on in ovo electroporation for chicken embryonic central nervous system

Organotypic slice culture is a living cell research technique which blends features of both in vivo and in vitro techniques. While organotypic brain slice culture techniques have been well established in rodents, there are few reports on the study of organotypic slice culture, especially of the central nervous system (CNS), in chicken embryos. We established a combined in ovo electroporation and organotypic slice culture method to study exogenous genes functions in the CNS during chicken embryo development. We performed in ovo electroporation in the spinal cord or optic tectum prior to slice culture. When embryonic development reached a specific stage, green fluorescent protein (GFP)‐positive embryos were selected and fluorescent expression sites were cut under stereo fluorescence microscopy. Selected tissues were embedded in 4% agar. Tissues were sectioned on a vibratory microtome and 300 μm thick sections were mounted on a membrane of millicell cell culture insert. The insert was placed in a 30‐mm culture dish and 1 ml of slice culture media was added. We show that during serum‐free medium culture, the slice loses its original structure and propensity to be strictly regulated, which are the characteristics of the CNS. However, after adding serum, the histological structure of cultured‐tissue slices was able to be well maintained and neuronal axons were significantly longer than that those of serum‐free medium cultured‐tissue slices. As the structure of a complete single neuron can be observed from a slice culture, this is a suitable way of studying single neuronal dynamics. As such, we present an effective method to study axon formation and migration of single neurons in vitro.     

Successful implantation of in vitro cultured rabbit embryos after uterine transfer: A role for mucin

The functional role of the mucin layer for development of rabbit embryos was examined by uterine transfer of embryos with different thicknesses of mucin. Embryos collected at various intervals after human chorionic gonadotropin (hCG) injection were cultured until 90 hr post-coitum (p.c.) and transferred to the uterus of synchronized recipients. When embryos collected at 20 or 25 hr p.c. were used for transfer, no implantation occurred. By contrast, embryos collected at 35 or 40 hr p.c. developed to term at high rates (53 and 80%, respectively). The thickness of the mucin layer on the embryos was different between these two groups. Embryos collected before 25 hr p.c. have less than 11.2 ± 0.2 μm of thickness of mucin and embryos collected after 35 hr p.c. have more than 34.3 ± 5.5 μm. To examine whether mucin deposition is required for in vitro cultured rabbit blastocysts to continue development after uterine transfer, embryos were collected at 20 hr p.c., cultured for 60 or 70 hr in vitro, and then temporarily transferred to the oviducts of recipient does to add mucin. These embryos were recovered from the oviducts at 24 hr after transfer, classified according to the thickness of mucin deposition, and transferred again to the uterus of synchronized recipients. Twenty live young were obtained from 67 embryos with a 20–40 μm thick mucin layer. No live young were obtained from 57 embryos with less than a 20 μm thick mucin. The thickness of the mucin layer appears to be an important factor for successful implantation of rabbit embryos. © 1996 Wiley-Liss, Inc.     

IN VITRO FERTILIZATION OF IN VITRO MATURED MINKE WHALE (BALAENOPTERA ACUTOROSTRATA) FOLLICULAR OOCYTES

In vitro fertilization of follicular oocytes harvested from ovaries and matured in vitro was attempted for 55 minke whales ( Balaenoptera acutorostrata ) captured for Japanese research purposes in the Antarctic Ocean during the period from November 1995 to March 1996. In Experiment 1, effects of culture duration (96 h or 120 h) on maturation of follicular oocytes and addition of caffeine (5 mM) and/or heparin (100 pg/ml) on sperm penetration and pro-nuclear formation were investigated. Spermatozoa recovered from the vasa deferentia of four mature males were diluted (5-fold) and frozen at - 80°C. The post-thawed and pooled spermatozoa were used for in vitro insemination. A higher ( P < 0.05) proportion of the oocytes cultured for 120 h (34.2% of 260) progressed beyond the second metaphase stage than of the oocytes cultured for 96 h (26.0% of 262). For the matured oocytes, higher rates of penetration ( P < 0.05) and pronuclear formation ( P < 0.01) were obtained in the oocytes cultured for 120 h (55.1% and 40.4%) than in those cultured for 96 h (32.4 % and 20.6%). Addition of caffeine and heparin did not show a significant effect. In Experiment 2, follicular oocytes matured for 120 h and then inseminated were cultured to examine the subsequent development in two culture systems (with and without co-cultured cumulus cells). Of 448 inseminated oocytes, cleaved embryos (2–16 cells) were observed with (5.8%) and without (4.9%) co-cultured systems. No cleavage was observed in 54 ova without insemination. These results indicate that in vitro fertilization of minke whale in vitro matured follicular oocytes with cryopreserved spermatozoa is possible, yielding cleaved embryos.     

Dynamics of differentiation in human epidermoid squamous carcinoma cells (A431) with continuous,long-term γ-IFN treatment

Summary We investigated the long-term effects of continuous gamma interferon (γ-IFN) treatment on A431, a human squamous carcinoma cell line. Cells were grown in an in vitro culture system, which over time produces cohesive cell masses (“tumoroids”) exhibiting three-dimensional, histotypically differentiated structures, e.g., keratin “pearls,” intercellular bridges (desmosomes), elongated flattened cells (squames) and stratification. The effects of γ-IFN on cell growth, morphology and stage of differentiation were assessed at different treatment times by light and electron microscopy and by immunohistochemical staining using antibodies to keratins 1 and 14 and to filaggrin, markers of specific stages of keratinocyte differentiation. Our results show that A431 cells have the capacity for spontaneous differentiation, that this capacity is significantly enhanced and accelerated by γ-IFN treatment leading to terminal differentiation and extensive cell death by 2 wk. Despite continuous exposure to IFN, a small number of viable, undifferentiated cells remain. Their proliferation, evident by 3 wk, reconstitutes the tumoroid which once again contains the full range of differentiating cell types.     

Co-culture of rabbit one-cell embryos with rabbit oviduct epithelial cells

Summary Rabbit 1-cell embryos were co-cultured with rabbit oviduct epithelial cells (ROEC) to determine if ROEC can enhance embryo development in vitro. Primary ROEC were cultured in serum-free media at 39°C in a 5% CO₂:95% air environment. In experiment 1, 1-cell embryos were co-cultured in Ham's F10 with freshly collected or 4-d-old cultures of ROEC seeded in plastic culture wells or on collagen membranes. One-cell embryos cultured without ROEC served as controls. After 65 h in culture, embryos were stained with Hoechst 33342 to determine the number of cells per embryo. Cell numbers were higher (P<0.035) in all co-culture treatments when compared to controls. Optimal development was obtained by co-culture with 4-d-old ROEC grown on plastic (P<0.003). In experiment 2, Ham's F10, Medium 199, and CZB with glucose medium were compared for their ability to support embryo development in the presence or absence of 4-d-old ROEC growth on plastic. Cell number and the percentage of embryos becoming blastocysts were significantly (P<0.002) higher for embryos cultured in Medium 199 compared to the other media tested. In Medium 199, co-culture with ROEC resulted in only a slight, nonsignificant increase in cell number over culture in Medium 199 alone (110 vs. 96 cells). However, the percentage of embryos reaching the blastocyst stage when co-cultured in Medium 199 with ROEC (49%) was nearly twice (P=0.01) that of embryos in Medium 199 without ROEC (26%). In experiment 3, transfer of embryos cultured in Medium 199 with or without ROEC of 24 or 48 h resulted in no significant differences in posttransfer development. These data indicate a beneficial effect of ROEC on blastogenesis and a salvage effect of ROEC on cell proliferation in embryos grown in a less supportive medium such as Ham's F10. This work was supported by a Multicenter Cooperative Program on Non-HumanIn Vitro Fertilization and Preimplantation Development and was funded by the National Institute of Child Health and Human Development, NIH, Bethesda, MD, through Cooperative Agreement HD 21939.     

In vitro growth of corpora allata fromDiploptera punctata

Summary An in vitro organ culture system was established to support growth of corpora allata from the cockroachDiploptera punctata. During a 1-wk incubation in L-15B medium supplemented with 10% fetal bovine serum (FBS) and 10% cockroach hemolymph, adult male corpora allata exhibited a cycle of de novo DNA synthesis followed by cell division. The number of S-phase cells and metaphase cells per corpus allatum were counted from whole-mount monolayers after labeling in vitro with 5′-bromo-2′-deoxyuridine and exposure to colchicine, respectively. While both FBS and cockroach hemolymph were essential for proliferation of allatal cells, the growth-promoting effect of insect hemolymph was not species-specific and adult female hemolymph was more potent than hemolymph from adult males. Furthermore, DNA synthesis of corpus allatum cells was stimulated in vitro by 20-hydroxyecdysone. This sensitive assay system will be of immense utility in the search for allatal growth factors.     

Adrenal cells in tissue culture. VIII. Dissociation of the steroidogenic and glycolytic effects of cyclic nucleotides and adrenocorticotropin

Summary In monolayer cultures of mouse adrenal cortex tumor cells, high concentrations of 3′,5′-cyclic adenosine monophosphate and 3′,5′-cyclic cytidine monophosphate (1.0 to 10.0mm produce steroidogenic responses equivalent to maximally stimulating levels of adrenocorticotropin. 3′,5′-Cyclic guanosine monophosphate and other cyclic nucleotides are not steroidogenic. Although the steroidogenic action of adrenocorticotropin is accompanied by an increased rate of glycolytic activity, the cyclic nucleotides stimulate steroidogenesis without increasing glycolysis. The data suggest that adrenocorticotropin can effect certain alterations in adrenal metabolism by a mechanism which does not involve the adenyl cyclase system. Supported by grants from the American Cancer Society (P-395) and the National Institutes of Health (R01-AM09901). Presented in part at the 1969 Laurentian Hormone Conference, Mt. Tremblant, Quebec, Canada, August 28, 1969.