Assessment of Histiotrophic Nutrition Using Fluorescent Probes.

Histiotrophic nutrition is a process whereby the rodent visceral yolk sac (VYS) internalizes exogenous macromolecules, degrades them, and sends the degradation products to the embryo. Quantification and visualization of histiotrophic nutrition can be accomplished using fluorescent tracer molecules such as fluorescein isothiocyanate-conjugated albumin (FITC-albumin). The methods are simple and can provide complimentary functional and structural information in studies of the effects of embryotoxicants on visceral yolk sac function.

Assessment of histiotrophic nutrition using fluorescent probes.

Histiotrophic nutrition is a process whereby the rodent visceral yolk sac (VYS) internalizes exogenous macromolecules, degrades them, and sends the degradation products to the embryo for use in de novo macromolecular biosynthesis. This process is important for embryonic development during early gestation prior to the formation of the functional placenta. Quantification and visualization of histiotrophic nutrition can be accomplished using fluorescent tracer molecules such as fluorescein isothiocyanate-conjugated albumin (FITC-albumin) that can be visualized using fluorescent microscopy and quantified using fluorescent spectroscopy. The methods are simple and can provide complementary functional and structural information in studies of the effects of embryotoxicants on yolk sac function.

Dietary myo-inositol therapy in hyperglycemia-induced embryopathy.

Dysmorphogenesis in diabetic mothers occurs more frequently than in the general population. This phenomenon is believed to be caused by the teratogenic effects of metabolic fuel mixtures with associated membrane injury and aberrations in the biochemical constituents. The present experiment was designed to determine: 1) if hyperglycemia-induced membrane injury is associated with intracellular and/or extracellular lipid disturbances; 2) if supplemental myo-inositol therapy prevents hyperglycemia-induced embryopathy; 3) if a correlation exists between dietary myo-inositol, serum and tissue levels of myo-inositol, and conceptus development; and 4) the cellular content of arachidonic acid following myo-inositol supplementation. Sixty-five female Sprague-Dawley rats were mated, and divided into three groups. One group was nondiabetic normal controls, and two groups had diabetes experimentally induced with streptozotocin. Of the diabetic groups, one received a normal diet, while the other received a myo-inositol-supplemented diet during the period of organogenesis. Blood samples were collected on days 0 and 12 of pregnancy. Embryos and yolk sacs were analyzed for myo-inositol and arachidonic acid levels, using mass spectrochromatography. Dietary myo-inositol supplementation of diabetic mothers resulted in a significant decrease in the incidence of neural tube defects when compared with diabetics not receiving supplements (9.5 vs. 20.4%; P < 0.05). This protective effect was incomplete, based on the incidence observed in the nondiabetic controls (9.5 vs. 3.8%; P < 0.05). The myo-inositol embryonic tissue levels in the diabetic group which had been fed a regular diet without supplementation were significantly lower than in the nondiabetic group. Dietary therapy successfully restored myo-inositol levels in the yolk sacs, as suggested by similar tissue levels in diabetics receiving myo-inositol supplementation and normal controls (18.7 +/- 1.3 vs. 19.1 +/- 2.0 ng/mg; P = ns). Dietary therapy, however, failed to restore myo-inositol levels in the embryos, suggesting hyperglycemia-induced faulty transport of nutrients from the yolk sac to the embryo. No correlation was noted between maternal blood levels of myo-inositol, with or without supplementation, and the clinical outcome. Tissue arachidonic acid levels were markedly reduced in the conceptuses of diabetic mothers with (0.4 +/- 0.1 micrograms/mg) or without (0.25 +/- 0.08 micrograms/mg) myo-inositol supplementation when compared to the nondiabetic controls (3.33 +/- 0.24 micrograms/mg). These data demonstrate that diabetes-induced embryopathy is associated with a deficiency state in both myo-inositol and arachidonic acid. The myo-inositol deficiency is not demonstrated at the serum level, but rather at the tissue level, suggesting a paracrine action. Dietary supplementation of myo-inositol is associated with an increase in tissue myo-inositol levels and a decrease in malformations. This therapy holds promise for use as a dietary prophylaxis against diabetic embryopathy.

Yolk sac failure in embryopathy due to hyperglycemia: horseradish peroxidase uptake in the assessment of yolk sac function.

We described previously the morphologic alterations of the visceral endodermal yolk sac cells of rat conceptuses cultured under hyperglycemic conditions which occurred concomitantly with major embryonic malformations. To determine whether the transport function of the yolk sac was impaired simultaneously as a result of these hyperglycemic conditions, horseradish peroxidase was used as a tracer protein to assess the transport function of the visceral endodermal yolk sac cells of conceptuses cultured in both control and hyperglycemic media. Cellular uptake of peroxidase, which was added to the culture medium for 3 or 24 hours, was observed in controls. This differed from the marked diminution in peroxidase uptake seen in conceptuses cultured in hyperglycemic medium. These results demonstrate that during hyperglycemia-induced embryopathy, there is concomitant yolk sac failure evidenced by morphologic alterations and impaired endocytosis. These findings therefore strengthen our hypothesis that diabetes-related malformations, as demonstrated experimentally in rat conceptuses, are associated with impairment in the structure and functions of the visceral yolk sac cells during a critical period of organogenesis.