Loren H. Hoffman

Inhibition of NF-kappaB activity results in disruption of the apical ectodermal ridge and aberrant limb morphogenesis.

In Drosophila, the Dorsal protein establishes the embryonic dorso–ventral axis during development. Here we show that the vertebrate homologue of Dorsal, nuclear factor-kappa B (NF-κB), is vital for the formation of the proximo–distal organizer of the developing limb bud, the apical ectodermal ridge (AER). Transcription of the NF-κB proto-oncogene c-rel is regulated, in part, during morphogenesis of the limb bud by AER-derived signals such as fibroblast growth factors. Interruption of NF-κB activity using viral-mediated delivery of an inhibitor results in a highly dysmorphic AER, reduction in overall limb size, loss of distal elements and reversal in the direction of limb outgrowth. Furthermore, inhibition of NF-κB activity in limb mesenchyme leads to a reduction in expression of Sonic hedgehog and Twist but derepresses expression of the bone morphogenetic protein-4 gene. These results are the first evidence that vertebrate NF-κB proteins act to transmit growth factor signals between the ectoderm and the underlying mesenchyme during embryonic limb formation.

Nutritional Provision to Embryos in a Predominantly Lecithotrophic Placental Reptile, Thamnophis ordinoides (Squamata: Serpentes)

Quantitative analysis of the composition of eggs and their sibling neonates in the viviparous natricine snake Thamnophis ordinoides revealed that yolk provided the principal source of organic nutrition but that embryos received a substantial allotment of inorganic nutrients from the placentas. The placental provision of water and sodium equaled or exceeded yolk supplies, and placental transport accounted for 23% of neonatal calcium composition. There was no difference between egg and newborn quantities of total phosphorus or total potassium, whereas neonates contained less total magnesium than eggs. The mode of embryonic nutrition in this species is characterized as predominantly lecithotrophic, yet placental nutrient provision contributes significantly to embryonic nourishment. Placental transport of sodium and embryonic uptake of water was greater in recently ovulated eggs that contained relatively low levels of sodium and water respectively. Thus, placental sources compensated for low yolk provision. Placental transport of calcium was independent of yolk calcium content and correlated positively with neonatal calcium content. This pattern of provision, in which placental sources determine neonatal content independent of egg content, has been described as facultative placentotrophy. A similar embryonic nutritional pattern was recognized previously in another predominantly lecithotrophic natricine snake.

Maternal-fetal transfer of organic and inorganic mercury via placenta and milk

The maternal-fetal transfer of organic and inorganic mercury across the placenta and through mothers milk has been investigated in the rat. It was found that organic mercury is more readily transferable across the placenta than inorganic mercury. However, both forms of mercury were transferred through milk with approximately the same efficiency. It was determined that the mercury concentrations in the placenta and the infant hair are directly related to the infants body burden of mercury.

The effects of testosterone, 5α-dihydrotestosterone, 3α-androstanediol, and 3β-androstanediol on the maturation of rabbit epididymal spermatozoa in organ culture

SummaryThe fertilizing ability of spermatozoa from epididymal tubules maintained in organ cultures from 1 to 7 days was assessed after artificial insemination into receptive does. It was found that spermatozoa from the distal corpus which were already capable of fertilizing eggs prior to the cultures retain this ability for 1 day without addition of hormone and for 3–4 days when testosterone (0.5 μg/ml) or 5α-dihydrotestosterone (0.5 μg/ml) is added to the culture medium. Spermatozoa from the proximal corpus which were not capable of fertilizing eggs prior to the cultures remain so after 1 day in cultures without addition of hormone. Testosterone, 5α-dihydrotestosterone, 3α-androstanediol, or 3β-androstanediol was added to cultures of proximal corpus at a concentration of 0.5 μg/ml. Only with 5α-DHT is the mean percentage of fertilization significantly higher than the percentage obtained without addition of hormone. Insulin does not potentiate the effect of 5α-DHT on sperm fertilizing ability. Epithelial growth factor is ineffective. Spermatozoa from the caput epididymidis kept in cultures for 1 to 4 days remain infertile. The results are discussed in light of the morphological findings presented in the preceding communication and in relation to the physiological requirement for sperm maturation in the epididymis.

Placental transfer of mercuric nitrate and methyl mercury in the rat.

Abstract Inorganic mercury tracer, 197 Hg(NO 3 ) 2 , and organic mercury tracer, CH 3 , 203 HgCl, were given to 15- and 20-day pregnant rats. The tissues investigated from 16 pregnant rats were decidua, yolk sac, placenta, and fetus. Tissues were taken as soon as 5 minutes after an intravenous maternal injection, as late as 120 hours for the 15-day-pregnant rats and as late as 24 hours for the 20-day-pregnant rats. The two mercury tracers were given simultaneously, and the percentages of the injected doses in the tissues were determined by gamma counting. Fetal uptake of methyl mercury was greater than that of mercuric nitrate at both 15 and 20 days gestation. Decidual tissue showed a comparatively low and constant uptake of both mercury tracers, while the percentage uptake of inorganic mercury by the yolk sac was higher than for methyl mercury.

Blastocyst-dependent upregulation of metalloproteinase/disintegrin MDC9 expression in rabbit endometrium

Abstract Blastocyst attachment to mammalian uteri at implantation involves the adhesion of the trophoblast to the uterine epithelial surface. In the rabbit, fusion between adjacent epithelial cells precedes the initial attachment phase and is followed by fusion between the trophoblast and the epithelium. The reverse transcription/polymerase chain reaction method has been used to prepare a partial cDNA (rbMDC9) from periimplantation-stage endometrium; this represents the rabbit ortholog of MDC9, a member of the cellular metalloproteinase/disintegrin (ADAM) gene family. We demonstrate here the reproductive stage-specific expression of rbMDC9 mRNA in uterine epithelium during the periimplantation period. Furthermore, this expression is upregulated at implantation sites, and in situ hybridization analysis has revealed that the epithelial cells with the most prominent signal are those apposed to blastocysts. Immunostaining with E-cadherin has been used to trace lateral membranes of epithelial cells and, together with nuclear staining, has enabled the identification of cells fusing to become multinucleated cells, and later, to become an epithelial syncytium (symplasma). These fusing cells express the highest level of rbMDC9 mRNA. The results suggest that MDC9, a transmembrane modular protein with domains having potential integrin-binding, metalloproteinase, and fusogenic functions, is probably critical for the cellular interactions accompanying blastocyst implantation.

Reduced expression of the adherens junction protein cadherin-5 in a diabetic retina

PURPOSE Transvascular leakage occurs in diabetic retinopathy. The tight junction proteins occludin and zonula occludens-1 (ZO-1) and adherens junction protein cadherin-5 are critical to the maintenance of endothelial barrier. We report a comparison of junction protein expression in the normal and diabetic retina. METHOD Case report. Postmortem retinal cryosections were prepared from the left eye of a 73-year-old woman with diabetic retinopathy. Cryosections were immunostained for cadherin-5, occludin, and ZO-1 and compared with retinal cryosections from the right eye of a 72-year-old man with no progression of retinal disease. RESULTS Immunofluorescence showed positive retinal vessel staining for occludin and ZO-1 in both eyes and cadherin-5 in the normal eye but reduced cadherin-5 staining in the retinal vessels of the diabetic eye. CONCLUSION Increases in transvascular leakage observed in diabetic retinal vasculature may be associated with reduction in the expression of the critical adherens junction protein, cadherin-5.

The effects of testosterone, 5α-dihydrotestosterone, 3α-androstanediol, and 3β-androstanediol on epithelial fine structure of the rabbit epididymis in organ culture

SummaryThe fine structure of the corpus epididymidis of the rabbit has been studied following organ culture. Various modifications of tissue preparation and culture conditions were examined to obtain good maintenance of cellular integrity as well as to preserve sperm fertilizing ability.After 5 to 7 days in culture in the absence of hormonal support, the epididymal epithelium showed signs indicative of cellular regression. Such changes included shrinkage of the cells, loss of the border of stereocilia, decrease in smooth endoplasmic reticulum, and an increase in autophagic vacuoles. The presence of androgens in culture media prevented cellular regression to varying degrees, depending on the hormone utilized. With regard to maintenance of cellular integrity, potency of the androgens tested was as follows: 5α-dihydrotestosterone >= 3α-androstanediol > testosterone > 3β-androstanediol. Addition of insulin to dihydrotestosterone-containing cultures resulted in no improvement in maintenance.Phagocytosis of spermatozoa by epithelial cells was observed in cultured tubules and the degree of spermiophagy was inversely proportional to successful maintenance of fine structural characteristics of epithelial cells.The morphological findings reported here correlate well with the fertilizing ability of spermatozoa from cultured epididymis as reported in an accompanying communication.

Crystalline inclusions in embryonic and maternal cells

A variety of cell types in early mammalian embryos and associated organs of the female genital tract contain inclusion bodies which are crystalline in nature. Although many of these structures were first seen by light microscopy, some have been further analyzed and others seen for the first time in more recent ultrastructural studies. Here, we will review structural aspects of such inclusions in several mammalian species. Evidence is scarce as to the chemical identity of most crystalline inclusions. A comparative look at structural features may allow us to come to tentative conclusions as to their functional importance and/or identity. Hopefully, such a review might stimulate new investigation on certain of these inclusions.

Uterine Receptivity to Implantation in the Rabbit: Evidence for a 42 kDa Glycoprotein as a Marker of Receptivity

The requirement for synchronous development of the mammalian embryo and uterus was evidenced by the failure of asynchronous embryo transfer to result in implantation except at the time of normal receptivity for each species (Chang, 1950; Finn, 1977). Evidence linking embryo-uterine synchrony to hormonal balance in the rabbit was based on steroid hormone-induced delay (Beier, 1976) or advancement (Adams, 1971; McCarthy et al., 1977) of uterine preparation relative to embryonic development; ovariectomy was directly lethal to contained blastocysts (Adams, 1958). That the epithelium constituted a major component of the “hostile uterine environment” resulting from inadequate hormone preparation was argued by Nilsson (1967) and Potts and Psychoyos (1967). Further support for this view was reported by Cowell (1969) who obtained “implantation”of mouse blastocysts in otherwise unreceptive uteri only after scraping of the epithelium to expose the underlying stroma. The uterine epithelium may be involved in a variety of implantation-related processes (absorption, secretion, barrier to trophoblast, message transduction to underlying tissues; Martin, 1980).