Toxic effect of heavy metals on cells isolated from the rat adrenal and testis

Summary Heavy metals including mercury, cadmium, cobalt, and copper (100 μM) exerted an adverse effect on the viability of isolated rat adrenal capsular (zona glomerulosa), adrenal decapsular (fasciculata and reticularis), and Leydig cells of the testis with mercury being the most potent. Due to the decreased cell viability there was a parallel reduction in corticotropin-stimulated, corticosterone production by adrenal decapsular cells and luteinizing hormone-stimulated testosterone production by Leydig cells. The results indicated a direct toxic action of these heavy metals on steroid-producing cell in the adrenal gland and the tectis. Other metals tested, including lead, zinc, aluminum, chromium, iron, nickel, and lithium, did not exert any deleterious effect on cell viability or hormone-induced steroidogenesis, in adrenal and Leydig cells when tested up to a concentration of 100 μM.     

Differential requirement for steroidogenic factor-1 gene dosage in adrenal development versus endocrine function

The importance of steroidogenic factor-1 (SF-1) gene dosage in endocrine function is evidenced by phenotypes associated with the heterozygous state in mice and humans. Here we examined mechanisms underlying SF-1 haploinsufficiency and found a striking reduction (12-fold) in SF-1 heterozygous (+/-) adrenocortical size at embryonic day (E) 12. Loss of one SF-1 allele led to a selective decrease in adrenal precursors within the adrenogonadal primordium at E10.0, without affecting the number of gonadal precursors, as marked by GATA-4. Beginning at E13.5, increased cell proliferation in SF-1 +/- adrenals allows these organs to approach but not attain a normal size. Remarkably, neural crest-derived adrenomedullary precursors migrated normally in SF-1 +/- and null embryos. However, later in development, medullary growth was compromised in both genotypes. Despite the small adrenal size in SF-1heterozygotes, an unexpected elevation in steroidogenic capacity per cell was observed in primary adult adrenocortical SF-1 +/- cells compared with wild-type cells. Elevated cellular steroid output is consistent with the up-regulation of some SF-1 target genes in SF-1 +/- adrenals and may partially be due to an observed increase in nerve growth factor-induced-B. Our findings underscore the need for full SF-1 gene dosage early in adrenal development, but not in the adult adrenal, where compensatory mechanisms restore near normal function.     

Lack of an adrenal cortex in Sf1 mutant mice is compatible with the generation and differentiation of chromaffin cells

The diversification of neural-crest-derived sympathoadrenal (SA) progenitor cells into sympathetic neurons and neuroendocrine adrenal chromaffin cells was thought to be largely understood. In-vitro studies with isolated SA progenitor cells had suggested that chromaffin cell differentiation depends crucially on glucocorticoids provided by adrenal cortical cells. However, analysis of mice lacking the glucocorticoid receptor gene had revealed that adrenal chromaffin cells develop mostly normally in these mice. Alternative cues from the adrenal cortex that may promote chromaffin cell determination and differentiation have not been identified. We therefore investigated whether the chromaffin cell phenotype can develop in the absence of an adrenal cortex, using mice deficient for the nuclear orphan receptor steroidogenic factor-1 (SF1), which lack adrenal cortical cells and gonads. We show that in Sf1-/- mice typical chromaffin cells assemble correctly in the suprarenal region adjacent to the suprarenal sympathetic ganglion. The cells display most features of chromaffin cells, including the typical large chromaffin granules. Sf1-/- chromaffin cells are numerically reduced by about 50% compared with the wild type at embryonic day (E) 13.5 and E17.5. This phenotype is not accounted for by reduced survival or cell proliferation beyond E12.5. However, already at E12.5 the 'adrenal' region in Sf1-/- mice is occupied by fewer PHOX2B+ and TH+ SA cells as well as SOX10+ neural crest cells. Our results suggest that cortical cues are not essential for determining chromaffin cell fate, but may be required for proper migration of SA progenitors to and/or colonization of the adrenal anlage.     

Characterization of the rat adrenal medulla cultured in vitro

Summary A wide variety of experimental animal models have been used to investigate the mechanisms of synthesis, storage, and release of catecholamines. Whereas in vivo experimental models are situated at one end of the spectrum, cell culture models are situated at the other end. In the present study, we have characterized various aspects of the rat adrenal medulla cultured in vitro as a whole tissue, aiming to establish a new experimental model in between in vivo animal models and cell culture models. We adapted a bottle rotator system commonly used for culturing rodent whole embryos. Changes in histology, activities and mRNA levels of catecholamine-synthesizing enzymes, and concentrations of catecholamines in the adrenal medulla were studied. In addition, the effects of cholinergic stimulation on catecholamine release from the adrenal medulla were examined. Overall the results indicate that various aspects of the adrenal medulla become stable after 4 d of culture and the adrenal medulla at this stage releases catecholamines in response to cholinergic stimulation. The whole adrenal medulla culture system may be a useful tool for investigating catecholamine-related functions dependent on intercellular reactions or communications.     

Secretion of 6beta-hydroxycortisol by normal human adrenals and adrenocortical adenomas

Although 6beta-hydroxycortisol (6betaOHF) is usually considered a cortisol metabolite produced by the liver, a few reports suggest that it may also originate from extrahepatic sources. To examine whether human adrenal cells are capable of 6beta-hydroxylating cortisol, we measured 6betaOHF secretion with a radioimmunoassay method in isolated human adrenal cell systems obtained from three normal adrenals, four nonhyperfunctioning adrenocortical adenomas, two adrenal adenomas causing Cushing's syndrome, and five aldosterone (Aldo)-producing adenomas. Cells were examined both under basal conditions and after stimulation with adrenocorticotrophic hormone (ACTH). In addition, 6betaOHF concentrations were determined in inferior vena cava and suprarenal vein plasma samples obtained from the side of nonhyperfunctioning adrenal adenomas (five patients) and aldosterone-producing adenomas (five patients). Under basal incubation conditions, 6betaOHF secretion, expressed as a percent of cortisol secretion, was between 0.5 and 2.0% in normal adrenal cells, between 1.0 and 7% in cells from nonhyperfunctioning adenomas, 12 and 15% in cells from Cushing's syndrome patients, and between 2.6 and 3.9% in cells from aldosterone-producing adenomas. In these cells, increasing doses of ACTH produced a dose-dependent stimulation of both 6betaOHF and cortisol secretion. The 6betaOHF concentration in suprarenal vein samples obtained from the side of adenomas was markedly increased; the suprarenal vein/inferior vena cava 6betaOHF ratios were 13.1+/-2.1 (mean+/-S.E.) in the case of nonhyperfunctioning adenomas and 17.8+/-4.5 in the case of aldosterone-producing adenomas. These results firmly suggest that 6betaOHF is not only a hepatic metabolite, but also a secretory product of human adrenals and that similarly to cortisol, its secretion may be controlled by ACTH.     

Immunocytochemical Detection of Structural and Regulatory Proteins in Rat Adrenal Nuclear Matrix

The nuclear matrix is a specific cell structure consisting of a residual nucleoskeleton that extends from the nucleoli to the nuclear envelope. The nuclear matrix of steroido-genic cells was isolated previously from a purified nuclear fraction. We present here an in situ extraction method, modified Lutz's method, for rat glandular adrenal cell nuclear matrix. This residual organelle was characterized and studied using immunocytochemical methods. The adrenal glands were removed, the cells prepared in suspension and deposited by cytospin onto Poly-L-lysine glass slides. The nuclear matrix was extracted with Nonidet P-40, DNase I and high and low ionic strength buffers. Structural proteins, nuclear lamins, coilin and fibrillarin were detected immunocytochemically. The adrenal fasciculata cells were easily identified by this method because of their large nuclei and abundant lipid droplets in the cytoplasm. After immunocytochemical detection by antibodies against lamins A and C, a marked brown layer at the periphery of the nucleus was observed. The intensity of the staining was lower using the antibody against nuclear lamin B. Immunocytochemical detection of the protein coilin revealed punctuated stained areas, 2-6 per nucleus, that probably correspond to the coiled bodies. The protein fibrillarin was detected at the nucleolus and coiled bodies. Our technique is simple, reveals well preserved adrenal nuclear matrices, and may be a useful method for immunocytochemical analysis and in situ hybridization.     

Cytophotometric assessment of Feulgen-DNA and coomassie-total cell protein in adrenocortical fasciculata cells of noise-exposed Wistar rats.

The present investigation was undertaken to examine possible noise-induced alterations in adrenal fasciculata cell (AFC) metabolism, and also to determine if the magnitude of these changes differs in male versus female rats. Wistar rats approximately 3 months old were exposed to intense noise for 60 min (100 dB, re 2 x 10(-5) N(m2)-1, 350-20,000 Hz); control rats were housed under identical conditions, at an ambient noise level of 40-60 dB. Adrenal fasciculata cells (AFC) from each animal were examined for noise-induced alterations in Feulgen-DNA reactivity (as an indicator of chromatin template activity) and Coomassie-total cell protein levels using scanning-integrating cytophotometry. The results provide evidence that intense noise elicited a marked AFC metabolic enhancement in both male and female rats; the degree of this enhancement was more pronounced in males. This disparity may be due to pre-existing differences in male versus female AFC enzymatic capability and subsequent responsiveness to noise-induced activation.     

Chronic stress and its effects on adrenal cortex apoptosis in pregnant rats

The model of chronic intermittent stress by immobilization during pregnancy may produce alterations in the mechanisms that maintain adrenal gland homeostasis. In earlier investigations using this model, significant variations in plasma prolactin and corticosterone levels, and adrenal gland weights were observed. We hypothesized that chronic stress causes changes in apoptosis in the adrenal glands of pregnant rats. We identified and quantified apoptotic cells in the adrenal cortex and examined their ultrastructural characteristics using transmission electron microscopy. Adrenal glands of pregnant rats at gestation days 12, 17 and 21 were studied for control and experimental (stressed) rats. Immunolabelling techniques, stereological analysis and image quantification of adrenal gland sections were combined to determine differences in apoptosis in the different cell populations of the adrenal cortex. The apoptotic index of the experimental rats showed a significant reduction at gestation day 17, while at days 12 and 21 there were no differences from controls. Moreover, the apoptotic index of the reticular zones in control and experimental animals showed a significant increase compared to the glomerular and fascicular zones at the three gestation times studied. Chronic stress by immobilization reduced the caspase-dependent apoptotic index at gestation day 17, which may be related to variations in plasma concentrations of estrogens and prolactin.     

An electron-microscopic stereological study of the compensatory hypertrophy of the rat adrenal zona fasciculata after unilateral adrenalectomy

Summary The ultrastructural changes associated with the compensatory hypertrophy of the zona fasciculata cells on monoadrenalectomized rats were investigated by stereological techniques. It was found that these subcellular changes display a different pattern from those underlying the ACTH-induced adrenocortical cell growth in the intact rats. This result supports the view that compensatory adrenal hypertrophy does not involve activation of the hypothalamo-hypophyseal-adrenal axis.     

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/- 8.1 cells, which increased to 84.4 +/- 5.7 and 125.5 +/- 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/- 6.0 and 40.3 +/- 5.0, respectively) and then doubled on day 7 (80.6 +/- 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/- 4.0 and 41.9 +/- 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/- 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.     

Development of chromaffin cells depends on MASH1 function

The sympathoadrenal (SA) cell lineage is a derivative of the neural crest (NC), which gives rise to sympathetic neurons and neuroendocrine chromaffin cells. Signals that are important for specification of these two types of cells are largely unknown. MASH1 plays an important role for neuronal as well as catecholaminergic differentiation. Mash1 knockout mice display severe deficits in sympathetic ganglia, yet their adrenal medulla has been reported to be largely normal suggesting that MASH1 is essential for neuronal but not for neuroendocrine differentiation. We show now that MASH1 function is necessary for the development of the vast majority of chromaffin cells. Most adrenal medullary cells in Mash1(-/-) mice identified by Phox2b immunoreactivity, lack the catecholaminergic marker tyrosine hydroxylase. Mash1 mutant and wild-type mice have almost identical numbers of Phox2b-positive cells in their adrenal glands at embryonic day (E) 13.5; however, only one-third of the Phox2b-positive adrenal cell population seen in Mash1(+/+) mice is maintained in Mash1(-/-) mice at birth. Similar to Phox2b, cells expressing Phox2a and Hand2 (dHand) clearly outnumber TH-positive cells. Most cells in the adrenal medulla of Mash1(-/-) mice do not contain chromaffin granules, display a very immature, neuroblast-like phenotype, and, unlike wild-type adrenal chromaffin cells, show prolonged expression of neurofilament and Ret comparable with that observed in wild-type sympathetic ganglia. However, few chromaffin cells in Mash1(-/-) mice become PNMT positive and downregulate neurofilament and Ret expression. Together, these findings suggest that the development of chromaffin cells does depend on MASH1 function not only for catecholaminergic differentiation but also for general chromaffin cell differentiation.     

Carbachol-Induced Cosecretion of Immunoreactive Atrial Natriuretic Peptides with Catecholamines from Cultured Bovine Adrenal Medullary Cells

The distribution and secretion of atrial natriuretic peptides (ANPs) were investigated in bovine adrenal medulla. (1) Cultured bovine adrenal medullary cells (2 x 10(6)/dish) contained 100.4 +/- 6.0 fmol of immunoreactive ANP (IR-ANP) and 207.3 +/- 6.6 nmol of catecholamines as epinephrine plus norepinephrine. (2) Stimulation of nicotinic but not muscarinic acetylcholine receptors caused a cosecretion of IR-ANP and catecholamines corresponding to the ratio of IR-ANP to catecholamines in cultured bovine adrenal medullary cells. (3) Carbachol-stimulated secretion of IR-ANP was dependent on the presence of extracellular Ca2+. (4) Chromaffin granules isolated from bovine adrenal medulla contained large amounts of IR-ANP and catecholamines, in the same ratio as did cultured adrenal medullary cells. (5) Reverse-phase HPLC analysis showed that both stored and secreted IR-ANP consisted of two components, which eluted at the position of ANP(99-126) or ANP(1-126). These results indicate that ANPs are stored as ANP(99-126) and ANP(1-126) in chromaffin granules, and are cosecreted in parallel with catecholamines in a Ca2+-dependent manner by the stimulation of nicotinic acetylcholine receptors.     

Distribution and morphology of ghrelin immunostained cells in the adrenal gland of the African ostrich

Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor. We investigated the distribution and morphological characteristics of ghrelin-immunopositive (ghrelin-ip) cells in the African ostrich adrenal gland. We found that the adrenal gland of the African ostrich consisted of three parts: capsule, inter-renal tissue and chromaffin cells. The inter-renal tissue and chromaffin cells interdigitated irregularly. The inter-renal tissue consisted of a peripheral zone and a central inner zone. The peripheral zone could be divided into an outer subcapsular zone and an inner zone. The subcapsular zone cells were arranged as a bow, while the inner area cells formed cords that were perpendicular to the capsule. The central inner zone exhibited irregular clumps and the cells were morphologically similar to chromaffin cells. Ghrelin-ip cells were located throughout the adrenal gland except the capsule. The majority of ghrelin-ip cells were found among the chromaffin cells. The number of ghrelin-ip cells in the inter-renal tissue decreased gradually from the central inner zone, to the inner zone to the subcapsular zone. The ghrelin-ip cells were oval or irregular in shape and exhibited cytoplasmic staining. Our findings suggest that ghrelin may play a role in regulating adrenal hormone secretion in the African ostrich.     

The influence of plasma lipoproteins on steroidogenesis of cultured ovine fetal and neonatal adrenal cells

The present study examined the effects of serum and lipoproteins on the function of cultured adrenal cells from 115-127-day-old ovine fetuses and from newborn lambs. On day 1 of culture, corticosteroid output was similar in medium containing 2% horse serum or in serum-free medium, both for fetal and neonatal cells. However, on day 5, cells cultured in the absence of serum produced smaller amounts of these steroids than cells maintained in medium containing serum; the difference was more marked under ACTH1-24 stimulation. Conversely, cAMP production was never lower in the absence than in the presence of serum. When stimulated by ACTH1-24 on day 2 of culture, fetal or neonatal adrenal cells incubated in the presence of a saturating concentration of ovine LDL produced more corticosteroids than cells incubated in serum-free medium; HDL also enhanced ACTH1-24-induced steroidogenesis, but to a lesser extent. VLDL was effective only with neonatal cells. In fetal and neonatal cells cultured for 6 days in ACTH-free medium, VLDL and LDL increased ACTH-induced steroidogenesis, but HDL did not. On the other hand, when cells were cultured in the presence of ACTH1-24, LDL and HDL were equipotent in supporting ACTH1-24-induced steroid output. Three major lipoprotein fractions were observed in serum of fetal and newborn lambs. The concentration of cholesterol was very low in the VLDL fraction of fetuses, but it was similar to that of newborns in LDL. Conversely, 4 times more cholesterol was present in HDL of newborns than in HDL of fetuses. These results suggest that: (i) after several days of cell culture, cholesterol availability is an important limiting factor for the steroidogenesis of cells maintained under serum-free conditions; (ii) both an "LDL pathway" and an "HDL pathway" are operating in adrenal cells from fetal as well as newborn sheep; (iii) LDL and HDL are important physiological sources of cholesterol to support steroidogenesis by fetal and neonatal adrenal cells.     

Effects of protease inhibitors on adenylate cyclase activation and aldosterone production in rat adrenal zona glomerulosa cells

It has been shown that serine proteases are involved in aldosterone and 18-hydroxycorticosterone production by the rat adrenal zona glomerulosa in response to a variety of stimulants. From evidence presented for various tissues, including the rat adrenal cortex, the observation that adenylate cyclase can be activated by proteolytic enzymes and inhibited by protease inhibitors has led to the suggestion that serine proteases may also be involved in the hormonal stimulation of adenylate cyclase. In studies designed to test this hypothesis using protease inhibitors, only high concentrations (greater than 10(-4) M) of TAME (p-tosyl-L-arginine methyl ester) inhibited ACTH stimulated steroid and cAMP production in rat adrenal glomerulosa cells. TPCK (tosyl-L-phenylalanine chloromethylketone) and TLCK (tosyl-L-lysine chloromethylketone) were found to have a similar effect at very high concentrations (10(-2) M) but had no effect at the serine protease inhibitory concentration of 5 X 10(-6) M. Other protease inhibitors tested had no effect on ACTH-stimulated cAMP but the inhibitory effect of high concentrations of protease inhibitors on ACTH-stimulated adenylate cyclase was duplicated by the polyanion dextran sulphate. The results suggest that the inhibitors act through non-specific membrane effects and that proteases are not involved in the activation of zona glomerulosa adenylate cyclase by ACTH. In view of these findings it is concluded that a more rigorous approach should be applied to the use of protease inhibitors in whole cell systems, and that the concept of hormonal activation of adenylate cyclase via proteolytic events, which is based on studies with such inhibitors, should be reconsidered.     

Difference in the Effectiveness of Ca2+ to Evoke Catecholamine Secretion Between Adrenaline-and Noradrenaline-Containing Cells of Bovine Adrenal Medulla

Abstract: Differential adrenaline (Ad) and noradrenaline (NA) secretions evoked by secretagogues were investigated using digitonin-permeabilized adrenal chromaffin cells, cultured adrenal chromaffin cells, and perfused adrenal glands of the ox. In digitonin-permeabilized cells, Ca²⁺ (0.8-160 μM) caused a concentration-dependent increase in catecholamine secretion, which was characterized by a predominance of NA over Ad secretion. Acetylcholine (10-1,000 μM), high K⁺ (14-56 μM), and bradykinin (0.1-1,000 μM) all were confirmed to induce the release of more NA than Ad at all concentrations used. There was no apparent difference in the ratios of NA/Ad between Ca²⁺-induced catecholamine secretion from digitonin-permeabilized cells and those induced by secretagogues from cultured cells. Qualitatively the same result was obtained in the secretory responses to acetylcholine and high K⁺ in perfused adrenal glands. These results indicate that the effectiveness of Ca²⁺ for catecholamine secretion is higher in the secretory apparatus of NA cells than in that of Ad cells of the bovine adrenal medulla. This may be one of the reasons why the secretagogues cause a predominance of NA secretion over Ad secretion in the bovine adrenal medulla.     

Autoradiographic localization of strychnine-sensitive glycine receptor in bovine adrenal medulla

The presence of an uptake system and a functional glycine receptor in adrenal medulla chromaffin cells was investigated using an autoradiographic technique in adrenal gland slices. Specific³[H]-glycine binding was observed in both adrenal cortex and medulla slices, while only specific binding of [³H]strychnine was seen only in chromaffin cells and was not associated with cortical cells. [³H]Glycine binding sites in the cortex are apparently different from those of [³H]strychnine binding sites in the medulla since excess strychnine does not displace [³H]glycine from adrenal cortex but does so from medulla. This difference supports biochemical evidence for glycine transport into medulla cells and glycine receptor sites on the chromaffin cell membrane.     

Macro- and microvascular endothelial cells in vitro: Maintenance of biochemical heterogeneity despite loss of ultrastructural characteristics

Summary Microvascular endothelial cells from bovine adrenal medulla and brain and macrovessel endothelial cells from bovine aorta were isolated and cultured under similar conditions in order to determine morphologic and biochemical heterogeneity in vitro. All three cell types exhibited nearly identical ultrastructural morphology and two-dimensional gel protein patterns of³⁵S-methionine-labeled whole cells. Two-dimensional gel analysis of³⁵S-methionine-labeled plasma membrane proteins however, revealed two-dimensional gel protein patterns unique to the tissue type from which the endothelial cells were isolated. This suggests that the functional significance of these specific endothelial cell types is manifested primarily in surface-associated proteins and that many of the differences are sustained in culture. To determine the potential of aorta, brain, and adrenal medulla endothelial cell (EC) cultures to respond to developmentally significant signals, morphology, growth pattern, and cell surface proteins were monitored in the presence and absence of growth factors. A 17 to 26% increase in cell density as well as an increase in the number of elongated and overlapping cells resulted when all three EC types were exposed to a mitogenic medium. Additionally, expression of specific glycoprotein profiles, as determined by Concanavalin A Western blotting of two-dimensional gels, was dependent on the presence or absence of growth factors in the medium. The ability to induce this morphologic and biochemical variation in the three endothelial cell types was maintained into later passage. Taken together, these data imply that endothelial cells isolated from different tissues exhibit and maintain biochemical heterogeneity and do not completely dedifferentiate into a common endothelial cell type in culture. Furthermore, expression of specific subsets of cell surface proteins is dependent on environmental conditions, and in some cases is both cell-type and media-type dependent. Thus, even though endothelial cells are considered terminally differentiated cells, there exists additional or “latent” heterogeneity in the ability of these different cells to respond to “developmental signals” (i.e. mitogenic medium) in vitro. This work was supported in part by a grant from the American Heart Association (860929), NIH (GM29127), and a Biomedical Research Grant from the University of Massachusetts.     

Endogenous peroxidase in the visceral endoderm of early mouse embryos

Endogenous peroxidase activity was demonstrated in early mouse embryos by means of the diaminobenzidine staining reaction. This enzyme was observed in visceral endoderm on the seventh to eighth day of gestation in vivo, but was no longer detected on the ninth day of development. In cell layers developing from blastocysts or isolated inner cell masses cultured for 96-144 h (developmental stage equivalent to 6-7.5-day-old embryos), diaminobenzidine product was also observed in visceral endodermal cells. Most of the endogenous peroxidase was localized inside or close to the numerous apical vacuoles in the endoderm. Ectoderm, mesoderm, ectoplacental cone, and trophoblast cells did not contain endogenous peroxidase.