Estrogen and progestin receptors in the reproductive tract of male and female primates.

With the aid of monoclonal antibodies specific to the estrogen and progestin receptors, we have examined the cellular localization of these proteins in the reproductive tract of male and female macaques. Two striking findings have resulted from our work with these new reagents. First, these receptors are detectable only in cell nuclei, regardless of hormonal treatment, and second, they are often detectable in stromal, but not epithelial cells when the epithelial cells undergo various estrogen or progestin-dependent events. The latter observation has led us to conclude that stromal cell-epithelial cell interactions may play previously unappreciated roles in the hormonal control of the primate reproductive tract. The lines of evidence that have drawn us to this conclusion will be reviewed.

Antibody against avian müllerian inhibiting substance (MIS) recognizes MIS on rat müllerian duct and human tumor cells.

Müllerian inhibiting substance (MIS) on rat Müllerian duct (Md), and four Müllerian-derived tumor cells: HeLa S-3, RL-95.2, A-431 and NIH:OVCAR-3 are recognized by the poly- and mono-clonal avian-MIS-antibodies (A-MIS-Abs) using avidin-biotin complex (ABC) immunolabeling techniques. Internalization of MIS-ligand complexes was successfully detected in HeLa S-3, OVCAR-3 and RL 95.2 cells. Control groups include: (i) the samples omitted primary antibody treatment, (ii) Wolffian duct by side of Md in the genital ridge, and (iii) another two MIS-negative tumor cell lines of non-Müllerian origin: Chang hepatoma ascites cell and mouse myeloma cell (X63-Ag 8.653). Genital ridges from rat embryos of 14d of gestation were removed under dissection microscope, fixed in 2.5% glutaraldehyde in D-PBS of pH 7.2 for 30 min, and sliced into 0.1-0.2 mm thick pieces. A-431, HeLa S-3 and NIH:OVCAR-3 were maintained in OPTI-MEM culture medium, RL-95.2 was cultured in F12 culture medium. The cells were transferred to 24-well flat bottom culture plates with Thermanox tissue culture coverslips. The immunolabeling of fixed and non-fixed samples were processed within the wells. These studies provide first immunocytochemical evidences for the similarity between A-MIS and M-MIS molecules by polyclonal and monoclonal A-MIS-Ab. It has also proved that the tumor cell lines, which were subjects of MIS inhibition of cell growth, showed MIS binding on cell surfaces.

The 56 kDa protein of human genital skin fibroblasts is identical to that radiolabelled by [3H]dihydrotestosterone 17 beta-bromoacetate.

Analysis of soluble proteins from human genital skin fibroblasts by two-dimensional polyacrylamide gel electrophoresis reveals an abundant protein doublet of mol. wt 56,000 with isoelectric points (pI) of 6.7 and 6.5. This protein is absent in non-genital skin fibroblasts as well as in genital skin fibroblasts of most patients with complete forms of androgen insensitivity. The protein specifically binds androgen. A protein of similar estimated molecular weight (58,000) from human genital skin fibroblasts has recently been found to be covalently radiolabelled by the affinity ligand dihydrotestosterone 17 beta-bromoacetate (DHT-BA). In the present study these proteins have been found to be indistinguishable on one- and two-dimensional gel electrophoresis. Antibodies raised against the 56 kDa pI 6.7/6.5 protein also recognized the protein covalently radiolabelled by DHT-BA. A third protein of estimated mol. wt 59,000 has been found to be associated with several steroid hormone receptor complexes but has no known ligand binding activity. This protein was found to be clearly separable from the 56/58 kDa protein on two-dimensional gel electrophoresis as it has a more acidic pI of approximately 5.4. Furthermore, antibodies against the 59 kDa protein do not recognize the 56 kDa species, and vice versa.

Occurrence and distribution of glycoconjugates in human tissues as detected by the Erythrina cristagalli lectin.

We applied a horseradish peroxidase-Erythrina cristagalli agglutinin (HRP-ECA) conjugate for histochemical staining of tissue sections from various formalin-fixed, paraffin-embedded human tissue specimens. The HRP-ECA conjugate showed broad reactivity, but there was a distinct distribution of native (not masked by sialic acid) and sialic acid-masked ECA binding sites in the various organs. Free ECA binding sites could be detected on red blood cells, lymphocytes of thymus, tonsil, lymph node, and in mucous substances of different organs. Independent of blood group type, the vascular endothelium exhibited strong ECA reactivity. Free ECA binding sites occurred in the cytoplasm of Kupffer's cells in liver, in histiocytic cells of thymus, lymph node, tonsil, and in bone marrow. Podocytes of kidney glomerulus, syncytiotrophoblasts of placenta, megakaryocytes in bone marrow, myelin sheath of nerve, medullary thymocytes, and hepatocytes, as well as islet cells of pancreas, contained only sialic acid-capped ECA binding sites. Inhibiting studies with galactose, lactose, and N-acetyl-lactosamine, as well as other sugars, revealed that this lectin is specific for galactosyl residues. In comparison to galactose and lactose, N-acetyl-lactosamine exhibited the highest inhibitory activity on lectin binding, supporting the concept that this lectin is most reactive with N-acetyl-lactosamine-type (type 2 chain) glycoconjugates.

A study of androgen-resistant subjects indicates that the 6.7 pI/56 kDa protein in genital skin fibroblasts is related to the androgen receptor.

Two-dimensional gel electrophoresis of cultured human skin fibroblast lysates reveals a silver-stained "spot" of molecular mass 56 kilodaltons (kDa) and isoelectric point (pI) 6.7, occasionally as part of a doublet with a minor pI 6.5 partner. Its presence in each of 23 genital skin fibroblast strains (6 labium majus, 17 prepuce) and its absence in 30 of 32 control non-genital skin fibroblast strains accords with the 3-fold greater concentration of androgen-receptor activity in the former. However, the size and intensity of the spot do not change when cells are preincubated for 48 hours with 3 nM methyltrienolone (MT, a non-metabolizable androgen), and it is pulse-labeled with [35S]methionine to an autoradiographically equal extent, with or without incubation in 3 nM MT for 2 or 16 hours. Furthermore, the protein identified by the spot is found in the labium majus skin fibroblast strains from 2 of 12 unrelated subjects with complete androgen resistance due to negligible androgen-receptor activity, but it is absent from those of 2 others who have the same phenotype despite a normal level of qualitatively abnormal androgen-receptor activity. Hence, it is very unlikely to be an androgen-induced protein, and it cannot be a functional version of the androgen receptor itself. Its absence in 12 of 14 labium majus strains of subjects with complete androgen resistance, regardless of 5 alpha-reductase activities, indicates that it is neither a constitutive cytotypic marker of genital skin fibroblast differentiation nor a reflection of that enzyme. When intact prepuce fibroblasts are covalently labeled by photolysis with 50 nM [3H]MT, the only specific labeling detectable after two-dimensional electrophoresis is in the 6.7 and 6.5 pI doublet of the 56 kDa protein. Considering the sensitivity of silver staining and the incomplete concordance between the androgen-receptor activity of a strain and the size/intensity of its 6.7 pI/56 kDa spot on the gels, we postulate the latter to be a comparatively abundant androgen-binding protein that is causally related to the androgen receptor. The precise nature of this relation remains to be elucidated by use of novel immunologic and/or nucleic acid probes for this protein and for the mature androgen receptor. In any event, the presence or absence of the 6.7 pI/56 kDa protein in genital skin fibroblast lysates is a new marker of genetic heterogeneity within the class of complete androgen resistance.

Anatomical distribution of vasoactive intestinal peptide binding sites in peripheral tissues investigated by in vitro autoradiography.

Vasoactive intestinal polypeptide has a widespread distribution in the body, occurring in both the central and peripheral nervous systems and considerable information is available on its distribution, physiology, and pharmacological actions. Receptors for VIP have been demonstrated previously in peripheral tissues by conventional binding techniques using isolated membrane preparations. However, information on their precise localization is limited. We therefore localized binding sites in a variety of guinea pig and rat tissues by in vitro autoradiography and made a parallel study of the distribution of VIP nerves in these tissues using immunocytochemistry. [125I]VIP was prepared by the chloramine T method and shown to be pharmacologically active. After a preincubation procedure to remove endogenously bound VIP, unfixed cryostat sections were incubated with 1 nM [125I]VIP. To determine specific binding, sections were incubated in the presence or absence of 1 microM unlabeled VIP. Autoradiograms were generated by exposing the sections to LKB-Ultrofilm or emulsion-coated coverslips. Dense binding occurred in discrete locations within the gastrointestinal, respiratory, and genital tracts, correlating with known actions of VIP and, to various extents, with the distribution of VIP nerves. For example, there was precise localization to respiratory epithelium, smooth muscle of airways and blood vessels, and alveolar walls, in keeping with the effects of VIP on vascular and airway smooth muscle and mucus secretion.

A solution hybridization assay for the quantitation of prodynorphin mRNA.

We have developed a RNA-RNA solution hybridization assay to quantitate the mRNA coding for prodynorphin precursor. This assay is extremely sensitive and highly specific. Using this assay we have measured the prodynorphin mRNA in various brain regions and reproductive tissues of rat. When we compared the distribution of prodynorphin mRNA with the dynorphin related peptides in these tissues, we found a general parallelism and a few noteworthy exceptions.

Cadmium in edible crabs (Cancer pagurus L.) from Scottish coastal waters.

Concentrations of cadmium in the hepatopancreas (0.1-61.3 mg kg-1), gonad (0.15-11.0 mg kg-1) and gills (0.2-10.7 mg kg-1) of the edible crab Cancer pagurus L. from 16 sampling sites round the Scottish coast are reported, and compared with published elevated concentrations in crabs from the Orkney Islands. Geographical variations in the distribution of cadmium between organs indicate that the dietary uptake of cadmium is predominant in northern mainland and Orkney crabs, but that uptake from the water is more important in the south of Scotland. Mean dissolved cadmium concentrations in eastern coastal water increase from approximately 10 ng dm-3 in northern waters to approximately 25 ng dm-3 in the south. It seems likely that a regional contamination of the environment by cadium of geological origin occurs in the extreme north coast of Scotland, and in the Orkney and Shetland areas.

The anatomy and functional morphology of the large hermaphroditic duct of three species of Aplysia, with special reference to the atrial gland.

The anatomy and functional morphology of the large hermaphroditic duct of three species of gastropod mollusc (Aplysia californica, A. dactylomela, and A. brasiliana) were examined. Each duct is composed of two parallel compartments, the red hemiduct (RHD) and the white hemiduct (WHD), which are distinguishable from the outside of the duct. Four secretory regions, all exocrine in morphology, are recognizable: the RHD secretory epithelium, the atrial gland (or atrial gland-like epithelium), the WHD secretory epithelium, and the accessory gland of the copulatory duct (AGCD). Of these regions, only the atrial gland (or atrial gland-like epithelium) contains egglaying activity and only the atrial gland (or atrial gland-like epithelium) is immunocytochemically labeled by serum antibodies generated against low molecular weight A. californica atrial gland peptides. The RHD is the functional oviduct: the egg cordon passes through a channel lined by the RHD secretory epithelium and bordered by the atrial gland (or atrial gland-like epithelium); the eggs are separated from both the WHD secretory epithelium and the AGCD by internal folds of the duct. The WHD is the functional copulatory duct: the penis, exogenous sperm, and endogenous sperm pass directly by the AGCD and in close proximity to the WHD secretory epithelium; they are separated from both the RHD secretory epithelium and the atrial gland (or atrial gland-like epithelium) by internal folds. The atrial gland (or atrial gland-like epithelium) is thus not likely to have a prostatic function or to be directly stimulated by the penis during copulation; it may play a role in oviductal function.

[Male pseudohermaphroditism caused by partial insensitivity to androgens. Clinical and biochemical heterogeneity]. / Pseudo-hermaphrodisme masculin par insensibilité partielle aux androgènes. Hétérogénéité clinique et biochimique.

Male pseudohermaphroditism due to partial androgen insensitivity (PAI) may be suspected clinically in case of incomplete masculinization of external genitalia in spite of age related plasma androgen levels. In 25 children or adolescents in whom PAI was suspected, the 5 alpha-reductase activity of external genitalia fibroblasts, the number of androgen receptor sites (Bmax) and the affinity of receptors for dihydrotestosterone (Kd) were studied. Clinical expression of PAI is highly polymorphic (Prader's type I to type IV), when most children (18/25) were considered as males. In a single patient the very low 5 alpha-reductase activity permitted the diagnosis of 5 alpha-reductase deficiency. The number of receptor sites (fmoles/mg DNA) varied from 0 to 730. Mean Bmax of patients (282 +/- 187 fmoles/mg DNA) was statistically lower than that of normal subjects (642 +/- 220 fmoles/mg DNA), p less than 0.05. The 5 cases in whom receptor concentrations were normal may be related to a qualitative abnormality of the androgen receptor or to a "post-receptor" defect. On the contrary no significant differences in Kd values were found. Correlation between sexual ambiguity and the number of measured receptors was not possible. These results emphasize the clinical and biochemical heterogeneity of PAI. Nevertheless, the decrease in number of androgen receptor sites remains the major data for the biochemical diagnosis of PAI. Study of post-receptor "markers" (3 alpha-reductase activity, aromatase, collagen) might allow better analysis of cases with PAI in whom androgen receptor concentrations are normal.

Identification of an acidic dipeptide, beta-aspartylglycine, in the CNS of Aplysia.

A novel dipeptide, beta-aspartylglycine (beta-DG), has been isolated from tissues of the marine gastropod mollusc Aplysia californica. This compound was detected only in Aplysia and not in other molluscs, such as Helix or Mercenaria, or in lobster or frog. Among the Aplysia tissues, the highest levels of beta-DG were in nervous tissue and in the reproductive tract. beta-DG was assayed by HPLC as the o-phthaldialdehyde derivative and found to be present in all individual, identified neurons at a concentration of approximately 40 pmol/microgram protein. The peptide was identified as beta-DG by gas chromatography-mass spectrometry (GCMS) using trimethylsilyl derivatives prepared before and after acid hydrolysis. It was further characterized as the beta-isomer by TLC, including Rf, atypical blue-gray color with ninhydrin, and a violet color with Cu2+-ninhydrin. A fractionation scheme is described whereby acid-soluble tissue constituents can be divided into acidic, neutral, and basic components using mini ion-exchange columns. This partial purification prior to TLC analysis was necessary to remove compounds that interfered with the isolation of beta-DG.

Localization of 3H-estradiol in the reproductive organs of male and female baboons.

The uptake and retention of radiolabeled estradiol by both the male and female reproductive organs were examined in the baboon. Two male and two female baboons were injected intracardially with 1 microgram/kg body weight of 3H-estradiol and two animals, one male and one female, were injected with both labeled and 100 micrograms/kg body weight of unlabeled estradiol. One and a half hours after the injections, the animals were sacrificed and the uterus, cervix, vagina, oviduct, seminal vesicles, and prostate gland were removed and processed for autoradiography. The stratified squamous epithelia of the cervix and vagina demonstrated a light uptake of the label in the germinative, but not in the superficial cell layers. The columnar cells lining the oviduct and uterine glands were labeled, whereas the luminal epithelium of the uterus and the glandular epithelia of the seminal vesicles and prostate gland did not sequester the tritiated steroid. The interstitial cells of all the organs studied demonstrated a moderate to heavy uptake of the radioactivity, whereas the smooth muscle cells were lightly labeled except in the vagina, in which these cells displayed a moderate number of silver grains.

Qualitative distribution of neutral lipids and phospholipids in Hymenolepis microstoma from the cysticercoid to the egg producing adult.

The distribution of neutral lipids and phospholipids in Hymenolepis microstoma has been studied using Fettrot, Sudan Black B, Sudan IV and copper phthalocyanin staining techniques. In the cysticercoid, neutral lipids are found in the outer membrane, the lining of the cysticercoid cavity, the tegument of the larval worm and the calcareous corpuscles. A decreasing gradient of phospholipids is found starting from the acellular layer, through the circular fibrous layer, the longitudinal fibrous layer, the adjacent dense zone and ending with the lining of the cysticercoid cavity. Phospholipids are also found in the calcareous corpuscles and the tegument of the larval worm. In the young adult (3 days p.i.) fat globules are first seen to accumulate in the last 2-3 proglottids. Until the 6th day p.i. they are found in the posterior third of the worm, surrounding developing gonads, but mostly concentrated along the transverse line. The mature proglottids contain fat, (a) in both granular and globular forms: in the folds of the uterus, sperm ducts, cirrus pouch and tegument (proximal cytoplasm), (b) in a diffuse form: in the vitellaria, ovary, testes and the tegument (distal cytoplasm). Pre-gravid and gravid proglottids show the largest fat globules. From the cleaving embryo to the fully developed oncosphere the concentrations of neutral lipids and phospholipids vary in form, intensity and location. In all strobilar forms of the parasite neutral lipids and phospholipids are found in the tegument and calcareous corpuscles. Although in H. microstoma lipid droplets are found in the excretory canals, all lipids in the proglottids are not absolutely waste products. From the results it would appear that they play a role in the maturation of gonads and transformation of the fertilized ovum to the oncosphere.