Prenatal exposure to betamethasone causes intergenerational impairment of epididymal development in the rat.

BACKGROUND: Studies on epididymal toxicology are scarce. Betamethasone (BM) is a glucocorticoid used in clinical practice for antenatal therapy. We previously reported changes to testicular morphology, altered sperm quality, and fertility in adult rats following intrauterine administration of BM. OBJECTIVES: Given that high levels of corticosteroids during gestation lead to fetal androgen depletion, and the essential role of testosterone during epididymal development, here we investigated epididymal morphology and physiology in the F1 and F2 male offspring of female rats treated with BM during gestation. MATERIALS AND METHODS: Pregnant rats were randomly divided into two experimental groups: control (saline vehicle, n = 11) and BM-treated group (0.1 mg/kg betamethasone 21-phosphate disodium, n = 13). Rats received an intramuscular injection of vehicle or BM on gestational days 12, 13, 18, and 19. This encompasses the beginning of the critical window of male rat reproductive tract development. A subset of three males from each litter (n = 5 litters/group) was used: One rat per litter was euthanized at puberty, one was euthanized at adulthood, while the others were mated with a non-treated female to obtain the F2 generation. The same protocol described for the F1 was applied for F2, except for the mating protocol. RESULTS: In both F1 and F2 generations, prenatal BM exposure resulted in delayed differentiation of the cauda epididymal epithelium, characterized by increased cribriform appearance on PND 45, and displayed weaker or non-detectable Cx43 immunostaining. Furthermore, in the F1 generation only, immunostaining of TP63, a transcription factor expressed in basal cells, appeared more intense with a greater number of TP63-positive cells observed in the cauda epididymis. In adults, the epithelial area was reduced in the F1 BM rats. The contractile activity of isolated epididymal ducts was comparable between groups. DISCUSSION AND CONCLUSION: Prenatal BM exposure leads to intergenerational impairment in the development and structure of the rat epididymis.

Tissue regeneration and the epididymal stem cell.

BACKGROUND: In most pseudostratified epithelia, basal cells represent a multipotent adult stem cell population. These cells generally remain in a quiescent state, until they are stimulated to respond to tissue damage by initiating epithelial regeneration. In the epididymis, cell proliferation occurs at a relatively slow rate under normal physiological conditions. Epididymal basal cells have been shown to share common properties with multipotent adult stem cells. The development of organoids from stem cells represents a novel approach for understanding cellular differentiation and characterization of stem cells. OBJECTIVE: To review the literature on tissue regeneration in the epididymis and demonstrate the presence of an epididymal stem cell population. METHODS: PubMed database was searched for studies reporting on cell proliferation, regeneration, and stem cells in the epididymis. Three-dimensional cell culture of epididymal cells was used to determine whether these can develop into organoids in a similar fashion to stem cells from other tissues. RESULTS: The epididymal epithelium can rapidly regenerate following orchidectomy or efferent duct ligation, in order to maintain epithelial integrity. Studies have isolated a highly purified fraction of rat epididymal basal cells and reported that these cells displayed properties similar to those of multipotent adult stem cells. In two-dimensional cell culture conditions, these cells differentiated into cells which expressed connexin 26, a marker of columnar cells, and cytokeratin 8. Furthermore, three-dimensional cell culture of epididymal cells resulted in the formation of organoids, a phenomenon associated with the proliferation and differentiation of stem cells in vitro. CONCLUSIONS: The rapid proliferation and tissue regeneration of the epididymal epithelium to preserve its integrity following tissue damage as well as the ability of cells to differentiate into organoids in vitro support the notion of a resident progenitor/stem cell population in the adult epididymis.

Male reproductive system defects and subfertility in a mutant mouse model of oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) is a dysmorphogenesis syndrome resulting from mutations in the GJA1 gene encoding the gap junction protein, connexin43 (CX43). In the testis this connexin localizes between Leydig cells, Sertoli cells and between Sertoli cells and germ cells. It is essential for Sertoli cell differentiation and spermatogenesis. This study explored male fertility in Gja1(Jrt) /+ mice which carry a dominant mutation that causes an amino acid substitution (G60S) in CX43. Gja1(Jrt) /+ mice mimic the phenotype of ODDD. Immunodetection methods revealed a reduction of both total CX43 and CX43 in membrane plaques in mutant testes. Correspondingly, intercellular coupling along the tubules was diminished as revealed by fluorescent dye transfer. Light and electron microscopy revealed loss of germ cells and sloughing of germ cells into the tubular lumen. There were also irregularities in size and shape of Sertoli cell nuclei. Analyses of cauda epididymal sperm indicated significant decreases in sperm count and sperm velocity parameters associated with sperm vigour, and significantly lower sperm head movement parameters associated with progressiveness. A significant decrease was also observed in total per cent motility. These results further confirm a critical role for CX43 in spermatogenesis and sperm maturation and support the possibility of subfertility in ODDD human males.

Effects of ozone, ultraviolet and peracetic acid disinfection of a primary-treated municipal effluent on the immune system of rainbow trout (Oncorhynchus mykiss).

Municipal sewage effluents are complex mixtures that are known to compromise the health condition of aquatic organisms. The aim of this study was to evaluate the impacts of various wastewater disinfection processes on the immune system of juvenile rainbow trout (Oncorhynchus mykiss). The trout were exposed to a primary-treated effluent for 28 days before and after one of each of the following treatments: ultraviolet (UV) radiation, ozonation and peracetic acid. Immune function was characterized in leucocytes from the anterior head kidney by the following three parameters: phagocytosis activity, natural cytotoxic cells (NCC) function and lymphocyte (B and T) proliferation assays. The results show that the fish mass to length ratio was significantly decreased for the primary-treated and all three disinfection processes. Exposure to the primary-treated effluent led to a significant increase in macrophage-related phagocytosis; the addition of a disinfection step was effective in removing this effect. Both unstimulated and mitogen-stimulated T lymphocyte proliferation in fish decreased dramatically in fish exposed to the ozonated effluent compared to fish exposed to either the primary-treated effluent or to aquarium water. Stimulation of T lymphocytes proliferation was observed with the peracetic acid treatment group. In conclusion, the disinfection strategy used can modify the immune system in fish at the level of T lymphocyte proliferation but was effective to remove the effects on phagocytosis activity.

Determination of p-tert-octylphenol in blood and tissues by gas chromatography coupled with mass spectrometry.

A sensitive and reproducible procedure using gas chromatography coupled with mass spectrometry is described for the determination of p-tert-octylphenol (OP), a persistent degradation product of alkylphenol ethoxylates that binds to the estrogen receptor in blood and tissues. The first step involved the extraction of blood (200 microL) or tissue homogenate (400 microL) with methyl tert-butyl ether, including p-tert-butylphenol (BP) as internal standard. After extraction, the sample was evaporated to dryness with a gentle stream of nitrogen at 45 degrees C, and OP and BP were derivatized with an acetylation reaction involving acetic anhydride and catalyzed by pyridine. Samples were then analyzed by a gas chromatograph equipped with a mass spectrometer (single ion monitoring) with a Varian VF-5ms capillary column. The limit of detection and the limit of quantification of the method in blood were 4.6 and 15.5 ng/mL, respectively. The linearity and reproducibility of the method were acceptable, with coefficients of variation of approximately 10% for blood and ranging between 9% and 27% for tissues. This method was applied to the determination of unchanged OP in blood and tissues obtained from Sprague-Dawley rats after oral and IV OP administration.

Phagocytic response of macrophages from the pronephros of American plaice (Hipoglossoides platessoides) exposed to contaminated sediments from Baie des Anglais, Quebec.

Sediments of Baie des Anglais on the St. Lawrence estuary have a history of environmental contamination, but little information exists regarding their toxicity. The purpose of the present study was to determine the effects of contaminated Baie des Anglais sediments on American plaice (Hippoglossoides platessoides) immune function. Three sites in Baie des Anglais were selected which vary in proximity to local industries and in their sediment contaminant load. Sites 1 and 2 (within the bay) are the closest to shore and most heavily contaminated while sediments at Site 3, which is outside the bay, are the least contaminated. In the first experiment, American plaice were placed in cages at each site for three weeks and immune function was assessed by measuring the phagocytic activity of pronephric macrophages. At the time of sampling, plaice displayed pronephros cell immune response disturbances indicating that Site 1 and 2 were most toxic and Site 3 the least toxic. The results obtained for phagocytosis revealed that contaminants present in the sediments are bioavailable to fish, which came in contact with them and significantly affected their immune system. In the second experiment, sediments from the most toxic site, Site 1, were collected for a laboratory controlled experiment in which plaice were exposed for up to 3 months to these contaminated marine sediments, while the control group was exposed to relatively uncontaminated beach sand. At the end of the exposure period, plaice were transferred from contaminated sediment to beach sand and sampled one month later in order to determine if immune function had returned to control levels. The total number of macrophages decreased following three months of exposure, while the active macrophages had already decreased after the first month of exposure. Following the rehabilitation period a significant trend toward normal response was noted. Sediments from Baie des Anglais contain primarily less highly chlorinated PCBs and lower concentrations of the intermediate and highly chlorinated PCBs. The total concentration of PCBs (sum of 20 congeners) in the contaminated sediments was 1500 ng/g while in the beach sand, the levels were 13.6 ng/g dry weight. Only the low chlorinated PCB congeners were efficiently transferred from the sediments to the plaice liver. Together, these results suggest that the effect of chemical exposure on the phagocytosis of plaice macrophages may be reversible if the fish are returned to a non-contaminated habitat.

Expression and regulation of metallothioneins in the rat epididymis.

Metallothioneins (MTs) are cytosolic proteins involved in cellular stress responses. The objectives of this study were to determine which epididymal cells express MTs, how they are regulated, and whether mRNA levels for 3 MT isoforms (MT I, MT II, and MT III) are modulated by heavy metals. MT expression was noted mainly in basal cells of all epididymal regions but not in all basal cells of any given region. MT I mRNA levels were highest in the testis, followed by levels in the corpus, cauda epididymidis, liver (positive control), caput epididymidis, initial segment, seminal vesicles, and ventral prostate. MT II mRNA levels were also highest in testis, followed by levels in the cauda, corpus, liver, caput, and initial segment, but they were undetectable in the seminal vesicles and ventral prostate. MT III mRNA levels were highest in the caput followed by testis and initial segment. Orchidectomy and orchidectomy with testosterone replacement experiments showed that immunoreactive MT in all epididymal segments was androgen dependent. Epididymal MT I mRNA levels were dependent on androgens in all segments except the corpus. MT II mRNA levels were androgen dependent only in the initial segment and corpus. MT III mRNA levels in the initial segment were not altered by orchidectomy but increased significantly in testosterone-treated rats. In the caput, MT III mRNA levels decreased following orchidectomy, but control levels were maintained by testosterone. In cadmium-injected rats, MT I mRNA levels were significantly increased in the testis and initial segment, but there were no effects in the liver and other epididymal regions. MT II mRNA levels were increased by more than eightfold in the liver and by three- to fourfold in the initial segment and caput. In the corpus, MT II mRNA levels were decreased by cadmium treatment. MT III mRNA levels were unaltered by cadmium treatment. In conclusion, all 3 MT transcripts are present in high abundance in the epididymis. Furthermore, MT is expressed mainly in basal cells with regulation by testosterone. Heavy metal induction appears to affect the proximal regions of the epididymis.

Quantification of rainbow trout (Oncorhynchus mykiss) estrogen receptor-alpha messenger RNA and its expression in the ovary during the reproductive cycle.

This study developed a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method to measure estrogen receptor-alpha (ERalpha) mRNA in the rainbow trout (Oncorhynchus mykiss). Using RT-PCR, and primers based on the known ERalpha DNA sequence in this species, cDNA sequences representing most of the protein coding region were obtained from ovary poly A(+) RNA. Using these DNA sequences as probes in Northern blot hybridizations confirmed that a single transcript of 4.2 kilobases in poly A(+) RNA could be detected in liver and ovary RNA. For the quantitative RT-PCR assay an internal standard RNA molecule was produced to control for inherent inter-tube differences in amplification efficiency and permit accurate quantification of ERalpha mRNAs. The quantitative RT-PCR assay proved to be highly specific for ERalpha mRNA with a detection limit of 6.9 fg, which corresponds to 273 fg ERalpha mRNA/microg total RNA. The quantitative RT-PCR assay was used to measure the levels of ERalpha mRNA in ovaries of rainbow trout at different stages of reproductive development. Ovarian ERalpha mRNA expression was found during two distinct periods of reproductive development, in pre-vitellogenic ovaries of fish with ovarian follicle diameters (OFDs) 1000 microm. ERalpha mRNA could not be detected in the ovaries of fish with OFDs >100 microm but 2000 microm.

Thyroid hormone deiodination in tissues of American plaice, Hippoglossoides platessoides: characterization and short-term responses to polychlorinated biphenyls (PCBs) 77 and 126.

We have described the tissue distribution and properties of thyroid hormone (TH) deiodination activities of the marine American plaice, Hippoglossoides platessoides. We then studied the 1- or 4-week responses of the plaice liver and brain deiodination activities and the plasma thyroxine (T4) and 3,5,3'-triiodothyronine (T3) levels to an intraperitoneal injection (5-500 ng/g) of the polychlorinated biphenyl (PCB) congeners 77 (3,3'-4,4'-tetrachlorobiphenyl) or 126 (3,3',4,4',5-pentachlorobiphenyl). T4 and 3,3'5'-triiodothyronine (rT3) outer-ring deiodination (ORD) activities were greater in liver than in kidney, gill, heart, brain, intestine or muscle; inner-ring deiodination (IRD) activity occurred in all tissues but was consistently higher in brain. Deiodination characteristics (optimal pH, optimal dithiothreitol concentration, responses to inhibitors and apparent Km values of 0.6-4 nM) fell in the same rage as those of low-Km deiodinases in other teleosts. Deiodination activities were maximal when assayed at 25 degrees C but uniformly low over the natural range of 0-9 degrees C. Neither PCB 77 nor PCB 126 altered brain T4ORD activity or plasma T4 levels (P < 0.05). However, at 1 week post injection hepatic T4ORD activity was increased and plasma T3 levels lowered by PCB 77 (5 and 25 ng/g), while hepatic IRD activity was increased by PCB 126 (50 and 500 ng/g). Neither PCB 77, PCB 126 nor selected hydroxylated. PCBs given in vitro compared with T4 for binding sites on plasma proteins or altered hepatic deiodination activity, indicating no direct action on plasma proteins or deiodinases We conclude that plaice TH deiodination tissue distribution and characteristics resemble those of other teleosts. Deiodination activities are low at natural assay temperatures but at 1 week show some responses to PCBs 77 and 126.

Effects of short-term methylmercury exposure on metallothionein mRNA levels in the testis and epididymis of the rat.

Methylmercury (MeHg) is a widespread environmental contaminant that causes reproductive dysfunction in men. Metallothioneins (MTs) are low-molecular-weight proteins that can bind heavy metals and protect the cell from metal toxicity. MT levels are increased by exposure to metals and physiological stressors. Although MTs have been identified in the testis and epididymis, little is known about their distribution and regulation in the epididymis or the effects of MeHg on MT levels in male reproductive tissues. The objective of this study was to determine whether MT I, II, and III mRNA are present in the epididymis, if their relative levels differ between epididymal segments, and if MeHg alters cellular mRNA levels for MT I, II, and III in the testis and epididymal segments of the rat. Northern blot analysis was done on total cellular RNA isolated from each of the four epididymal segments (initial segment [IS], caput [CT], corpus [CS], and cauda [CA] epididymidis) using a cDNA probe for MT I and MT II. MT I transcripts were present in all epididymal segments. The lowest mRNA levels were observed in the IS; these levels were 4-fold less than in the CT and CS and 5.5-fold less than in the CA. MT II mRNA levels were similar in the IS and CT but were eightfold higher in the CS and CA. A cDNA probe for MT III was generated by reverse transcription-polymerase chain reaction using testicular RNA. MT III mRNA was detected only in the IS and CT and not in the CS and CA. To assess whether exposure to MeHg alters MT mRNA levels, rats were exposed for 14 days to one of five MeHg doses (0, 25, 50, 100, and 200 [microg/kg/day] via a subdermal osmotic pump. No changes were observed in either body weight or in the weights of the testis, epididymis, seminal vesicles, or ventral prostate between MeHg-treated and control rats. Serum testosterone levels were significantly decreased only at the highest MeHg dose. In the testis, MeHg treatment resulted in 2.5- to 7-fold increases in MT I mRNA levels. There were no changes in either MT II or MT III mRNA levels. In the initial segment of the epididymis, MT I mRNA levels were significantly increased only at the 50 microg/kg/ day dose, whereas there were no significant differences in MT II mRNA levels. In the caput epididymis, MT I mRNA levels were significantly lower at the 50 and 100 microg/kg/day dose. MT II mRNA levels were also lower, with the exception of the 50 microg/kg/day dose. Although MT III mRNA levels were lower at the two lower doses, levels were not different from controls in the two highest doses tested. In the corpus epididymidis, MeHg did not alter MT I mRNA levels, and MT II was higher only in the 50 microg/kg/day group. In the cauda epididymidis, MT I mRNA levels were decreased in a dose-dependent manner by up to 63%. MT II levels were unaltered. Together these data indicate that exposure of adult rats to MeHg can modulate MT mRNA levels in both the testis and epididymal segments. Furthermore, changes in MT mRNA levels following exposure to MeHg differ between epididymal segments, suggesting either differences in MeHg accumulation or differences in MT modulation.

Cellular immunolocalization of occludin during embryonic and postnatal development of the mouse testis and epididymis.

Cellular junctions in the testis and epididymis play crucial roles for the development and maturation of spermatozoa. In the testis, tight junctions between Sertoli cells form a functional blood testis barrier between 10 and 16 days of age, whereas the tight junctional blood epididymal barrier between adjacent epithelial cells is formed between days 18 and 21. In the present study, occludin, a constituent integral membrane protein of tight junctions, was localized by immunofluorescent confocal microscopy in embryonic (days 13.5-18.5), postnatal (days 5-23) and adult (day 70) mouse testes and epididymides to correlate its expression with the onset of tight junctions and eventual formation of these barriers. At embryonic days 13.5 and 16.5, low diffuse cytoplasmic levels of occludin were observed in cells of the testicular cords. By embryonic day 18.5, the level of occludin was still low but appeared as a filiform-like network streaming toward the center of the cord. At postnatal days 5 and 7 immunostaining became more intense and appeared to outline the periphery of Sertoli cells of seminiferous tubules. Postnatal day 14 marked the appearance of an intense, focal band-like localization of occludin at the base of the tubules, correlating with the appearance of a functional blood-testis barrier. By day 23 and in adults, expression of occludin was noted at the base of the tubule appearing as intense, wavy, discontinuous bands similar in appearance irrespective of the stage of the seminiferous epithelium cycle. In the developing epididymis, intense cytoplasmic immunostaining was present in epithelial cells of many epididymal tubules at embryonic day 13.5. By embryonic day 16.5, intense occludin immunostaining appeared along the lateral plasma membranes of epithelial cells, whereas at embryonic day 18.5, immunostaining was punctate and apically located, suggesting the presence of tight junctions by this age; similar immunostaining was noted at postnatal days 5 and 7. In the adult epididymis, distinct punctate apical staining was observed between adjacent principal cells of all epididymal regions except the proximal initial segment, where occludin was found only in association with narrow cells. These results indicate that in the epididymis, the appearance of occludin at apical sites between adjacent epithelial cells occurs during embryonic development suggesting that tight junctions form earlier than in the testis. While occludin was expressed in a similar pattern between Sertoli cells at all stages of the cycle in the adult testis, its expression in the adult epididymis was cell- and region-specific. Taken together these data suggest that different factors regulate occludin expression in the testis and epididymis.

Toxicological characterization of sediments from Baie des Anglais on the St. Lawrence Estuary.

The sediments of Baie des Anglais on the St. Lawrence Estuary have a history of environmental contamination, but no information exists relating to their toxicity. The purpose of the present study was to characterize three sites in and near Baie des Anglais, in terms of sediment toxicity and contaminants. Sites 1 and 2 within the Baie des Anglais are relatively close to local industry and municipal sewage discharge points, while Site 3 is outside the bay. Three microscale bioassays, Microtox, echinoderm fertilization and Toxi-ChromoPad, showed that sediments from Site 1 were the most toxic, followed by Site 2. Site 3 was non-toxic. While the solid phase Microtox test did indicate that Site 1 was most toxic, the absolute response was weak. Liver cytochrome P450 1A1 mRNA in American plaice (Hippoglossoides platessoides), captured at Site 1 in the bay was significantly induced compared to the P450 system of plaice captured at Sites 2 and 3. Hepatic metallothionein mRNA levels were not significantly different between plaice captured at all three sites. Sediment chemical analyses revealed a gradient in polycyclic aromatic hydrocarbons, polychlorinated biphenyls and dibenzofurans (PCDF) with the highest levels recorded at Site 1, about 10-fold less at Site 2 and 100-fold less at Site 3. Amongst the organochlorines the PCDF group were deemed the most important due to their prevalence and known toxicity. Heavy metal concentrations were low and representative of background levels for the St. Lawrence Estuary.

Effects of long-term temperature acclimation on thyroid hormone deiodinase function, plasma thyroid hormone levels, growth, and reproductive status of male Atlantic cod, Gadus morhua.

The recent collapse of the Northwestern Atlantic cod fisheries has coincided with a cooling of water temperatures. During this time the condition factor of cod has been poor. The objective of the present study was to determine the effects of long-term temperature acclimation on growth reproduction and thyroid function in laboratory held Atlantic cod (Gadus morhua). One of the key parameters used to assess thyroid function is the peripheral metabolism of L-thyroxine (T4) by microsomal deiodinase enzymes. Deiodinase function has not been described for gadid fish. T4 outer-ring deiodinating activity (apparent K(m) 1-2 nM) was confined primarily to liver. Its properties resembled those for hepatic T4ORD activity of other teleosts and the mammalian type II deiodinase. The T4ORD activity of cod liver exceeded that of salmonids and could explain the high plasma T3 levels (10-18 ng/ml), which were 2-5 times greater than T4 levels. T4 and T3 inner-ring deiodination was confined mainly to brain. In order to determine the effects of long-term temperature acclimation on cod, somatic growth, reproduction, and thyroidal status were assessed monthly in 400-900-g satiation-fed male Atlantic cod captured in June from the St. Lawrence Estuary and then acclimated from August to the following June under a natural photoperiod at 2-4 degrees C (LT) or 6-10 degrees C (HT). Reproductive status was determined from the gonadosomatic index (GSI), plasma testosterone (T) and 11-ketotestosterone (11-KT) levels, and the appearance of milt; thyroidal status was determined from plasma T4 and 3,5,3'-triiodo-L-thyronine (T3) levels and hepatic T4ORD activity to produce biologically active T3. Testis maturation (high levels of 1 and 11-KT, and milt release) occurred in April and May and was uninfluenced by acclimation temperature. LT cod grew more slowly than HT cod. Differences in body weight were particularly evident from December to February. In conclusion, (i) cod possess outer- and inner-ring deiodinase activities, predominating respectively in liver and brain, and with properties resembling those of other teleosts, (ii) T4ORD activity of liver is unusually high and may account for the high plasma T3 levels in this species, (iii) T4ORD activity tends to increase during periods of increased somatic growth, and (iv) chronic acclimation of male cod to 2-4 degrees C, as opposed to 6-10 degrees C, decreases somatic growth but does alter circulating levels of thyroid hormones and androgens and it does not change the time of sexual maturation.

Immunocytochemical localization and regulation of connexin43 in the adult rat epididymis.

Connexin43 (Cx43) is one of the most predominant gap junction proteins found in vivo, and although present in the testis, it has not been examined in the epididymis. Immunocytochemistry using an anti-Cx43 antibody revealed a punctate immunoperoxidase reaction at the apical margins between adjacent epithelial cells of the efferent ducts. In the epididymis, punctate reactive sites were observed at the base of the epithelium between basal and principal cells. There was no staining between adjacent principal cells at their apical or lateral margins. Cx43 immunostaining was also seen between myoid cells surrounding the tubules but only in the cauda epididymidis. Using a Cx43 cDNA probe, Northern blot analysis of total cellular RNA revealed a single hybridizing band of approximately 3.0 kb in all regions of the epididymis. Throughout the epididymis of bilaterally orchidectomized rats at 7 or 14 days, an immunoperoxidase reaction for Cx43 persisted at the base of the epithelium between principal and basal cells. However, in the initial segment only, immunolocalization of Cx43 was also observed apically between adjacent principal cells. This apical staining was lost in rats that received testosterone replacement. Myoid cells in the cauda epididymidis of control rats expressed Cx43, however, orchidectomized rats did not express Cx43 in this cell layer. Western blots revealed the presence of a major protein band at 43 kDa corresponding to unphosphorylated Cx43 as well as Cx43 species at 44 and 46 kDa, which were more prominent in orchidectomized rats. Together these data represent one of the first examples of Cx43 gap junctions between heterologous cell types (i.e. principal and basal cells). Moreover, Cx43 expression in myoid cells of the cauda epididymidis is androgen-dependent and in the initial segment of the epididymis only, the intracellular targeting of Cx43 towards the principal-principal cell interface under normal conditions is regulated by androgens.

Structure and turnover of junctional complexes between principal cells of the rat epididymis.

The epididymal junctional complex between adjacent principal cells is composed of apically located gap, adherens and tight junctions. Tight junctions between adjacent epithelial cells lead to the formation of the blood-epididymal barrier. The objectives of this study were to examine the structure of the epididymal junctional complex in the different regions of the epididymis and to review the regulation of epithelial cadherin in the rat epididymis. Changes in the structure of the junctional complex, at the level of the electron microscope, were evident when comparing the initial segment to other regions of the epididymis. In the initial segment, the tight junction spanned a considerable length of the apical plasma membrane but had few desmosomes. In the other regions of the epididymis, the span of merging plasma membranes was considerably reduced, but in these regions, numerous desmosomes were present in the apical region. Several examples of what appeared to be a loss of portions of the plasma membrane of adjacent principal cells were evident along the entire epididymis. Such images as the invagination of a portion of the lateral plasma membrane of one principal cell into another, constriction of the invaginated area and eventual detachment leading to the formation of annular junctions suggest that there is a turnover of plasma membranes. The formation of cellular junctions involves the interactions of cell adhesion proteins followed by the addition of junctional proteins which assemble into tight and gap junctions. Epithelial cadherin (E-Cad), a calcium-dependent cell adhesion protein, was localized to the principal cells of the epididymis. Immunocytochemistry at the level of the electron microscope showed that E-Cad was present between the lateral plasma membranes of adjacent principal cells, both in the region of the junctional complex and in the deeper lying areas. E-Cad was also present in annular junctions located in close proximity to the junctional complex, indicating that these structures were related to the plasma membrane. E-Cad mRNA levels are regulated during postnatal epididymal development. In the caput-corpus epididymidis, E-Cad mRNA concentrations increase to peak at 42 days of age. This is well correlated with the conversion of testosterone to dihydrotestosterone in the epididymis. In the cauda epididymidis, however, E-Cad mRNA concentrations do not increase as a function of age, indicating that this protein is regulated in a segment-specific manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of apoptosis in mediating phosphoramide mustard-induced rat embryo malformations in vitro.

Phosphoramide mustard, an active metabolite of the anticancer drug cyclophosphamide, causes malformations in rat embryos undergoing organogenesis in vitro. The purpose of the present study was to investigate the hypothesis that apoptosis plays an important role in mediating the teratogenicity of phosphoramide mustard. Apoptosis is a process of active or programmed cell death which is characterized by internucleosomal DNA fragmentation and de novo RNA and protein synthesis. Sulphated glycoprotein-2 (SGP-2) or clusterin is induced in some models of apoptosis and is one of the proteins likely to be involved in the maintenance of cell integrity. In the present study, day 10 rat embryos were cultured for 6, 12, 24, and 45 hr, with or without the addition of 10 microM phosphoramide mustard. After culture for 24 or 45 hr with exposure to 10 microM phosphoramide mustard, the embryos were both growth-retarded and malformed. Exposure to phosphoramide mustard for 6 or 12 hr did not significantly alter the relative amounts of either the mRNA or protein for SGP-2; this treatment also had no effect on DNA fragmentation in embryos or their yolk sacs. After 24 hr in culture, the relative amounts of SGP-2 protein, but not mRNA, were increased 2-fold in the yolk sacs of the phosphoramide mustard-exposed embryos, but not in the embryos themselves. At this time, DNA fragmentation was detected in phosphoramide mustard-exposed embryos, but not in their yolk sacs or in control embryos. After 45 hr in culture, SGP-2 protein and mRNA levels were increased 2-4-fold above the controls in the phosphoramide mustard-exposed embryos and their yolk sacs. Immunohistochemical analysis revealed that in control embryos cultured for 45 hr, the SGP-2 reaction product was localized in the heart, hindgut, and yolk sac. In contrast, in phosphoramide mustard-treated embryos cultured for 45 hr, SGP-2 immunostaining was found throughout the embryo, with a strong immunoreaction in the mesenchyme and ectoplacental cone. DNA fragmentation in the embryos exposed to phosphoramide mustard for 45 hr was more extensive than that found after 24 hr, but fragmentation was still not detected in the yolk sac. Thus exposure in vitro to a teratogenic concentration of phosphoramide mustard resulted in DNA fragmentation and an increased expression of SGP-2 in the embryo. These data suggest that apoptosis is involved in mediating the teratogenicity of phosphoramide mustard.

Mechanism of trimethylamine-induced inhibition of macromolecular synthesis by mouse embryos in culture.

The effects of trimethylamine (TMA) on uptake mechanisms and lysosomal function were studied in mouse embryos, isolated yolk sacs and limb buds. TMA at 0.75 mM did not inhibit uptake of [14C]sucrose by yolk sacs of day 9 embryos or by day 15 isolated yolk sacs but did inhibit uptake of 125I-labelled bovine serum albumin ([125I]BSA) by day 15 isolated yolk sacs. Concentrations of TMA up to 2.5 mM did not inhibit lysosomal degradation of [125I]BSA by isolated yolk sacs, as judged by the release of trichloroacetic acid (TCA)-soluble radioactivity into the culture media. The inhibition of [125I]BSA uptake induced by TMA was reversible on removal of TMA. When day 8 embryos were cultured in serum containing [3H]leucine-labelled proteins, uptake and incorporation of radioactivity in 0.75 mM TMA-treated embryos was 47 and 44%, respectively, of that in untreated controls. TMA at 0.75 mM did not inhibit the uptake and incorporation of free [3H]leucine into embryonic protein nor the amount of free [3H]leucine taken up or incorporated into protein by day 12 isolated limb buds. It is concluded that the reduced macromolecular synthesis in embryos exposed to TMA is due to an inhibition of receptor-mediated uptake of nutrients by the yolk sac.

Developmental changes in epithelial cadherin messenger ribonucleic acid and immunocytochemical localization of epithelial cadherin during postnatal epididymal development in the rat.

The formation of junctional complexes between adjacent epithelial principal cells leads to formation of the blood-epididymal barrier; this barrier is complete by 21 days of postnatal age. Cadherins are cell surface proteins that mediate intercellular adhesion and are involved in the formation of adherence, gap, and tight junctions between epithelial cells. In the adult rat epididymis, epithelial cadherin (E-Cad) is localized in principal cells; E-Cad mRNA concentrations are androgen dependent in this tissue. The objectives of this study were to determine the regulation of E-Cad mRNA concentrations and the pattern of immunocytochemical localization of E-Cad during epididymal development. Using Northern blot analysis, we noted that in the caput-corpus epididymidis, there was a 3-fold increase in E-Cad mRNA concentrations between 7-14 days; an additional 3-fold increase between days 35-42, when E-Cad mRNA concentrations reached their peak, was noted. A dramatic decrease in E-Cad mRNA was observed between 42-49 days of age. This effect was transitory as E-Cad mRNA concentrations returned to almost 80% of peak concentrations on day 56 and remained constant thereafter. In the cauda epididymidis, E-Cad mRNA concentrations increased by only 1.6-fold between days 7-21. E-Cad mRNA concentrations then decreased by 70% to their lowest concentrations on day 56. There was a 2-fold increase in E-Cad mRNA concentrations between postnatal ages 56-91 days. These results suggest that the developmental regulation of E-Cad mRNA concentrations is segment specific. A subsequent study on the longitudinal distribution of E-Cad mRNA levels in six epididymal segments at 21, 42, and 56 days of age revealed that the relative proportion of E-Cad mRNA along the epididymis changes as a function of age. An immunocytochemical study with the light microscope, using an anti-E-Cad antibody, demonstrated that the localization and relative concentrations of E-Cad varied as a function of age. On day 15, the immunoperoxidase staining of the entire epididymal epithelium was apical, with the weakest staining in the cauda epididymidis. By day 21, the reaction spread to cover the supranuclear region of the principal cells in all segments, while on day 39, it covered the entire cytoplasm of these cells, suggesting a high rate of synthesis or storage of the protein. At later time intervals, the intensity of staining over the principal cells appeared to increase with age.(ABSTRACT TRUNCATED AT 400 WORDS)

Clinical significance of type 5 patterns in platform posturography.

Several abnormal patterns have been identified on the sensory portion of the computerized dynamic posturography test. The vestibular deficit pattern, also known as the "5-6" pattern, is frequently seen in patients with either uncompensated unilateral vestibular lesions, severe bilateral peripheral vestibular loss, or dysfunction involving the vestibular pathways in the brain stem and/or cerebellum. In both sensory conditions 5 and 6, the patient's balance/equilibrium is determined primarily by the vestibular system. A subgroup of the vestibular deficit pattern has been identified, in which only sensory condition 5 is abnormal. This article presents findings in several cases identified with the 5 pattern. Implications for diagnosis and for monitoring the recovery phase after treatment are discussed.