Alterations in gap junction protein expression in human benign prostatic hyperplasia and prostate cancer.

PURPOSE: Gap junctions composed of connexin proteins have an essential role in intercellular communication and differentiation. Dysregulation of connexin expression is believed to have a role in carcinogenesis. The human prostate has been reported to express connexin 32 and 43. However, the expression pattern in prostate cancer is controversial, while to our knowledge connexin expression has not been reported in benign prostatic hyperplasia (BPH). To understand the potential involvement in prostate disease connexin 32 and 43 expression was evaluated in a series of normal prostate, BPH and prostate cancer specimens that were surgically removed due to bladder outlet obstruction. MATERIALS AND METHODS: Frozen sections of 23 normal, 43 BPH and 40 cancer involved prostates were evaluated for the presence, staining intensity and pattern of connexin 32 and 43 by immunocytochemical testing. RESULTS: In all specimens examined connexin 43 stain was punctate along the borders of the basal epithelial cells, whereas connexin 32 immunolocalized to luminal epithelial cells. In normal prostate connexin 43 and 32 were present in 87% and 65% of specimens, respectively, at low to moderate stain intensity. Importantly none of the normal samples were negative foreach connexin. In BPH specimens there was a marked increase in the incidence and intensity of connexin 43 and 32 immunostaining within epithelial cells. In addition, 23% of BPH samples showed strong connexin 43 expression in stromal cells. In contrast, connexin was decreased in prostate cancer specimens, of which 65% and 38% were negative for connexin 43 and 32, respectively, and 28% were negative for each type. In poorly differentiated tumors connexin 43 and 32 were present in only 10% and 40% of tumors, respectively, at low immunostaining intensity. CONCLUSIONS: In normal human prostate basal cells communicate via connexin 43 gap junctions, whereas luminal cells communicate via connexin 32 gap junctions. In BPH gap junctional intercellular communication is increased in epithelial and stromal cells, which may have a role in BPH pathogenesis. In prostate cancer gap junctional intercellular communication is decreased, is as indicated by decreased expression of connexin 43 and 32 with severe loss in poorly differentiated prostate cancer. These alterations in connexin expression may have a role in dedifferentiation and tumor progression.

Thyroid hormones and thyroid antibodies in infertile males.

OBJECTIVE: To investigate the incidence of thyroid dysfunction, thyroid antibodies, and the correlation with semen and hormonal parameters in infertile men. DESIGN: Prospective study. SETTING: University-based andrology laboratory. PATIENT(S): Three hundred five infertile men with idiopathic infertility. INTERVENTION(S): Medical history, clinical examination, semen analysis, measurement of free thyroxin (fT4), free triiodothyronine (fT3), basal thyroid-stimulating hormone (bTSH), LH, FSH, T, free testosterone (fT), PRL, E2, sex hormone-binding globulin (SHBG), DHEAS, and the thyroid antibodies thyreoglobulin antibody (TGA), thyroid peroxidase antibody (TPO-Ab), and thyroid receptor antibody (TRAK). MAIN OUTCOME MEASURE(S): Incidence of thyroid dysfunction and thyroid antibodies, as well as the correlation with hormones and the results of semen analyses. RESULT(S): No manifest thyroid dysfunction was observed. Latent thyroid dysfunction and latent hypothyroidism were diagnosed in 11.5% and 3% of infertile men, respectively. No correlation between thyroid dysfunction and semen parameters was detected. bTSH correlated significantly with PRL (P<.001). Thyroid antibodies were elevated in 7.5%. Elevated TPO-Ab were significantly correlated with pathozoospermia (P=.036) and asthenozoospermia (P=.049). CONCLUSION(S): Latent thyroid dysfunction had no impact on semen parameters. In patients with elevated TPO-Ab levels, pathozoospermia or asthenozoospermia should be considered.

Developmental exposure to estrogens alters epithelial cell adhesion and gap junction proteins in the adult rat prostate.

Brief exposure to estrogens during the neonatal period interrupts rat prostatic development by reducing branching morphogenesis and by blocking epithelial cells from entering a normal differentiation pathway. Upon aging, ventral prostates exhibit extensive hyperplasia and dysplasia suggesting that neonatal estrogens may predispose the prostate gland to preneoplastic lesions. To determine whether these prostatic lesions may be manifested through aberrant cell-to-cell communications, the present study examined specific gap junction proteins, Connexins (Cx) 32, and Cx 43, and the cell adhesion molecule, E-cadherin, in the developing, adult and aged rat prostate gland. Male rat pups were given 25 microgram estradiol benzoate or oil on days 1, 3, and 5 of life. Prostates were removed on days 1, 4, 5, 6, 10, 15, 30, or 90 or at 16 months, and frozen sections were immunostained for E-cadherin, Cx 43, and Cx 32. Colocalization studies were performed with immunofluorescence using specific antibodies for cell markers. Gap junctions in undifferentiated epithelial cells at days 1-10 of life were composed of Cx 43, which always colocalized with basal cell cytokeratins (CK 5/15). Cx 32 expression was first observed between days 10-15 and colocalized to differentiated luminal cells (CK 8/18). Cx 43 and Cx 32 never colocalized to the same cell indicating that gap junction intercellular communication differs between basal and luminal prostatic cells. While epithelial connexin expression was not initially altered in the developing prostates following estrogen exposure, adult prostates of neonatally estrogenized rats exhibited a marked decrease in Cx 32 staining and an increased proportion of Cx 43 expressing cells. In the developing prostate, E-cadherin was localized to lateral surfaces of undifferentiated epithelial cells and staining intensity increased as the cells differentiated into luminal cells. By day 30, estrogenized prostates had small foci of epithelial cells that did not immunostain for E-cadherins. In the adult and aged prostates of estrogenized rats, larger foci with differentiation defects and dysplasia were associated with a decrease or loss in E-cadherin staining. The present findings suggest that estrogen-induced changes in the expression of E-cadherin, Cx32 and Cx43 may result in impaired cell-cell adhesion and defective cell-cell communication and may be one of the key mechanisms through which changes toward a dysplastic state are mediated. These findings are significant in light of the data on human prostate cancers where carcinogenesis and progression are associated with loss of E-cadherin and a switch from Cx32 to Cx43 expression in the epithelium.

Influence of neonatal estrogens on rat prostate development.

Brief exposure of rodents to estrogens during early development alters prostate branching morphogenesis and cellular differentiation in a dose-dependant manner. If estrogenic exposures are high, these disturbances lead to permanent imprints of the prostate, which include reduced growth, differentiation defects of the epithelial cells, altered secretory function and reduced responsiveness to androgens in adulthood. This process, referred to as neonatal imprinting or developmental estrogenization, is associated with an increased incidence of prostatic lesions with aging, which include hyperplasia, inflammation and dysplasia. To better understand how early estrogenic exposures can permanently alter prostate growth and function and predispose the gland to neoplasia, the effects of estrogens on prostatic steroid receptors, cell-cell communication molecules and key developmental genes were examined. Transient and permanent alterations in the expression of prostatic androgen receptors, estrogen receptors alpha (ERalpha) and beta, and retinoic acid receptors are observed. It is proposed that the estrogen-induced alterations in these critical transcription factors play a fundamental role in initiating prostatic growth and differentiation defects. Down-stream effects of the altered steroid receptor expression include disruption of TGFbeta paracrine communication, altered expression of gap junction connexin molecules and loss of epithelial cadherin on epithelial cells. Additionally, specific disruptions in the expression of prostatic developmental genes are observed in response to neonatal estrogen. An extended developmental period of hoxa-13 expression, a lack of hoxd-13 increase with maturation, and an immediate and sustained suppression of hoxb-13 was noted within prostatic tissue. A transient decrease in Nkx3.1 expression in the developing prostate was also observed. Thus subtle and overt alterations in Hox-13 and Nkx3.1 genes may be involved in the altered prostate phenotype in response to neonatal estrogen exposure. In summary, estrogen imprinting of the prostate gland is mediated through up-regulated levels of stromal ERalpha, which initiates alterations in steroid receptor expression within the developing gland. Rather than being an androgen-dominated process, as occurs normally, prostatic development is regulated by alternate steroids, including estrogens and retinoids, in the estrogenized animal. This, in turn, leads to disruptions in the coordinated expression of critical developmental genes including TGFbeta, Hox-13 genes and Nkx3.1. Since a precise temporal expression pattern of these and other molecules is normally required for appropriate differentiation of the prostatic epithelium and stroma, the estrogen-initiated disruption in this pattern would lead to permanent differentiation defects of the prostate gland. It is hypothesized that these molecular and cellular changes initiated early in life predispose the prostate to the neoplastic state upon aging.

In vitro fertilization outcomes after intracytoplasmic sperm injection with fresh or frozen-thawed testicular spermatozoa.

OBJECTIVE: To compare the outcomes of intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) with fresh and cryopreserved testicular spermatozoa in patients with obstructive and nonobstructive azoospermia. DESIGN: Retrospective analysis of consecutive ICSI cycles. SETTING: Large urban reproductive medicine program. PATIENT(S): Twenty-nine patients with obstructive and nonobstructive azoospermia undergoing testicular sperm extraction for a total of 46 IVF-ICSI cycles (12 fresh, 34 frozen). INTERVENTION(S): Testicular sperm extraction, cryopreservation, and IVF-ICSI with fresh or frozen-thawed spermatozoa. MAIN OUTCOME MEASURE(S): Fertilization rates, embryo cleavage rates, embryo implantation rates, clinical pregnancy rates per cycle and per embryo transfer, and delivery and spontaneous abortion rates. RESULT(S): No statistically significant differences were noted in any of the parameters examined between IVF-ICSI cycles from fresh or frozen-thawed testicular spermatozoa. Fertilization rates were 56% with fresh vs. 61% with frozen-thawed testicular sperm, cleavage rates 92% vs. 95%, implantation rates 26% vs. 17%, clinical pregnancy rates per cycle 33% vs. 41%, and pregnancy rates per embryo transfer 33% vs. 45%, respectively. Delivery rates were 75% with fresh vs. 69.2% with frozen-thawed testicular sperm, and spontaneous abortion rates 25% and 30.8%, respectively. CONCLUSION(S): No differences were found in IVF-ICSI outcomes between cryopreserved and fresh testicular sperm. In addition, cryopreservation provides several advantages for the patients and reproductive team.

Copper deficiency in Helianthus annuus L.: developmental and metabolic consequences, stomatal function and senescence.

The effects of copper deficiency on developmental morphology and on age related metabolic activity, water utilization, stomatal response to light and leaf senescence in the sunflower, Helianthus annuus L., were monitored by sampling single leaves representing each leaf pair at designated harvest times during much of the vegetative lifespan. Plants grown in nutrient solutions lacking copper exhibited no apparent abnormalities during early vegetative growth and leaf morphology differed little from controls. There were no significant differences in rates of respiratory oxygen uptake of basal leaves between minus copper and controls. The effects of copper deficiency on rates of photosynthetic oxygen evolution were related to leaf age: rates were lower than controls in young basal leaves but higher as these leaves approached senescence. Both respiration and photosynthesis were more susceptible to KCN inhibition in minus copper leaves than in controls. There were no differences in cumulative water use per plant during the first five weeks after transfer of seedlings to nutrient solutions and only minor differences in stomatal aperture and guard cell potassium levels in ambient greenhouse light. Both minus copper and control leaves exhibited a sequential loss of stomatal function. Decay in the turgor driven opening response to light preceded loss of light dependent uptake of potassium into guard cells. Both changes in stomatal response to light occurred before major breakdown of chlorophyll. Basal leaves of sunflowers grown in minus copper nutrient solution tended to be retained longer and exhibited delayed loss of chlorophyll.

Evidence for Two Photoreactions and Possible Involvement of Phytochrome in Light-dependent Stomatal Opening.

Leaves of the xantha mutant of Helianthus annuus have a higher rate of transpiration and a lower diffusive resistance in the light than in the dark. Stomates of this nonphotosynthetic mutant open in the light and close in the dark.Comparative studies of tobacco, xantha mutant, and wild-type sunflower stomatal opening over a range of light intensities in isolated portions of the spectrum reveal two patterns of response: (a) a low intensity opening in the green and far red characterized by partial opening, absence of a threshold, and saturation of the response at low light intensities; (b) a high intensity response in the blue characterized by a threshold (intensities greater than 100 microwatts per square centimeter needed for opening) and a linear opening response at higher incident light intensities. In xantha mutant stomates only the low intensity system appears to be operational, while both low and high intensity systems are present in the wild-type sunflower and tobacco.Red light has an inhibiting effect on stomatal opening in both mutant and wild-type sunflowers. They require prior exposure to far red for opening to occur in red light. This redfar red antagonism suggests the involvement of phytochrome.

Reversal of copper inhibition in chloroplast reactions by manganese.

In the Mehler reaction, a Hill reaction utilizing molecular oxygen as the electron acceptor, rates of net oxygen uptake are stimulated by added manganous ions. Both whole cell photosynthesis and the Mehler reaction are inhibited by copper. Copper inhibition of the Mehler reaction can be reversed by manganese salts. Glutathione. which alone has no effect on Mehler reaction rates, enhances the effect of manganese in reversing copper inhibition. The effects of added Cu(2+), Cu(2+) and Mn(2+), or Cu(2+), Mn(2+), and glutathione exhibit no induction phenomena when measured manometrically. Furthermore, the order of addition of these factors is unimportant: final rates are dependent only on the composition of reaction mixtures. Compared to the Mehler reaction, conventional Hill reactions are less sensitive to copper poisoning, while certain chloroplast mediated photoxidations (e.g. the photoxidation of diketogulonic acid) are far more sensitive. In all of the chloroplast mediated photoreactions tested, manganese is effective in reducing the sensitivity to copper poisoning.

Allagochrome II. Effects of Light and Substrate on Allagochrome and Chlorogenic Acid Levels of Incubated Sunflower Leaf Discs.

Sunflower leaf discs incubated in the light on carbohydrate substrates exhibit several-fold increases in amounts of extractable allagochrome and chlorogenic acid. These changes are linear with time, and oxygen is required. The light effect saturates at approximately 600 muW/cm(2) "white" light, roughly the compensation point for photosynthesis. Red light is as effective as white light. Incubation in the dark, or in far red light, produces negligible changes in allagochrome and chlorogenic acid content.Sucrose (0.2 m) has been used as the standard substrate. At this concentration, glucose and fructose are slightly more effective. The optimum temperature range for incubation is 20 to 30 degrees . Allagochrome and chlorogenic acid values of both light and dark incubated samples decrease between 30 and 50 degrees , approaching zero at 50 degrees . Net light effects decrease to zero between 40 and 50 degrees .Of the inhibitors tested, 2,4-dinitrophenol, hydroxylamine and salicylaldoxime have no effect on light enhanced allagochrome and chlorogenic acid values except at high concentrations (which are generally deleterious to leaf tissues and cause decreased values for both dark and light incubated samples). Net light effects are completely inhibited, without changes in values of dark incubated discs, by azide (0.1 to 1 mm) and dichlorophenyldimethylurea (1 to 10 mum).Leaf tissues from the xantha mutant of Helianthus annuus do not exhibit a light effect. The absence of light effects in nonphotosynthetic leaf tissue and the inhibiting effects of photosynthetic poisons suggest that the photosynthetic apparatus is somehow involved.An hypothesis for light regulated metabolism via phenolic synthesis is discussed.