The uterusmasculinus of the Eurasian beaver (Castor fever L.) - The appraisal of fast hormone regulation by membrane androgen and estrogen receptors involvement.

The phenomenon of remaining paramesonephric ducts (uterus masculinus) in males of some animal species concerning its role is still an unresolved issue. Now it is well-recognized that sex hormonal regulation of reproductive physiology involves also fast nongenomic control of cellular processes through noncanonical signaling. Herein, in the uterus masculinus of Eurasian beaver membrane androgen receptor (metal ion transporter Zrt- and Irt-like protein 9; ZIP9) and membrane estrogen receptor (G protein-coupled estrogen receptor; GPER) were studied. Scanning electron microscopy together with anatomical analysis revealed that Eurasian male beavers possess one double uterus (uterus duplex). Two odd parts open into the vagina but do not form a common lumen. The length of the horns is the most differential feature of this organ in studied animals. Uterus masculinus is not a tightly closed tubular structure. Histological analysis showed an analogy to the female uterus structure however no glands but gland-like structures were observed. The presence and abundant localization of ZIP9 and GPER proteins in cells of uterus masculinus was confirmed by immunohistochemistry while their expression was measured by western blotting. GPER expression in remnants was lower (P < 0.001) than those in the female uterus. Parallelly, the concentration of progesterone and estradiol but not testosterone was lower (P < 0.05 and P < 0.01, respectively) in comparison to the female uterus. Our study, for the first time, reports the involvement of fast hormonal regulation in the uterus masculinus of Eurasian beavers reflecting the participation of this organ in the creation local hormonal environment. Moreover, the uterus masculinus seems to be a useful research model for understanding and resolving urgent biological problems such as gender identities and having children by women with a lack of uterus or anatomical barriers on this level.

The role of G-protein-coupled membrane estrogen receptor in mouse Leydig cell function-in vivo and in vitro evaluation.

In this study, G-coupled estrogen receptor (GPER) was inactivated, by treatment with antagonist (G-15), in testes of C57BL/6 mice: immature (3 weeks old), mature (3 months old) and aged (1.5 years old) (50 µg/kg bw), as well as MA-10 mouse Leydig cells (10 nM/24 h) alone or in combination with 17ß-estradiol or antiestrogen (ICI 182,780). In G-15-treated mice, overgrowth of interstitial tissue was found in both mature and aged testes. Depending on age, differences in structure and distribution of various Leydig cell organelles were observed. Concomitantly, modulation of activity of the mitochondria and tubulin microfibers was revealed. Diverse and complex GPER regulation at the mRNA level and protein of estrogen signaling molecules (estrogen receptor α and ß; ERα, ERß and cytochrome P450 aromatase; P450arom) in G-15 Leydig cells was found in relation to age and the experimental system utilized (in vivo and in vitro). Changes in expression patterns of ERs and P450arom, as well as steroid secretion, reflected Leydig cell heterogeneity to estrogen regulation throughout male life including cell physiological status.We show, for the first time, GPER with ERs and P450arom work in tandem to maintain Leydig cell architecture and supervise its steroidogenic function by estrogen during male life. Full set of estrogen signaling molecules, with involvement of GPER, is crucial for proper Leydig cell function where each molecule acts in a specific and/or complementary manner. Further understanding of the mechanisms by which GPER controls Leydig cells with special regard to male age, cell of origin and experimental system used is critical for predicting and preventing testis steroidogenic disorders based on perturbations in estrogen signaling.

Craniofacial structure in patients with obstructive sleep apnoea.

BACKGROUND: Obstructive sleep apnoea (OSA) is characterised by at least five 10-s episodes of apnoea or markedly shallow breathing per 1 h of sleep, which can lead to severe, sometimes life-threatening complications. It is essential to determine the specific features of the affected patients' craniofacial structure, thus enabling their allocation to risk groups. The aim of the study was to assess the craniofacial structure in OSA patients, comparing the findings with Hasund's and Segner's cephalometric normal values. In addition, the sagittal dimensions of the upper airways, measured at two levels, were compared to McNamara's normal values. MATERIALS AND METHODS: The study covered 41 patients diagnosed polysomno-graphically with OSA. Lateral cephalograms with cephalometric analysis and the measurements of the upper and lower sagittal dimensions of the upper airways were taken for each patient. RESULTS: The only feature of the patents' facial skeleton that significantly diverged from the normal range was the SNB angle (p = 0.004). Other angles, i.e. SNA, ANB, NL/NSL, NL/ML and NSL/ML, were not significantly different from normal. The average upper cross-sectional area of the upper airways was 10.4 mm; in 97.6% patients, this measurement was below McNamara's normal values. In the majority of patients (75.6%), the average lower sagittal dimension of the upper airways (10.4 mm) was also below the normal. CONCLUSIONS: Mandibular retrognathia, manifested by the reduced SNB angle, and the narrowed upper and lower sagittal dimensions of the upper airways can be considered one of OSA prognostic factors.

Is membrane androgen and estrogen receptor signaling imperative in the governing function of the adrenal cortex in the Eurasian beaver (Castor fiber L.)?

There is a need to fully know the physiology of Eurasian beaver due to its essential role in environmental homeostasis. However, a "human factor" impacts this, including stress conditions and environmental pollution. Adrenal glands protect these all. The regulation of endocrine processes by nonclassical androgen and estrogen signaling, the first and fastest control, is still a matter of research. The specific analyses performed here in mature female and male beaver adrenals contained: anatomical and histological examinations, expression and localization of membrane androgen receptor (zinc transporter, Zinc- and Iron-like protein 9; ZIP9) and membrane estrogen receptor coupled with G protein (GPER), and measurement of zinc (Zn2+) and copper (Ca2+) ion levels and corticosterone levels. We revealed normal anatomical localization, size, and tissue histology in female and male beavers, respectively. Equally, ZIP9 and GPER were localized in the membrane of all adrenal cortex cells. The protein expression of these receptors was higher (p < 0.001) in male than female adrenal cortex cells. Similarly, Zn2+ and Ca2+ ion levels were higher (p < 0.05, p < 0.01) in male than female adrenal cortex. The increased corticosterone levels (p < 0.001) were detected in the adrenal cortex of females when compared to males. The present study is the first to report the presence of nonclassical androgen and estrogen signaling and its possible regulatory function in the adrenal cortex of Eurasian beavers. We assume that this first-activated and fast-transmitted regulation can be important in the context of the effect of environmental physical and chemical stressors especially on adrenal cortex cells. The beaver adrenals may constitute an additional supplementary model for searching for universal mechanisms of adrenal cortex physiology and diseases.

Evaluation of upper airways depth among patients with skeletal Class I and III.

BACKGROUND: The aim of this study was to determine the value of upper and lower pharyngeal depth among patients with skeletal Class III malocclusion on lateral cephalograms, as well as to examine the relationship between SNA, SNB, and ANB angles, along with Wits appraisal and the cross-sectional value of upper airway space at the level of the soft palate and tongue base among patients with skeletal Class I and III. MATERIALS AND METHODS: The material consisted of lateral cephalograms taken from 80 patients living in the Lubelskie voivodeship. The study group consisted of cephalograms of 50 patients with skeletal Class III malocclusion (17 male and 33 female), whereas the control group consisted of 30 roentgenograms of patients with Class I malocclusion with proper jaw to mandible relation (14 maleand 16 female). The study and the control group shared no statistically significant differences considering basic sociographic data such as gender (chi = 1.267, p = 0.26)and age (U = 727.5, p = 0.82). The upper and lower pharyngeal depths were assessed with the use of McNamara's method. Spearman's rho test, Mann--Whitney's U test, and chi test were used for statistical analysis. RESULTS: Among both males and females the pharyngeal depths were greater considering patients with skeletal Class III in comparison to patients with Class Imalocclusion (p < 0.001). Furthermore, it was determined that the lower as well as the upper pharyngeal width is statistically significantly dependent on ANB and SNB angles and Wits appraisal (p < 0.001). CONCLUSIONS: Pharyngeal width at the level of the soft palate and tongue base depends on skeletal class, namely ANB angle and Wits appraisal; it increases with the increase of SNB angle (forward movement of the mandible). The SNA angle (position of the maxilla) does not influence the anterior-posterior nasopharyngeal dimension.

Effect of indoxyl sulfate on the morphology and function of the thyroid gland - ex vivo studies in rabbits.

Indoxyl sulfates are uremic indolic toxins known to participate in the pathogenesis of cardiovascular diseases during chronic kidney disease in humans and some animal species. However, nothing is known about the indoxyl sulfate effect on the thyroid gland which is especially responsible for the general organism metabolism. This study determines the morpho-functional status of the thyroid gland after exposure to indoxyl sulfate (10, 25, and 50 mM) with the use of an ex vivo system and rabbit (n=10) as an experimental model thyroid gland histology, immunoexpression of thyrotropin receptor (TSHR), and concentrations of thyroxine (T4) and triiodothyronine (T3) were evaluated. Statistical analyses were performed using one-way analysis of the variance (ANOVA) followed by Tukey's post hoc comparison test. Minor alterations in thyroid tissue structure e.g. very rare exfoliated epithelial cells, condensed colloid fluid, or slight loosening of the epithelium were found. In addition, modulated dose dependent-expression of TSHR (p<0.01, p<0.001) together with a decreased level of T4 and T3 (p<0.001, p<0.01) exception of an increased level of T4 after the middle dose of indoxyl sulfate were revealed. We report here, for the first time, that indoxyl sulfate affects the thyroid gland mainly at the molecular level. The rabbit thyroid gland ex vivo system seems to be suitable for further studies on the thyroid gland in health and disease. However, the effect of TSH-TSHR signaling at ultrastructural, and epigenetic levels needs supplementary appraisal.

In vitro culture Muller cell model to study the role of inverted internal limiting membrane flap technique in macular hole closure.

Inverted internal limiting membrane (ILM) flap may constitute a scaffold for Muller cells whose migration and proliferation on its surface begin the process of macular hole closure. The goal of the study was to establish an in vitro model of the interaction between ILM and the Muller cells. Vitrectomy with inverted ILM flap was performed in 23 patients due to a full-thickness macular hole (FTMH). After dissection of the inverted flap, the area of ILM peeling was extended and material was collected for cell culture experiments. Muller cells cultured on adherent cell plates showed significantly better growth than on suspension plates. Our results reveal that the presence of the ILM can overcome the growth inhibitory effect of the non-adhesive surface. Moreover, the ILM appears to be the optimal growth surface under normoxia conditions mimicking the microenvironment after vitrectomy and hypoxia which is natural state for Muller cells. The closure rate of FTMH was 100%. Our study revealed that in non-adhesive culture conditions patient derived ILM constitutes an optimal growth surface for Muller cells. We have demonstrated that the ILM effectively stimulates attachment, proliferation, and survival of Müller cells in conditions of normoxia which is the case after vitrectomy. The results strongly advocate for the use of inverted ILM flap method in macular hole closure surgeries.

Leydig cell tumorigenesis - implication of G-protein coupled membrane estrogen receptor, peroxisome proliferator-activated receptor and xenoestrogen exposure. In vivo and in vitro appraisal.

The etiology and molecular characteristics of Leydig cell tumor (LCT) are scarcely known. From the research data stems that estrogen can be implicated in LCT induction and development, however it is not investigated in detail. Considering the above, herein we analyzed the relation between G-protein coupled membrane estrogen receptor, peroxisome proliferator-activated receptor and insulin-like family peptides (insulin-like 3 peptide; INSL3 and relaxin; RLN) expressions as well as estrogen level with impact of xenoestrogen (bisphenol A; BPA, tetrabromobisphenol A; TBBPA, and tetrachlorobisphenol A; TCBPA). While in our previous studies altered GPER-PPAR partnership was found in human LCT being a possible cause and/or additionally effecting on LCT development, here mouse testes with experimentally induced LCT and mouse tumor Leydig cell (MA-10) treated with BPA chemicals were examined. We revealed either diverse changes in expression or co-expression of GPER and PPAR in mouse LCT as well as in MA-10 cells after BPA analogues when compared to human LCT. Relationships between expression of INSL3, RLN, including co-expression, and estrogen level in human LCT, mouse LCT and MA-10 cells xenoestrogen-treated were found. Moreover, involvement of PI3K-Akt-mTOR pathway or only mTOR in the interactions of examined receptors and hormones was showed. Taken together, species, cell of origin, experimental system used and type of used chemical differences may result in diverse molecular characteristics of LCT. Estrogen/xenoestrogen may play a role in tumor Leydig cell proliferation and biochemical nature but this issue requires further studies. Experimentally-induced LCT in mouse testis and MA-10 cells after BPA exposure seem to be additional models for understanding some aspects of human LCT biology.

Does signaling of estrogen-related receptors affect structure and function of bank vole Leydig cells?

To get a deeper insight into the function of estrogen-related receptors (ERRs) and dissect underlying mechanism in Leydig cells, ERRs (type α, ß and γ) were blocked or activated in testes of adult bank voles (Myodes glareolus) which show seasonal changes in the intratesticular sex hormones level. Both actively reproducing animals (long day conditions; LD) and those with regression of the reproductive system (short day conditions; SD) received intraperitoneal injections of selective ERRα antagonist 3-[4-(2,4-Bis-trifluoromethylbenzyloxy)-3-methoxyphenyl]-2-cyano-N-(5-trifluoromethyl-1,3,4-thiadiazol-2-yl)acrylamide (XCT 790) or selective ERRß/ERRγ agonist N-(4-(Diethylaminobenzylidenyl)-N'-(4-hydroxybenzoyl)-hydrazine (DY131) (50 µ/kg bw; six doses every other day). Markedly more, XCT 790 (P < 0.05) but also DY131 affected interstitial tissue histology whose volume increased in both LD and SD males while seminiferous epithelium structure was untouched. Ultrastructure analysis revealed alterations in mitochondria number as well as endoplasmic reticulum and Golgi complexes volume and structure especially after ERRα blockage. Diverse and complex ERRs regulation at mRNA level and protein expression (P < 0.05; P < 0.01 and P < 0.001) of steroidogenic (lutropin receptor (LHR), translocator protein (TSPO), steroidogenic acute regulatory protein (StAR)) and secretory (insulin-like protein 3 (INSL3) and relaxin (RLN)) molecules were revealed in relations to endogenous estrogen level in treated males. Notably, immunolocalization of ERRs and above proteins, exclusively in Leydig cells, indicated their involvement in Leydig cell function control based on interactions with endogenous estrogen level and/or estrogen signaling via ERRs. Treatment with XCT 790 or DY131 significantly decreased (P < 0.05; P < 0.01 and P < 0.001) intratesticular estrogens concentration, with exception in SD DY131 males. In addition, androgens level was decreased, but not in LD DY131 voles. Similarly, ERRßγ activation significantly reduced (P < 0.05; P < 0.01 and P < 0.001) cAMP and calcium ions (Ca2+) concentrations particularly in DY131 voles. Overall, for the first time, we have shown that ERRs are involved in maintenance of Leydig cell architecture and supervision of its steroidogenic and secretory activity that is closely related to endogenous estrogen status in the testis. Further understanding of mechanism(s) by which individual types of ERRs can control Leydig cell function is relevant for predicting and preventing steroidogenic and spermatogenic disorders.

Flutamide alters the distribution of c-Src and affects the N-cadherin-ß-catenin complex in the seminiferous epithelium of adult rat.

This study was undertaken to explore interactions between c-Src kinase and the N-cadherin-ß-catenin complex in seminiferous tubules of flutamide-treated rats. An anti-androgen flutamide (50 mg/kg bw) was injected daily into adult rats from postnatal days 82 to 88. Testes from 90-day-old control and flutamide-treated rats were used for experiments. Flutamide did not affect testis morphology, but impaired connexin43 immunoexpression between Sertoli cells at the blood-testis barrier (BTB) region, indicating the BTB as a sensitive target for flutamide. Real-time RT-PCR and western blot analyses revealed upregulation of N-cadherin at the mRNA and protein level after flutamide exposure (p < 0.05), whereas no changes in ß-catenin and c-Src expression were observed. Notably, membranous ß-catenin immunolocalization indicated its involvement in the cell adhesion complex rather than its contribution to the Wnt signaling pathway. As we used an exposure regime which avoided germ cell loss, it is likely that changes in the N-cadherin-ß-catenin complex are a primary effect of androgen signaling disruption by flutamide. Immunohistochemistry revealed a diffusion of N-cadherin and ß-catenin signals away from the BTB with concomitant disruption of c-Src staining pattern. As detected by immunofluorescence and coimmunoprecipitation, flutamide promoted disassembly of the N-cadherin-ß-catenin complex, induced N-cadherin to dissociate from c-Src at the BTB site, and altered interactions between the cell junction proteins and/or c-Src. Equally important, increased levels of p-N-cadherin-Tyr860 and p-ß-catenin-Tyr654 (p < 0.05) pointed to a mechanism related to adhesion complex disassembly and suggested a potential role of c-Src in the control of the protein-protein dynamics. Overall, for the first time we have shown that flutamide alters the distribution of c-Src and affects N-cadherin-ß-catenin interactions at the BTB. Understanding mechanism(s) by which anti-androgens can affect intercellular adhesion within the testis is relevant for predicting and preventing reproductive disorders affecting male fertility.

The value of postoperative radiotherapy in advanced renal cell cancer.

The study reviews experience with the treatment of advanced renal cell cancer at Bydgoszcz Regional Cancer Center within a 10-year period from 1985 to 1996. The aim of this paper was to evaluate the value of postoperative radiotherapy. The medical records of 186 patients with locally advanced renal cell cancer were reviewed retrospectively. Postoperative radiation therapy with a median dose of 50.0 Gy/t was given in 114 patients. The overall and disease-free survival, the pattern of recurrences, time interval to recurrence were assessed. For all patients, the 5-year overall and disease-free survival rates were 36.2% and 30.5%, respectively. Non significant difference was observed in terms of 5-year overall and disease-free survival between the group of patients with postoperative radiotherapy and without, 37.9%/29.5% vs. 35.5%/31.3%, respectively. A total of 29 patients (15.6%) developed local recurrences. Local failure by stage was as follows: T3N0 without postoperative radiation therapy--15.8%, with irradiation--8.8%; T3N(+) without radiation therapy--33.3%, with irradiation--33.3%; T4N0 without radiation therapy--33.3%, with irradiation--33.3%, T4N(+) without radiation therapy--33.3%, with irradiation--25.0%. 73 patients (39.3%) had distant metastases as a first symptom of renal cell cancer relapse. The median time to relapse for local recurrence or distant metastases were approximately two times longer in patients with adjuvant radiotherapy compared to those without, 27.0/21.0 months vs. 16.0/12.5 months, respectively. In our opinion postoperative radiotherapy reduces the probability of local recurrences in selected patients, mainly with pathologic stage T3N0, but its impact on survival is minimal.

Postnatal exposure to flutamide affects CDH1 and CTNNB1 gene expression in adult pig epididymis and prostate and alters metabolism of testosterone.

In both epididymis and prostate the dynamic cross-talk between the cells is hormonally regulated and, in part, through direct cell-to-cell interactions. Functionality of the male reproductive organs may be affected by exposure to specific chemicals, so-called 'reprotoxicants'. In this study we tested whether early postnatal and prepubertal exposure to anti-androgen flutamide altered the expression of adherens junction genes encoding E-cadherin (CDH1) and ß-catenin (CTNNB1) in adult pig epididymis and prostate. In addition, the expression of mRNAs and proteins for 5α-reductase (ST5AR2) and aromatase (CYP19A1) were examined to show whether flutamide alters metabolism of testosterone. Thus, flutamide was injected into male piglets between Days 2 and 10 and between Days 90 and 98 postnatally (PD2 and PD90; 50 mg/kg bw), tissues that were obtained on postnatal Day 270. To assess the expression of the genes and proteins, real-time RT-PCR and Western blot were performed respectively. Moreover, adherens junction proteins were localized by immunohistochemistry. In response to flutamide, CDH1 and CTNNB1 expressions were down-regulated along the epididymis, mostly in PD2 group (p < 0.001, p < 0.01). In the prostate, CDH1 mRNA and protein expressions were significantly down-regulated (p < 0.01), whereas CTNNB1 mRNA was slightly up-regulated in both flutamide-treated groups. CTNNB1 protein level was markedly elevated in both PD2 (p < 0.001) and PD90 (p < 0.01) groups. In the epididymis, the expression of ST5AR2 and CYP19A1 was down- and up-regulated, respectively (p < 0.05), whereas in the prostate evident decrease in CYP19A1 expression (p < 0.001, p < 0.01, p < 0.05) was demonstrated. In both tissues, membranous immunolocalization of CTNNB1 suggests its involvement in cell-cell adhesion. Overall, flutamide administration resulted in suppression of androgen action in the epididymis and prostate leading to deregulation of CDH1 and CTNNB1 gene expressions which is probably caused by the alterations in the expression of ST5AR2 and CYP19A1 in both reproductive organs.