Is there a relationship between infertility and fertilin β protein distribution? / ¿Existe relación entre la infertilidad y la distribución de la proteína fertilin β?

Introduction: Fertilin β is a sperm surface protein that can mediate sperm-egg membrane interaction. This study was conducted to determine whether the expression of fertilin β after intrauterine insemination (IUI) in donors with normal parameters after standard semen analysis is related to low success rate or failure of fertilization. Methods: We examined the sperm of 30 male donors who have normal as controls, oligozoospermia, and unexplained infertility as the clinically indication for IUI. Fertilin β has been labeled with the ADAM2 antibody by indirect immunofluorescence (IF) assay. To evaluate the reproducibility of the test, we selected four sperm samples scale of 0 to +++ according to the distribution of fluorescence label. Results: The results were highly correlated with the corrected total cell fluorescence (CTCF) (Rp=0.9972, P<0.05). We suggest that the relationship between infertility and fertilin β may be due to the distribution of this protein on the sperm surface. Male partners of couples with unexplained infertility showed a low distribution of fertilin β by a decrease of the fluorescence signal in the IF labeling (scale of +++ by 7.4±10.32%, P<0.0001, ±SD). Discussion: Abnormal fertilin β function may be a potential mechanism that could lead to fertilization failure. (AU)

Introducción: La fertilinβ es una proteína de la superficie del esperma que puede mediar entre la interacción del espermatozoide y la membrana del óvulo. Este estudio se realizó para determinar si la expresión de fertilinβ después de la inseminación intrauterina (IIU) en donantes con parámetros normales después del análisis estándar de semen está relacionada con una baja tasa de éxito o fracaso de la fertilización. Métodos: Examinamos los espermatozoides de 30 donantes masculinos que tenían controles normales, oligozoospermia e infertilidad inexplicable como indicación clínica de IIU. La fertilinβ ha sido etiquetada con el anticuerpo ADAM2 mediante un ensayo de inmunofluorescencia indirecta (IF). Para evaluar la reproducibilidad de la prueba, seleccionamos cuatro muestras de esperma en una escala de 0 a +++ según la distribución de la etiqueta de fluorescencia. Resultados: Los resultados estuvieron altamente correlacionados con la fluorescencia celular total corregida (Rp=0,9972, p<0,05). Sugerimos que la relación entre la infertilidad y la fertilinβ puede deberse a la distribución de esta proteína en la superficie del esperma. Los compañeros masculinos de parejas con infertilidad inexplicable mostraron una baja distribución de fertilinβ por una disminución de la señal de fluorescencia en el etiquetado IF (escala de +++ en 7,4 ±10,32%, p<0,0001, ±DE). Conclusión: La función anormal de la fertilinβ puede ser un mecanismo potencial que podría conducir al fracaso de la fertilización. (AU)

Is there a relationship between infertility and fertilin ß protein distribution?

INTRODUCTION: Fertilin ß is a sperm surface protein that can mediate sperm-egg membrane interaction. This study was conducted to determine whether the expression of fertilin ß after intrauterine insemination (IUI) in donors with normal parameters after standard semen analysis is related to low success rate or failure of fertilization. METHODS: We examined the sperm of 30 male donors who have normal as controls, oligozoospermia, and unexplained infertility as the clinically indication for IUI. Fertilin ß has been labeled with the ADAM2 antibody by indirect immunofluorescence (IF) assay. To evaluate the reproducibility of the test, we selected four sperm samples scale of 0 to +++ according to the distribution of fluorescence label. RESULTS: The results were highly correlated with the corrected total cell fluorescence (CTCF) (Rp=0.9972, P<0.05). We suggest that the relationship between infertility and fertilin ß may be due to the distribution of this protein on the sperm surface. Male partners of couples with unexplained infertility showed a low distribution of fertilin ß by a decrease of the fluorescence signal in the IF labeling (scale of +++ by 7.4±10.32%, P<0.0001, ±SD). DISCUSSION: Abnormal fertilin ß function may be a potential mechanism that could lead to fertilization failure.

A comparative study of the effects of gutta-percha solvents on human osteoblasts and murine fibroblasts.

We aimed to investigate the in vitro physiologic effects of xylene, chloroform, orange oil and eucalyptus oil solvents for dissolving gutta-percha on L929 and HOB cell lines; 2.5 and 10 µL mL-1 of these solvents were tested for 24, 48 and 72 h. Gutta-percha solvents inhibited the proliferation rate of fibroblasts in a dose- and time-dependent manner; however, no inhibition was detected in HOB (evaluated using MTT assay). None of the solvents induced apoptosis/necrosis in HOB cells at ≤2.5 µL mL-1 concentration in contrast to L929 (determined using acridine orange/ethidium bromide dual staining). Each solvent tested reduced the migration rate of both L929 and HOB cell lines in a dose-dependent manner (evaluated using a scratch assay). Gutta-percha solvents can damage fibroblast-rich tissues. Osteoblasts seemed to be more resistant to the tested solvents, and excessive extrusion of solvents from the root canal may also damage the periradicular tissues and reduce the ability to repair.

Immunolocalization of Fertilin ß, IZUMO1, and P34H in Ram Spermatozoa.

According to various reports, current methods of sperm freezing destroy the integrity of the sperm plasma membrane and acrosome. This study aimed to determine the changes in the existence and location of three proteins, namely fertilin ß, IZUMO1, and P34H, in ram spermatozoa. By using frozen-thawed spermatozoa, ejaculated fresh spermatozoa, and testicular and epididymal spermatozoa (obtained from caput, corpus, and caudal regions), the localizations of the mentioned proteins were performed using signal labeling with indirect immunofluorescence, and the quantification of these proteins was compared using Western blot analyses. Moreover, protein localization and signal labeling in fresh and frozen-thawed spermatozoa subjected to in vitro capacitation and acrosome reaction were compared. Using chlortetracycline (CTC) staining, as expected, it was detected that after incubating for 4 hours under capacitating conditions related to the control sample (0 hour), capacitated and acrosome-reacted sperm were increased (p < 0.001). Frozen-thawed samples had a lower density and expression than the ejaculate samples. Expression was not obtained, except for IZUMO1, from samples that underwent in vitro capacitation/acrosome reactions. Expression of IZUMO1 was seen as an increasing band formation from the equatorial region through the acrosome, after in vitro capacitation. However, after the acrosome reaction, the band formation was only on the equatorial region. Region-specific differences of proteins at the kDa level were obtained using Western blot analysis and possible isoforms specific to ram spermatozoa or proteins with similar epitopes were expressed. Considering the changes in surface proteins in frozen-thawed sperm, it is suggested that fertilin ß and P34H can be used as fertility or freezability markers.

Investigation of Physiological Effects Induced by Dehydroepiandrosterone in Human Endothelial Cells and Ovarian Cancer Cell Line

Objectives: Dehydroepiandrosterone (DHEA) is an endogenous hormone that acts as a ligand for several cellular receptors. An age-dependent decline in circulating levels of DHEA is linked to changes in various physiological functions. In gynecological clinical practice, DHEA is commonly prescribed to induce ovulation. Some clinical studies report a positive association between high serum concentrations of DHEA and an increased risk of developing ovarian cancer. However, the in vitro physiological effects of DHEA on ovarian cancerous cells have not been explored thus far. In this study, we aimed to investigate the physiological effects of DHEA treatment (0-200 µM, 24-72 hours) on MDAH-2774 human ovarian cancer cell line and primary HuVeC human endothelial cells. Materials and Methods: The physiological effects of DHEA treatment (0-200 µM, 24-72 hours) on MDAH-2774 human ovarian cancer cell line and primary HuVeC human endothelial cells were investigated with the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, acridine orange/ethidium bromide staining, and scratch assay. Results: DHEA treatment promoted proliferation of the MDAH-2774 cancer cell line in a dose-dependent manner (r=0.6906, p<0.0001, for 24 hours) (r=0.6802, p<0.0001, for 48 hours) (r=0.7969, p<0.0001, for 72 hours). In contrast, DHEA inhibited proliferation of the primary HuVeC cells (r=0.9490, p<0.0001, for 24 hours) (r=0.9533, p<0.0001, for 48 hours) (r=0.9584, p<0.0001, for 72 hours). In agreement with these observations, DHEA treatment resulted in a dose-dependent increase in the number of necrotic cells in the primary HuVeC cells (r=0.97, p<0.0001). However, the number of necrotic or apoptotic cells did not change significantly when the MDAH-2774 cells was exposed to DHEA. Moreover, we found that DHEA treatment reduced the migration rate of HuVeC cells in a dose-dependent manner (r=0.9868, p<0.0001), whereas only a slight increase was observed in the MDAH-2774 ovarian cancer cell line (r=0.8938, p<0.05). Conclusion: Our findings suggest that DHEA promotes the proliferation of ovarian cancer cells in a dose-dependent manner in vitro. Moreover, DHEA induced necrosis and inhibited proliferation in endothelial cells. Although mechanistic evidence is required, our preliminary findings imply that exposure to high doses of DHEA may be associated with an increased risk of developing ovarian cancer.

In Vitro Physiological Effects of Betahistine on Cell Lines of Various Origins

Objectives: Betahistine is a histamine analog commonly prescribed for symptomatic treatment of vertiginous symptoms. In vitro studies have shown that betahistine was not toxic at the prescribed doses in a nasal epithelial cell line. However, the effect of betahistine on other cell types has not been studied. In this study, we aimed to investigate some of the physiological effects of betahistine on L929 fibroblast, A549 lung cancer, human umbilical vein endothelial (HUVEC), and Ishikawa endometrial cell lines. Materials and Methods: Cellular proliferation was assed assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, apoptosis was evaluated by acridine orange-ethidium bromide staining, and cellular migration was assed assessed by scratch assay. Results: Betahistine treatment (0.1-0.5 mg/mL, 24 hours) can inhibit cell proliferation and induce apoptosis in HUVEC, A549, Ishikawa, and L929 cell lines. Betahistine (≥0.1 mg/mL) significantly increased the number of apoptotic cells (HUVEC: 26.3%, A549: 17.3%, L929: 8.6%, and Ishikawa: 2.3%). Betahistine at doses over 0.1 mg/mL significantly suppressed the cell migration rate in all of the cell lines. In contrast, exposure to a low dose of betahistine (0.025 mg/mL) induced migration rates of HUVEC and Ishikawa cells by 81% and 48%, respectively. Conclusion: Betahistine may alter the processes of cellular proliferation, apoptosis, and cellular migration in a cell line- and dose-dependent manner. In this sense, proliferative and metastatic properties of certain cancer cells can potentially be altered in response to betahistine treatment.

Cell-Free Lactobacillus sp Supernatants Modulate Staphylococcus aureus Growth, Adhesion and Invasion to Human Osteoblast (HOB) Cells.

The increase of antibiotic resistance has become a problem. Probiotic bacteria play an important role in preventive/supportive medicine. Therefore, we examined the inhibitory effects of four different Lactobacillus species' (L. acidophilus-La, L. plantarum-Lp, L. fermentum-Lf and L. rhamnosus-Lr) cell-free supernatants (CFSs) on growth, adhesion, invasion, and biofilm formation of Staphylococcus aureus and effects of S. aureus, CFSs, and S. aureus-CFSs co-existence on human osteoblast (HOB) cell viability. Growth alterations were measured spectrophotometrically. Adhesive/invasive bacterial counts were detected by colony counting. Biofilm was evaluated using microtiter plate assay. The MTT assay was used for detection of HOB cell viability. The growth of MSSA significantly (P < 0.01) decreased in the presence of two CFSs (Lf and Lr) (P < 0.01); the growth of MRSA significantly (P < 0.05) reduced in the presence of La CFSs. All tested CFSs were found to reduce adhesion and invasion of MSSA (P < 0.0001). The adhesion of MRSA was enhanced (P < 0.0001) in the presence of all CFSs except La and the invasion of MRSA was decreased (P < 0.01) in the presence of Lr and Lf CFSs. All tested CFSs were shown to inhibit biofilm formation significantly (P < 0.0001). The reduction of S. aureus infected HOB cell viability and exposed to all CFSs except Lr that was found to be significant (P < 0.0001). The viability of HOB cell during co-incubation with MSSA and CFSs was shown to be decreased significantly. However co-existence of MRSA and CFSs did not alter HOB cell viability. These results suggested that lactobacilli as probiotics have low protective effects on MRSA-infected host cells.

A simple and inexpensive method for evaluation of in vitro cell adhesion on screws.

Only a limited number of techniques are available for assessing the effect of different coating materials on cell adherence to screws. In this study, we describe a simple and inexpensive method for evaluation of cell adhesion on irregular surfaces such as the surgical or implant screws. For this purpose, we prepared semi-submerged screws in the petri dishes using agar. Using BSA- or HA-coated screws, we tested whether BSA or HA could improve cell adherence when used as coating materials. Agar-coated screws were used as internal control. Then the "ratio of cell adherence" was calculated by subtracting the reference RCA value obtained from the agar coated screws (internal control). When compared to that of the non-coated screws both the HA- and BSA-coating improved cell adherence on the screws by 2.34 and 2.72 fold respectively. Similarly, MTT assay data revealed that the metabolic capacities of cells on HA- or BSA-coated screws were improved by 2.36 and 2.86 fold respectively. These findings suggest that this protocol can be used for comparing the ability of cells to attach on irregular surfaces such as dental or orthopedic screws and assessing their viability.

Antimuscarinic-induced convulsions in fasted mice after food intake: No evidence of spontaneous seizures, behavioral changes or neuronal damage.

Prolonged or repeated seizures have been shown to cause spontaneous recurrent seizures, increased anxiety­related behavior, locomotor hyperactivity, impaired functions of learning and memory, and neuronal damage in the hippocampus and other brain regions in animals. Mice and rats treated with antimuscarinic drugs after fasting for two days or less develop convulsions after being allowed to eat ad libitum. To address whether such behavioral and neuroanatomic changes occur following these convulsions, mice treated i.p. with saline (control) or 2.4 mg/kg atropine and given food after 24 h of fasting were grouped according to seizure scores for behavioral and histological analysis. Following convulsions, the occurrence of spontaneous recurrent seizures was observed for 30 days. Motor activity and grooming behavior were assessed in the open field, and memory was assessed using the novel object recognition test 4 and 7 days after onset of convulsions, respectively. Animals allocated for the histological analysis were decapitated 7 days after onset of convulsions and hippocampal slices were evaluated for the percentage of degenerating neurons stained with Fluoro­Jade C. Spontaneous recurrent seizures, locomotor alterations, anxiety­related behavior, memory impairment, and neuronal loss in the granular layer of the dentate gyrus were not detected in the animals with seizure score 1-2 or 3-5. These results are in accordance with those related to the absence of behavioral changes, cognitive deficits, and hippocampal neuronal damage after single brief seizures in animals and patients with epilepsy.