MiRNAs secreted by human blastocysts could be potential gene expression regulators during implantation.

BACKGROUND: Micro RNAs (miRNAs) are small non-coding RNAs known as essential regulators of cell-cell communication. Recent studies have revealed that miRNAs are secreted by a blastocyst in culture media. We hypothesized that endometrial epithelial cells take up embryo-derived miRNAs as well as other soluble factors and regulate their receptivity-related gene expression. METHODS AND RESULTS: Blastocyst culture media (BCM) were collected from the individually cultured embryos, while human endometrial epithelial cells (HEECs) were collected from healthy fertile volunteers. To evaluate the effect of BCM on the endometrial receptivity gene expression, HEECs were co-cultured with implanted BCM, non-implanted BCM, and a control culture medium. After determining altered gene expression in the HEECs, the miRNAs-related genes through bioinformatics databases were identified and evaluated in the BCM. Co-culture of primary HEECs with BCM significantly stimulated the expression levels of VEGFA, HBEGF, HOXA10, and LIF in the implanted group compared with non-implanted and control groups. The fold changes of miR-195 significantly diminished in the implanted BCM group compared with the non-implanted BCM group. Reduced fold changes of miR-29b, 145 and increased miR-223 were also observed in the implanted BCM group compared with the non-implanted ones. CONCLUSION: miRNAs could function as potential gene expression regulators during implantation. These molecules are secreted by human blastocyst, taken up by endometrial epithelial cells, and cause a change in the endometrial function. We found that BCMs can be effective in implantation process by stimulating related receptivity gene expression.

The therapeutic potential of adipose tissue-derived mesenchymal stromal cells in the treatment of busulfan-induced azoospermic mice.

PURPOSE: The generation of germ cells from mesenchymal stromal cells (MSCs) provides a valuable in vitro platform for infertility modeling. The establishment of these cells is a new approach for assisted reproductive technology (ART) to help infertile patients who lack functional gametes. METHODS: Human adipose-derived MSCs were isolated and then characterized for multipotency by flow cytometry, differentiation capacity, and cytogenetic assays. These cells were used in a male germ cell differentiation study. The expression of male germ cell markers was evaluated at day 21 of differentiation using an immunofluorescence assay, flow cytometry, and RT-qPCR. Undifferentiated MSCs were used for transplantation in busulfan-induced azoospermic mice. RESULTS: In this study, MSCs were successfully isolated from human adipose tissues which were positive for cell markers such as CD90, CD105, CD73, and CD29 but negative for CD34 and CD45. The results of flow cytometry, immunocytochemistry, and RT-qPCR analysis at day 21 of differentiation showed that the undifferentiated adipose-derived MSCs are able to differentiate into male germ cells. Additionally, transplantation of undifferentiated MSCs in busulfan-induced azoospermic mice caused spermatogenesis recovery in the majority of seminiferous tubules. CONCLUSION: In this study, we showed that differentiation of human adipose-derived MSCs into male germ cells is a useful tool for in vitro study of human germ cell development. Our results demonstrated that cell therapy with adipose-derived MSCs could help the repair of pathological changes in testicular seminiferous tubules. Therefore, it may have a clinical application for the treatment of azoospermia in infertile patients.

Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold.

Electrospun nanofiber matrices sufficiently mimic the structural morphology of natural extracellular matrix. In this study, we aimed to examine the effects of agar/polyvinyl alcohol nanofiber (PVA) scaffold on the proliferation efficiency and differentiation potential of neonate mouse spermatogonial stem cells (SCCs). Testicular cells were isolated from testes of 40 mouse pups and were seeded in: 1) 2D cell culture plates in the absence (2D/-GF) or presence (2D/+GF) of growth factors and 2) onto agar/PVA scaffold in the absence (3D/-GF) or presence (3D/+GF) of growth factors. The cells were subsequently cultured for 4 weeks. First 2 weeks were dedicated to proliferative phase, whereas the next 2 weeks emphasized the differentiation phase. The identity of the SCCs was investigated at different time-points by flow cytometry and quantitative reverse transcription PCR (qRT-PCR) analyses against the germ cell markers, including PLZF, Id-4, Gfrα-1, Tekt-1, and Sycp-3. After 2 weeks of culture, the 3D/+GF group showed the highest percentage of PLZF-positive cells among culture systems (P < 0.05). The expression levels of pre-meiotic markers (Id-4 and Gfrα-1) decreased significantly in all groups, particularly in 3D/+GF group after 28 days of culture. Additionally, the cells in the 3D/+GF group displayed the highest expression of meiotic (Sycp-3) and post-meiotic markers (Tekt-1) 14 days after differentiation induction. Seemingly, the combination of the agar/PVA scaffold and growth factor-supplemented medium synergistically increased the differentiation rate of mouse SSCs into meiotic and post-meiotic cells. Thus, agar/PVA nanofiber scaffolds may have the potential for applications in the restoration of infertility, especially in azoospermic males. ABBREVIATIONS: 2D: two dimentional; 3D: three dimentional; bFGF: basic fibroblast growth factor; BMP-4: bone morphogenetic protein 4; DMEM: Dulbecco's modified Eagle's medium; ECM: extracellular matrix; FCS: fetal calf serum; FTIR: Fourier-transform infrared spectroscopy; GDNF: glial cell line-derived neurotrophic factor; GF: growth factors; Gfrα-1, GDNF family co-receptor α1; Id-4, Inhibitor of DNA Binding 4; MTT: methylthiazoltetrazolium; PLZF: promyelocytic leukemia zinc finger; PVA: polyvinyl alcohol; qRT-PCR: quantitative reverse transcription PCR; RA: retinoic acid; SACS: soft agar culture system; SD: standard deviation; SEM: scanning electron microscope; SSCs: spermatogonial stem cells; Sycp-3, Synaptonemal complex protein 3; Tekt-1, Tektin 1.

Three-dimensional decellularized amnion membrane scaffold promotes the efficiency of male germ cells generation from human induced pluripotent stem cells.

Cells grow differently in conventional 2D cell culture than when they grow in the physiological microenvironment. In this study, we developed a 3D cell culture model for generating male germ cells from human iPSCs using a human decellularized amnion membrane (DAM) scaffold. To this end, human iPSCs were generated using retroviral vectors and characterized for pluripotency properties by immunofluorescence assay, flow cytometry, ALP staining, cytogenetic assay, and differentiation capacity. The iPSCs were used for investigating male germ cells differentiation efficiency in both conventional 2D culture and 3D-DAM scaffold. The expression of male germ cell markers was evaluated at day 21 of differentiation using immunofluorescence assay, flow-cytometry, and RT-qPCR. The results indicated a successful reprogramming of human foreskin fibroblast cells into pluripotent iPSCs. The reprogrammed cells were positive for pluripotency markers and differentiated into the three germ layers. During male germ cell differentiation, the cells tend to aggregate and form colony-like structures in both 2D and 3D conditions. However, significant expression of VASA, DAZL, PLZF, STELLA, and NANOS3 markers and more efficient haploid male germ cell production were observed in the 3D condition when compared to the 2D model. Considering the effect of the 3D-DAM scaffold in prompting male germ cell-specific markers and increased efficiency of germ cell differentiation in 3D culture, it appears that DAM scaffold is a useful tool for in vitro studies of human germ cell development and ultimately future clinical application.

Role of epigenetic modifications in the aberrant CYP19A1 gene expression in polycystic ovary syndrome.

INTRODUCTION: In this study, the global DNA methylation, histone acetylation and methylation levels of cumulus cells (CCs) in infertile polycystic ovary syndrome (PCOS) patients and the correlation of these epigenetic modifications with the expression of the ovarian aromatase gene (as an important marker in the etiology of PCOS) were investigated. MATERIAL AND METHODS: A cross-sectional study was conducted on 24 patients (12 PCOS patients and 12 healthy women), who underwent ovarian stimulation. Nucleosome ELISA was performed, in order to identify the global occupancy level of Mecp2 (as a marker of DNA methylation) and H3K9me2/H3K9ac as histone modification markers in chromatin fractions obtained from CCs. The CYP19A1 gene expression was measured by qRT-PCR. The level of DNA incorporation of MeCP2, histone modification markers and binding of estrogen receptor ß (ERß) to CYP19A1 regulatory sequences were examined by ChIP-QPCR assay. RESULTS: The data demonstrate a significant increase in global occupancy levels of MeCP2 and H3K9ac markers and a decrease of H3K9me2 to chromatin in CCs of PCOS patients vs. control group. Furthermore, CYP19A1 gene expression, and the incorporation of H3K9ac in PII, PI.3, and PI.4 promoters of CYP19A1 in PCOS, were higher than those of controls. Also, significant hypomethylation of H3K9 at PII and DNA hypomethylated at PII and PI.3 promoters and differential binding of ERß to three promoters were observed in PCOS patients (p < 0.05). CONCLUSIONS: Aromatase expression can be affected by epigenetic modifications and differential ERß binding to the proximal CYP19A1 promoters. These mechanisms may be involved in the enhanced aromatase transcription during ovarian stimulation in PCOS patients.

Three-dimensional decellularized amnion membrane scaffold as a novel tool for cancer research; cell behavior, drug resistance and cancer stem cell content.

BACKGROUND: Cancer is the second leading cause of human death. Therefore, comprehensive research and the appropriate tools are needed in this field. Animal models and cell culture studies are the most important preclinical tools in cancer research. In 2D cell culture models, cells are forced to grow in a 2D environment, which differs from their natural physiology. Recently, 3D cell culture models were developed to fill the gap between 2D cell culture and animal models. MATERIALS AND METHODS: Human amniotic membranes were obtained, decellularized, characterized and used as a natural 3D scaffold to investigate cancer cell behavior in 2D compared to 3D conditions. Time-lapse imaging of cells was used, and cell proliferation, velocity and migration were evaluated. Cisplatin was applied in 2D and 3D conditions, followed by evaluation of viability, apoptosis and cancer stem cell proteins by flow cytometry and western blot analysis. RESULTS: The results showed that in the decellularized amnion membrane (DAM) scaffold most cells did not spread and remain rounded and then penetrated into the scaffold with no cytotoxicity. Significant differences in migration, velocity, morphology and proliferation of cancer cells were observed between the 3D DAM scaffold and 2D model. Furthermore, the cells in the 3D DAM scaffold showed much more resistance to apoptosis and higher CSC content. CONCLUSION: In conclusion, considering the effect of the 3D DAM scaffold in cell behavior, apoptosis resistance and CSC content as well as the short processing time for decellularizing the AM, it appears that the 3D DAM scaffold offers an appropriate tool for in vitro cancer research.

Epigenetic alterations of CYP19A1 gene in Cumulus cells and its relevance to infertility in endometriosis.

PURPOSE: The purpose of the present study was to investigate the epigenetic mechanisms responsible for the aberrant aromatase expression (CYP19A1) in Cumulus Cells (CCs) of infertile endometriosis patients. METHOD: Cumulus cells were obtained from 24 infertile patients with and without endometriosis who underwent ovarian stimulation for intracytoplasmic sperm injection. Expression of CYP19A1 gene was quantified using Reverse Transcription Q-PCR. DNA methylation, histone modifications, and binding of Estrogen Receptor, ERß to regulatory DNA sequences of CYP19A1 gene were evaluated by Chromatin ImmunoPrecipitation (ChIP) assay. RESULTS: CYP19A1 gene expression in CCs of endometriosis patients was significantly lower than the control group (P = 0.04). Higher incorporation of MeCP2 (as a marker of DNA methylation) on PII and PI.4 promoters, and hypoacetylation at H3K9 in PII and hypermethylation at H3K9 in PI.4 were observed in CYP19A1 gene in endometriosis patients (P < 0.05). Moreover, a decreased level of ERß binding to PII and an increased level of its binding to PI.3 and PI.4 promoters of CYP19A1 were observed in endometriosis patients when compared to control. CONCLUSION: Significant reduction of CYP19A1 gene expression in CCs of endometriosis patients may be the result of epigenetic alterations in its regulatory regions, either by DNA methylation or histone modifications. These epigenetic changes along with differential binding of ERß (as a transcription factor) in CYP19A1 promoters may impair follicular steroidogenesis, leading to poor Oocyte and embryo condition in endometriosis patients.

Migration of Bone Marrow-Derived Very Small Embryonic-Like Stem Cells toward An Injured Spinal Cord.

OBJECTIVE: Bone marrow (BM) is one of the major hematopoietic organs in postnatal life that consists of a heterogeneous population of stem cells which have been previously described. Recently, a rare population of stem cells that are called very small embryonic-like (VSEL) stem cells has been found in the BM. These cells express several developmental markers of pluri- potent stem cells and can be mobilized into peripheral blood (PB) in response to tissue injury. In this study we have attempted to investigate the ability of these cells to migrate toward an injured spinal cord after transplantation through the tail vein in a rat model. MATERIALS AND METHODS: In this experimental study, VSELs were isolated from total BM cells using a fluorescent activated cell sorting (FACS) system and sca1 and stage specific embryonic antigen (SSEA-1) antibodies. After isolation, VSELs were cultured for 7 days on C2C12 as the feeder layer. Then, VSELs were labeled with 1,1´-dioctadecyl-3,3,3´,3´- tetramethylindocarbocyanine perchlorate (DiI) and transplanted into the rat spinal cord injury (SCI) model via the tail vein. Finally, we sought to determine the presence of VSELs in the lesion site. RESULTS: We isolated a high number of VSELs from the BM. After cultivation, the VSELs colonies were positive for SSEA-1, Oct4 and Sca1. At one month after transplantation, real-time polymerase chain reaction analysis confirmed a significantly increased expres- sion level of Oct4 and SSEA-1 positive cells at the injury site. CONCLUSION: VSELs have the capability to migrate and localize in an injured spinal cord after transplantation.

Different effects of olive leaf extract on antioxidant enzyme activities in midbrain and dopaminergic neurons of Substantia Nigra in young and old rats.

OBJECTIVES: Study of the effects of olive leaf extract on antioxidant enzyme activities in midbrain and dopaminergic neurons of Substantia Nigra in young and old rats. METHODS: Male wistar rats age 4 and 18 months were randomized into control and experimental groups. A single daily dose of 50 mg/kg of olive leaf extract was administered orally by gavage to each rat for 6 months. The control group received only distilled water. All rats were sacrificed 2 hours after the last gavage and their midbrains were separated for Malondialdehyde (MDA) and antioxidant enzyme activitiy analysis. TUNEL assay and immunohistochemical (IHC) staining were used for evaluation of the number of neurons in the Substantia Nigra. RESULTS: The level of Catalase, Glutathione Peroxidase and Superoxide Dismutase enzyme activity were significantly increased in experimental young and old groups compared to their control groups. However the level of Superoxide Dismutase enzyme activity was significantly increased in experimental old group when compared to control group (P< 0.05), the level of Superoxide Dismutase enzyme activity was not significantly changed in young groups. MDA level was decreased significantly in experimental young and old rats compared to their control groups. Histological analysis demonstrated that the number of neurons in Substantia Nigra of experimental old group was more than the control group (P<0.05). The number of apoptotic cells was significantly decreased in experimental old group compared to the corresponding control group (P<0.05). In IHC and TUNEL assay, no change was observed in the number of neurons between experimental and control young groups. CONCLUSION: Long term treatment with olive leaf extract increases antioxidant enzyme activity and protects the neurons in Substantia Nigra against oxidative stress.

Transplant of Very Small Embryoniclike Stem Cells to Spinal Cord Injury in a Rat Model Promotes Movement Recovery.

OBJECTIVES: Very small embryoniclike stem cells are a population of small stem cells with embryonic characteristics that were identified in adult murine bone marrow. During the past decade, researchers have examined different alternatives for functional recovery after spinal cord injury. The aim of this study was to evaluate transplant of small embryoniclike stem cells in a spinal cord injury rat model, and investigate cell migration to the lesion sites and the effects of cells on lesion size and overall functional recovery of the injured rats. MATERIALS AND METHODS: Small embryoniclike stem cells were isolated from bone marrow and injected intravenously to rats with spinal cord injury. RESULTS: Quantification of size of cavities in injured spinal cord tissue revealed significant reduction of the size of cavities in rats transplanted with very small embryoniclike stem cells (P < .05). Florescence microscopic images from injured spinal cord tissue showed localization of DiI-labeled small embryoniclike stem cells at the lesion site at 7 weeks after transplant. Real-time reverse transcription polymerase chain reaction analyses indicated higher expression of neural markers in the rats transplanted with small embryoniclike stem cells than in the other rats that did not receive small embryoniclike stem cells (P < .05). Assessment of improvement of locomotor function in rats transplanted with small embryoniclike stem cells was noticeable at 7 weeks after injury. CONCLUSIONS: Small embryoniclike stem cells may be a good source of stem cells for transplant into injured tissue of rat spinal cord for regeneration because they contain both embryonic and adult stem cell characteristics.

Evaluation of effect of oleuropein on skin wound healing in aged male BALB/c mice.

OBJECTIVE: Olive oil and olive leaf extract are used for treatment of skin diseases and wounds in Iran. The main component of olive leaf extract is Oleuropein. This research is focused on the effects of Oleuropein on skin wound healing in aged male Balb/c mice. MATERIALS AND METHODS: In this experimental study, Oleuropein was provided by Razi Herbal Medicine Institute, Lorestan, Iran.Twenty four male Balb/c mice, 16 months of age, were divided equally into control and experimental groups.Under ether anesthesia, the hairs on the back of neck of all groups were shaved and a 1 cm long full-thickness incision was made.The incision was then left un-sutured. The experimental group received intradermal injections with a daily single dose of 50 mg/kg Oleuropein for a total period of 7 days.The control group received only distilled water. On days 3 and 7 after making the incision and injections, mice were sacrificed, and the skin around incision area was dissected and stained by hematoxylin and eosin (H&E) and Van Gieson's methods for tissue analysis.In addition, western blot analysis was carried out to evaluate the level of vascular endothelial growth factor (VEGF) protein expression. The statistical analysis was performed using SPSS (SPSS Inc., Chicago, USA). The t test was applied to assess the significance of changes between control and experimental groups. RESULTS: Oleuropein not only reduced cell infiltration in the wound site on days 3 and 7 post incision, but also a significant increase in collagen fiber deposition and more advanced re- epithelialization were observed (p<0.05) in the experimental group as compared to the control group. The difference of hair follicles was not significant between the two groups at the same period of time. Furthermore, western blot analysis showed an increased in VEGF protein level from samples collected on days 3 and 7 post-incision of experimental group as compared to the control group (p<0.05). CONCLUSION: These results suggest that Oleuropein accelerates skin wound healing in aged male Balb/c mice. These findings can be useful for clinical application of Oleuropein in expediting wound healing after surgery.

Antioxidant role of oleuropein on midbrain and dopaminergic neurons of substantia nigra in aged rats.

BACKGROUND: Oleuropein is a phenolic compound which is present in the olive leaf extract. The purpose of the present study was to investigate the neuroprotective effect of oleuropein as an antioxidant agent on the substantia nigra in aged rats. METHODS: Twenty 18-month-old Wistar rats (450-550 g) were randomly divided into control and experimental groups. The experimental group received a daily single dose of 50 mg/kg of oleuropein by oral gavage for 6 months. The control group received only distilled water. All rats were sacrificed two hours after the last gavage and the brains were removed and midbrains were cut. One part of the midbrains were homogenized and centrifuged. The tissue supernatant was assayed for lipid peroxidation (LPO) and antioxidant enzyme activities. The other part of midbrains fixed and embedded in paraffin, then processed for Nissl and immunohistochemistry (IHC) staining. Data was analyzed using SPSS by t-test. Differences were considered significant for P<0.05. RESULTS: The level of LPO in midbrain of the rats was decreased significantly in the experimental group, but superoxide dismutase, catalase and glutathione peroxidase activities were increased in experimental group compared to control group (P<0.05). Morphometric analyses showed significantly that the experimental group had more neurons in substantia nigra pars compacta (SNc) either in Nissl or IHC staining when compared to control (P<0.05). CONCLUSION: The results of the present study indicate that treatment of the old rats with oleuropein reduces the oxidative damage in SNc by increasing the antioxidant enzyme activities.

Therapeutic effects of oleuropein on wounded skin in young male BALB/c mice.

INTRODUCTION: Oleuropein is generally the most abundant phenolic compound in olive leaves. In this study, therapeutic effects of oleuropein were studied on wounded skin in young male Balb/c mice. METHODS: Four-month-old male Balb/c mice were randomized into 2 groups, a control and an experimental group. Under ether anesthesia, hair on the neck of mice in both groups was shaved and 1-cm long full-thickness incisions were made and left unsutured. The experimental group was injected intradermally with a daily single dose of oleuropein (50 mg/kg) for a total of 7 days. The control group received only distilled water. On days 3 and 7 post-incision, mice were sacrificed and skin around the area of the incisions were dissected and processed for hematoxylin and eosin and Van Gieson's staining. Portions of dissected tissues were also lysed and used for western blot analysis to evaluate the level of vascular endothelial growth factor (VEGF) protein expression. RESULTS: The analyses showed oleuropein reduced cell infiltration into the wound sites on day 3 and 7 postincision; however, it significantly increased collagen fiber deposition and caused faster reepithelialization when compared to the control group (P < 0.05). Furthermore, western blot analysis showed a significant increase in VEGF protein level compared to the control group (P < 0.05). CONCLUSION: In summary, oleuropein showed healing effects on wounded skin by accelerating the reepithelialization process, enhancing collagen fiber generation, and increasing the blood supply to the wounded area by upregulation of VEGF protein expression.

Neuroprotective effect of exogenous melatonin on dopaminergic neurons of the substantia nigra in ovariectomized rats.

BACKGROUND: Melatonin has receptors in substantia nigra pars compacta (SNc) and regulates development of dopaminergic (DA) neurons. This study was undertaken to determine ability of melatonin to protect SNc dopaminergic neuron loss induced by estrogen deficiency in ovariectomized rats. METHODS: Female rats were randomized into four groups of seven each: control, ethanol sham, ovariectomy (ovx) and ovx with melatonin (ovx + m). In ovx, ovaries were removed. Ovx + m group was intraperitoneally injected with melatonin for 10 days, while the ethanol sham group received only ethanol. All rats were perfused with 4% paraformaldehyde, midbrains removed, fixed and paraffin embedded, then processed for Nissl and tyrosine hydroxylase staining (IHC). Ten sections of SNc in Nissl and IHC staining were analyzed in each animal, Nissl stained and tyrosine hydroxylase (TH) immunoreactive cells were counted in five experimental groups randomly. Data was analyzed using SPSS by ANOVA and t-test. Differences were considered significant for P<0.05. RESULTS: There was less cell number in ovx compared to control and ethanol sham groups significantly (P<0.001). The ovx + m group had more cells than the ovx group in the SNc significantly (P<0.001). Furthermore, there was significant decrease of TH positive cell number in the ovx group compared to control and ethanol sham groups (P<0.05). The number of TH immunoreactive cells was higher in ovx + m compared to the ovx group (P<0.05). CONCLUSION: These findings can be compared with human and used in clinical application for prevention of DA neuron death of SNc after ovariectomy.

The effects of melatonin on open wounds of aged mice skin.

UNLABELLED: Objective. To study the effects of melatonin on open wounds of aged mice skin by morphological and morphometric evaluations. MATERIALS AND METHODS: Forty 16-month-old male Suri mice were used for this study. They were divided into two groups: 20 mice for control and 20 mice for the experimental group. Under ether anesthesia, the hair on the back of the neck of the two groups was shaved and a 2-cm long, full-thickness incision was made and left unsutured. The experimental group was injected intraperitoneally with a daily single dose of 10 mg/kg melatonin in saline for a total period of 12 days, while the control group received only saline for the same period of time. The mice of experimental and control groups were sacrificed on days 9 and 12 after making the incision, and the skin in the area of the incision was dissected and processed for light microscopy analysis. The epithelization, the mean diameter of nucleus in fibroblasts, the level of hydoxyproline, and the amount of collagen fibers were evaluated. Statistical analysis was performed using SPSS software and Mann- Whitney U test to assess the significance of changes between control and experimental groups. RESULTS: The dissected skin from the experimental group 9 and 12 days after making the incisions showed a significant increase of mean diameter of nucleus in fibroblasts (P < 0.001). The epithelization, the amount of collagen fibers, and the level of hydroxyproline were also increased significantly compared to control group (P < 0.001). CONCLUSION: The results of this study suggest that melatonin may be beneficial to the healing process of open wounds in aged mice. .

Morphometric evaluation of seminiferous tubules in aged mice testes after melatonin administration.

OBJECTIVE: Melatonin, the pineal gland hormone as a direct or indirect antioxidant and free radical scavenger, is involved in the process of both aging and age-related diseases. This study investigates the effects of melatonin on the histology of testicular seminiferous tubules in aged mice. MATERIALS AND METHODS: Twenty male, white mice, aged 16 months, that weighed 20-23 gr were equally divided into control and experimental groups. The experimental group was intraperitoneally injected with a daily single dose of 10 mg/kg melatonin for 14 days. The control group received only saline. Six days after the last injection, all mice were sacrificed and the testes were excised and processed for light microscope observation. In the morphometric study, we evaluated testicular seminiferous tubule parameters such as height of germinal epithelium, seminiferous tubule diameter, thickness of interstitial connective tissue and spermatogenesis index (SI). SPSS software and student's t-test analyzed all parameters to assess the significance of changes between control and experimental groups. RESULTS: Melatonin-treated mice had seminiferous tubules with a wide lumen lined by low height germinal epithelium. The interstitial connective tissue thickened significantly in the experimental group (p<0.05), tubular diameter and germinal epithelium height decreased significantly (p<0.01), and the SI reduced compared to the control group (p<0.001). CONCLUSION: The results of this study showed the disadvantages of melatonin on seminiferous tubules of aged mice testes.

Morphological changes of rabbit lacrimal gland cells from amiodarone administration.

BACKGROUND: Amiodarone is a drug that is used for treatment of cardiac arrhythmia after cardiac ischemia. This drug as beta blocker decreases arrhythmia rate but it has many side effects on different tissues. Since there are rare reports about changes of lacrimal glands, this research has been carried out to study the morphological and ultrastructural changes of lacrimal gland cells after amiodarone administration. METHODS: Male rabbits (n = 14) were divided into control and experimental groups. Experimental group were intra peritoneally injected with a daily single dose of 80 mg/kg amiodarone for two weeks. The control group only received normal saline. At the end of the injection period, the two groups were anesthetized and perfused with Karnovsky's fixative. The lacrimal glands were removed, fixed and then prepared for light and electron microscopic studies. Quantitative studies on lacrimal gland cell micrographs were performed by point counting method. The results were statistically compared between the two groups. RESULTS: Light microscopic observation showed many secretory granules in the cytoplasm of the lacrimal gland cells, which were also seen in the lumen of acini. Ultrastructure study of these cells showed the presence of inclusions in their cytoplasm with homogenous and dense structure. In quantitative analysis, the volume fractions (Vv) of mitochondria and nucleus to the cell showed no differences between the two groups but the Vv of euchromatin to the nucleus was different (P<0.05 ). CONCLUSION: The presented results show adverse effects of amiodarone on rabbit lacrimal gland cells.