Sertoli cell-conditioned medium can improve blood-testis-barrier function and spermatogenesis in azoospermia mice induced by scrotal hyperthermia: An experimental study.

Background: An increase in the temperature of the testis is associated with damage to the epithelium of seminiferous tubules and disruption of sperm production. Objective: The current study aimed to investigate the effect of the Sertoli cell-conditioned medium (SCCM) on the blood-testis-barrier associated genes and spermatogenesis process following scrotal hyperthermia. Materials and Methods: In this experimental study, 40 adult NMRI mice (8 wk, 25-30 gr) were allocated into 4 groups: I) control, II) DMEM (10 µl Dulbecco's Modified Eagle Medium), III) scrotal hyperthermia, and IV) scrotal hyperthermia+SCCM (10 µl SCCM). Hyperthermia was induced by placing the mice scrotum in water at 43 C for 20 min every other day for 10 days. Mice were treated every other day for 5 wk. Then the animals were euthanized, and the tails of epididymis were removed to analyze sperm parameters, testis were taken for stereological assessment, reactive oxygen spices and glutathione levels, and the expression of Ocln, Gja1, Cdh2, and Itgb1. Results: The results of sperm analysis indicated that SCCM-treated mice significantly increased sperm count and motility and reduced DNA fragmentation. In addition, histological and molecular findings showed that the volume of testicular tissue, the number of germ cells, the glutathione level, and the expression of Ocln, Gja1, Cdh2, and Itgb1 genes were significantly increased in the SCCM-treated mice. Conclusion: Findings suggest that growth factors of SCCM stimulate the proliferation and differentiation of germ cells through paracrine effects and upregulate the blood-testis-barrier-associated genes in mice subjected to scrotal hyperthermia.

Exploring Intercellular Dynamics: Ultra-Weak Biophoton Emission as a Novel Indicator of Altered Cell Functions and Disease in Oligospermia Mice.

Introduction: Biophoton emission, the spontaneous release of photons from living cells, has emerged as an attractive field of research in the study of biological systems. Scientists have recently discovered that changes in biophoton emission could serve as potential indicators of pathological conditions. This intriguing phenomenon suggests that cells might communicate and interact with each other through the exchange of these faint but significant light signals. Therefore, the present study introduces intercellular relationships with biophoton release to detect normal and abnormal cell functions to further achieve cellular interactions by focusing on cell and cell arrangement in disease conditions. Methods: Twenty male mice were assigned to control and busulfan groups. Five weeks after the injection of busulfan, the testis was removed, and then the stereological techniques and TUNEL assay were applied to estimate the histopathology of the testis tissue sections. Results: The findings revealed that the ultra-weak biophoton emission in the control group was significantly lower than in the busulfan group. The oligospermia mice model showed that it significantly changed the spatial arrangement of testicular cells and notably decreased the testis volume, length of seminiferous tubules, and the number of testicular cells. The results of the TUNEL assay showed that the percentage of apoptotic cells significantly increased in the busulfan group. Conclusion: The ultra-weak biophoton emission from testis tissue was reduced in oligospermia mice. As a result, the decline of ultra-weak biophoton can indicate a change in cell arrangement, a decrease in intercellular interaction, and eventually disease.

Impaired spermatogenesis caused by busulfan is partially ameliorated by treatment with conditioned medium of adipose tissue derived mesenchymal stem cells.

Busulfan (BSU) is a chemotherapeutic drug that can cause subfertility or sterility in males. We investigated the effects of adipose tissue-derived mesenchymal stem cells (AT-MSC) conditioned medium (CM) (AT-MSC-CM) on histopathological and molecular characteristics of mouse testes exposed to BSU using stereology. We used adult male mice divided randomly into five groups: control, Dulbecco's modified Eagle's medium (DMEM), dimethyl sulfoxide (DMSO), BSU, and BSU + CM. Thirty-five days following BSU injection, sperm and testis tissues were harvested for stereological and molecular studies. The BSU group exhibited significantly reduced testis volume, interstitium and tubules compared to the other groups, although the volume of the testis remained unchanged for BSU and CM groups. The number of testis cells was reduced in the BSU group compared to the other groups. The CM group exhibited a significantly increased number of testis cells compared to the BSU group. Sperm count and motility, and length density of seminiferous tubules were increased in CM group compared to the BSU group. AT-MSC-CM exhibited ameliorative effects on histopathologic changes of mouse testes exposed to BSU.

Chronic scrotal hyperthermia induces azoospermia and severe damage to testicular tissue in mice.

Scrotal hyperthermia leads to altered spermatogenesis due to heat-related oxidative stress. One of the main causes of infertility in men is oxidative stress, which refers to an imbalance in the levels of reactive oxygen species (ROS) and antioxidants. Therefore, this study aimed to evaluate the effects of chronic scrotal hyperthermia on testicular tissue structure, sperm parameters, and oxidative stress in adult mice. Thirty adult NMRI male mice were divided into three groups: Control (n = 10), Sham (n = 10), and Hyperthermia (n = 10). At the end of the study animals were sacrificed for evaluations of biochemical, cellular and histological analysis. The Hyperthermia group revealed a significant reduction in sperm count and weight of testis when compared to the control and sham groups. Also, succinate dehydrogenase (SDH) activity, ROS, ATP production, glutathione disulfide (GSH), tiols metabolism and stereological parameters in the hyperthermia group showed a significant reduction compared to the control and sham groups. Our results also revealed that scrotal hyperthermia significantly increases ROS production, mitochondrial membrane permeability (MMP), malondialdehyde (MDA), oxidized glutathione (GSSG) and apoptotic cells in testicular tissue in the hyperthermia groups in comparison with the control and sham groups. Overall, our result indicated that chronic scrotal hyperthermia causes complete spermatogenic arrest, probably mainly throughout the induction of oxidative stress.

Related Fluoxetine and Methylprednisolone Changes of TNF-α and IL-6 Expression in The Hypothyroidism Rat Model of Spinal Cord Injury.

OBJECTIVE: Spinal cord injury (SCI) is a serious clinical condition that leads to disability. Following primary injury, proinflammatory cytokines play an important role in the subsequent secondary events. The thyroid hormone (TH) is known as the modulator of inflammatory cytokines and acts as a neuroprotective agent. Methylprednisolone (MP) is used for the early treatment of SCI. Fluoxetine (FLX), also is known as a selective serotonin reuptake inhibitor (SSRI), has therapeutic potential in neurological disorders. The aim of the present study was to investigate the combined effects of MP and FLX on SCI in the rat hypothyroidism (hypo) model. MATERIALS AND METHODS: In this experimental study, 48 male Wistar rats with hypothyroidism were randomly divided into 6 groups (n=8/group): control (Hypo), Hypo+Surgical sham, Hypo+SCI, Hypo+SCI+MP, Hypo+SCI+FLX, and Hypo+SCI+MP+FLX. SCI was created using an aneurysm clip and Hypothyroidism was induced by 6-Propyl-2-thiouracil (PTU) at a dose of 10 mg/kg/day administered intraperitoneally. Following SCI induction, rats received MP and FLX treatments via separate intraperitoneal injections at a dose of 30 and 10 mg/kg/day respectively on the surgery day and FLX continued daily for 3 weeks. The expression levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were quantified by Real-time polymerase chain reaction (PCR) and Western blotting. Myelination and glutathione (GSH) levels were analyzed by Luxol Fast Blue (LFB) staining and ELISA respectively. RESULTS: Following combined MP and FLX treatments, the expression levels of TNF-α and IL-6 significantly decreased and GSH level considerably increased in the trial animals. CONCLUSION: Our results show the neuroprotective effects of MP and FLX with better results in Hypo+SCI+MP+FLX group. Further study is required to identify the mechanisms involved.

Photobiomodulation restores spermatogenesis in the transient scrotal hyperthermia-induced mice.

OBJECTIVE: Heat stress shock affects the generation of free radicals and can have a harmful effect on spermatogenesis. Photobiomodulation (PBM) is very effective in andrology for treating male infertility. This research aimed at the evaluation of the impacts of PBM on spermatogenesis on the transient scrotal hyperthermia-induced oligospermia mouse model. MATERIALS AND METHODS: This experimental research divided 24 mice into the following four groups: (1) Control, (2) Scrotal hyperthermia, (3) Scrotal hyperthermia receiving laser 0.03 J/cm2 for 30 s for each testis, 35 days after induction of scrotal hyperthermia every other day for 35 days, and (4) Scrotal hyperthermia receiving laser 0.03 J/cm2 for 30 s for each testis, immediately after induction of scrotal hyperthermia every other day for 35 days. Scrotal hyperthermia was induced by water bath with 43 °C for 30 min. Then, the mice were euthanized, and their sperm samples were collected for sperm parameters analysis. Then, we took the testis samples for histopathological experimentations, serum testosterone level, reactive oxygen species (ROS), RNA extraction for the examination of IL1-α, IL6 and TNF-α genes expression as well as production and glutathione disulfide (GSH) activity. KEY FINDINGS: Our outputs indicated that PBM could largely improve the sperms parameters and stereological parameters, like spermatogonia, primary spermatocyte, round spermatid and Leydig cells together with an increasing level of the serum testosterone and GSH activity compared to the scrotal hyperthermia induced mice. In addition, it was found that the diameter of seminiferous tubules, ROS production, as well as the expression of IL1-α, IL6, and TNF-α genes significantly decreased in the treatment groups by PBM compared to the scrotal hyperthermia induced mice, but there was not a significant difference in terms of testis weight and Sertoli cells between the studied groups. SIGNIFICANCE: It could be concluded that PBM may be regarded as an alternative treatment for improving the spermatogenesis process in the scrotal hyperthermia induced mice.

The Combined Effects of Levothyroxine and Low Level Laser Therapy on Wound Healing in Hypothyroidism Male Rat Model.

Introduction: Hypothyroidism is caused by inadequate production and storage of thyroid hormones. Hypothyroidism is associated with delayed wound healing. Laser therapy may stimulate wound regeneration. The aim of this study was to determine the combined effects of levothyroxine and low level laser therapy during the wound healing process on skin of hypothyroidism male rat model. Methods: Thirty male Wistar rats were randomly divided into 5 groups: control group, hypothyroidism group, hypothyroidism group treated by laser, hypothyroidism group treated by levothyroxine, and hypothyroidism group treated by laser and levothyroxine. To induce hypothyroidism, methimazole was given at a dose of 4 mg/100 mL in their drinking water. After hypothyroidism was proven through immunoassay commercial kit, rats were generally anesthetized with ketamine and xylazine, then, an incisional skin wound was created in a length of 1.2 cm on the back of the ribcage. The surgical day is considered as the zero day. The third and fifth groups were treated with a pulse laser, 810 nm wavelength 80 Hz frequency and 0.2 J/cm2 energy densities for 200 seconds. Levothyroxine was injected to the fourth and fifth groups intraperitoneally. On the 14th day, a normal sample of each healing skin wound was harvested for biomechanical examination. The obtained data were analyzed by the SPSS software 21 and reported as a mean ± standard error of mean (SEM). P < 0.05 was considered statistically significant. Results: The results showed that the mean maximum force and the accomplished work (energy) made a significant difference in the group receiving both laser and levothyroxine synchronously rather than the other groups (P ≤ 0.05). The elasticity of the wound healing in the groups that received laser and levothyroxine synchronously was significantly higher in comparison with the control and hypothyroidism groups but the difference was not significant in comparison with the laser or levothyroxine groups. Conclusion: The results of our study showed that the application of laser and levothyroxine synchronously improves the biomechanical parameters of wound during healing in comparison to the use of laser and levothyroxine solely.

The Effects of In Vitro Maturation Technique on The Expression of Genes Involved in Embryonic Genome Activation of Human Embryos.

OBJECTIVES: In vitro maturation technique (IVM) is shown to have an effect on full maturation of immature oocytes and the subsequent embryo development. Embryonic genome activation (EGA) is considered as a crucial and the first process after fertilization. EGA failure leads to embryo arrest and possible implantation failure. This study aimed to determine the role of IVM in EGA-related genes expression in human embryo originated from immature oocytes and recovered from women receiving gonadotrophin treatment for assisted reproduction. MATERIALS AND METHODS: In this experimental study, germinal vesicle (GV) oocytes were cultured in vitro. After intracytoplasmic sperm injection of the oocytes, fertilization, cleavage and embryo quality score were assessed in vitro and in vivo. After 3-4 days, a single blastomere was biopsied from the embryos and then frozen. Afterwards, the expression of EGA-related genes in embryos was assayed using quantitative reverse transcriptase-polymerase chain reaction (PCR). RESULTS: The in vitro study showed reduced quality of embryos. No significant difference was found between embryo quality scores for the two groups (P=0.754). The in vitro group exhibited a relatively reduced expression of the EGArelated genes, when compared to the in vivo group (all of them showed P=0.0001). CONCLUSIONS: Although displaying the normal morphology, the IVM process appeared to have a negative influence on developmental gene expression levels of human preimplanted embryos. Based on our results, the embryo normal morphology cannot be considered as an ideal scale for the successful growth of embryo at implantation and downstream processes.

Combined Effect of Low-Level Laser Treatment and Levothyroxine on Wound Healing in Rats With Hypothyroidism.

Introduction: Hypothyroidism delays wound healing by reducing the synthesis of keratinocytes, fibroblast cells, and collagen. Methods for enhancement of wound healing include laser therapy and hormone therapy. The current study evaluated the combined effect of laser and levothyroxine therapy to cure wounds in male rats with hypothyroidism. Methods: Sixty male Wistar rats were randomly divided into 5 groups: (1) healthy controls; (2) controls with hypothyroidism; (3) hypothyroidism + laser treatment; (4) hypothyroidism + levothyroxine treatment; (5) hypothyroidism + laser + levothyroxine treatment. Hypothyroidism was induced by dissolving 4 mg of methimazole in 100 mL of drinking water daily for 28 days. After hypothyroidism had been confirmed, a longitudinal incisional wound was created on the dorsal rib cages of the rats. The wounds that received laser treatment were divided into 12 sections and treated at 810 nm wavelength and 0.2 J/cm2 of energy density for 200 seconds. Levothyroxine was administrated in doses of 20 µg/kg/d i.p. All groups were divided into 3 subgroups for testing on days 4, 7 and 14. Samples were collected in all the subgroups. Results: The results showed that hypothyroidism reduced fibrous tissue volume, fibroblasts, and basal cell numbers. The combined effect of laser and levothyroxine improved all parameters. Conclusion: Combined laser and levothyroxine treatment showed the best effect on wound healing and accelerated the closure of the wounds.

Does sperm DNA fragmentation affect the developmental potential and the incidence of apoptosis following blastomere biopsy?

Common methods employed in assisted reproduction technology (ART) include intracytoplasmic sperm injection (ICSI) with an unspecified level of sperm DNA fragmentation (SDF) and preimplantation genetic diagnosis (PGD). The aim of this study was to investigate the impact of SDF on human preimplantation embryo development and the incidence of apoptosis following a single blastomere biopsy. Using sperm chromatin dispersion (SCD) to assess SDF, a total of 20 processed semen samples were categorized into two groups; group I: SDF ≤30% and group II: SDF >30%. After ICSI, fertilization, cleavage, and embryo quality score were assessed. A single blastomere was biopsied from day 3 embryos and development was monitored on day 4. The frequency of apoptosis in biopsied embryos was assayed by TUNEL and the level of BCL-2, BAX, hsa-mir-15a, and hsa-mir-16-1 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). SCD was found to be negatively correlated with sperm motility and normal form spermatozoa (p < 0.05). The rate of fertilization, cleavage, and embryo quality score were not significantly different between the two groups (all p > 0.05). SDF >30% had no negative effect on potential development and did not increase the proportion of apoptotic cells and the level of apoptosis-related genes and microRNAs (miRNAs) in group II vs. group I (p > 0.05). It appears that at the levels assessed paternal genome damage had little if any negative effect on preimplantaton embryo development and apoptosis following single blastomere biopsy. This may reflect the selection of morphologically normal sperm for ICSI and the repair capacity of the oocyte.

Effect of L-carnitine supplementation on maturation and early embryo development of immature mouse oocytes selected by brilliant cresyle blue staining.

PURPOSE: The present study was designed to investigate the effect of L-carnitine treatment during IVM on nuclear and cytoplasmic maturation of immature oocytes selected by Brilliant Cresyle Blue (BCB) staining, and their subsequent developmental competence. MATERIALS & METHODS: Compact cumulus-oocyte complexes (COCs) were collected from NMRI mice ovaries and stained with BCB staining. BCB+ (colored cytoplasm) oocytes were then cultured in tissue culture medium (TCM) 199 with 0.0, 0.3 and 0.6 mg/ml L-carnitine. RESULTS: The both L-carnitine concentrations significantly increased the intracellular glutathione (P<0.001), nuclear maturation (P<0.01) and expression levels of cyclin-dependent kinase1 (CDK1) (P<0.05). Moreover, treated oocytes with 0.6 mg/ml L-carnitine showed increased (P < 0.05) expression of mitogen-activated protein kinase1 (MAPK1) mRNA. Also, adding L-carnitine (0.6 mg/ml) to IVM medium significantly increased the cleavage rate (P<0.05). The blastocyst development rate (BDR) in the both L-carnitine treated groups was significantly higher (P<0.001) than the control group. L-carnitine had no significant effect on total blastocyst cell numbers. CONCLUSIONS: These data indicated that L-carnitine supplementation during IVM of immature BCB+ oocytes improved preimplantation developmental competence of oocytes after IVF, probably by accelerating cytoplasmic and nuclear maturation of oocytes. It may provide a novel approach to improving ART outcomes in infertile couples.

Association between increased expression of endothelial isoform of nitric oxide synthase in the human fallopian tube and tubal ectopic pregnancy.

BACKGROUND: Tubal ectopic pregnancy (tEP) is the most common type of extra-uterine pregnancy and the most common cause of maternal mortality. Nitric oxide (NO) is a molecule that incorporates in many physiological processes of female reproductive system. Recent studies have demonstrated the possible role of endothelial isoform of nitric oxide synthase (eNOS) enzyme in the regulation of many reproductive events that occur in the fallopian tube (FT). OBJECTIVE: The aim of this study was to evaluate the expression of eNOS in the FTs of women with tEP. MATERIALS AND METHODS: In this case-control study, a total number of 30FTs samples were obtained from three groups including: 10 FTs of women that bearing an EP, 10 FTs from the non-pregnant women at luteal phase of the menstrual cycle, and 10 FTs of healthy pregnant women (n=10). Samples were fixed in 10% buffered formalin and then were evaluated by immunohistochemistry. RESULTS: Localization of eNOS was seen in secretory and ciliated luminal epithelium and vascular endothelium of all groups. However, we did not observed the expression of eNOS in smooth muscle cells of all groups. Expression of eNOS in luminal epithelium of women with EP compared to non-pregnant women at luteal phase of menstrual cycle and healthy pregnant group showed statistically significant increase (p=0.00). Significant difference in expression of eNOS was not observed in luminal epithelium of FTs of women at luteal phase compared to healthy pregnant groups (p=0.78). CONCLUSION: This study indicates that changes in expression of eNOS in luminal epithelium of FT may lead to development of EP. This article extracted from M.Sc. thesis. (Leyla Fath Bayati).

The effects of insulin-like growth factor-1 gene therapy and cell transplantation on rat acute wound model.

BACKGROUND: Wound healing is a complex process. Different types of skin cells, extracellular matrix and variety of growth factors are involved in wound healing. The use of recombinant growth factors in researches and production of skin substitutes are still a challenge. OBJECTIVES: Much research has been done on the effects of gene therapy and cell therapy on wound healing. In this experimental study, the effect of insulin-like growth factor (IGF-1) gene transfer in fibroblast cells was assessed on acute dermal wound healing. MATERIALS AND METHODS: Fibroblasts were cultured and transfected with IGF-1. Lipofectamine 2000 was used as a reagent of transfection. Transgene expression levels were measured by the enzyme linked immunosorbent assay (ELISA). To study in vivo, rats (weighing 170-200 g) were randomly divided into three groups (five/group) and full-thickness wounds were created on the dorsum region. Suspensions of transfected fibroblast cells were injected into the wound and were compared with wounds treated with native fibroblast cells and normal saline. For the microscopic examination, biopsy was performed on day seven. RESULTS: In vitro, the maximum expression of IGF1 (96.95 pg/mL) in transfected fibroblast cells was 24 hours after gene transfer. In vivo, it was clear that IGF-1 gene therapy caused an increase in the number of keratinocyte cells during the wound healing process (mean of group A vs. group B with P value = 0.01, mean of group A vs. group C with P value = 0.000). Granulation of tissue formation in the transfected fibroblast group was more organized when compared with the normal saline group and native fibroblast cells. CONCLUSIONS: This study indicated that the optimization of gene transfer increases the expression of IGF-1. High concentrations of IGF-1, in combination with cell therapy, have a significant effect on wound healing.