Spirulina platensis (Arthrospira platensis) attenuates Cyclophosphamide-induced reproductive toxicity in male Wistar rats: Evidence for sperm apoptosis and p53/Bcl-2 expression.

Cyclophosphamide is an antitumor agent that causes disorders in fertility. This study aimed to evaluate the protective effects of Spirulina platensis against Cyclophosphamide-induced testicular toxicity. 42 male Wistar rats were randomly divided into six groups. Experimental groups included three groups. The first experimental group received Cyclophosphamide at a dose of 5 mg/kg body weight (BW) orally. The second and third experimental groups received 5 mg/kg BW Cyclophosphamide and 500 and 1,000 mg/kg BW S. platensis orally, respectively. The control groups included a control group, and two S. platensis control groups. Following 28 days, two flow cytometry techniques were used to determine sperm apoptosis and testicular protein expression of tumor protein (p53) and B-cell lymphoma 2 (Bcl-2). p53 is a tumor suppressor protein that causes the cell to enter the apoptosis cycle after DNA damage and Bcl-2 is an anti-apoptotic protein that acts through the mitochondrial pathway of apoptosis. FITC-Annexin V assay was used for sperm apoptosis evaluation. For protein expression assay, primary and secondary antibodies staining were performed. The Cyclophosphamide group showed a significant increase in sperm apoptosis compared to the control group. Cyclophosphamide significantly increased p53 and decreased Bcl-2 expression compared to the control group. S. platensis co-treated groups exhibited a significant decrease in sperm apoptosis compared to the Cyclophosphamide group. Moreover, S. platensis co-treated groups displayed a significant decreasing in p53 and increasing in Bcl-2 expression compared to the Cyclophosphamide group. The results of this study indicated that S. platensis protected rats against Cyclophosphamide-induced reproductive toxicity. PRACTICAL APPLICATIONS: Cyclophosphamide is the chemotherapy agent used to treat different cancers. Cyclophosphamide has side effects on the male reproductive system. Spirulina plantesis has a protective effect because of its antioxidant and anti-apoptotic properties. Co-administration of Spirulina plantesis with Cyclophosphamide reduces sperm apoptosis also decreases P53 protein expression and increases Bcl-2 protein expression. This study validated the anti-apoptotic potential of Spirulina plantesis against Cyclophosphamide-induced male reproductive toxicity.

Effects of liraglutide on sperm characteristics and fertilization potential following experimentally induced diabetes in mice.

The current study was conducted to analyze the dose-dependent effects of liraglutide against the diabetes-induced detrimental impact on sperm parameters and fertilization potential. For this purpose, 42 adult male mice were randomly divided into control (with no intervention) and experimental groups. Next, the experimental group was subdivided into diabetic, 1.20 mg kg-1 liraglutide-received diabetic, 1.80 mg kg-1 liraglutide-received diabetic, 1.20 mg kg-1 liraglutide-received non-diabetic and 1.80 mg kg-1 liraglutide-received non-diabetic groups. All chemicals were administrated subcutaneously. Following 42 days, the animals were euthanized, and sperm samples were collected. The sperm count, motility, viability, DNA integrity, and maturity were analyzed and compared between groups. Moreover, the sperm fertilization potential was investigated by in vitro fertilization (IVF). For this purpose, the preimplantation embryo development at 2-cell, 4-cell, morula, and blastocyst stages was investigated and compared. Observations revealed that diabetes significantly diminished sperm count, motility, viability, chromatin condensation, and DNA integrity percentages versus a control group. On the other hand, 1.20 mg kg-1 and 1.80 mg kg-1 of liraglutide did not improve sperm motility and viability, while ameliorated sperm count and chromatin condensation and DNA integrity in diabetic animals. The diabetic animals represented diminished preimplantation embryo development, which was not altered in liraglutide-received groups. In conclusion, at least in administrated doses, liraglutide could not improve the sperm viability and motility and, via this mechanism, could not induce an appropriate/beneficial effect on IVF outcome.

Experimental diabetes negatively affects the spermatogonial stem cells' self-renewal by suppressing GDNF network interactions.

The present study was done to analyse the time-dependent effects of diabetes on Sertoli cells-spermatogonial stem cells' (SSCs) network interaction by focusing on glial cell line-derived neurotrophic factor (GDNF) and its special receptors, gfrα1 and c-RET as well as the Bcl-6b. In total, 40 Wistar rats were considered in; control, 20, 45 and 60 days diabetes-induced groups. An experimental diabetes was induced by STZ. The GDNF, gfrα1, c-RET and Bcl-6b expressions were evaluated. The serum level of testosterone, tubular repopulation (RI) and spermiogenesis (SPI) indices, general histological alterations, germ cells, mRNA damage, sperm count and viability were assessed. The diabetes, in a time-dependent manner, diminished mRNA and protein levels of GDNF, gfrα1, c-RET and Bcl-6b versus control group (p < .05), enhanced percentage of seminiferous tubules with negative RI, SPI, and diminished Leydig and Sertoli cells distribution, serum levels of testosterone, sperm count and viability. Finally, the number, percentage of cells and seminiferous tubules with normal mRNA content were significantly (p < .05) diminished. In conclusion, as a new data, we showed that the diabetes by inducing severe mRNA damage and suppressing GDNF, gfrα1, c-RET and Bcl-6b expressions, potentially affects the Sertoli-SSCs' network and consequently inhibits the SSCs' self-renewal process.

Deoxynivalenol reduces quality parameters and increases DNA damage in mice spermatozoa.

This study was performed to investigate in vitro effects of deoxynivalenol (DON) on mice sperm quality parameters including viability, motility and DNA damages at various concentrations and exposure times. Mice spermatozoa were exposed to DON at 0, 2.5, 5 and 10 µM for 1, 3 and 6 hr, motility parameters were evaluated by computer-assisted analysis and viability was examined by colorimetric metabolic activity assay and HOS test. DNA damage was examined by acridine orange staining, and sperm damages via lipid peroxidation pathway were determined by malondialdehyde (MDA) content measurement. DON affected sperm parameters in a concentration- and time-dependent manner. In all test groups, the average path velocity and progressive motile spermatozoa were remarkably reduced. In comparison with the controls, after 1, 3 and 6 hr exposure to DON, viability of spermatozoa was reduced 25, 30 and 49% respectively. DON exposure at 10 µM for 6 hr resulted in 15% DNA damage and 2.5-fold more MDA generation, when compared with nonexposed spermatozoa. Our data suggest that DON causes sperm quality parameters decline in concentration- and time-dependent fashion, which attribute to the reduction in sperm metabolic activity and membrane integrity and equally to increase in lipid peroxidation rate and DNA damage.

Antioxidant effects of Spirulina platensis (Arthrospira platensis) on cyclophosphamide-induced testicular injury in rats.

Cyclophosphamide (CP) is known to reduce fertility. The protective effects of Spirulina plantesis (SP) against CP-induced testicular toxicity were investigated. Male Wistar rats were categorized into eight groups (n = 7). Four groups of rats were administered CP at a dose of 5 mg in 5 mL distilled water kg-1 per day orally. Two of these groups were received SP (500 and 1000 mg kg-1 per day) orally after CP administration. One of these groups was also received vitamin E (100 mg kg-1 per day) intraperitoneally. A vehicle treated control group, two SP control groups (500 and 1000 mg kg-1 per day) and a vitamin E control group were also included. Body and testes weights, sperm count, serum levels of glutathione peroxidase (GPx), malondialdehyde (MDA), histological and histomorphometric alternations in testes were investigated after four weeks. The CP-treated group exhibited significant decreases in the body and testes weights and spermatogenic activities. Several histological alterations were observed in this group. The CP treatment caused a significant reduction in sperm count, in serum level of GPx, as well increased serum concentration of MDA. The SP co-administration caused an increase in GPx serum level, a decrease in MDA serum level and improvements in histological and histomorphometric alternations. Vitamin E co-treatment showed partial recovery in above-mentioned parameters. These results suggest that SP due to a reduction in oxidative stress has more effective protection against CP-induced reproductive damages in rat than vitamin E.

Ameliorative effects of Achillea millefolium inflorescences alcoholic extract on nicotine-induced reproductive toxicity in male rat: Apoptotic and biochemical evidences.

Nicotine (NIC) adversely influences male reproductive system. Achillea millefolium (Achm) as a medicinal plant is highly regarded for its antioxidant and anti-inflammatory properties. The present study was conducted to assess whether Achm inflorescences alcoholic extract could serve as a protective agent against reproductive toxicity in NIC-exposed male rats. Adult male rats were randomly divided into six groups. Two groups received NIC at doses of 0.20 and 0.40 mg kg-1 per day in 0.50 mL sterile distilled water for 48 days intraperitoneally, respectively. The further two groups received NIC at doses of 0.20 and 0.40 mg kg-1 per day in 0.50 mL sterile distilled water for intraperitoneally along with Achm extract at a dose of 1.20 g kg-1 per day in 1 mL sterile distilled water orally for 48 days, respectively. A vehicle treated control group and an Achm-only treated group were also included. The NIC-exposed groups showed significant reductions in epididymal sperm count, motility, viability and serum levels of FSH, LH and testosterone as well as testicular antioxidant capacity. Moreover, the incidence of apoptosis and abnormality in spermatozoa along with testicular malondialdehyde and total nitrite levels were significantly higher in NIC-treated rats. The above-mentioned parameters were restored to near normal levels by Achm co-administration. These findings indicated that Achm may partially be protective against NIC-induced testicular toxicity.

Nicotine-induced damages in testicular tissue of rats; evidences for bcl-2, p53 and caspase-3 expression.

OBJECTIVES: Present study was performed in order to uncover new aspects for nicotine-induced damages on spermatogenesis cell lineage. MATERIALS AND METHODS: For this purpose, 36 mature male Wistar rats were divided into three groups as; control-sham (0.2 ml, saline normal, IP), low dose (0.2 mg/kg BW-1, IP) nicotine-received and high dose (0.4 mg/kg BW-1, IP) nicotine-received groups. Following 7 weeks, the expression of bcl-2, p53 and caspase-3 at mRNA and protein levels were investigated by using reverse-transcriptase PCR (RT-PCR) and immunohistochemical (IHC) analyses, respectively. Moreover, the serum level of FSH, LH and testosterone were evaluated. Finally, the mRNA damage was analyzed by using special fluorescent staining. RESULTS: Nicotine, at both dose levels, decreased tubular differentiation, spermiogenesis and repopulation indices and enhanced cellular depletion. Animals in nicotine-received groups exhibited a significant (P<0.05) reduction at mRNA and protein levels of bcl-2. More analyses revealed a remarkable (P<0.05) enhancement in expression of p53 and caspase-3 in comparison to control-sham animals. Finally, nicotine resulted in a significant (P<0.05) reduction in serum level of testosterone and elevated mRNA damage. CONCLUSION: Our data showed that, nicotine by suppressing the testosterone biosynthesis, reducing mRNA and protein levels of bcl-2 and up regulating the p53 and caspase-3 mRNA and protein levels adversely affects the spermatogenesis and results in cellular depletion.

Crataegus monogyna fruit aqueous extract as a protective agent against doxorubicin-induced reproductive toxicity in male rats.

OBJECTIVE: Doxorubicin (DOX) is a broad spectrum chemotherapeutic agent used in the treatment of several malignancies. The use of DOX in clinical chemotherapy has been restricted due to its diverse toxicities, including reproductive toxicity. Crataegus monogyna (C. monogyna) is one of the oldest medicinal plants that have been shown to be cytoprotective because of scavenging free radicals. The present study was undertaken to determine whether C. monogyna fruits aqueous extract could serve as a protective agent against reproductive toxicity during DOX treatment in a rat model through antioxidant-mediated mechanisms. MATERIALS AND METHODS: Male Wistar rats were allocated to four groups. Two groups of rats were treated with DOX at a dose of 4 mg/kg intraperitoneally on days 1, 7, 14, 21, and 28 (accumulated dose of 20 mg/kg). One of the groups received C. monogyna fruits aqueous extract at a dose of 20 mg/kg per day orally for 28 days along with DOX. A vehicle-treated control group and a C. monogyna control group were also included. RESULTS: The DOX-treated group showed significant decreases in the body and organ weights and spermatogenic activities as well as many histological alterations. DOX treatment also caused a significant decrease in sperm count and motility with an increase in dead and abnormal sperms. Moreover, significant decrease in serum levels of testosterone and increased serum concentrations of FSH, LH, LDH, CPK, and SGOT were observed in DOX-treated rats. Notably, Crataegus co-administration caused a partial recovery in above-mentioned parameters. CONCLUSION: These findings indicated that doxorubicin can adversely damage the testicular tissue, while Crataegus co-administration could effectively prevent these adverse effects by effective inhibiting oxidative processes and restoration of antioxidant defense system.

The effects of pyridaben pesticide on the histomorphometric, hormonal alternations and reproductive functions of BALB/c mice.

OBJECTIVE(S): The adverse effects of pyridaben on reproductive system in male animals are not well established. This study was designed to elucidate how pyridaben can effects the histomorphometric, hormonal alternations and reproductive functions of BALB/c mice. Materials and Methods : For this study, 80 adult and apparently healthy male BALB/c mice were divided into three groups Viz, control, test group 1 and test group 2. Test groups 1 and 2 were received the toxin at doses of 53 mg/kg. BW, and 212 mg/kg. BW, respectively. The experiment period for both groups was 10, 25 and 45 days. Results : The levels of FSH, LH and testosterone were significantly (P<0.05) decreased on the dose and time dependant means. The levels of the ROS and NOS were significantly (P<0.05) increased in all test groups. The percent body weight gains significantly (P<0.05) reduced, whereas weights significantly (P<0.05) increased in test groups in a dose and time dependant manner. The histomorphometric and stereologic findings, including diameters of somniferous tubules, thickness of somniferous tubules epithelium, the leydig's cell distribution, TDI, SI, RI revealed that, all these parameters are also significantly (P<0.05) reduces in test groups in a dose and time dependant manner. C onclusion : Pyridaben causes histomorphometric and stereologic changes in testis, as well as hormonal and reproductive functional alternations in BALB/c mice.

Decapeptyl ameliorates cyclophosphamide-induced reproductive toxicity in male Balb/C mice: histomorphometric, stereologic and hormonal evidences.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) is a reproductive key hormone. The GnRH analogues are widely used in in vitro fertilization and treatment of sex hormone-depended cancers induced by the materials used in chemotherapeutic agents. OBJECTIVE: The aim of this study is to evaluate the effects of cyclophosphamide and decapeptyl (analogues of GnRH) on histomorphometry and stereology of testicular tissue as well as gonadotropic and gonadal hormones indices in mice. MATERIALS AND METHODS: For this study, 24 adult male Balb/C strain mice were divided in four groups; first, cyclophosphamide (65 mg/kg/body weight (BW)), second, decapeptyl (0.05 mg/kg/BW), third, decapeptyl at first, and after 10 days of cyclophosphamide injection, and control group was received same volume of sterile saline. In order to evaluate the tissue changes in testes of the mice, sections were prepared and stained with Hematoxylin-Eosine, Periodic Acid Schief's (PAS) and Oil-Red-O staining techniques. RESULTS: The cyclophosphamide causes histomorphologic changes in the testicular tissue; whereas such changes by decapeptyl were comparatively mild. The morphometric results revealed significant reduction in diameters of seminiferous tubules (p=0.02), and the stereological results confirmed significant differences in spermatogenesis (SI) as well as rate of tubal differentiation (TDI) indices between experimental and control groups (p=0.001). In addition, the morphometric findings proved that, there are significant decrease (p=0.001) in thicknesses of epithelia and stereologic result revealed reduction in number of cell layers in both decapeptyl and chemotherapy groups, but the decrements of these parameters were significant (p=0.02) in later group. In groups that had received cyclophosphamide, and decapeptyl alone, the LH and testosterone levels were decreased significantly (p=0.03), whereas in those that had received decapeptyl along with cyclophosphamide, the LH and FSH levels showed a decline but the level of testosterone increased. CONCLUSION: These results demonstrated that, analogue of GnRH i.e., decapeptyl protect morphologic, morphometric, and stereologic alterations of the testes tissue, as well as gonadotropic and gonadal hormonal changes preceding cyclophosphamide treatment in male mice. This article extracted from M.Sc. thesis. (Afsaneh Niakani).

The effects of pyridaben pesticide on the DNA integrity of sperms and early in vitro embryonic development in mice.

BACKGROUND: Pyridaben, a pyridazinone derivative, is a new acaricide and insecticide for control of mites and some insects such as white flies, aphids and thrips. OBJECTIVE: This study was designed to elucidate how pyridaben can affect the sperms' morphological parameters, its DNA integrity, and to estimate the effect of various quantities of pyridaben on in vitro fertilization rate. MATERIALS AND METHODS: In this study, 80 adult male Balb/C strain mice were used. Animals were divided into control and two test groups. Control group received distilled water. The test group was divided into two subgroups, viz, high dose (212 mg/kg/day) and low dose (53 mg/kg/day) and they received the pyridaben, orally for duration of 45 days. The spermatozoa were obtained from caudae epididymides on day 45 in all groups. Sperm viability, protamin compression (nuclear maturity), DNA double-strand breaks, and in vitro fertilizing (IVF) ability were examined. RESULTS: The pyridaben treatment provoked a significant decrease in sperm population and viability in epididymides. The data obtained from this experiment revealed that, the pyridaben brings about negative impact on the sperm maturation and DNA integrity in a time-dependent manner, which consequently caused a significant (p<0.05) reduction in IVF capability. Embryo developing arrest was significantly (p<0.05) higher in treated than the control group. CONCLUSION: Theses results confirmed that, the pyridaben is able to induce DNA damage and chromatin abnormalities in spermatozoa which were evident by low IVF rate. This article extracted from Ph.D. thesis. (Ghodrat Ebadi Mans).

Crataegus monogyna aqueous extract ameliorates cyclophosphamide-induced toxicity in rat testis: stereological evidences.

Cyclophosphamide (CP) is extensively used as an antineoplastic agent for the treatment of various cancers, as well as an immunosuppressive agent. However, despite its wide spectrum of clinical uses, CP is known to cause several adverse effects including reproductive toxicity. Crataegus monogyna is one of the oldest pharmaceutical plants that have been shown to be cytoprotective by scavenging free radicals. The present study was conducted to assess whether Crataegus monogyna fruits aqueous extract with anti-oxidant properties, could serve as a protective agent against reproductive toxicity during CP treatment in a rat model. Male Wistar rats were categorized into four groups. Two groups of rats were administered CP at a dose of 5 mg in 5 ml saline/kg/day for 28 days by oral gavages. One of these groups received Crataegus monogyna aqueous extract at a dose of 20 mg/kg/day orally four hours after cyclophosphamide administration. A vehicle treated control group and a Crataegus monogyna control group were also included. The CP-treated group showed significant decreases in the body, testes and epididymides weights as well as many histological alterations. Stereological parameters and spermatogenic activities (Sertoli cell, repopulation and miotic indices) were also significantly decreased by CP treatment. Notably, Crataegus coadministration caused a partial recovery in above-mentined parameters. These findings indicate that Crataegus monogyna may be partially protective against CP-induced testicular toxicity.

The ultrastructural changes of the sertoli and leydig cells following streptozotocin induced diabetes.

OBJECTIVES: This investigation was conducted to evaluate the effects of diabetes on the structure and function of testicular tissue. MATERIALS AND METHODS: Diabetes was induced in male adult rats by a single intraperitoneal injection of streptozotocin. Body and testicular weight, hormonal analyses, histological and ultrastructural analyses were measured. RESULTS: The body and testicular weights were dropped significantly (P< 0.05) in diabetic rats in comparison with control rats. On the other hand, in diabetic rats, the blood glucose level increased significantly (P< 0.05). The blood plasma levels of testosterone, 17-ß estradiol, progesterone, FSH and LH were reduced in diabetic rats. Histomorphological studies were revealed reduction in diameter of seminiferous tubules and germinal epithelium height, edema in interstitial tissue, germ cell depletion, decrease in cellular population and activity with disruption of spermatogenesis in diabetic rats. Ultrastructural study showed the mitochondrial change and reduction of smooth endoplasmic reticulum in Sertoli and presence of lipid droplets in Leydig cells of diabetic rat's testes. CONCLUSION: The results of the present study confirmed that, the ultrastructural changes of Sertoli and Leydig cells, brought about by streptozotocin induced diabetes, because of the alterations in pituitary gonadotropins, and these changes influence the normal spermatogenesis in rats.

Defense cells profile of cervical mucous during follicular and luteal phases of estrus cycle in river buffalo.

The aim of this study was to evaluate the defense cells changes of cervical mucous during follicular and luteal phases of estrus cycle in river buffalo. Reproductive organs of the adult and apparently healthy female buffaloes were collected from the slaughterhouse. By visual investigation of both the ovaries for presence of corpus luteum and growing follicles, the luteal and follicular phase of each buffalo was specified. Cervical discharge samples were collected by sterile swabs and then spread over the glass slides, dried and fixed with methanol. The specimens were undergone Giemsa staining. The percentage of lymphocytes, neutrophils, monocytes (macrophages), eosinophils and basophils in each case (for both the follicular and luteal phases) were obtained at 20 microscopic fields. The percentage of lymphocytes, neutrophils and basophils in luteal phase were higher than the follicular phase. The percentage of eosinophils in follicular phase was higher than the luteal phase. The percentage of monocytes (macrophages) in luteal and follicular phases was nearly equal. The statistical analysis showed that the differences of all cells between follicular and luteal phase were not significant (P > 0.05). The most defense cells in discharges of external os of cervix (both follicular and luteal phases) were neutrophils and lymphocytes.

An investigation on body weights, blood glucose levels and pituitary-gonadal axis hormones in diabetic and metformin-treated diabetic female rats.

Diabetes is a metabolic disorder which affects whole body systems including reproductive system. Diabetes is also a contributing factor to infertility. Metformin is one of the most common drugs to control hyperglycemia. In this study, 36 adult Sprague-Dawley female rats (170-210 g) were divided into 3 groups (control, diabetic and diabetic-treated by metformin). In second and third groups, diabetes was induced by streptozotocin injection (45 mg kg(-1), IP) and the third group was treated by metformin hydrochloride (100 mg kg(-1) day(-1), PO) for 8 weeks. Body weights were compared and blood glucose, gonadotropins and sexual hormones were measured. In diabetic group the blood glucose level significantly (P < 0.05) increased in comparison with that of control and metformin-treated diabetic rats. The results also revealed that, in the untreated diabetic rats, the mean body weights and pituitary-gonadal axis hormones were significantly (P < 0.05) reduced in comparison with the control. Although there were significant (P < 0.05) reduction in mean body weights in metformin-treated diabetic rats, reduction in pituitary-gonadal axis hormones was not as sharp as in untreated diabetic rats and only level of progesterone was significantly (P < 0.05) reduced in comparison with the control. The results of this investigation revealed that there was a clear relationship between experimental diabetes with body weight and pituitary-gonadal axis hormones, and treatment with metformin relatively restored diabetic complications.