Effect of Glyphaea brevis twigs extract on cell viability,apoptosis induction and mitochondrial membrane potential in TM3 Leydig cells

Glyphaea brevis twigs (Spreng) Monach. (GBT) are used by local herb healers to manage male sexual fertility disorders. The aim of this study was to examine the effects of G. brevis twigs on TM3 Leydig cells. GBT was extracted using methanol solvent, and Leydig cells were exposed to the respective concentrations of GBT extract (0.1, 1, 10, 100 and 1,000 μg/ml) for 24 and 72 hr respectively. Parameters evaluated include cell morphology, viability (MTT assay), mitochondrial membrane potential (TMRE dye), apoptosis (Annexin V Alexa Fluor 488 binding) and RT‐qPCR analyses of the mRNA expression. Results revealed that GBT had no cytotoxic effect on cell viability and the cell morphology. GBT also revealed a considerable elevation (p < 0.05) in fluorescence intensity, accompanied by intact mitochondria in TM3 Leydig cells. Furthermore, GBT resulted in the reduction of necrotic and apoptotic cells. The mRNA StAR was upregulated markedly with the effect prominent at 100 μg/ml. This study showed that GBT might be useful for managing male infertility ailments.     

Androgenic effect of aqueous leaf extract of Moringa oleifera on Leydig TM3 cells in vitro

Moringa oleifera (MO) is an excellent source of dietary antioxidant. MO is used traditionally to enhance libido and as an aphrodisiac in the treatment of sexual dysfunction. This study aimed to investigate the direct effect of aqueous leaf extract of MO on Leydig cell in vitro. Specifically, the effect of MO on viability, testosterone production, antioxidant activity and lipid peroxidation on TM3 cells were evaluated. TM3 cells seeded for 24 hr were exposed to aqueous leaf extract of MO (0, 10, 50, 100, 250, 500 and 1,000 µg/ml) for 24 hr, in the absence or presence of human chorionic gonadotropin (hCG; 6 mIU/200 µl). Cell viability remained unchanged while testosterone production significantly increased at 500 and 1,000 µg/ml of the extract under stimulatory conditions by 34 and 45% respectively. Glutathione level substantially increased at 250 µg/ml, while lipid peroxidation, catalase and superoxide dismutase activity, and total antioxidant capacity remained unchanged. Our results demonstrate the androgenic effect of MO especially at high concentrations in TM3 cells. The androgenic effect may be attributed to its antioxidant enzyme activities.     

Feeding hydroalcoholic extract powder of Lepidium meyenii (maca) increases serum testosterone concentration and enhances steroidogenic ability of Leydig cells in male rats

Although Lepidium meyenii (maca), a plant growing in Peru's central Andes, has been traditionally used for enhancing fertility and reproductive performance in domestic animals and human beings, effects of maca on reproductive organs are still unclear. This study examined whether feeding the hydroalcoholic extract powder of maca for 6 weeks affects weight of the reproductive organs, serum concentrations of testosterone and luteinising hormone (LH), number and cytoplasmic area of immunohistochemically stained Leydig cells, and steroidogenesis of cultured Leydig cells in 8‐week‐old male rats. Feeding the extract powder increased weight of seminal vesicles, serum testosterone level and cytoplasmic area of Leydig cells when compared with controls. Weight of prostate gland, serum LH concentration and number of Leydig cells were not affected by the maca treatment. The testosterone production by Leydig cells significantly increased when cultured with 22R‐hydroxycholesterol or pregnenolone and tended to increase when cultured with hCG by feeding the extract powder. The results show that feeding the hydroalcoholic extract powder of maca for 6 weeks increases serum testosterone concentration associated with seminal vesicle stimulation in male rats, and this increase in testosterone level may be related to the enhanced ability of testosterone production by Leydig cells especially in the metabolic process following cholesterol.     

Taurine ameliorates cytotoxic effects of Di(2-ethylhexyl) phthalate on Leydig cells

It has been revealed that di(2-ethylhexyl)phthalate (DEHP) has toxic impacts on the male reproductive system. Taurine (TAU) is an amino acid with antioxidant property and beneficial impacts on the male reproductive system. In this study, protective impacts of Taurine (TAU) on DEHP-induced Leydig TM3 cell toxicity were investigated. The cells exposed to DEHP (0.8 µmol) or TAU (100 mg/ml) for 24 hr. Cell viability (MTT assay), apoptosis, oxidative stress and testosterone level were examined. DEHP could significantly decrease the cell viability percentage, reduce testosterone level, increase apoptosis, elevate Bax/ Bcl-2 ratio and enhance caspase-3 and -9 activity in the TM3 cells. Additionally, DEHP significantly elevated malondialdehyde contents and reactive oxygen species levels. It also augmented superoxide dismutase and catalase activity in the Leydig cells. Co-treatment of DEHP with TAU increased viability and testosterone level, while oxidative stress and apoptosis significantly reduced. TAU could decrease Bax/Bcl-2 ratio and caspase-3 and -9 activity in the DEHP-intoxicated cells. Our results have clearly shown that TAU protects TM3 cells against oxidative stress and apoptosis induced by DEHP.     

Long‐term feeding of hydroalcoholic extract powder of Lepidium meyenii (maca) enhances the steroidogenic ability of Leydig cells to alleviate its decline with ageing in male rats

This study examined whether feeding hydroalcoholic extract of Lepidium meyenii (maca) to 8‐week‐old (sexually maturing) or 18‐week‐old (mature) male rats for more than a half year affects serum testosterone concentration and testosterone production by Leydig cells cultured with hCG, 22R‐hydroxycholesterol or pregnenolone. Testosterone concentration was determined in the serum samples obtained before and 6, 12, 18 and 24 weeks after the feeding, and it was significantly increased only at the 6 weeks in the group fed with the maca extract to maturing rats when it was compared with controls. Testosterone production by Leydig cells significantly increased when cultured with hCG by feeding the maca extract to maturing rats for 27 weeks (35 weeks of age) and when cultured with 22R‐hydroxycholesterol by feeding it to mature rats for 30 weeks (48 weeks of age). Overall testosterone production by cultured Leydig cells decreased to about a half from 35 to 48 weeks of age. These results suggest that feeding the maca extract for a long time to male rats may enhance the steroidogenic ability of Leydig cells to alleviate its decline with ageing, whereas it may cause only a transient increase in blood testosterone concentration in sexually maturing male rats.     

Effect of Cissampelos capensis rhizome extract on human spermatozoa in vitro

Cissampelos capensis is commonly known by the Afrikaans name ‘dawidjies’ or ‘dawidjieswortel’. C. capensis is the most important and best‐known medicinal plant of the family Menispermaceae used by the Khoisan and other rural people in the western regions of South Africa. Among numerous other ailments, it is traditionally taken to treat male fertility problems. Yet, no studies have investigated the effects of this plant or its extracts on human spermatozoa. The aim of study was to investigate the effects of C. capensis extracts on sperm function. A total of 77 semen samples were collected. Spermatozoa were washed with HTF‐BSA medium and incubated with different concentrations of C. capensis (0, 0.05, 0.5, 5, 50, 200 μg ml^?1) for 1 h at 37 °C. Sperm motility, vitality, acrosome reaction, reactive oxygen species (ROS), capacitation, Annexin V binding, DNA fragmentation and mitochondrial membrane potential (Δψ_m) were determined. While viability, Annexin V positivity and Δψ_m were not affected, the percentages of ROS‐positive, TUNEL‐positive, capacitated and hyperactivated spermatozoa increased significantly and dose‐dependently. It is concluded that the alkaloids present in the extract of C. capansis rhizomes triggered sperm intrinsic superoxide production leading to sperm capacitation and DNA fragmentation.     

Anti-androgenic,anti-oxidant and anti-apoptotic effects of the aqueous and methanol extracts of Pterorhachis zenkeri (Meliaceae): Evidence from in vivo and in vitro studies

The aim of this study was to evaluate the effects of Pterorhachis zenkeri (Meliaceae) on sex organ growth in immature male rats and, oxidative stress and apoptosis markers in CCL-97 (R2C) Leydig cells. For the in vivo studies, 70 immature male Wistar rats (n = 10/group) were treated for 2 or 4 weeks with: distilled water (10 ml/kg, per os) plus soya oil (1 ml/kg, sc), bicalutamide (10 mg/kg, per os), aqueous or methanol extract of P. zenkeri (10 mg/kg or 62 mg/kg, per os) or testosterone propionate (3 mg/kg, sc). After each treatment period, body and sexual organ weights, plasmatic testosterone, total proteins and total cholesterol levels were measured. In the in vitro test, the effects of the methanol extract of P. zenkeri on cell viability, apoptosis, reactive oxygen species (ROS) production, intracellular calcium release and caspases 3/9 were assessed using CCL-97 Leydig cells. Pterorhachis zenkeri extracts decreased sex organ weights, plasmatic testosterone and protein levels in rats. In the in vitro studies, P. zenkeri inhibited apoptosis, ROS production, calcium release and caspase 3/9 activities. These results suggest that P. zenkeri has anti-androgenic, anti-oxidant and anti-apoptotic activities with methanol extract being the most active and could be an effective alternative for the management of androgen-related diseases.     

Effects of perfluorooctanoic acid exposure during pregnancy on the reproduction and development of male offspring mice

This study was conducted to explore the effects of maternal exposure to perfluorooctanoic acid (PFOA) on reproduction and development of male offspring mice. Pregnant mice were given 1, 2.5 or 5 mg/kg BW PFOA daily by gavage during gestation. The results showed that the survival number of offspring mice at weaning was significantly decreased. There were no differences in the testicular index of offspring mice between PFOA exposure groups and non‐PFOA group. Maternal exposure to PFOA reduced the level of testosterone in the male offspring mice on PND 21 (p < 0.01) but increased in 1 mg/kg group and decreased in 2.5 and 5 mg/kg groups on PND 70 (p < 0.01). There were different degrees of damage to testis in a dose‐dependent manner, and the number of Leydig cells markedly decreased (p < 0.01) in 2.5 and 5 mg/kg PFOA groups on PND 21 and PND 70. The expression of Dlk1‐Dio3 imprinted gene cluster showed a decreasing trend, where Glt2, Rian and Dio3 gene expressions were significantly reduced (p < 0.05) on PND 21. Therefore, PFOA exposure during pregnancy reduces the number of survival offspring mice, damages testis, disrupts reproductive hormones and reduces the mRNA expressions of the Dlk1‐Dio3 imprinted cluster in testis.     

Preventive effects of Aframomum melegueta extracts on the reproductive complications of propylthiouracil‐induced hypothyroidism in male rat

Recent studies have demonstrated that hypothyroidism is associated with infertility. This work was undertaken to evaluate the protective effects of Aframomum melegueta on testicular functions and fertility of hypothyroid male rats. Male rats were orally treated with propylthiouracil (PTU: 10 mg/kg) in combination with plant aqueous or methanol seed extract (20 and 100 mg/kg) for 56 days. Vitamin E and clomiphene citrate served as positive controls. On day 47 of treatment, each male was mated with two adult females for fertilization potential evaluation. At the end of the treatment, genital sex organ weights, sperm characteristics, testicular histology, oxidative status, plasmatic hormones and fertility potential were evaluated. Results indicated that PTU created hypothyroidism characterised by a significant increase in TSH with reduction of T3 and T4. PTU also lowered genital sex organ weights, sperm count, viability and motility, plasmatic levels of luteinising hormone, follicle‐stimulating hormone and testosterone, and increased prolactin, cholesterol and testicular oxidative stress. Alteration in sperm morphology, testis and epididymis histology, and fertilization potential was also noticed. Co‐administration with A. melegueta extracts successfully reversed PTU‐induced infertility without any effect on thyroid hormones. These results provide evidence that A. melegueta has a protective effect on fertility in hypothyroid condition.     

Comparison of the efficacy of three concentrations of retinoic acid for transdifferentiation induction in sheep marrow‐derived mesenchymal stem cells into male germ cells

Recent studies have shown the unique role of retinoic acid (RA) in the induction of transdifferentiation in mesenchymal stem cells (MSCs) into germ cells (GCs). This study is the first study that compares the efficacy of three different concentrations of RA for the production of male GCs in vitro. Male sheep marrow‐derived MSCs (MMSCs) were treated with the following concentrations of RA: 1 μm (RA1), 5 μm (RA2) and 10 μm (RA3) for a period of 21 days. The production of male GCs was evaluated by the assessment of expressions of GC‐specific markers (by RT‐PCR, qRT‐PCR and immunocytochemistry), morphological characteristics and changes in alkaline phosphatase (ALP) activity. All three concentrations created male GC features. RA treatment upregulated the expressions of VASA and beta1 INTEGRIN and downregulated PIWIL2 and OCT4. DAZL was not expressed by RA treatment. Interestingly, immunocytochemistry detected PGP 9.5 expression in all treatment groups, with the highest expression noted in the RA3 group (P < 0.05). GC‐like cells along with increased ALP activity were observed in all treated cultures, too. Finally, results showed that 10 μm RA has the most efficiency for transdifferentiation induction in MMSCs and production of male GCs in vitro.     

Protective efficiency of Ashrasi date palm hydroalcoholic extract against diabetes‐induced testicular toxicity: A biochemical and stereological study

The present study was designed to investigate the protective effects of hydroalcoholic extract of Ashrasi date palm (ADP) on diabetes‐induced testicular injuries. Adult male rats were randomly allocated into five groups (n = 8 in each group): 1: control; 2: diabetic; 3: diabetic + 30 mg/kg of ADP extract; 4: diabetic + 90 mg/kg of ADP extract; and 5: diabetic + 270 mg/kg of ADP extract. Diabetes was induced by a single dose of streptozotocin (55 mg/kg) intraperitoneally. Testicular changes were assessed quantitatively using stereological method followed by measuring antioxidant enzymes including catalase, superoxide dismutase and glutathione peroxidase and serum testosterone level. Malondialdehyde (MDA) and Bcl‐2 expression were also evaluated in tissue samples. Diabetes resulted in significant deleterious alterations in the architecture of testicular tissue, suppressed antioxidant enzymes and testosterone levels and increased lipid peroxidation. The expression of Bcl‐2 was downregulated in diabetic testis and resulted in enhanced apoptosis. Eight weeks of ADP extract treatment especially at higher doses could markedly improve structural changes of testis and restore the antioxidant defence and testosterone levels in testicular tissue. In conclusion, this findings showed that ADP extract can play as a potent antioxidant and can attenuate the adverse effects of diabetes on male reproduction.     

Corrective role of Eugenia jambolana on testicular impairment in streptozotocin‐induced diabetic male albino rat: An approach through genomic and proteomic study

The present study was conducted to explore the effect of ethyl acetate fraction of hydro‐methanolic (40 : 60) extract of seed of Eugenia jambolana on testicular impairment in diabetic rats. In this respect, biomarkers of oxidative stress, genomics and proteomics in testicular tissue were assessed. Side by side, glycated haemoglobin, serum testosterone, activities of glutamate oxaloacetate transaminase and glutamate pyruvate transaminase in serum, epididymal sperm count including reproductive organosomatic indices were evaluated. Results indicate that a significant recovery (P < 0.05) in the levels of these parameters in fraction‐treated diabetic group in comparison with diabetic control. A significant recovery was noted (P < 0.05) in the expression of Bax and Bcl‐2 gene towards the control after the treatment of said fraction. Histological study also focused a significant recovery (P < 0.05) in the number of different generation of germ cells at stage VII of spermatogenesis in fraction‐treated diabetic group. The said fraction treatment to diabetic rat can recover the activities of serum glutamate oxaloacetate transaminase and glutamate pyruvate transaminase significantly towards the control (P < 0.05). Finally, it may be concluded that ethyl acetate fraction of seed of E. jambolana has a promiseable remedial effect on diabetes‐induced testicular dysfunctions in male rat without inducing any metabolic toxicity.     

Reduced testosterone production in TM3 Leydig cells treated with Aspalathus linearis (Rooibos) or Camellia sinensis (tea)

Flavonoids are major compounds of Aspalathus linearis and Camellia sinensis. They are classified as endocrine disruptors and some have been shown to inhibit testosterone production. TM3 Leydig cell cultures were treated with 250–5000 μg mL^?1 A. linearis (unfermented or fermented rooibos) or Camellia sinensis (green or black tea) for 24 h in the absence or presence of 6 mIU/200 μl human chorionic gonadotropin (hCG). Under nonstimulated conditions, all teas tend to decrease testosterone production (3.9–31.8%). However, under hCG‐stimulation, a significant reduction in testosterone production was observed at all concentrations by both rooibos and tea (16.3–37.9%). MTT assay and phase contrast microscopy, revealed that at 250–1000 μg ml^?1, both plants maintained the viability, proliferation and morphology of the cells, while 5000 μg ml^?1 was cytotoxic to the cells (P < 0.05). In conclusion, the results here demonstrate the anti‐androgenic property of A. linearis and C. sinensis.     

Differential expression of microRNAs in luteinising hormone‐treated mouse TM3 Leydig cells

Testosterone is primarily produced by Leydig cells of the mammalian male gonads. The cellular functions of Leydig cells are regulated by the hypothalamus–pituitary–gonad axis, whereas the microRNA (miRNA) changes of LH‐treated Leydig cells are unknown. Mouse TM3 Leydig cells were treated with LH, and deep sequencing showed that 29 miRNAs were significantly different between two groups (fold change of >1.5 or <0.5, p < .05), of which 27 were upregulated and two were downregulated. The differential expression of miR‐29b‐3p, miR‐378b, miR‐193b and miR‐3695 was confirmed by quantitative real‐time polymerase chain reaction. Bioinformatic analysis revealed that miRNAs regulated a large number of genes with different functions. Pathway analysis indicated that miRNAs were involved in the Wingless and INT‐1, adenosine 5′‐monophosphate‐activated protein kinase, NF‐kappa B and Toll‐like receptor signalling pathways. Results showed that miRNAs might be involved in the regulation of LH to Leydig cells.     

The methanolic extract of Guibourtia tessmannii (caesalpiniaceae) and selenium modulate cytosolic calcium accumulation,apoptosis and oxidative stress in R2C tumour Leydig cells: Involvement of TRPV1 channels

This study evaluated the effects of the methanolic extract of Guibourtia tessmannii (GT) and selenium (Se) on cell viability, intracellular calcium concentration ([Ca²⁺]_i), apoptosis and oxidative stress through transient receptor potential vanilloid 1 (TRPV1) channel activity in CCL‐97 (R2C) tumour Leydig cells. The cells were divided into nine groups and treated as follows: (a)‐Control, (b)‐Capsazepine (CPZ, 0.1 mM, a TRPV1 channel blocker), (c)‐Capsaicin (CAP, 0.01 mM, a TRPV1 channel activator), (d)‐GT (500 μg/ml), (e)‐GT+CPZ, (f)‐GT+CAP, (g)‐Se (200 nM), (h)‐Se+CPZ and (i)‐Se+CAP. After treatments, cell viability, [Ca²⁺]_i, apoptosis, caspase 3/9, reactive oxygen species (ROS) and mitochondrial membrane depolarisation (MMD) were evaluated. The [Ca²⁺]_i, apoptosis, caspase 3/9, MMD and ROS levels were significantly (p < 0.001) increased in CAP group, but lowered in CPZ group. Interestingly, these parameters were significantly (p < 0.001) improved by GT and Se, compared to the CAP group. Moreover, the co‐administration of GT+CAP or Se+CAP inhibited the cytotoxicity of CAP. Thus, the modulatory properties of GT and Se on Ca²⁺ influx, apoptosis and oxidative stress require the integrity of TRPV1 channel in CCL‐97 Leydig cells. These results suggest that GT and Se might be used in the management of cytotoxicity in the testes, involving TRPV1 channel activity.     

Hypobaric hypoxia causes impairment of spermatogenesis in developing rats at pre‐puberty

The effect of hypoxia on the spermatogenesis of male Wistar rats (n = 32) at pre‐puberty was studied using a hypobaric chamber simulating an altitude of 5,000 metres above sea level. Persistent hypoxic exposure with brief interruption for 3 weeks caused significant decreases in body and testis weights and testosterone level compared to the normobaric controls. Histologically, spermatogenic development was arrested; arrays of spermatids were misshaped; numbers of spermatogonia, Sertoli and Leydig cells were reduced; and apoptotic spermatocytes were increased substantially in the germinal epithelium of testis in the hypoxic‐exposed group. These hormonal and histopathological changes did not improve remarkably after 3 weeks of normobaric conditions. There was a significant decrease in sperm production when the rats in the hypoxia/oxygen‐resuming group were examined at 63 days of post‐natal age. Exposing rats to hypoxic conditions at pre‐puberty induced damages on spermatogenesis, which could affect sperm production after sex mature.     

Brysocarpus coccineus (Schum & Thonn) root reinstates sexual competence and testicular function in paroxetine‐induced sexual dysfunction in male Wistar rats

The effects of aqueous extract of Brysocarpus coccineus roots (AEBCR) were studied on sexual behaviour and testicular function of paroxetine‐induced sexual dysfunction (SD) in male rats. Ninety, sexually matured male rats (150.88 ± 5.53 g) were assigned into two groups: A and B. Fifteen SD animals from group B were each allotted to B1, B2, B3, B4 and B5 and received distilled water (DW), Powmax M (7.14 mg/kg body weight, b.w.) 50, 100 and 150 mg/kg b.w of AEBCR, respectively, for 7 days while the non‐SD animals (group A) received DW. Eleven secondary metabolites were present in AEBCR. The lowered (p < .05) ejaculation frequency, penile erection index and penile grooming, higher mount and intromission frequencies, prolonged (p < .05) latencies of mount, intromission, ejaculation, and post‐ejaculatory interval, reduced (p < .05) serum luteinising hormone, follicle stimulating hormone, testosterone, nitric oxide and testicular function indices, degenerated seminiferous tubules and low luminal spermatozoa contents by paroxetine were significantly (p < .05) attenuated and/or reinstated by AEBCR and Powmax M. The restoration of androgen‐dependent sexual and testicular functions in SD male rats by AEBCR validates its folkloric use as aphrodisiac. Clinical studies are desirable to ascertain the efficacy of AEBCR in SD.     

In vitro effects of nicotine on human spermatozoa

Washed human spermatozoa from 12 normozoospermic donors were treated with different concentrations of nicotine 0.1, 1.0, 5.0 and 10.0 mm and were compared to spermatozoa suspended in nutrient medium only (control). Computer‐aided sperm analysis was used to assess sperm kinematic properties after 30, 60, 120 and 180 min of incubation. Viability was assessed by means of a dye exclusion staining technique (eosin/nigrosin), while acrosome‐reacted cells were identified under a fluorescent microscope using fluorescein isothiocyanate–Pisum sativum agglutinin as a probe. Nicotine significantly reduced total motility, progressive motility, curvilinear velocity, amplitude of lateral head displacement, beat cross‐frequency, viability and caused spontaneous acrosome reaction at concentrations of ≥5.0 mm after 2 and 3 h of exposure. Nicotine concentrations of 0.1 and 1.0 mm had no significant effect (P < 0.05) on spermatozoa except that 1.0 mm significantly decreased (P < 0.05) sperm progressive motility at 2 and 3 h of incubation as well as viability after 3 h of incubation. This study concludes that the occurrence of high levels of nicotine in the body and seminal fluid might adversely affect fertilisation capacity of human spermatozoa through a mechanism that involves decreased motility, viability and premature induction of the acrosome reaction.     

Icariin improves the sexual function of male mice through the PI3K/AKT/eNOS/NO signalling pathway

We aimed to investigate the effect and mechanism of icariin on male sexual function. Forty‐eight Crl:CD1(ICR) male mice were randomly divided into control, low‐, medium‐ and high‐dose icariin group (intragastric administration of 50, 100 and 200 mg/kg/d for 21 days). Mating experiment was then performed at a ratio of 1: 3 (male: female). The mating behaviours of male mice were recorded. The genital indexes and serum testosterone, nitric oxide (NO), hypothalamic dopamine (DA) and 5‐ hydroxy tryptophan (5‐HT) concentrations were measured. The expression of endothelial nitric oxide (eNOS), phosphatidylinositol tallow alcohol 3‐kinase (PI3K) and phosphorylated protein kinase (p‐AKT) in penile tissue was detected by Western blot. All icariin groups exhibited shorter capture latency and ejaculation latency, increased number of capture and ejaculation, higher capture and ejaculation rate, and higher testicular and prostate indexes compared with controls (p < .001). These groups had higher serum testosterone and NO concentrations (p < .001), hypothalamic DA and 5‐HT levels, and eNOS, PI3K and phosphorylated AKT expressions in penile tissue (p < .05). The effect of icariin was dose‐dependently increased. Our study suggests that icariin improves the sexual function of male mice, which might be associated with the hypothalamic–pituitary–gonadal axis and the PI3K/AKT/eNOS/NO signalling pathway.