Infertility affects at least 20% of married couples and growing evidence suggest an increasing incidence of male reproductive problems (Kolettis, 2003; Nayernia et al., 2004). In Africa, infertility prevalence rates are feared to be higher and range from 20 to 35% (Eze and Okonofua, 2015). The “infertility belt,” geographical regions with high infertility prevalence, is well-known to Africa, stretching from West Africa, through Central to East Africa (Irvine, 1998; Larsen, 2000; Etuk, 2009). Sperm count declined to a mean of 71.2 million/ml in Ibadan, Nigeria, 54.6 million/ml in Lagos, Nigeria, 65.0 million/ml in Salem, Libya, 66.9 million/ml in Dar Es salaam and Tanzania (Elkin and Fenster, 1997). Nigeria has about twelve million infertile persons (Giwa-Osagie, 2003). Although there is a general documented belief that the most common cause of infertility in Nigeria is infection (Cates et al., 1985; Orisakwe et al., 2004), cases abound where infection have been treated without correction of infertility (Giwa-Osagie, 2003). Environmental pollution and indiscriminate use of herbal medications have tended to add burden on male infertility in sub Sahara Africa (Amadi et al., 2011; Orisakwe et al., 2014). In Nigeria there are higher rates of irreversible oligospermia or azoospermia than most other causes of infertility and less resources for the management of infertility (Osegbe and Amaku, 1985).

Mushrooms are regarded as functional foods and have been in use for decades in folk medicine. Most of the edible mushrooms belong to two major superfamily; Ascomcota and Basidiomycota (Dandapat and Sinha, 2015) with over 600 species possessing therapeutic activity (Wasser and Weis, 1999). Diabetic rats fed some mshroom fruiting bodies exhibit significant anti-glycemic and anti-hypercholesterolemic effects (Jeong et al., 2010; Volman et al., 2010). Similarly, mushrooms show positive influence on lipid metabolism, liver function and decreased severity of streptozotocin-induced diabetes in rats with considerable protective effects on the pancreas and apparent repopulation. Many edible mushrooms contain pharmacological active agents which are mainly secondary metabolites like alkaloids, tannins, polysaccharides, phenolics, flavonoids, etc. (Dandapat and Sinha, 2015). Pleurotus tuber-regium is an edible mushroom found in Nigeria (Okjuoya, 1991) with several medicinal and nutritional properties (Alobo, 2003; Hu et al., 2006; Ngai and Ng, 2006).

This study has evaluated the ameliorative effects of P. tuber-regium in carbon tetrachloride induced testicular damage in Sprague Dawley rats.

Table 1 shows the effect of P. tuber-regium on the testis and body weight CCl₄ treated rats administration and treatment on the body weight of rats. Carbon tetrachloride significantly decreased the body weight [from 221 ± 2.24 (control) to 193 ± 1.87 g (CCl₄ treated groups)] and increased the absolute (from 1.73 ± 0.40 (control) to 2.74 ± 0.12 g CCl4 group and relative weight of the testis [0.91 ± 0.20 (control) to 1.40 ± 0.02 CCl4 treated group p < 0.01]. Treatment with 100, 200, and 500 mg P. tuber-regium gave significant and dose-dependent reversal in the body weight and testis weight in CCl₄ only treated group.

The effect of P. tuber-regium on sperm motility, epididymal count, vitality and (MDA of CCl₄ treated rats is shown on Table 2. Carbon tetrachloride significantly reduced the sperm motility (54.33 ± 3.79%), epididymal count (28.73 ± 2.86 × 10⁶/ml and vitality (4.96 ± 0.62) when compared with control (89.33 ± 9.01), 91.91 ± 1.92 × 10⁶/ml and 13.12 ± 0.19, respectively. There was significant increase in the sperm motility, epididymal count and vitality following P. tuber-regium administration. The reversal effect of P. tuber-regium on these seminal parameters were dose-dependent. There was significant difference between the MDA level (14.00 ± 3.50) in the CCl₄ treated rats and the untreated control (1.40 ± 0.32 μg/mg protein). Following the co-administration of 100, 200, and 500 mg P. tuber-regium plus CCl₄ there was significant reduction in the level of the MDA (3.50 ± 0.61, 1.60 ± 0.36, and 1.70 ± 0.15 μmol/mg protein, respectively) in dose-dependent manner.

Table 3 shows the effect of P. tuber-regium on LH, FSH, Testosterone, Prolactin, and Estradiol of CCl₄ treated rats. Administration of carbon tetrachloride significantly decreased the levels of LH (0.88 ± 0.14), FSH (2.04 ± 0.33), and Testosterone (2.02 ± 0.24) compared to control 2.74 ± 0.32, 3.64 ± 0.62, and 4.16 ± 0.23, respectively. The CCl₄, however, increased significantly the levels of prolactin from 1.06 ± 0.21 to 3.58 ± 0.48 and oestradiol from 0.81 ± 0.10 (control) to 2.89 ± 0.18 in the CCl₄ treated group. There was dose-dependent reversal of the effects of CCl4 on these hormonal parameters in the P. tuber-regium treated groups.

Infertility which is usually defined as a couple’s inability to achieve pregnancy following 1 year of unprotected intercourse and considered one of the main public health issues (Jungwirth et al., 2012). Male factor infertility is present in approximately half of all infertile couples (Thonneau et al., 1991). Spermatogenesis takes place in the seminiferous tubular structures of the testis which are lined with germ and sertoli cells. Spermatogenesis consists of three major phases; Proliferation, reduction-division and differentiation with distinct and specific germ cell types being associated with each phase (Hess, 1990). The present study has investigated the effect of P. tuber-regium on carbon tetrachloride induced testicular injury in animal model. After hepatic handling CCl₄ generates chloride and trichloromethyl radicals CCl₃, which reacts with oxygen to produce CCl₃ O₂. CCl₃ bind to fatty acids to generate alkoxy and peroxy radicals (Bruckner et al., 2002). The increased lipid peroxidation provoke destruction of sperm (Aitken et al., 2014). P. tuber-regium co-administered with carbon tetrachloride produced a significant dose dependent decrease in the level of MDA. Reduced lipid peroxidation will promote spermatogenesis.

There are estrogen receptors in the hypothalamic nuclei and in pituitary gonadotropes, which act on the hypothalamus to affect gonadotrophin releasing hormone GnRH pulses and at the pituitary level to regulate FSH and LH secretion (Cabler et al., 2010). The excessive conversion of testosterone to estrogen in peripheral adipose tissue has been known to cause secondary hypogonadism through hypothalamic-pituitary-gonadal axis inhibition (Cabler et al., 2010). Estrogens affect spermatogenesis directly within the testis by alterations in gonadotropin secretion by the pituitary gland. High levels of circulating oestradiol and/or elevated oestradiol/testosterone ratios which is a feature of male infertility (Cabler et al., 2010), was observed in rats treated with CCl₄ in this study. P. tuber-regium significantly reversed the oestradiol level in this study. Although there is a school of thought that normal levels of gonadotropins in the context of low free testosterone may signify the effective suppression of the hypothalamic-pituitary axis, resulting in subclinical hypogonadotropic hypogonadism (Agarwal et al., 2006). In this study, however, P. tuber-regium significantly reversed the CCl₄, LH, FSH, and testosterone lowering and oestradiol increase. P. tuber-regium may be competitively bind to estrogen receptors on the hypothalamus and pituitary gland to decrease estrogenic firing to increase the release of LH, which increases testosterone production by the testes.

Luteinizing hormone binds to its receptors to activate G-proteins and, in turn, adenylate cyclase, which can increase cyclic AMP formation. cAMP will then stimulate protein kinase A (PKA), which will phosphorylate proteins. The phosphorylated proteins will further phosphorylate other proteins or induce new protein synthesis, i.e., steroidogenic acute regulatory protein (Manna et al., 2013). The function of steroidogenic acute regulatory protein is to transfer free cholesterol from the cytoplasm into the inner membrane of mitochondria, where cytochrome P450 side-chain cleavage enzyme converts cholesterol to pregnenolone (Stocco, 2002; Manna and Stocco, 2005). Pregnenolone will then be transported to smooth endoplasmic reticulum for testosterone synthesis, which is an essential steroid hormone for reproduction in males (Liu et al., 2007). It has also been shown that activation of the protein kinase C (PKC) signal pathway can strongly modulate Leydig cell steroidogenesis (Hirakawa et al., 2002). Chen et al. (2005) explained that Clonorchis sinensis activated both PKA and PKC signal transduction pathways to stimulate cell steroidogenesis. Although there seem to be no significant association between semen parameters and prolactin levels (Lotti et al., 2013), prolactin can affect steroidogenesis by modulating the expression of LH receptors (Dombrowicz et al., 1992), or by regulating the activity of steroidogenetic enzymes (Chandrashekar and Bartke, 1988) and has a trophic effect on male seminal accessory glands.

Histological examination suggests the consequences of oxidative stress in spermatogenesis with sperm cell maturation arrest. The result conform with the sperm parameter result of decreased sperm count since maturation arrest will ultimately decrease the number of sperm reaching the epididymis (El-kholy et al., 2013).

In this study has shown that P. tuber-regium may protect the testis from CCl₄ induced damage as evidenced by the by the ameliorative effects on the epididymal sperm count, motility, viability, sex hormones, MDA, and testicular histomorphology. Since biochemical hypogonadism is considered a main feature of male infertility, dietary supplementation with P. tuber-regium with its significant reversal of all the deleterious testicular effects in CCl₄ treated rats may hold some promise in the management of involuntary childlessness amongst married couples.

KO: Designed study, carried out the bench work and analysed data, IS: Design, and OO: Designed the study, analysed data and write up.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.