Scholarly article on topic 'Spermatotoxic effects of galactose and possible mechanisms of action'

Spermatotoxic effects of galactose and possible mechanisms of action Academic research paper on "Veterinary science"

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Abstract of research paper on Veterinary science, author of scientific article — Toyin Muhammed Salman, Luqman Aribidesi Olayaki, Isiaka Abdullateef Alagbonsi, Adeoye Oyetunji Oyewopo

Abstract While numerous studies have documented the ovotoxic effect of galactose, few available studies on male gonad are of the opinion that it seems to fully escape the toxic effects galactose exerts on the ovary. The present study was therefore designed to further investigate the effects of galactose on male sperm parameters and some reproductive hormones. Thirty male albino rats (200–250g) were randomly divided in a blinded fashion into 6 groups (n =5). Group A received normal saline and served as control. Groups B, C, D, E and F received 3mg/kg, 10mg/kg, 20mg/kg, 30mg/kg, and 40mg/kg of galactose respectively through oral gavage for 42days. The results showed that chronic administration of galactose promotes sperm toxicity by reducing epididymal sperm count, motility and percentage of morphologically normal sperm. Moreover, galactose increased luteinizing hormone but slightly decreased testosterone and had no effect on follicle stimulating hormone. Galactose also caused a slight decrease in superoxide dismutase and increase in lactate dehydrogenase activity but no effect on catalase. The present study thus showed that chronic administration of galactose could promote sperm toxicity which could be mediated partly by oxidative stress. Moreover, the response of the hormones is similar to that in premature ovarian insufficiency (POI) in female galactosemic model.

Academic research paper on topic "Spermatotoxic effects of galactose and possible mechanisms of action"

Middle East Fertility Society Journal (2016) 21, 82-90

Middle East Fertility Society Middle East Fertility Society Journal

www.mefsjournal.org www.sciencedirect.com

ORIGINAL ARTICLE

Spermatotoxic effects of galactose and possible mechanisms of action

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Toyin Muhammed Salman a, Luqman Aribidesi Olayakia, Isiaka Abdullateef Alagbonsi a'b'*, Adeoye Oyetunji Oyewopo

a Department of Physiology, College of Health Sciences, University of Ilorin, Ilorin, Kwara, Nigeria

b National Health Insurance Scheme, North Central A Zonal Office, Kwara State Ministry of Health Premises, Ilorin, Kwara, Nigeria c Department of Anatomy, College of Health Sciences, University of Ilorin, Ilorin, Kwara, Nigeria

Received 12 July 2015; accepted 14 September 2015 Available online 18 November 2015

KEYWORDS

Galactose; Hormones;

Lactate dehydrogenase; Oxidative stress; Sperm

Abstract While numerous studies have documented the ovotoxic effect of galactose, few available studies on male gonad are of the opinion that it seems to fully escape the toxic effects galactose exerts on the ovary. The present study was therefore designed to further investigate the effects of galactose on male sperm parameters and some reproductive hormones. Thirty male albino rats (200-250 g) were randomly divided in a blinded fashion into 6 groups (n = 5). Group A received normal saline and served as control. Groups B, C, D, E and F received 3 mg/kg, 10mg/kg, 20 mg/kg, 30 mg/kg, and 40 mg/kg of galactose respectively through oral gavage for 42 days. The results showed that chronic administration of galactose promotes sperm toxicity by reducing epi-didymal sperm count, motility and percentage of morphologically normal sperm. Moreover, galactose increased luteinizing hormone but slightly decreased testosterone and had no effect on follicle stimulating hormone. Galactose also caused a slight decrease in superoxide dismutase and increase in lactate dehydrogenase activity but no effect on catalase. The present study thus showed that chronic administration of galactose could promote sperm toxicity which could be mediated partly by oxidative stress. Moreover, the response of the hormones is similar to that in premature ovarian insufficiency (POI) in female galactosemic model.

© 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Middle East Fertility Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-

nd/4.0/).

* Corresponding author at: Department of Physiology, College of Health Sciences, University of Ilorin, Ilorin, Kwara, Nigeria. Tel.: + 234 8067509458.

E-mail address: easylat@gmail.com (I.A. Alagbonsi).

Peer review under responsibility of Middle East Fertility Society.

1. Introduction

Glucose and fructose are two of the most commonly found monosaccharides in mammalian seminal plasma, although other sugars, such as sorbitol or mannose, can also be detected (1). The presence of either glucose or fructose can affect the function of mammalian spermatozoa in several ways. Glucose concentrations of about 5mmoll~1 produce much higher

http://dx.doi.Org/10.1016/j.mefs.2015.09.004

1110-5690 © 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Middle East Fertility Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

penetration rates than do fructose or mannose in human spermatozoa (2). Moreover, glucose, but not fructose, produces a high fertility rate and capacitation-like changes in the chlorte-tracycline fluorescence pattern of mouse spermatozoa subjected to 'in vitro' capacitation (3).

The beneficial (4-8) and detrimental effects (9,10) of gly-colysable sugars in male fertility have been well reported. Supplementation of glucose in drinking water improved semen qualities, plasma total protein, cholesterol, and globulin; but decreased abnormal and dead sperm in rabbit (11). It also improved ejaculate volume in broiler breeders (12) and sperm motility in dog (13). Previous reports on the effect of glucose on plasma testosterone are controversial. For instance, increase in testosterone level in rabbit (11) and decrease in human (14) following oral glucose ingestion have been reported. However, Attia et al. (12) and Hotzel et al. (15) reported no effect in broiler breeders and Merino rams respectively. Decrease in luteinizing hormone following oral glucose ingestion has also been reported (14).

Galactose is a monosaccharide similar to glucose and fructose but the three sugars have different stereochemistry. The impairment of ovarian function in classical galactosemia has been known for over 35 years (16). Galactosemia, an inherited inborn error of the major galactose assimilation pathway caused by galactose-1-phosphate uridyltransferase (GALT) deficiency, produces wide phenotypes of ovarian dysfunction (17). The prevalence of premature ovarian insufficiency (POI) in galactosemic population is 1 in 10,000 for women between 15 and 29 years of age, and 7.6 in 10,000 for women aging between 30 and 39 (18). In some women, ovarian failure is a consequence of premature depletion of follicular reserve (afollicular or follicle depletion type of POI), while the other galactosemic women do exhibit the presence of follicles that are refractory to gonadotropin stimulation and therefore suffer from arrested growth and maturation (follicle dysfunction type of POI or resistant ovary syndrome) (19). Despite more than four decades of intense research, the cause and effect relationships between galactosemia and POI, and the molecular mechanisms of galactose toxicity remain elusive; however, the general consensus is that the ovarian pathology is the aftermath of toxic effects of galactose and its metabolites both at the ovarian and extra-ovarian levels (20-22).

Previous studies have used rats placed on high dose of galactose as a model for galactose toxicity. For instance, experimental galactose toxicity in female rats produced an array of ovarian dysfunctions that characterize the basic tenets of diverse phenotypes of POI (23). Moreover, embryos exposed to high galactose in utero suffer from significant attenuation of germ cell migration and develop ovaries with deficient follicular reserve (24). Liu et al. (25) reported that high galactose diet down regulated the oocyte specific growth factor, GDF-9, an obligatory factor for folliculogenesis, leading to inhibition of follicular development. Lai et al. (26) have also reported an apoptotic effect of high galactose diet on the ovary.

However, despite all the documented findings on the toxic effect of galactose on ovary, there seem not to be any published reports on a possible impairment of the reproductive outcome in human galactosemic males, nor is there any gona-dal toxicity in male offspring of galactose-fed rats (27). Forges et al. (17) concluded that the male gonad seems to fully escape the toxic effects galactose exerts on the ovary. This has been underlined since the initial descriptions of ovarian failure in

galactosemic patients: in eight male galactosemics aged between 13 and 28 years, pubertal development occurred normally, and serum gonadotropin and testosterone levels were in the normal range for all patients (28). Normal testosterone levels were also detected by other authors in 10 galactosemic males; however, in the three oldest (21-24 years) of them, elevated serum FSH was measured (29).

A recent study by Yu et al. (30) changed the impression that the male gonad seems to fully escape the toxic effects galactose exerts on the ovary. For instance, the authors reported that galactose administration caused down-regulation of sperm motility, which was regained with diosgenin (a material for the synthesis of dehydroepiandrosterone which is a precursor of testosterone) treatment. However, testosterone production was not affected by galactose treatment but was decreased in diosgenin rats exposed to galactose. They concluded that the down-regulation of reproductive function in galactose-induced aging model of rat is via reduction in sperm motility but not via testosterone production.

However, the study of Yu et al. (30) was not comprehensive enough to erase previous long-standing belief that the male gonad seems to fully escape the toxic effects that galactose exerts on the ovary as the parameters measured (sperm motil-ity and testosterone) were not sufficient to do so. For instance, other characteristics of sperm such as count, morphology and viability all of which will help to ascertain whether or not normal spermatogenesis takes place in galactose-treated rats were not investigated by Yu et al. (30). Moreover, the study did not provide any information on the mechanism of galactose-induced sperm toxicity. Hence, there is a need for further information on the spermatotoxic effect of galactose in male rats, which this study aimed to provide.

2. Materials and methods

2.1. Animals and treatment protocol

Thirty male albino rats (200-250 g) were obtained from the Animal House of the Department of Biochemistry, Faculty of Life Sciences, University of Ilorin, Kwara State, Nigeria. They were housed at room temperature and allowed free access to food and water ad libitum. ''Principles of laboratory Animal care (NIH publication No. 85-23, revised 1985)" were followed. All experiments have been examined and approved by our institutional ethics Committee.

After 2 weeks of acclimatization to their new environment, 30 animals were randomly divided in a blinded fashion into 6 groups (5 rats per group) that received treatment as described below:

(A) Treated with normal saline for 42 days through oral gavage and served as control.

(B) Treated with 3 mg/kg of galactose through oral gavage for 42 days.

(C) Treated with 10 mg/kg of galactose through oral gavage for 42 days.

(D) Treated with 20 mg/kg of galactose through oral gavage for 42 days.

(E) Treated with 30 mg/kg of galactose through oral gavage for 42 days.

(F) Treated with 40 mg/kg of galactose through oral gavage for 4 days.

A day after the treatment, blood sample from each rat was collected (by cardiac puncture) into lithium heparinized capillary tubes. It was spun using centrifuge at the rate of 3000 revolutions per minute for 15 min. Plasma was collected from each sample and preserved. The testis of each rat was harvested and preserved in separate formalin bottles. The testis was removed, washed in the washing buffer and weighed with electronic weighing balance to know the ratio of the homogenizing buffer to the organ. The constituent of the washing buffer is 11.5 g of KCl in 1000 ml of distilled water. The homogenizing buffer {pH = 7.4} contains 11.5 g of KCl and 7.88 g of Tris HCl in 1000 ml of distilled water. NaOH was added to correct the pH. The homogenizing buffer was added at a ratio of 1:4. The testis was ground in the homogenizing buffer and centrifuged and the homogenate was refrigerated.

2.2. Determination of epididymal sperm parameters

The testes from each rat were carefully exposed and one of them was removed together with its epididymis. For each separated epididymis, the caudal part was removed and placed in a beaker containing 1 ml of normal saline solution. It was macerated with a pair of sharp scissors and left for few minutes to liberate the sperm cells into the normal saline. Semen drops were placed on a clean grease-free glass slide and two drops of warm 2.9% sodium citrate were added. The improved Neubauer counting chamber was charged with the semen solution and the number of sperm cells, appearing as black dots, was counted in 25 small squares within the central counting area of the counting chamber as earlier described (31,32).

Sperm motility, estimated as the percentage of sperm that manifests progressive motility, was determined as previously described (33,34). Briefly, the sperm suspension was diluted in 1 ml of normal saline solution. About 10 L was pipetted onto a clean grease free glass slide. A cover slip was lowered onto the sample on the slide, avoiding air bubbles, and the slide was examined using a microscope with a 40 x objective. At least, six widely spaced fields were examined to provide an estimate of the percentage of the progressively motile sperm cells. The sperm cells with progressive motility were estimated and recorded as (N) while the total number of all the sperm cells counted was recorded as (T). Sperm motility (%) was calculated using (N/T x 100%).

Percentage of morphologically normal sperm was estimated by the method previously described (33,35). The principle is based on the ability of morphologically normal sperm to appear white in color as the plasma membrane will prevent the dye to enter, while abnormal sperms take up the dye and stain dark color. The microscope slides and the eosin stain were pre-warmed to room temperature. One milliliter of the sperm suspension - normal saline solution was transferred to a test tube and 2 drops of 1% eosin were added and mixed gently for agitation. This was incubated for 45-60 min to allow its proper staining and then re-suspended with a Pasteur pipette. A clean grease-free glass slide was used. Potential damage to the sperm cells should be avoided. One or 2 drops of the stained sperm were placed approximately 1 cm from the end of the slide lying on a flat surface. A second slide was held with the slide's long edge gently touching across the width of the sperm slide and pulled across to produce a sperm smear. After air-drying the slide, using a microscope at

100 x objective, the sperm cells were examined. The sperm along the periphery was normally excluded from the examination because there is a greater tendency for artifacts to occur in these regions. At least, five fields were viewed covering the whole slide. Examples of morphological abnormalities are double-headed, elongated head, pyriform head, bent head, bent tail, bent mid-piece, coiled tail, double tail, headless, tailless, etc. All those with normal morphology were recorded as N while the total number of the counted spermatozoa was recorded as T. The percentage sperm morphology was calculated as (N/T x 100%).

2.3. Estimation of plasma biochemical parameters

Testosterone and gonadotropins (luteinizing hormone and follicle-stimulating hormone) were measured by enzyme-linked immunosorbent assay method using commercial kits (IBL-Hamburg GmbH, Germany). The ELISA is a standardized method used by WHO and part of its program for human reproduction research. The procedures for the assay as contained in the manufacturer's manual were strictly followed.

Lactate dehydrogenase (LDH) assay was done spectropho-tometrically with LDH kit (Fortress diagnostics Limited, UK). This kit uses lactate as a substrate for the reaction, with a concomitant generation of NADH + from NAD in the presence of lactate dehydrogenase.

Superoxide dismutase (SOD) estimation was done as previously described (33,36). The principle is based on the ability of SOD to inhibit the auto-oxidation of adrenaline at pH of 10.2. Superoxide radical {O~} generated causes the oxidation of adrenaline to adrenochrome. The yield of adrenochrome increases per O~ introduced with increasing concentration of adrenaline. Briefly, 0.1 ml of blood plasma was diluted with 0.9 ml of distilled water. Then 0.1 ml of the resulting solution was added to 2.5 ml of the carbonate buffer. Then 0.3 ml of the adrenaline was added. This was read in a spectrophotometer at wavelength of 480 nm. Blank cuvette contained 2.5 ml of carbonate buffer, 0.3 ml of adrenaline and 0.1ml of distilled water. The absorbance at 0 s and 150 s was recorded.

Malondialdehyde (MDA) was estimated as previously described (33,37). The principle is based on the reaction of malondialdehyde {MDA} with thiobarbituric acid {TBA}, forming a MDA-TBA complex which absorbs strongly at a wavelength of 532 nm. Small amounts of MDA are produced during lipid peroxidation, which react with TBA to give a pink colored complex and absorb light when in an acidic solution at 532 nm. Briefly, 0.4 ml of the blood plasma was mixed with 0.5 ml of 30% TCA, and 1.6 ml of Tris KCl was added. TBA (0.5 ml) was then added and the solution was incubated for 45 min at 80 0C. This produced pink colored reaction mixtures which were read at 532 nm.

The catalase activity was determined spectrophotometri-cally according to the standard protocol described by Beers and Sizer (38). Briefly, 0.05 mL of the plasma was added to 1.95 mL of 10 mM H2O2 in 60 mM phosphate buffer (pH = 7.0). Degradation of H2O2 was read at 240 nm wavelength per min and the rate of decomposition of H2O2 was calculated using the formula k = 2.303/Dt x log (A1/A2) followed by calculation of catalase in terms of units/mg of protein. One unit of catalase is defined as the quantity that

decomposes 1.0 imole of H2O2 per min at pH = 7.0 at 25 0C, while this H2O2 concentration falls from 10.3 to 9.2 mM.

2.4. Statistical analysis

Data were analyzed using SPSS version 16.0 for windows (IBM Corporation, Armonk, NY, USA). All values given were the Mean ± S.E.M of the variables measured. Significance was assessed by the one-way analysis of variance (ANOVA), followed by a post-hoc Tukey's test for multiple comparisons. p-Values of 0.05 or less were taken as statistically significant.

3. Results

3.1. Galactose reduced epididymal sperm parameters in rats

Epididymal sperm count was significantly lower (p < 0.05) in all the galactose-treated groups than the control. There were no significant differences between the effects caused by different galactose doses except for 3 mg/kg group that had lower sperm count than the 40 mg/kg group (p < 0.05) (Fig. 1).

The percentage of motile sperm was low in rats treated with galactose, with the four higher doses producing more significant reductions (p < 0.01, p < 0.001) than 3 mg/kg (p < 0.05) when compared to the control rats (Fig. 2).

In addition, the life-death ratio, also referred to as the sperm viability, was significantly reduced by the administration of all the doses of galactose, although 10 mg/kg and 30 mg/kg caused more significant reductions (p < 0.01, p < 0.001) than other doses (p < 0.05) when compared to the control (Fig. 3).

The percentage of morphologically normal sperm demonstrated a very much identical trend to the sperm motility. The four higher doses produced more significant reductions (p < 0.01, p < 0.001) than 3 mg/kg (p < 0.05) when compared to the control rats (Fig. 4).

£ 60-

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<u a. 30-

Control 3mg/kg 10mg/kg 20mg/kg 30mg/kg 40mg/kg

Figure 1 Effects of galactose administration on epididymal sperm count in male rats. Values are expressed as Mean ± S.E.M (n = 5). *p < 0.05 vs control; bp < 0.05 vs 3 mg/kg.

Control 3mg/kg 10mg/kg 20mg/kg 30mg/kg 40mg/kg

Figure 2 Effects of galactose administration on epididymal sperm motility in male rats. Values are expressed as Mean ± S.E. M (n = 5). *p < 0.05; **p < 0.01; ***p < 0.001 vs control.

Control 3mg/kg 10mg/kg 20mg/kg 30mg/kg 40mg/kg

Figure 3 Effects of galactose administration on epididymal sperm viability in male rats. Values are expressed as Mean ± S.E. M (n = 5). *p < 0.05; **p < 0.01; ***p < 0.001 vs control.

3.2. Galactose increased luteinizing hormone but decreased testosterone and had no effect on follicle stimulating hormone

Galactose caused reductions in plasma testosterone levels that were not significant for all the groups (p > 0.05) except 3 mg/kg (p < 0.05) when compared to control. However, the response of plasma testosterone was dose-dependent when the treated groups were compared, as the plasma testosterone in rats that

E 60»

■¡5 50

Control 3mg/kg 10mg/kg 20mg/kg 30mg/kg 40mg/kg

Figure 4 Effects of galactose administration on epididymal sperm morphology in male rats. Values are expressed as Mean ± S.E.M (n = 5). *p < 0.05; **p < 0.01; ***p < 0.001 vs control.

received either 10 mg/kg or 40 mg/kg was significantly higher (p < 0.01, p < 0.001 respectively) when compared to those that received 3 mg/kg galactose. Similarly, testosterone was higher in rats that received 40 mg/kg galactose than those that received either 20 mg/kg or 30 mg/kg (p < 0.05) (Table 1).

Galactose also caused a general increase in the plasma luteinizing hormone that was significant in groups that received 3 mg/kg, 10 mg/kg or 30 mg/kg (p < 0.01) but not significant in 20 mg/kg and 40 mg/kg groups (p > 0.05) when compared to the control group (Table 1).

Plasma follicle stimulating hormone was not significantly different between all the galactose-treated groups and the control (p > 0.05) (Table 1).

3.3. Effects of galactose administration on lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD) and catalase activity in male rats

Lactate dehydrogenase (LDH) activity showed an increasing pattern in all treated groups when compared with the control. However, the increase was only significant (p < 0.001) in the groups treated with 3-, 20- and 30 mg/kg of galactose (Table 2).

Malondialdehyde (MDA) concentration in animals that received 3 mg/kg, 10 mg/kg and 20 mg/kg was increased (p < 0.001, p > 0.05, p > 0.05 respectively) while 30 mg/kg and 40 mg/kg showed insignificant decrease (p > 0.05) when compared to control. Moreover, 3 mg/kg galactose caused significantly higher lipid peroxidation than the other doses (p < 0.01; p < 0.001) (Table 2).

Moreover, superoxide dismutase activity was decreased in all the galactose-treated groups, but the decreases were only significant in rats treated with 20 mg/kg (p < 0.05) and 30 mg/kg (p < 0.01) when compared to control (Table 2).

There were also apparent decreases, albeit insignificant (p > 0.05), in the catalase activities of all the galactose-treated rats when compared to the control (Table 2).

4. Discussion

The ovotoxic effect of galactose in females has been extensively documented. Chen et al. (27) observed that there seem not to be any published reports on a possible impairment of the reproductive outcome in human galactosemic males, nor is there any gonadal toxicity in male offspring of galactose-fed rats. Consequently, Forges et al. (17) concluded that the male gonad seems to fully escape the toxic effects galactose exerts on the ovary. On the contrary, a recent study of Yu et al. (30) reported that sperm motility but not testosterone was down-regulated in galactose-induced aging model of rat. Our study further provides convincing and interesting data that chronic administration of galactose, a non-glycolysable monosaccha-ride, could cause sperm toxicity by reducing epididymal sperm count; and percentages of motile, viable and morphologically normal sperm in rats. This spermatotoxic effect of galactose in male rats provides additional information to that earlier provided by Yu et al. (30) and changed the impression that the male gonad fully escapes the toxic effect that galactose exerts on the ovary.

Glycolysis is widely considered to be a key pathway in mammalian sperm energy production (39-42). In some experimental animal models including bull, ram, dog and mouse, glycolysis can solely fuel sperm motility when oxidative metabolism is absent (40,43), whereas motility cannot be maintained by respiration alone in human and rhesus macaque (44,45). Even in species capable of sustaining motility by oxidative metabolism alone (e.g., mouse), glycolysis may be required for sperm capacitation (3,5,43), hyperactivation (46,4), the acrosome reaction (4), zona binding, or fusion with the oocyte plasma membrane (47). The fact that microtubule sliding occurs in the distal flagellum, far from the site of mito-chondrial activity, is another reason to believe that glycolysis may be an obligate energy source for cellular motility. Moreover, lactate dehydrogenase (LDH) and some other glycolytic enzymes are found in the principal piece of sperm flagellum (48) in many species including rabbit, boar, bull, rat, stallion, mouse, fox, and human (49-51).

Having established the spermatotoxic effect of galactose, is it related to oxidative stress? Galactose has recently been shown to cause oxidative stress, especially by increasing serum lipid peroxidation (52), but the mechanism involved is poorly understood. Reports on D-galactose-treated rodents (53,54) and flies (55) suggest that galactose metabolism in these systems leads to oxidative stress and that the resulting oxidative damage accounts for the life-shortening effect of the exposure. Galactose-dependent free-radical generation observed in rat brain homogenates was also reversible after antioxidant administration (56). Recently, Jumbo-Lucioni et al. (57) investigated whether oxidative stress and response contribute to the mechanism of acute galactose toxicity in galactose-1-phosphate uridyltransferase deficiency using Drosophila melanogaster genetic model of classic galactosemia, and found the answer to be yes.

The quest for answer to this question made us to estimate malondialdehyde (a marker of lipid peroxidation) and anti-oxidant enzymes such as superoxide dismutase and catalase. Close observation of our data weakly supports this claim due to slight lipid peroxidation recorded for the lower doses of galactose but not the high doses. The insignificant reduction

Table 1 Effects of galactose administration on plasma testosterone and gonadotropins in male rats. Values are expressed as Mean ± S.E.M (n = 5).

Control 3 mg/kg galactose 10 mg/kg galactose 20 mg/kg galactose 30 mg/kg galactose 40 mg/kg galactose

Testosterone 0.72 ± 0.07 0.1 ± 0.00*** 0.63 ± 0.11№ 0.43 ± 0.06 0.46 ± 0.05 0.78 ± 0.05№bA£

LH 4.64 ± 0.18 8.50 ± 0.50** 7.5 ± 0.67** 6.00 ± 0.32 7.5 ± 0.74** 6.5 ± 0.22

FSH 4.34 ± 0.18 4.00 ± 0.00 4.26 ±0.11 4.34 ± 0.18 4.5 ± 0.16 4.62 ± 0.19

p <0.01 vs control. p < 0.001 vs control. p < 0.01 vs 3 mg/kg. p < 0.001 vs 3 mg/kg. D p < 0.05 vs 20 mg/kg. £ p < 0.05 vs 30 mg/kg.

Table 2 Effects of galactose administration on plasma lactate dehydrogenase (LDH), malondialdehyde dismutase (SOD) and catalase (CAT) activity in male rats. Values are expressed as Mean ± S.E.M (n = 5). (MDA), superoxide

Control 3.0 mg/kg galactose 10 mg/kg galactose 20 mg/kg galactose 30 mg/kg galactose 40 mg/kg galactose

LDH 41.48 ± 5.88 216.4 ± 56.1*** 53.01 ± 7.32bbb 161.6 ± 13 2***,@@@ 124.22 ± 8 23***,bb,@@@ 48.6 ± 5 38ßßß,AAA,£££

MDA 2.12 ± 0.31 4.48 ± 0.02*** 2.22 ± 0.18bb 2.60 ± 0.16bb 1.71 ± 0.40bbb 1.51 ± 0. 19bbb

SOD 0.33 ± 0.02 0.31 ± 0.04 0.29 ± 0.01 0.26 ± 0.02* 0.24 ± 0.02** 0.28 ± 0.02

CAT 0.31 ± 0.00 0.35 ± 0.02 0.30 ± 0.02 0.31 ± 0.02 0.26 ± 0.01 0.28 ± 0.01

* p < 0.05 vs control.

p < 0.01 vs control.

p < 0.001 vs control.

p < 0.01 vs 3 mg/kg.

ß№ p < 0.001 vs 3 mg/kg.

@@@p < 0.001 vs 10 mg/kg.

AAA p < 0.001 vs 20 mg/kg.

£££ p < 0.001 vs 30 mg/kg.

(except for SOD in some groups) in the activities of antioxidant enzymes observed by us is also similar to the previous submission by others (55,58,57) that the oxidative stress induced by galactose is followed by a compromised normal defense mechanism.

The adverse effects of ROS on sperm function have been well-documented. For instance, reactive oxygen species (ROS) can cause peroxidation of the sperm membrane, which are vulnerable to this type of damage as they contain large amounts of unsaturated fatty acids, thereby decreasing its flexibility and tail motion (59). Direct ROS damage to mitochondria, decreasing energy availability, may also impede sperm motility (60).

Previous study has reported that a unique isozyme of LDH expressed specifically in germ cells (61,62), LDH type C (LDHC), is abundant in spermatids and spermatozoa. It has also been shown that targeted disruption of the LDHC gene resulted in male infertility due to sperm having a decrease in progressive motility, a failure to develop the hyperactivated motility pattern essential for fertilization, and a rapid decline in ATP levels (63). In order to establish the link between lactate dehydrogenase activity and sperm toxicity, which may be a consequence of oxidative stress, this study estimated lactate dehy-drogenase activity by a method that uses lactate as a substrate. The lactate then reduces NAD + to yield pyruvate and NADH in a reaction catalyzed by LDH. This means that an increase in LDH activity is a pointer to an accumulation of NADH, a molecule that is used to fuel generation of ROS via NADPH

oxidase located within the sperm membrane (64,65). This study thus suggests that the increase in LDH activity by galactose could have caused an accumulation of NADPH, and consequently ROS, which might not have necessarily led to a much significant increase in MDA level. Thus, galactose-induced oxidative stress earlier proposed by Ghanbari et al. (52), the mechanism of which was not understood could actually be due to LDH-dependent generation of ROS.

It is known that conversion of pyruvate to lactate with the concomitant oxidation of NADH to NAD + is essential for the continued production of ATP by glycolysis; this reaction is also catalyzed by lactate dehydrogenase (LDH) (66). This then means that the reverse reaction also catalyzed by LDH as in this study will hinder ATP production, an indication for the reduced sperm motility in galactose-treated rats observed in this study. This is similar to previous report that showed that incubation of sperm with human tubal fluid containing either pyruvate or lactate caused reductions in the percentage of motile and hyperactive sperm (67).

Recently, it has been shown in domestic cats and cheetah that teratospermia (a condition where males produce P60% structurally abnormal spermatozoa) was linked to remarkable reduction in sperm lactate production rate, suggesting that ATP production is impaired in them (68). The concomitant reduction in lactate level in the presence of high LDH following galactose ingestion in this study supports the contention that ATP is required for normal sperm morphology. These evidences suggest that the spermatotoxic effect induced by

galactose in male rats could have been partly mediated by oxidative stress.

Again, could the spermatotoxic effect of galactose be related to any alteration in the gonadotropic and gonadal hormones? Premature ovarian failure, currently referred to as premature ovarian insufficiency (POI), is a frequent finding in women with galactosemia (69-71). In some women, ovarian failure is a consequence of premature depletion of follicular reserve (afollicular or follicle depletion type of POI), while the other galactosemic women do exhibit the presence of follicles that are refractory to gonadotropin stimulation and therefore suffer from arrested growth and maturation (follicle dysfunction type of POI or resistant ovary syndrome) (19). The pattern of some of our data in this study closely relates to what has been described for POI in females by others. For instance, there was a reduction in sperm count and at the same time there was an increase in luteinizing hormone (LH) in male rats chronically exposed to galactose in this study. Apparently, the increase in LH was meant to stimulate Leydig cells to produce testosterone in response to the low sperm count but could not, probably because the Leydig cells were insensitive to LH. This is consistent with the report that galactosemic women do exhibit the presence of follicles that are refractory to gonadotropin stimulation (19). Moreover, the insignificant effect of galactose on follicle stimulating hormone (FSH) in this study closely agrees with that of Gubbels et al. (72) and Sanders et al. (73) that demonstrated no significant differences in either FSH isoforms or FSH bioactivity respectively in the galactosemic patient.

Finally, what could be responsible for the oxidative stress and consequently spermatotoxic effects of galactose in male rats? Could it be a direct effect of galactose or that of its metabolites? It should be noted that the metabolism of galactose begins in the Leloir's pathway with the conversion of galactose to galactose-1-phosphate in the reaction catalyzed by ATP-dependent galactokinase. This is then followed by the conversion of galactose-1-phosphate to UDP galactose by galactose-1-phosphate uridyltransferase. The third step is the conversion of UDP-galactose to UDP-glucose by UDP-galactose-4-epimerase. UDP-glucose is then converted to glucose-1-phosphate by UDP-glucose pyrophosphorylase. Finally, glucose-1-phosphate is converted to glucose-6-phosphate by a phosphoglucomutase and moves through the glycolytic pathway and the tricarboxylic acid pathway to be oxidized to CO2 (74).

Galactose-1-phosphate and galactitol are known to be the major toxic metabolites of galactose. The accumulation of galactose-1-phosphate has been thought to alter the energy production of the cell by inhibiting several enzymes of glucose metabolism, such as phosphoglucomutase, glucose-6-phosphate dehydrogenase and glycogen phosphorylase (75). Furthermore, the role of galactitol in ovarian toxicity has been demonstrated in rats fed with 40% galactose (76). In these animals, an abnormal oocyte maturation and a decreased ovarian response to an exogenous gonadotropin stimulation were observed. The simultaneous administration of an aldose reduc-tase inhibitor, however, prevented all of these abnormalities, suggesting a determining role of galactitol in ovarian galactose toxicity. In fact, it has been known for a long time that in galactose-fed rats, galactitol concentration in the ovary (77) or testis (78) is even higher than in the liver. Galactitol does not cross cell membrane easily; therefore, its accumulation

within the cells leads to an osmotic imbalance and a water influx, thus altering membrane permeability and cell functions. Moreover, galactitol-induced membrane alteration also accounts for a loss in cellular glutathione, which leads to an increased sensitivity to oxidative stress (79). It is therefore not unreasonable to speculate that galactitol or galactose-1-phosphate might have accumulated in the rats chronically exposed to galactose in this study, and that the toxic effects of galactose observed could be the effects of one or both of these metabolites. Measurement of galactitol and gal-1-P in future study, which is a limitation of this study, will better our understanding of the spermatotoxic effect of chronic galactose administration in rats.

5. Conclusion

In conclusion, the present study further showed that chronic administration of galactose could promote sperm toxicity by reducing epididymal sperm count, motility and percentage of morphologically normal sperm. It also suggests that chronic galactose administration in male rats could cause hormonal disturbances closely similar to those found in POI in female galactosemia. Lastly, oxidative stress might partly play a role in the observed effects of galactose in male rats.

Conflict of interest

None declared. Author contributions

All authors made substantial contributions in the conception and design of the study, acquisition of data, analysis and interpretation of data, drafting and revising the article critically, and finally approving the version to be submitted.

Funding

None. References

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