Mn2+: A potent antioxidant and stimulator of sperm capacitation and acrosome reaction in crossbred cattle bulls

Manganese is a well known antioxidant and is a potent inhibitor of in vitro oxidative stress. The aim of this work was to study the influence of Mn (60 μM) on capacitation and acrosome reaction of crossbred cattle bull spermatozoa. Fresh semen was centrifuged and pellet so obtained was suspended in TALP medium (pH 7.4). The sperm suspension was divided into four equal fractions(four tubes). In the control tube, only TALP was added; whereas the remaining three tubes were supplemented with ferrous ascorbate (FeAA – 150 μM FeSO4 : 750 μM ascorbic acid) , 60 μM Mn and FeAA+ Mn , respectively All fractions were incubated incubated (37°C) for 6 hr in CO2 incubator. These fractions were assessed for morphology, % hyperactivity, lipid peroxidation (LPO) and % acrosome reaction. Morphology of the capacitated spermatozoa remained unaltered with FeAA/Mn/FeAA+Mn treatments. FeAA treatment non-significantly (p≥ 0.05) decreased % sperm hyperactivity % acrosome reaction, but, increased the LPO level. Supplementation of Mn increased % hyperactivity and % acrosome reaction significantly (p≤ 0.05) in FeAA untreated fractions, but, non-significantly (p≥ 0.05) in FeAA treated fractions. Malondialdehyde (MDA – end product of LPO) decreased significantly (p≤ 0.05) with the Mn supplementation both in FeAA treated and untreated fractions. It is concluded that Mn protects the bull sperm against LPO/oxidative stress and facilitates the occurrence of capacitation and acrosome reaction.


Introduction
For successful fertilization, mammalian spermatozoa must undergo a preparation period known as capacitation (O'FLAHERTY et al., 1999).In physiological terms, capacitation can be considered as the sum of biochemical and biophysical modifications that take place in sperm cell during its transport through the female genital tract (PARRISH et al., 1999).These modifications regulate temporary changes in the pattern of sperm motility referred to as hyperactivation.This process culminates in an exocytotic event called acrosome reaction, an essential step to fertilize oocytes (O'FLAHERTY et al., 1997).Mammalian spermatozoa represents a growing list of cell types that exhibit a capacity to generate highly reactive oxygen species (ROS).Controlled generation of ROS has a physiological role in spermatozoal functions such as hyperactivation, capacitation and acrosome reaction (de LAMIRANDE et al., 1993;GRIVEAU et al., 1997;O'FLAHERTY et al.,2005).Uncontrolled and excessive ROS production or exposure of spermatozoa to fatty acidperoxides or the combination of ferrous ions( Fe 2+ ) and ascorbic acid (AA) at high concentrations induces lipid peroxidation (LPO) / oxidative stress, resulting in decreased sperm motility , viability and increased mid-piece defects that impair capacitation and acrosome reaction ( de LAMIRANDE et al., 1993( de LAMIRANDE et al., & 1995;;BANSAL and BILASPURI, 2007).Therefore, an antioxidant that reduces oxidative stress and improves spermatozoal functions could be useful in the management of male infertility ( VERMA and KANWAR, 1999).Antioxidants are the agents, which break the oxidative chain reaction, thereby, reducing the oxidative stress (MILLER and SLEBODZINSKA, 1993;KUMAR and MAHMOOD,2001).A variety of biological antioxidants that attack ROS and LPO are presently under investigation (SIKKA, 1996).
The antioxidative action of Mn (II) on different peroxidizing systems has been studied.It inhibits LPO produced by free radical producing system but not the one induced by singlet oxygen (CAVALLINI et al., 1984).Manganese in very -very small amount affects human health and participates in many enzymatic activities ( SINGH, 2008).It is an essential component of several enzymes and some of them (superoxide dismutase, pseudo-catalase and the photosynthetic oxygen evolving centre) are involved in redox processes.Manganese has also been assigned as a chain breaking antioxidant, as it is able to quench the peroxyl radicals (COASSIN et al., 1992).
The present study was undertaken to examine the effect of 60 µM Mn2+ on morphology, % hyperactivity, % acrosome reaction and changes in lipid peroxidation (LPO) level during capacitation and acrosome reaction of crossbred cattle bull spermatozoa.

Morphology and percentage acrosome reaction:
Smears of each fraction were prepared at 2 hr interval, stained with Giemsa and examined under oil emulsion using binocular microscope and percentage acrosome reaction and morphology of spermatozoa of all fractions were compared.
'Analysis of Factorial Experiment in CRD'(computer software programme) or 'one way variance analysis' was used to evaluate the significance levels between the parameters studied.Bull to bull variations were negligible , therefore, the effect of factor bull was not considered.The critical difference (CD) of three factors-A (incubation period), B (control and treatments) and AB (interaction between incubation periods and treatments) obtained were used to find the level of significance.A 'P' value of 0.05 was selected as a criterion for statistically significant differences.

Morphology:
Morphology of bull spermatozoa remains unaffected with FeAA/Mn 2+ /FeAA + Mn 2+ treatments, however, the spermatozoa undergo normal process of capacitation and acrosome reaction.The stages during these processes observed in bull spermatozoa are clearly seen in Figs. 1 to 4.

Lipid peroxidation and % acrosome reaction:
Corresponding to the incubation period, LPO of all fractions increased significantly (P≤0.05) after 4 hr and 6 hr as compared to 0 hr of incubation (Table 2).FeAA treatment to the bull sperm increased the LPO non-significantly (P≥0.05).However, supplementation of Mn 2+ decreased the MDA production significantly (P≤0.05) in both FeAA treated and untreated spermatozoal samples (Table 2).Statistical analysis shows non-significant interaction (P≥0.05) between treatments and incubation periods, thus, increase or decrease in LPO with treatments is not affected by incubation periods or viceversa.
The % acrosome reaction of bull spermatozoa increased significantly (P≤0.05) from 4 hr to 6 hr (Table 3).FeAA treatment decreased the % acrosome reaction non-significantly (P≥0.05) as compared to the control.Subsequently, supplementation of 60 µM Mn 2+ increased % acrosome reaction significantly (P≤0.05) in FeAA untreated, but, nonsignificantly (P≥0.05) in FeAA treated fractions (Table 3).Statistical analysis shows nonsignificant (P≥0.05)interaction between the treatments and incubation periods.Thus, increase or decrease in acrosome reaction with various treatments is not affected by incubation periods or vice-versa.

Discussion
In our study, % sperm hyperactivity decreases from 3 hr to 6 hr of incubation.It indicates that during capacitation, motility of spermatozoa decreases gradually and significantly (P≤0.05).Similar observations were made by DHANJU et al., 2006, who reported a 15 to 21% and 55 to 62% decline in the percentage motility during incubation of spermatozoa in TALP medium and 0.85 % saline, respectively.Corresponding to the treatments, by inducing oxidative stress, FeAA decreases the % hyperactivity.It may be explained on the basis of oxidative stress caused by FeAA which deteriorates the membrane permeability and integrity, thereby, decreasing the % hyperactivity or motility (KODAMA et al., 1996).The present study assumes that supplementation of Mn 2+ to FeAA treated/untreated samples increases the % hyperactivity by activating a single transduction cascade.
Following model of signal transduction for the role of Mn 2+ in enhancing % hyperactivity is suggested based on our study and relevant literature (LAPOINTE et al., 1996;GARBERS and KOPF, 1980;ROJAS et al., 1992;TASH and MEANS, 1983).Messenger systems such as adenylate cyclase, guanylate cyclase and calmodulin are highly affected by intracellular salts, such as Mn 2+ , Mg 2+ and Ca 2+ .Out of these, Mn 2+ is a well known potent stimulator of adenylate cyclase activity, which in turn enhances the level of cAMP (LAPOINTE et al., 1996).Increase in the level of cAMP with the Mn 2+ supplementation phosphorylates many proteins that are involved in the movements or flagellar beating of spermatozoa.Increase in the level of cAMP also stimulates the Ca 2+ uptake by the cell, thus, increasing the level of intracellular calcium (Ca i 2+ ) (GURAYA, 1999).At a higher level, Ca i 2+ increase the membrane integrity and viability.These properties of the sperm are required for its optimal functioning under normal and oxidative stress conditions.Elevation of Ca i 2+ in flagellum of spermatozoa drives hyperactivation and this action of calcium could be at the level of sperm flagella as revealed by experiments with demembranated rat (LINDEMANN and GOTZ, 1988) and bull (LINDEMANN et al., 1991) spermatozoa.
The previous literature suggest that the intake of Ca 2+ is slow during capacitation; but rapid at the time of acrosome reaction.Adequate level of Ca 2+ is required in sperm, once the capacitation gets completed (GURAYA,1999).LARSEN (1994) found that high concentration of Ca i 2+ is related to the cell death.Based on the present and above studies, it is suggested that supplementation of Mn 2+ to the bull spermatozoa permits the rise in Ca i 2+ level without decreasing their viability.Further, Mn 2+ has beneficial effects on sperm survival and % hyperactivity during capacitation and acrosome reaction.
The present study also shows that Mn 2+ possess the antioxidative property as they decrease the MDA production significantly in both FeAA treated as well untreated samples.60 µM Mn 2+ supplementation increases the rate of acrosome reaction under normal and oxidative stress conditions.Thus, on correlating the results of LPO and acrosome reaction, it is suggested that supplementation of Mn 2+ to the bull sperm enhances the % acrosome reaction by decreasing the oxidative stress.As manganese inhibits LPO both in vitro (TAM and McCAY, 1970) and in vivo (SHUKLA and CHANDRA, 1981), it is suggested that its antioxidative property stabilizes the plasma membrane, thereby, maintaining membrane integrity and viability.Therefore, Mn 2+ enhance the rate of acrosome reaction in the present study.Similar observations on acrosome reaction have been made, when bull sperms are incubated with 0.1 mM MnCl 2 (LAPOINTE et al., 1996).Another possible explanation for the increase in sperm capacitation and acrosome reaction with Mn 2+ supplementation is related to the increase in intracellular calcium (Ca i 2+ ) content.
The following model for the role of Mn 2+ in sperm capacitation and acrosome reaction is suggested based on our study and relevant literature (GARBERS and KOPF, 1980;TASH and MEANS, 1983;SIDHU et al., 1984;SIDHU and GURAYA, 1989).According to this model, calmodulin or calmodulin-like proteins loosely bind to the plasma membrane and/or Ca 2+ or Mg 2+ -ATPase.These bindings enhance the extrusion of intracellular calcium (Ca i 2+ ) and interfere with the capacitation and acrosome reaction processes.However, Mn 2+ supplementation stimulates the calmodulin removal from its receptors, thereby, enhancing the Ca i 2+ level.As more and more Ca i 2+ depositing leads to vesiculation of the acrosome, it causes the fusion of outer acrosomal membrane with the plasma membrane, thus, resulting in acrosome reaction.Extracellular addition of Mn 2+ ions also enhance the level of cAMP by stimulating Ca 2+ or Mg 2+ -ATPase which lead to the activation of calcium channel openings, thereby, depositing more Ca i 2+ .Thus, Mn 2+ promotes the acrosome reaction.
Further studies are required to understand the antioxidant strategies or the mechanisms whereby ROS and endogenous antioxidants produced in sperm cells influence the reproductive processes, and ,thereby, promote the fertility of cattle bull spermatozoa.
It is concluded that Mn 2+ protects the crossbred cattle bull sperm against LPO / oxidative stress and facilitates the occurrence of capacitation and acrosome reaction.

Fig. 1 :
Fig. 1: Cattle bull spermatozoa incubated for 2hr in Fig. 2: Cattle bull spermatozoa incubated for 4hr in Mn 2+ (60μM) at 37° C. The capacitation has Mn 2+ (60μM) at 37° C. The capacitation has not started yet and spermatozoa show normal has started as shown by swelling (S)in the (N)morphology.headregion.