V. R. Upadhyay, Atul Walunj and Nishtha Kushwah
ICAR-National Dairy Research Institute, Karnal, Haryana-132001Introduction:Under intensive Dairy cattle farming, optimum milk production heavily rests upon efficient reproductive management. High fertility of bull plays an important role in cow reproduction and in genetic improvement. This includes availability of good quality semen straws. Many factors affect semen qualities and fertility of bull, season is one such factor affecting semen quality and fertility. With rising ambient temperature, semen and its further processing for making straws becomes more vulnerable to damaging effects of free radicals/ reactive species generation thereby contributing to impaired sperm viability and fertility. Apart from the conventional causes for male infertility a new and important cause has been identified due to toxic effect of these reactive species. Infliction of such stress and sperm damage caused by reactive nitrogen species (RNS) is referred to as nitrosative stress. Thus, the purpose of this article is to address the impact of RNS on patho-physiology of seminal quality and consequently on male infertility.Nitrosative stress:Nitrosative stress is part of oxidative stress which has become a real concern for the dairy reproduction in recent years because of their potential toxic on sperm quality and function. High environmental temperature during summer season affect male reproduction in deleterious ways, nitrosative stress is one of which deteriotes semen quality thus hampering bull fertility during dry and humid hot season especially in crossbred bull. Also nitrosative stress is a result of the imbalance between reactive nitrogen species and antioxidants in the seminal plasma of the animal. It is a powerful mechanism that can lead to sperm damage, deformity and eventually, male infertility.
Although Physiological levels of RNS are necessary to maintain the reproductive functions such as cell signaling, tight junction regulation, capacitation, acrosomal reaction, sperm motility and zona pellucida binding however excess generation of RNS can adversely affect reproductive potential by causing testicular dysfunction and impaired semen quality. Thus nitrosative stress results from an imbalance of free radicals and antioxidants defence mechanisms in the bull reproductive system can seriously inflict sperm pathology.
Seminal plasma is a vital source of antioxidants such as glutathione peroxidase, superoxide dismutase and catalase etc. seminal plasma efficiently counteracts RNS and maintains a physiological range of RNS through antioxidants present in the seminal plasma. Therefore antioxidants are good indicators of semen quality. But with the progressive rise in the hot dry and hot humid conditions, the seminal enzymatic antioxidants system becomes inefficient due to micro and trace mineral deficiency coupled with reduced feed intake in breeding bulls.
Fig. Types of reactive nitrogen species(RNS).
In recent years, nitrosative stress in semen has been increasingly been recognized as potent stress type adversely affecting semen quality. Reactive nitrogen species are a potent oxidant and nitrating agent formed by reaction between nitric oxide and superoxide produced via enzymatic activity of nitric oxide synthase. They are highly unstable and react with a variety of lipids, proteins, and components of semen. Of the most common, Nitric oxide reacts with various chemicals mediators to form much harmful potent reactive nitrogen species eg. peroxynitrite. All this gets initiated during spermatogenesis in testis. High concentration of nitric oxide in semen has significantly been correlated with sperm motility and semen variables. Available literature suggests that Nitric oxide being the progenitor of various RNS can be recognized as biomarker of nitrosative stress in semen.
Sources of reactive nitrogen species
It has been observed that immature, defective, damaged and dead sperms are the major source of RNS. Leucocytes and other inflammatory cells during pathological conditions of male reproductive tract such as affections of seminal vesicles and other accessory sex glands are other sources for nitrosative stress in bovine semen. Mitochondria are also the prime source of RNS, spermatozoa containing dysfunctional mitochondria sources into increased production of ROS affecting mitochondrial function and ultimately motility.Effect on seminal attributes:RNS, specially nitric oxide plays a bimodal action on semen parameters giving positive response at physiological level while at toxic levels, it can cause a variety of pathological effects on sperm parameters and normal cellular functions.
Fig. Detrimental role of oxidative and adjoining nitrosative stress on semen parameters.Sperm morphology:Pathologic concentration has strongly been implicated in the lipid peroxidation of polyunsaturated fatty acids within the sperm plasma membrane. This NO mediated chain reaction leads to disturbance in structural integrity of sperm resulting into abnormal sperm morphology and impaired sperm functions.Sperm motility:Nitric oxide exerts a bimodal action on sperm motility; specifically at low concentrations of NO there is increase in the motility of spermatozoa while at higher concentrations of NO there is reduction in the motility of spermatozoa through formation of protein nitrotyrosine. Another reason for suppressed Sperm motility is reduced ATP generation caused by dysfunction of mitochondria transmembrane proton gradient and leakage of electrons and free radical formation. Moreover, high concentrations of NO have been reported in the semen of infertile male with decreased sperm motility. Post thaw sperm motility was improved at lower concentrations of NO.Sperm DNA integrity:In particular, the reaction of NO with the superoxide anion results in the formation of a more noxious oxidant, peroxynitrite which are highly toxic to sperm. Peroxynitrite and its breakdown product, Peroxynitrous acid (HONOO), are capable of inducing peroxidative damage and sperm DNA damage. Damage to DNA can affect sperm functions like motility by declining the ATP production required for the sperm movement. NO reacts with O2 to form the nitrosating agent nitrous anhydride which then nitrosate primary amine functionalities on DNA bases, leading to direct DNA damage via Deamination. Nitrosative damage leads to DNA damage by down-regulation of various genes involved in DNA repair.Sperm apoptotic rate:High levels of NO damage the mitochondrial membrane of the sperm, inducing the release of cytochrome-c and activation of the caspase cascade attributing to enhanced sperm apoptotic rate. Activation of caspase cascade leads to expression of phosphotidylserine – sperm apoptosis marker. ROS, RNS and the consequential cellular redox imbalance work as strong stimulants in apoptotic signal transduction in damaged sperm.Sperm capacitation and acrosome reaction:Physiologically nitric oxide is required for the event of capacitation and acrosome reaction to occur. Level of NO influence the rate of initiation of above physiologically important events. In this regard supra-physiological level of nitric oxide production results into early capacitation during crypreservation referred to as cryocapacitation which reduces fertilizing ability of viable sperm. Production of RNS during freeze-thaw procedure is one of the major cause of cryocapacitation. RNS was reported to activate membrane targets i.e. adenyl cyclase causing an intracellular increase in cAMP sufficient to trigger the intracellular mechanisms involved in sperm capacitation. Similar observations are recorded about the acrosome reaction. With respect to seasonal variation lowest acrosome integrity percent is observed during summer and highest during winter season, thus greatly hampering the fertilizing quality of sperm.Conclusion:Even though the controlled generation of ROS and RNS may have physiologic functions as signaling molecules (second messengers) in many different cell types, their uncontrolled production is considered an important factor and invoke profound changes in semeninal quality parameters and gene expression. Heat stress damage in bovines is mediated by nitrosative stress in semen as it serves as a significant entity responsible for reducing semen motility and viability. Inherent antioxidant system is insufficient to protect spermatozoa from oxidative damage during cryopreservation. Spermatozoa are highly vulnerable to these oxidative and nitrosative attacks since they lack significant antioxidant protection and appropriate armory of defensive enzymes during this storage period. Thus in countries like India, where lack of quality semen straws has been the main hurdle in dairy animal improvement, demand of fertilizable semen straws are continuously increasing. Thus in this regard nitric oxide can serve as a diagnostic biomarker of nitrosative stress in bovine semen and its physiological level can be maintained by adding optimum quantity of NO inhibitors like L-NAME and RNS scavengers (antioxidants) in extender.
