EVALUATION OF IN VITRO SPERM CHARACTERS
ICAR-NDRI, Eastern Regional Station
Kalyani, West Bengal
Part of the complexity dealing with the spermatozoa came from the fact that each spermatozoon was a multi-compartmental cell that must possess different attributes to be able to fertilize an oocyte. Each spermatozoon must possess motility, active mitochondria to supply the energy necessary for motility, intact acrosomal membranes that were capable of undergoing capacitation changes thereby permitting the acrosome reaction to occur, plasma membranes that permitted fusion with the oolemma and a nucleus that was capable of proper decondensation and nuclear reorganization to maintain zygotic and embryonic development.
The traits of a semen sample that were important for fertility were divided into compensable and uncompensable. Compensable traits were those that did not affect fertility if high numbers of spermatozoa were used during insemination (Ballachey et al., 1988). For example, spermatozoa with defects in compensable traits (i.e., motility and morphology) might have difficulty crossing the barriers of the female reproductive tract to reach the site of fertilization. Because an excessive number of spermatozoa were usually inseminated, compensable traits were not as closely related to fertility as uncompensable traits. Uncompensable traits were those that were not overcome by increasing the number of spermatozoa in the inseminate because these defects affected the function of spermatozoa during later stages of fertilization and embryonic development. Nuclear vacuoles and defective chromatin structure were examples for uncompensable traits (Ballachey et al., 1988).
Sperm cell abnormalities were classified based on the location of defects (head, tail, mid piece) or site of origin (primary: testis; secondary: epididymis; tertiary: accessory glands/post ejaculation). The significance of specific sperm abnormalities were better understood from the results of mating trials, analysis of non-return rates to artificial insemination and in vitro fertilization with semen containing high percentages of sperm with individual classes of abnormalities.
Accurate morphological screening of the ejaculates allowed elimination of bulls with a potential low fertility, prior to the entrance of bulls to progeny testing program and the preservation of semen, thus contributing to a major saving for AI enterprises (Padrick and Jaakma, 2002; Esteso et al., 2006).. There was undoubtedly a correlation between motility and fertility as well as for morphology and fertility; however these correlations were reduced concomitantly with an increase in the lower limit set to accept an ejaculate for further processing. When the acceptable range for these parameters was narrow, motility and gross morphology only had limited value for separating ejaculates in respect to the expected fertility of the ejaculate.
Plasma membrane integrity
Structural and functional integrity of sperm outer membrane (plasmalemma) was essential for sperm metabolism, capacitation, ova binding and acrosome reaction. Membrane exerted role in the maintenance of the sperm fertilizing capability (Oura and Toshimori, 1990). The plasma membrane was responsible for the mechanism of maintaining the cell osmotic equilibrium, acting as a barrier between intra and extra cellular mediums. Damages in this structure conduced to homeostasis loss, leading to cellular death. Consequently, plasma membrane integrity was crucial to sperm survival inside the female reproductive tract (Oura and Toshimori, 1990).
The sperm plasma membrane was the primary site where lesions occured during freezing-thawing of semen (Hammerstedt et al., 1990). It was recognized during the last several decades that one of the major features discriminating dead from live cells was a loss of the transport function and physical integrity of the plasma membrane. For example, since the intact membrane of live cells excluded a variety of charged dyes, such as trypan blue or propidium iodide (PI), incubation with these dyes resulted in selective labeling of dead cells, while live cells showed no or minimal dye uptake. A combination of supra vital staining dyes such as trypan blue/giemsa, eosin/aniline blue and some other classical dyes were widely used for differential live/dead staining of spermatozoa. For light microscopic evaluation a relatively high concentration of the dye (in mg/ml) was required. At these concentrations, eosin and many other dyes were toxic, which can lead to under estimation of the proportion of live cells (Woelders, 1991).
The development of staining technology using fluorophores for nucleic acid, intra cytoplasmic enzymes or membrane potential provided new tools for assessing the functionality of frozen-thawed spermatozoa. Single fluorophores or in combinations were used to determine sperm membrane integrity. The combination of carboxyfluoroscein diacetate (CFDA) with propidium iodide (PI), or CYBR-14 with PI was commonly used. PI was membrane-impermeant red fluorescent molecule that entered the nucleus of a cell in which the plasma membrane was damaged. CFDA was a membrane-permeant colourless substrate that was rapidly converted by intracellular esterases into a membrane-impermeant green fluorescent derivative (Garner et al., 2001; Colenbrander et al., 2003). Used in combination, cells with damaged membranes fluoresced red as PI entered the cell, and intracellular esterases leaked from the cell, therefore, CFDA was not converted into its green derivative. Cells with intact membranes fluoresced green as the membranes prohibited PI entry and the retained esterases converted CFDA into a green derivative. In contrast, SYBR-14 was a membrane-permeant green fluorescent probe that bound to the DNA in the nucleus of all cells, both membrane intact and membrane compromised. When used in combination with PI, cells with intact plasma membranes only stained with SYBR-14 and fluoresced green, while cells with damaged plasma membranes stained with both SYBR- 14 and PI and fluoresced red, or red-orange as the fluorescence of the PI was brighter than that of the SYBR-14 (Garner and Johnson, 1995).
Functional membrane integrity
Vital staining of sperm evaluated the structural integrity of the plasma membrane which indicated whether the sperm cell was viable or dead. But in the viable cells the functional integrity of plasma membrane had to be evaluated since sperm required an active membrane during fertilization and will fail to fertilize ovum if plasma membrane was physically intact but biochemically / functionally inactive. Functional integrity of the plasma membrane was evaluated by measuring the resistance of sperm membranes to swelling in a hypo-osmotic medium. This much simpler method was based on the ability of the membranes to allow passage of water in order to establish equilibrium between the fluid compartment within the spermatozoon and the external surroundings (Drevius and Eriksson, 1996). It was suggested that the ability of spermatozoa to swell in the presence of hypo-osmotic medium reflected normal water transport across the sperm membrane, which was a sign of normal membrane integrity and functional activity (Jeyendran, et al., 1984). Spermatozoa with compromised or inactive membranes were unable to regulate water influx and remain not swollen. Brito et al. (2003) concluded that HOST was the only plasmalemma functional evaluation method that significantly contributed to conventional sperm quality tests in predicting in vitro fertilization rate. The use of this inexpensive and simple assay was recommended as an additional fertility indicator to be incorporated in the routine semen analysis.
Mitochondrial membrane potential of sperm cells
Energy was stored in mitochondria as a proton concentration gradient maintained by the inner mitochondrial membrane which drives the synthesis of ATP. Membrane permeable lipophilic cations accumulated in the mitochondria and exhibited a negative interior membrane potential. The lipophilic cationic fluorescent carbocyanine dye, JC-1, was used to differentially label mitochondria with high and low membrane potential. When JC-1 formed monomers in mitochondria with low potential, the JC-1 stain emited a green fluorescence at 510-520 nm when the JC-1 formed multimers known as J-aggregates after accumulation in mitochondria with high membrane potential, the JC-1 stain emited a bright red-orange fluorescence at 590 nm. Any changes in mitochondrial membrane potential were a good indicator of sperm motility. These dyes were used to study the effect of cryopreservation on bovine sperm organelle function and viability (Thomas et al., 1998). They showed that fluorometric measurement of mitochondrial function after thawing correlated with SYBR-14-assessed sperm viability and with microscopic assessment of motility.
Sperm chromatin integrity was vital for successful pregnancy and transmission of genetic material to the offspring. The nuclear chromatin of mammalian sperm had a peculiar organizational status characterized by a remarkable process of remodeling or condensation (Govin et al., 2004; Caron et al., 2005). The sperm DNA was organized in a specific way that kept the chromatin compact and stable in the nucleus. It was packed into a tight, almost crystalline condition and occupied nearly the whole nucleus (Fuentes-Mascorro et al., 2000). This process of condensation occured in two main phases. The first phase, which occurred in the testis, involved the substitution of somatic histones by testis-specific protamines (Caron et al., 2005). The protamines contained numerous cysteine residues, which generated disulphide cross-links between adjacent protamine molecules during the condensation of the chromatin. The formation of large numbers of disulphide cross-links between protamine molecules occurred in the second main phase of chromatin condensation, when the sperm left the caput epididymis and were en route to the cauda epididymis (Caron et al., 2005).
Sperm DNA fragmentation resulted from aberrant chromatin packaging during spermatogenesis (Gorczyca et al., 1993; Manicardi et al., 1995; Sailer et al., 1995). The sperm DNA integrity was reported to be both unaltered (Evenson et al., 1994; Evenson et al., 2002; Van der Schans et al., 2000) and impaired after cryopreservation (Hamamah et al., 1990; Bochenek et al., 2001; Baumber et al., 2000; Peris et al., 2004).
Several methods were developed to evaluate the DNA integrity. The sperm chromatin structure assay (SCSA), which was considered the most efficient and successful, used flow cytometric analysis (Evenson et al., 2002). TUNEL and Comet assays were also used to measure DNA fragmentation (Duty et al., 2002; Sakkas et al., 2002; Evenson and Wixon 2006). The acridine orange staining was a simple microscopic procedure based on the same principle as the SCSA (Chohan et al., 2004). Acridine orange intercalated into native DNA and fluoresced green upon exposure to blue light and red when related with single stranded DNA.
Sperm with chromatin abnormalities were associated with reduced fertility or abortions (Chemes and Rawe 2003). Fertilization by sperm with fragmented DNA resulted in poor embryonic development, decreased implantation, lower pregnancy rates, and recurrent pregnancy losses in human (Henkel et al, 2003; Virro et al, 2004) and reduced fertility in bulls (Kasimanickam et al., 2006).
Motility and gross morphology were most used parameters for semen quality assessment. Variations of 30–60 per cent have been reported in subjective microscopic evaluations of motility characters of human and animal semen in the same ejaculates (Amann, 1989; Auger et al., 1993). Despite a close match between subjective and objective evaluations of sperm motility, subjective estimation of motility was affected by numerous factors (Verstegen et al., 2002; Rijsselaere et al., 2003).
The development of computer-assisted semen analysis (CASA), using software that analyzed every sperm track characteristics, had strongly improved the semen evaluation. The availability of data recorded by CASA facilitated the comparison of results and made it possible to find subtle differences between bulls or treatments (Verstegen et al., 2002). Furthermore, CASA systems appeared to have high accuracy and repeatability (Farrell et al., 1995). CASA was an objective method that gave extensive information about the kinetic property of the ejaculate based on measurements of the individual sperm cells. However, CASA were not ready-to-use devices, thus results depended largely on the expertise of the user and the technical settings (Mortimer et al., 1995). Numerous variables like the frequency of frame acquisition, number of fields checked, concentration of semen sample and dilution rate affected motility resulted in semen evaluation even with the same CASA device (Rijsselaere et al., 2003).
Correlation coefficients estimated between post thaw motility and in vitro fertilization rate were of very low magnitude. The lower correlations may be because motility reflected the ability of sperm to reach fertilization site, but not its ability to undergo capacitation, acrosome reaction and oocyte penetration (Graham et al.,1987; Brahmkshtri et al., 1999).
Capacitation status of sperm cells
The process of capacitation was originally recognized by Austin (1952) and Chang (1951) independently, who reported that spermatozoa had to reside in the female reproductive tract for acquisition of fertilizing competence. Capacitation was a continuous biochemical change associated with the functional and structural changes in the sperm. Removal of cholesterol from sperm membrane increased membrane fluidity (Langlais et al., 1988), resulted in increased calcium influx (Singh et al., 1978), cAMP level (White and Aitken, 1989) and changed some enzymatic activities such as protein kinase C (Furuya et al., 1993). These biochemical modifications lead to a transient change in the pattern of sperm motility called hyperactivation (Yanagimachi and Usui, 1974). The preparation process ended with an exocytotic event called the acrosome reaction, an essential stage for oocyte fertilization (O’Flaherty et al., 1999). The physiological mammalian acrosome reaction was experienced only by sperm that had been previously capacitated.
Cryopreservation resulted in a loss of lipids from the sperm membranes and a rearrangement of lipids and proteins within the membrane. Destabilization of sperm membranes following cooling resembled that of the physiological sperm capacitation (Collin et al., 2000) and resulted in a ‘‘precapacitated’’ spermatozoa with a reduced fertilizing lifespan. Therefore, assays to evaluate sperm capacitation were used to evaluate the normalcy of spermatozoa after freezing and thawing.
The destabilization of sperm membranes were evaluated by tracking the distribution of Ca2+ in spermatozoa. There were two basic classes of Ca2+ indicators. The main example of the first class was antibiotic chlortetracycline (CTC), which accumulated in organelles containing high concentrations of Ca2+. The second class consisted of molecules that resided in aqueous compartments such as cytosol and changed their spectra when bound to Ca2+. Indicators of cytosolic free Ca2+ concentrations are quin-2, fura-2, indo-1 and fluo-3 (Tsien, 1989). Neutral uncomplexed CTC easily crossed the membranes where it ionized to an anion and chelated Ca2+. The latter complex bound preferentially to hydrophobic site, such as membrane and showed increased fluorescence as a result. The extent of the binding to membranes depended on the surface-to-volume ratio of the vesicle and the properties of the lipid. Due to the compartmentalization of the plasma membrane of spermatozoa, several distinct staining patterns were evaluated which were associated with a functional status of spermatozoon (Fraser et al., 1995). A number of investigators found that human, mouse, bull (Cormier et al, 1997), boar and several other species exhibited similar patterns of CTC staining. The three patterns were F Pattern, with uniform fluorescence on the head that indicated uncapacitated, acrosome intact spermatozoa; B Pattern, with a fluorescence-free band on the post acrosomal region that indicated capacitated, acrosome intact spermatozoa; and AR pattern with uniformly fluorescence-free head and with a fluorescence band on the equatorial region that indicated acrosome reaction. Several studies (Thomas et al., 1997) were performed aiming to find the key to the hypothesis that freezing/thawing destabilized sperm membranes and the extent of destabilization was inversely related to the fertility values.
The acrosome, a large lysosome- like vesicle overlying the sperm nucleus contained large array of powerful hydrolyzing enzymes including hyaluronidase and acrosin (Zaneveld and De Jonge, 1991). The acrosome of spermatozoa was to be maintained intact up to the time it bound to zona pellucida of the oocyte and underwent the acrosome reaction to release acrosomal enzymes (Graham et al., 1987). Therefore an intact acrosome was a must before and during the transit of the sperm to the isthmus until zona binding was accomplished.
Two types of acrosome reaction were described for mammalian spermatozoa; a true acrosome reaction that consisted of progressive vesiculation of the outer acrosome membrane and overlying plasma membrane, and a false acrosome reaction in which the acrosome was lost following cell death. Procedures for the detection of true acrosome reaction in spermatozoa included triple stain technique (trypan blue, bismark brown and rose bengal stains), lectins labeled with flurochromes (Cummins et al., 1986) and antibiotic chlortetracycline (CTC) which stained the sperm surface differently depending upon the stage of capacitation (Kim and Gerton, 2003). The use of Coomassie blue G- 250 staining was another reliable method for the assessment of acrosomal status in variety of mammalian species (Larson and Miller, 1999) by simple microscopy.