1. Introduction
Sperm performance, a major determinant of male fertility, can be dissected into a series of traits that are intricately connected to sperm fertilizing potential [
1,
2]. Therefore, a combination of various performance parameters is usually assessed to evaluate the quality of sperm samples and predict its fertilizing potential in man and other animals [
2,
3,
4,
5,
6,
7,
8,
9,
10]. Sperm viability, motility and kinetics, as well as acrosome integrity, are all linked to sperm survival. The proportion of motile sperm and the velocity of spermatozoa are essential for sperm to swim along the cervix, uterus and the uterotubal junction, and cells that show higher values in these parameters also have higher chances to reach and fertilize the ovum [
11]. The assessment of sperm motility may be carried out subjectively, estimating the percentage of motile sperm by microscopic visualization, and objectively through the quantification of sperm swimming parameters by computer aided sperm analysis (CASA) [
1,
2,
10,
12,
13].
The sperm acrosome contains enzymes that are released during exocytosis, an essential step required for penetration of the oocyte’s vestments [
14]. The timing of release is important, so the integrity of the acrosome during sperm transport in the female tract and, in particular, along the oviduct, has to be preserved in order to ensure fertilizing potential [
15]. Controversy still exists as to which is the site where acrosomal exocytosis takes place or which is the physiological ligand (or ligands) responsible for initiating exocytosis [
16,
17,
18,
19]. Nevertheless, since this is a key event in the processes leading to completion of fertilization, acrosomal status has become a valuable assessment of fertilizing potential [
20].
Other relevant sperm parameters relate to sperm bioenergetics, with sperm ATP content serving as an indicator of the balance between sperm ATP production and consumption [
21]. High ATP levels are positively correlated to sperm swimming velocity in rodents [
22] and mammals in general [
23]. Moreover, assessments of the variation in sperm ATP content and sperm traits over time among rodent species revealed that maintenance of high performance in species with high competitive ability is associated with high concentrations of intracellular ATP over time [
22,
24,
25].
Exogenous factors could influence sperm performance in vivo and under in vitro conditions [
26]. Besides the provision of energetic substrates that are fundamental for the production of ATP for sperm motility and survival, conditions in the female tract promote the acquisition of sperm’s fertilizing ability, a process known as capacitation [
27]. Among physical factors, extracellular pH, temperature, and viscosity are known to affect the survival and performance of sperm cells [
28,
29,
30,
31,
32,
33,
34,
35]. Several biological factors are also known to have important roles in sperm survival in vivo and in vitro. Early efforts to achieve in vitro fertilization had to rely on homologous or heterologous serum to ensure sperm survival and the acquisition of fertilizing ability [
36,
37]. Better definition of media was possible with replacement of serum by bovine serum albumin. However, these media were still not completely defined [
38]. Components of the extracellular milieu may be required to sustain sperm motility, and variations between species may exist regarding the nature of such components. For golden hamster (
Mesocricetus auratus) spermatozoa, one set of important motility factors are catecholamines, which maintain and stimulate sperm motility in vitro [
39]. Within the group of catecholamines, epinephrine is an essential co-factor for golden hamster sperm as it activates motility as well as Na
+/K
+ ATPase and Ca
2+-ATPase [
40], and it is also involved in the acquisition of fertilizing ability [
41]. However, epinephrine is not able to maintain golden hamster sperm motility on its own, and a second factor, hypotaurine, is also essential. Hypotaurine is a superoxide scavenger that functions inhibiting lipid peroxidation and superoxide dismutase [
42], which prevents motility loss. Hypotaurine and epinephrine together also cause a mild increase of the acrosome reaction in hamster sperm [
43]. Other factors have been shown to influence hamster sperm function [
44,
45,
46].
Another molecule of interest, with regards to hamster sperm survival, is D-penicillamine. This is an α-amino acid which acts as a cation chelator, protecting sperm from oxidation in several species [
40,
47,
48,
49,
50]. As a zinc chelator, D-penicillamine facilitates capacitation, acting in the early step of this process because it removes most of the zinc within the first ten minutes of its addition, but it is not enough to support full capacitation [
51]. D-penicillamine also prolongs hyperactivated motility [
52]. This amino acid together with epinephrine and hypotaurine (PHE: penicillamine + hypotaurine + epinephrine) act as motility factors, necessary for the maintenance of golden hamster sperm motility in vitro [
51]. The addition of PHE to incubation media maintains golden hamster sperm motility within the first hour [
53,
54] and also promotes sperm capacitation in this [
54] and other species [
55]. The synergistic effect of the three components of PHE can reactivate immotile spermatozoa of golden hamsters [
56].
Many early studies of sperm function have used the golden hamster as a model, particularly because of the ease of examining acrosomal status in motile spermatozoa [
57]. Studies of sperm behavior in a related species, the Chinese hamster (
Cricetulus griseus), have been performed [
58,
59,
60] and these studies have also identified the need to support sperm viability and motility over time to achieve fertilization. Similar requirements seem to exist for Siberian hamster (
Phodopus sungorus) spermatozoa because low success was achieved in in vitro fertilization with a variety of media and supplements [
61], but characterization of these requirements has not been undertaken. Hamsters are a valuable model for studies of sperm-oocyte recognition and interaction. Hybridization has been observed among hamsters (
Mesocricetus species: [
62,
63,
64,
65,
66];
Phodopus species: [
67];
Allocricetulus species: [
68]), and cross-fertilization in vitro between golden and Chinese hamsters has been reported [
37]. Despite the potential of this group of species as a model for sperm biology, little is still known about the spermatozoa and fertilization of most species in this group. Comparative and evolutionary studies are needed to understand diversity in function and underlying mechanisms of sperm survival, capacitation and acrosomal exocytosis. Hamster species are attractive because they are at the higher end of the range of mammalian sperm dimensions [
69,
70], have high sperm swimming velocities [
22] and exhibit high levels of sperm ATP [
22,
24]. Such traits, characterizing these species as high performers, may be the result of intense sperm competition [
71]. Detailed comparative studies of sperm performance may therefore be rewarding in hamsters of the subfamily
Cricetinae for which good background information of phylogenetic relations currently exists based on molecular studies [
72] and chromosome evolution [
73,
74].
In the present study, we evaluated whether several biological molecules had a role in the performance of spermatozoa from five different species of hamster:
Mesocricetus auratus,
Cricetulus griseus,
Phodopus campbelli,
P. sungorus and
P. roborovskii, focusing on sperm motility, viability, sperm kinematics, acrosome integrity, and bioenergetics. Spermatozoa from these species are at the higher end of the range of sperm quality parameters among rodent species [
22,
24]; thus, a detailed analysis of modulators of sperm function in these species could help understand determinants of male fertility.
4. Discussion
The results of this study show that the addition of D-penicillamine, hypotaurine and epinephrine (PHE) to the incubation medium seems to be necessary to sustain the performance (motility, sperm swimming velocity and trajectory) and, to a lesser extent, acrosome integrity and viability, of M. auratus sperm throughout incubation. Moreover, PHE appears to have a positive effect on the maintenance of sperm quality (motility, viability, and ATP content) in C. griseus. In contrast, the presence of PHE had no consistent effect on spermatozoa of the Phodopus species.
Golden hamster sperm is known to be very susceptible to in vitro dilution for more than 15–30 min [
81,
82] unless the medium is supplemented with motility factors. The addition of epinephrine, taurine, hypotaurine, penicillamine, or their combination, to hamster sperm has been studied mainly in
M. auratus [
39,
40,
56,
81,
82,
83,
84,
85]. In the present work, a combination of D-penicillamine (20 µM), hypotaurine (100 µM) and epinephrine (1 µM) was selected to analyze their effects on sperm performance. Our results showed an improvement in motility, integrity of the acrosome and velocity and trajectory parameters from 2 h onwards after addition of PHE in
M. auratus. Several studies showed that epinephrine and hypotaurine have a synergistic effect in maintaining sperm motility and promoting the acrosome reaction [
40,
43]. These two molecules caused a slight enhancement in motility, acrosome reaction and fertility when added separately, and these effects were increased when used together [
83]. Moreover, the effect of hypotaurine occurs within the first 2 h [
83], in agreement with the present study, where sperm motility, velocity and trajectory showed better results 2 h after the addition of PHE in
M. auratus.
Epinephrine is a catecholamine that stimulates Na
+/K
+ ATPase and Ca
2+-ATPase, and inhibits certain phosphodiesterases [
40,
86,
87]. The inhibition of Na
+/K
+ ATPase decreased the acrosome reaction, whereas the inhibition of phosphodiesterase increased it [
85]. Hypotaurine is a β-amino acid present in ejaculated sperm and oviductal fluid of mammals [
43]. It is an intracellular scavenger that protects from lipid peroxidation and inactivation of superoxide dismutase by superoxidation, preventing sperm motility loss [
84]. D-penicillamine acts as a divalent cation chelator, increasing the effects of epinephrine by protecting it from oxidation [
40]. It was found that this α-amino acid could maintain sperm motility in hamsters at lower concentrations (10 µM) and stimulate sperm capacitation at higher concentrations (50 µM) [
51]. In the present study, the concentration of penicillamine was 20 µM, which seems to be adequate in
M. auratus to maintain or improve sperm traits for 4 h, in combination with epinephrine and hypotaurine. Taken together, results lead to the conclusion that the addition of PHE to
M. auratus spermatozoa improves sperm quality over time, probably through the interaction with ATPases and phosphodiesterases and by protecting sperm from oxidation.
Changes in sperm traits in
C. griseus after the addition of PHE are different from those observed in
M. auratus. In
C. griseus sperm motility and ATP content are enhanced at a later time, i.e., after 4 h of incubation with PHE. Moreover, sperm viability improves from 2 h onwards. In an earlier study [
58], it was found that only 20–30% of cauda epididymal
C. griseus sperm exhibit motility, which is significantly lower than the results in our study. This discrepancy may be due to differences in media used to collect and incubate spermatozoa because in the earlier study [
58] the medium contained epinephrine and taurine (instead of hypotaurine, as in our study), and lacked penicillamine. The improvement in sperm survival and performance observed in our work may be useful to improve the reduced in vitro fertilization success observed in this species [
58,
88]. Indeed, better fertilization rates were obtained when sperm were pre-exposed to PHE and to the Ca
2+ ionophore A23187, while doubling the usual Ca
2+ concentrations [
60].
Different patterns of sperm motility have been found when comparing
M. auratus with
C. griseus [
58]. Whereas
M. auratus sperm flex the entire length of the tail when moving,
C. griseus sperm move by vibrating the tails firmly. This difference in swimming patterns may explain the diverse results in both species. Despite these differences, the addition of PHE seems to promote an improvement of sperm performance in both species. There does not seem to be a relation between the presence or absence of PHE, ATP levels, and motility when comparing these two species. In
M. auratus PHE sustains motility better and for longer times than in controls, but there does not seem to be an effect on ATP levels. In
C. griseus, there seems to be a parallel improvement of PHE on both motility and ATP levels, but only at the end of incubation.
The results in
Phodopus species are somewhat surprising because we did not find differences over time in variables assessed in sperm incubated in the absence or presence of PHE. To the best of our knowledge, this is the first report examining the effects of PHE in these three species of hamsters. Since previous studies found that epinephrine acts through α and β-adrenergic receptors [
41] and hypotaurine activates ATPases and inhibits phosphodiesterases, differences between sperm from
Phodopus,
C. griseus and
M. auratus may be explained by differences in receptors or signaling mechanisms. Thus, one possibility could be that targets of PHE action vary in concentration between species, and thus effects are not visible. Another possibility is that responses to each factor vary, since different concentrations of penicillamine, hypotaurine and epinephrine are known to have different effects in somatic and sperm cells [
51,
86,
87]. More studies are necessary to elucidate if changes in concentrations of PHE may affect sperm traits in
Phodopus species. In any case, sperm quality in these hamsters is extremely high when compared to other hamster species after 4 h of incubation without PHE, indicating that the addition of these molecules or other motility factors may not be required to sustain sperm performance.