What provides energy for sperm movement

Role of Osmosensing for Fish Sperm Motility

Fish sperm motility is controlled tightly by osmolality and the concentration of cations. Therefore, fish sperm represents an excellent model for studying general mechanisms of cellular osmosensing in fish. Depending on habitat salinity and osmoregulatory strategy, sperm motility is either activated or inhibited by increased osmolality. In marine teleosts, sperm motility increases when released into a plasmahyperosmotic environment (SW), whereas in FW teleosts it increases upon release into a plasmahypoosmotic environment (FW). In striped bass sperm motility is promoted by Ca2+-free solutions and suppressed when [Ca2+] exceeds 10 mM. The effect of [Ca2+] is transient and irreversible; once sperm motility has been activated, it cannot be suppressed by high [Ca2+] (80 mM) and reactivation by Ca2+-free medium after suppression is also not possible. Verapamil, a Ca2+ channel inhibitor, and cAMP had no effect on sperm motility in striped bass suggesting that Ca2+ acts through other pathways. These data reinforce the notion that calcium is of central importance for osmosensing (see above). Because of the rapid time course of initiation of sperm motility, the study of sensory and signaling mechanisms underlying this event in marine versus FW fishes may provide critical clues about mechanisms of osmosensing in fish. In addition to calcium, intracellular [K+] has also been shown to be critical for the regulation of sperm motility in FW (zebrafish) and marine (pufferfish) species. Thus, intracellular inorganic cation concentrations are critical signals for osmosensory signal transduction in fish sperm.

2.3.3 Quality of Sperm and Eggs

Sperm motility is a reliable predictor for sperm quality and fertilization success (Au et al., 2002). After exposure to hypoxia for 12 weeks, sperm motility (measured by their curvilinear velocity VCL, straight‐line velocity VSL, and angular path velocity VAP) was significantly decreased in male carp (Table 3.2), indicating that sperm quality was impaired. All of the normoxic and hypoxic male carp could be induced to spawn using carp pituitary extract; however, the percentage of spawning success in the hypoxic male carp was drastically reduced from 71.4% to 8.3%, clearly demonstrating that the sperm quality produced by males was impaired by hypoxia (Wu et al., 2003).

Table 3.2. Sperm motility of carp after exposure to normoxia (7.0 mg O2/L, ∼81% saturation) and hypoxia (1.0 mg O2/L, ∼12% saturation) for 12 weeks

7.0 mg O2/L1.0 mg O2/LVCL77.42 ± 29.1346.25 ± 10.83*VSL38.83 ± 21.0110.65 ± 3.89*VAP47.69 ± 5.3821.12 ± 11.41*

Mean ± SD; n = 6. The velocity is expressed as micrometers per second.

VCL, mean curvilinear velocity; VSL, mean straight‐line velocity; VAP, angular path velocity (Wu et al., 2003).

Sperm Motility

Sperm motility is a functional measurement of the sperm themselves. Sperm are sampled either from the cauda or the vas deferens, and assessed for motility either manually or by CASA. In rats, motility is generally high and fairly consistent between animals: the normal range for percent motile varies between different laboratories, but control values should be in the 85–96% range for rodents, dogs, and monkeys. There is an extensive literature on the methodologic requirements for measuring motility and obtaining acceptable values (a minimum acceptable value for sperm motility in control groups is generally considered as 70%). It is recommended that the process of making these measures be assigned to a group within the research organization with the interest and skill to focus on obtaining the best and appropriately representative data from each sample.

Sperm motility can be affected by disturbances in testicular spermatogenesis or by effects on the epididymis. One way to consider these endpoints in the laboratory is that the testis provides the “hardware” (the substrate) for motility. The epididymis modifies that substrate and provides the motility-conferring “software” factors for the sperm. Thus, in a laboratory study in rodents, if normal numbers of well-formed sperm were immotile in the absence of testis pathology, one might reasonably suspect a treatment effect on the epididymis.

The Biological Relevance of Sperm Motility

Sperm motility alone, expressed using simple parameters such as average sperm velocity or estimates of the proportion of motile spermatozoa in a sample, is a poor predictor of conception rate, whether estimated in humans or agricultural animals. Experimental approaches to determine the predictive value of sperm motility have to take account of the potential confounding influence of inconsistent numbers of inseminated spermatozoa, where there are positive correlations between sperm numbers and fertility outcomes. Natural experiments involving reproductive skews and sperm competition can, however, provide useful indications about the value of sperm motility and other parameters. Critically important clues about the relevance of sperm motility have come from a series of papers that focused on mouse reproduction (e.g., the t-haplotype mouse [80]) and transmission-ratio distortion (TRD). This is an effect whereby genetic mosaicism resulting from meiotic recombination during spermatogenesis leads to the development of genetically distinct sperm subpopulations within a single ejaculate that are either functionally advantaged or disadvantaged with respect to flagellar activity. While spermatozoa with normal flagellar activity are able to cross the uterotubal junction, enter the oviduct, and reach the oocyte, those spermatozoa with abnormal flagellar function are unable to do so. Mechanistically, TRD in the case of the t-haplotype occurs because the cell signaling cascades that control flagellar function and motility operate incorrectly. The protein kinases controlling flagellar function in these mice are overexpressed and cause abnormal sperm motility; however, those spermatozoa carrying the t-haplotype also possess a t-complex responder gene (Tcr) that corrects the overexpression of the sperm motility kinase gene (smok) and restores normal flagellar action [81]. During meiosis, the Tcr cosegregates with the Y-chromosome and causes 95% skewing of the offspring sex ratio in favor of males by promoting unbalanced fertilization success. This extreme example, which results in non-Mendelian inheritance, is paralleled by non-Mendelian transmission of retinoblastoma in humans. Girardet et al. [82] proposed that TRD and sex-ratio distortion among the offspring of males affected with sporadic bilateral retinoblastoma could be explained by the existence of a defectively imprinted gene located on the human X chromosome that produces a subpopulation of spermatozoa that show defective motility.

These data can be viewed as natural sperm competition experiments that provide an intriguing insight into possible mechanisms of sperm selection. They indicate how genetic traits, not overtly reproductive in nature, can be influenced directly by their association with protein kinase-regulated signaling cascades that affect sperm motility. Reproductive skews detected in heterospermic artificial insemination experiments [39,41,83], where spermatozoa from two or more males are mixed in equal proportions, may be partly attributable to similar mechanisms. This suggestion is supported by observations that when porcine sperm populations are activated by bicarbonate [84], a stimulator of adenylate cyclase and hence protein kinase A, heterogeneous responses are seen both within single semen samples and between individual boars [85,86] (Fig. 1.1). Subpopulations of boar spermatozoa respond to bicarbonate in different ways; some are quiescent in the absence of bicarbonate but are rapidly stimulated to maximal progressive motility, some are refractory to stimulation, and others lie somewhere in between.

4.1 Stroboscopic imaging

Sperm motility and navigation in a chemical gradient have been studied using caged cGMP and caged resact in combination with stroboscopic techniques allowing visualization of the rapidly beating flagellum (Fig. 10A). Motility in 2D has been mostly studied on sperm swimming in shallow recording chambers. Interaction of sperm with the glass surface of the chamber can generate Ca2 + signals that alter motility or interfere with swimming. Therefore, we used the surfactant Pluronic F-127 (0.5%) that prevents sperm sticking to the glass surface.

The sperm flagellum beats about 50 times per second. Imaging such rapidly moving objects requires very brief exposure times that can be accomplished by short light pulses produced by LED light sources (stroboscopic illumination). Using this technique, it has been possible to decipher the steering mechanism of sea urchin sperm (Fig. 10A). Specifically, the beating flagellum adopts different waveforms allowing for straight swimming or swimming along an arc. Sperm use different flagellar waveforms for navigation in a gradient.

(b) Sperm motility and sperm morphology

Good sperm motility in general is very important for active swimming along certain sections of the female tract and for penetration of physical barriers, such as the uterotubal junction and ova vestments. The percentage of motile sperm is one of the main factors lnfluenclng fertility rates (Drobnis and Overstreet 1992).

Sperm motility and sperm morphology are intricately linked because morphologically abnormal sperm swim more slowly or less efficiently (Katz et al. 1982) and are thus selected against at various levels: cervical mucus (Katz et al. 1989), uterotubal junction (Krzanowska 1974), oviduct and ova vestments (Krzanowska and Lorenc 1983; Meistrich et al. 1994). Thus, the percentage of morphologically normal sperm is also a good predictor of fertility rates in humans both in vivo and in vitro (Fig. 16.5; Liu and Baker 1994; Mortimer 1994).

Recent evidence suggests that even some morphologically normal spermatozoa may swim inefficiently and in these cases morphologically normal sperm may be unable to fertilize. Morales et al. (1988) found that human patients suffering from infertility not only had a lower proportion of normal sperm, but also their normal sperm were less likely to be motile and swam less efficiently than normal donor sperm.

Oligospermia-Asthenospermia

Low sperm motility is observed in 25% of abnormal semen analyses; an isolated low sperm concentration is much less common. Low sperm motility may be due to antisperm antibodies (>50% of sperm bound) or excessive white blood cells in the ejaculate (leukocytospermia), the latter resulting in overproduction of reactive oxygen species that can damage sperm.21 Because “round cells” in the infertile ejaculate are more commonly immature germ cells (65%) rather than leukocytes,22 special leukocyte stains are recommended before treatment. Semen cultures are uninformative in asymptomatic infertile men with leukocytospermia as 83% are normally positive with multiple organisms.23 It is important to evaluate sexually transmitted diseases, penile discharge, prostatitis, or epididymitis. An expressed prostatic secretion is examined for leukocytes, and urethral cultures for Chlamydia and Mycoplasma can be considered. As a reference, the prevalence rate for PCR-based Chlamydia antigen detection is 0.3% among infertile men in general.24 Although the findings vary by both methodology and laboratory, in general, more than 50% of sperm bound with antibodies is considered significant and merits treatment.

Low sperm concentration may be due to an endocrinopathy such as prolactinoma, varicocele, or genetic causes, as outlined in Figure 24.4. Although eternally debated, there is substantial evidence to support the role of varicocele in male infertility (Table 24.3). Increasingly, genetic abnormalities should be considered in men with sperm concentrations less than 5 million/mL.25 Deletion of regions on the Y chromosome (microdeletions) occur in 6% of men with severely low sperm counts and in 15% of men with no sperm counts. In addition, 2% of men with low counts and 15% to 20% of men with no sperm counts will harbor chromosomal abnormalities detected by cytogenetic analysis (karyotype). These conditions include Klinefelter syndrome and translocations of non-sex chromosomes. Box 24.3 outlines current indications for genetic testing of infertile males. Importantly, varicocele repair for oligospermia in the setting of positive genetic findings is unlikely to improve semen quality or result in natural pregnancy.26

ii. Flow Cytometric Sperm Analysis.

Although sperm motility and morphology are the most commonly used parameters for the assessment of sperm viability, neither is an accurate indicator of function. Spermatozoa gross morphology is insufficient to detect damage to the plasma membrane, mitochondria, and acrosome. Flow cytometric analysis using specific fluorescent probes is a valuable method of determining these structural changes that may hamper normal fertilization and subsequent embryo development. This method provides a rapid and accurate means of determining the functional status of large numbers of spermatozoa.

Gravance et al. (2001) used flow cytometric analysis of JC-1 staining patterns of rat spermatozoa to detect chemical-induced alterations of sperm mitochondrial membrane potential. They found that the mid-piece (mitochondrial location) of live, highly motile spermatozoa stained bright orange, whereas the mid-piece of live, non-motile spermatozoa stained green. The mid-piece of slightly or non-progressively motile spermatozoa stained a faint orange-green. Gravance et al. (2003) were able to assess frozen-thawed rat sperm viability using SYBR-14 and propidium iodide. Sperm mitochondria were differentially labeled with JC-1. Motile sperm stained with JC-1 appeared orange in the mid-piece, indicating a high mitochondrial membrane potential, whereas immotile sperm with a low membrane potential stained green.

Assessment of Sperm Motility

The assessment of sperm motility should be done within a couple of minutes of semen collection whenever possible. In the event that semen cannot be assessed immediately, then it should be appropriately extended and stored. For those species that produce highly concentrated semen (ram, bull), a small drop of semen is often first placed on a warm slide and examined directly using the 4x lens (with the condenser turned down) for evidence of mass activity (often scored from no swirling wave to rapid swirling wave on a 1–5 or 1–10 scale). This examination enables rapid determination of whether the sample collected is consistent with the findings from examination of the scrotal contents (i.e., is representative of what the male would ejaculate if mated). However, it is important to note that mass activity is a function of sperm concentration and percentage motile sperm, and when samples have low sperm concentration, typical of some species and in bulls and rams due to variable response to electroejaculation, a swirl may not be observed even though the sample contains a high proportion of motile sperm. Further, observing a swirl does not allow the veterinarian to be able to always conclude that the sample contains a high proportion of progressively motile sperm, as a swirl can be observed in samples containing a high proportion of sperm swimming backwards due to the distal-midpiece reflex defect or looped tails.

To assess the percentage of progressively motile sperm, a small drop (~3 mm in diameter) of semen is placed on a prewarmed slide, and a warm coverslip is placed over it. It is critical that the droplet is small enough to ensure that a single layer of sperm can be observed. If the microscope being used only has bright field capacity, then the condenser must be turned down, the light reduced, and the 10x or 40x lens used. If the semen sample is quite concentrated, then it is advisable to dilute a drop of semen with several drops of either phosphate buffered saline or 2.9% sodium citrate solution on a warm slide, and then draw up a drop of the diluted semen and make a wet mount preparation as described previously. It is very important that the diluent is in-date and has been appropriately stored; otherwise, the pH or osmolality may have changed, and this can negatively bias the estimate of percentage progressively motile sperm. Some experience is required to accurately estimate the percentage progressively motile sperm. The veterinarian should focus on estimating the proportion of sperm that are not moving forward and initially broadly categorise the sample as having poor, fair, or good progressive motility (< 30%, 30%–59%, and ≥ 60%, respectively). The ‘golden rule’ when assessing sperm motility is, if in doubt, think ‘there might be a problem with how I have managed the sample, conducted the evaluation or, there could be a problem with the sample itself or the bull’. Therefore, if the mass activity or percentage progressively motile sperm is inconsistent with the findings of the physical examination of the scrotal contents, examine one or more additional droplets/wet mount preparations and/or collect another sample of semen. Also, sometimes with males who are examined out of season or have not been serving, the first sample/ejaculate may contain a relatively high number of dead sperm. In these cases the motility observed in the second sample is usually much better than initially observed.

What provides energy for sperm movement in mammals?

What provides energy for movement?