Respiration and Motility in Starfish Spermatozoa at Various Times in the Breeding Season

In the spermatozoa of Asterias amurensis , patterns of changes in the respiratory rate and motility following dilution of dry sperm in sea water varied among batches and were classified into three types. The type I spermatozoa were immotile and exhibited quite low respiratory rate. Ethylenediamine tetraacetic acid made the type I spermatozoa motile and elevated their respiratory rate. The type II spermatozoa were also immotile and the respiratory rate remained quite low for about 5 min after the dilution. Thereafter, they spontaneously became motile and the respiratory rate increased. The type III spermatozoa became motile upon their dilution and exhibited high respiratory rate. Differences in the motility and respiratory rate of spermatozoa among batches probably result from degree of their maturation. In moving spermatozoa, the ADP and AMP levels increased at the expense of ATP. 2,4-Dinitrophenol elevated the respiratory rate only in immotile spermatozoa, which showed a high ATP level and quite low ADP level, but did not made them motile. Oligomycin inhibited the respiration of both motile and immotile spermatozoa. Probably, the respiratory rate is made low by a shortage of ADP in immotile spermatozoa and is enhanced by ADP production due to the initiation of their movement.     

Motility of spermatozoa from shovelnose sturgeon and paddlefish

The spermatozoa in the seminal plasma from shovelnose sturgeon Scaphirhynchus platorynchus and paddlefish Polyodon spathula were immotile with only a few spontaneously motile spermatozoa for 5-10 and 10-20 s, respectively. Spermatozoa of shovelnose sturgeon were observed to be 100% motile immediately after sperm dilution in 10 m m NaCl and 20 m m Tris-HCl, pH 8.5. The duration of mass progressive movement was 2-3 min; and 1 to 5% of spermatozoa remain active after 360 s (P<0.01). Spermatozoa of paddlefish demonstrated the best motility 10 s after dilution in 10 m m NaCl with 20 m m Tris-HCl, pH 8.5. The duration of mass progressive movement was 2-3 min and 1 to 5% of spermatozoa remained active after 370 s ( p <0.01). The spermatozoa of shovelnose sturgeon and paddlefish were motile in a range of osmotic pressure from 0 to 100 mosmol kg⁻¹ and 0 to 120 mosmol kg⁻¹, respectively. The best results with short-term storage of sperm from shovelnose sturgeon and paddlefish were observed in 100 m m glucose + 20 m m Tris-HCl, pH 8.5 and 150 m m glucose + 20 m m Tris-HCl, pH 8.5.     

Sperm movement in the ooplasm, dithiothreitol pretreatment and sperm freezing are not required for the development of porcine embryos derived from injection of head membrane-damaged sperm

The present study investigated the correlation of sperm movement in the ooplasm, pretreatment of sperm with dithiothreitol (DTT) and sperm freezing with the development of porcine embryos derived from modified intracytoplasmic sperm injection (ICSI). In vitro, matured gilt oocytes without centrifugation were injected with head membrane-damaged spermatozoa aspirated tail-first. In Exp. 1, frozen-thawed sperm were categorized into three groups: impaired, immotile or motile. Oocytes injected with motile sperm (43.6%) showed a higher (P < 0.05) fertilization rate compared to oocytes injected with impaired or immotile sperm (34.5 or 37.2%). The survival rate was significantly higher (P < 0.05) in oocytes injected with impaired sperm (92.9%) than in oocytes injected with immotile or motile sperm (84.8 or 86.7%). No differences were observed in the rates of cleavage or blastocyst formation, and in total cell number of blastocysts among three groups of oocytes. In Exp. 2, motile frozen-thawed sperm were pretreated with DTT before injection and non-treated sperm served as controls. Higher rates (P < 0.05) of fertilization, male pronucleus (MPN) and decondensed sperm head (DSH) formation were observed in oocytes injected with control sperm (41.1, 50.0 and 91.1%, respectively) than in oocytes injected with DTT-treated sperm (22.1, 30.2 and 72.1%, respectively). No differences in embryo development and total cell number of blastocysts were observed between two groups of oocytes. In Exp. 3, motile frozen-thawed or fresh sperm without DTT pretreatment were injected into oocytes. The rates of fertilization and MPN formation were significantly higher (P < 0.05) in oocytes injected with fresh sperm (59.8 and 73.5%) than in oocytes injected with frozen-thawed sperm (36.7 and 59.2%). No differences in embryo development and total cell number of blastocysts were observed between two groups of oocytes. In conclusion, the present study clearly demonstrated that sperm movement in the ooplasm, use of DTT and fresh spermatozoa did not significantly affect on embryo development in porcine modified ICSI.     

Quantitative comparison of the passage of homologous and heterologous spermatozoa through the uterotubal junction of the golden hamster

A quantitative method was used to determine whether the spermatozoa of foreign species could pass through the uterotubal junction (UTJ) of the hamster as efficiently as homologous (hamster) spermatozoa. Estrous female hamsters were artificially inseminated with epididymal spermatozoa of homologous and heterologous (foreign) species. The number and distribution of spermatozoa in the oviduct were determined several hours after insemination (shortly before ovulation). The passage of immotile (dead) hamster spermatozoa through the UTJ was also examined. It was found that the spermatozoa of all foreign species tested (rat, mouse, guinea pig, and rabbit), as well as immotile hamster spermatozoa, could pass through the UTJ but did so in much smaller numbers compared to live hamster spermatozoa. This was not specifically due to poor survival of foreign spermatozoa in the hamster uterus, as the viability of all inseminated spermatozoa (including hamster spermatozoa) was considerably reduced by 1 h after insemination. While a large number of live hamster spermatozoa were distributed throughout the caudal isthmus at the time of examination, none or only a very few foreign spermatozoa had advanced this far. The few foreign and immotile spermatozoa that reached the caudal isthmus were confined to the first ascending loop of this segment. Some possible causes for the small number and retarded advance of foreign spermatozoa in the hamster oviduct were discussed.     

Enzyme distribution in "naturally-decapitated" bull spermatozoa: acetylcholinesterase, adenylpyrophosphatase and adenosinetriphosphatase

Naturally-decapitated spermatozoa were separated into motile flagella and head and immotile flagella by differential and density gradient centrifugation. In preparations microscopically free of cross-contamination after repeated centrifugation, the heads appeared to be enzymatically inert, while there was virtually no change in the specific activity of the immotile flagella which had been subjected to as much manipulation as the heads. The non-motile flagella had almost twice the acetylcholinesterase and about one-third the apyrase activity of the motile flagella. The flagella appear to contain a structurally-bound adenosinetriphosphatase which may be identical with the “spermosin” extracted from bull sperm.     

On the integrity of sperm DNA

Ejaculated rabbit spermatozoa washed with buffer prior to decondensation by Triton X-100 and dithiothreitol were good templates for DNA synthesis by Escherichia coli DNA polymerase. This result agrees with the observations of Zirkin and Chang [1977], and implies that the sperm DNA is nicked. Template activity, however, was reduced if spermatozoa were extensively washed before decondensation, and if DNase inhibitors EDTA or Na₂SO₄ were present during decondensation. Template activity was also low if decondensation was induced with DNase inhibitors thioglycollic acid, Na₂SO₃ or sodium dodecylsulphate and dithiothreitol instead of with Triton X-100 and dithiothreitol. Calf thymus DNA was completely degraded when incubated with rabbit seminal plasma or buffer-washed spermatozoa, but much less degradation was observed if EDTA, Na₂SO₄, thioglycollic acid, Na₂SO₃ or sodium dodecylsulphate were also present, or if spermatozoa were extensively washed with buffer. Centrifugation of spermatozoa through 2.05 M sucrose completely removed contaminating DNase, and such spermatozoa were inactive as DNA templates after decondensation. The DNA template activity of swollen rabbit sperm nuclei thus parallels the activity of a contaminating seminal plasma DNase. This suggest that the nicks in sperm DNA enabling it to act as a template for DNA synthesis were generated by the DNase during decondensation and thus are not a natural structural feature of the DNA. The presence of breaks in the DNA of decondensed buffer-washed spermatozoa (DNase contaminated) was confirmed by their incorporation of phosphate from [γ^?32 P] ATP in the presence of the enzyme polynucleotide kinase. These spermatozoa were found to contain as few as two breaks/mole of DNA, but sucrose-washed spermatozoa (DNase free) were free of breaks. The possible use of this enzymic procedure for the assessment of sperm genome damage and the evaluation of the quality of a sperm population are discussed.     

Energy pathways associated with sustained spermatozoon motility in the endangered Siberian sturgeon Acipenser baerii

Sturgeon spermatozoa are unique for their sustained motility. We investigated the relative importance of bioenergetic pathways in the energy supply of Siberian sturgeon Acipenser baerii spermatozoa during motile and immotile states. Spermatozoon motility and oxygen consumption rate (OCR) were analysed following exposure to inhibitors of oxidative phosphorylation (sodium azide, NaN₃), glycolysis (2-deoxy-D-glucose, DOG) and β-oxidation of fatty acids (sodium fluoride, NaF), and to an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenyl hydrazine, CCCP). No significant difference in curvilinear velocity was observed after addition of these reagents to activation medium (AM) or nonactivation medium (NAM) for incubation. Incubation of spermatozoa in NAM containing CCCP or NaN₃ resulted in significantly decreased motility duration compared to controls. The OCR of sturgeon spermatozoa in AM (11.9 ± 1.4 nmol O₂ min⁻¹ (10⁹ spz)⁻¹) was significantly higher than in NAM (8.2 ± 1.5 nmol O₂ min⁻¹ (10⁹ spz)⁻¹). The OCR significantly declined with addition of NaN₃ to AM and NAM. No significant difference in motility parameters or OCR was observed with NaF or DOG. These results suggest active oxidative phosphorylation in both immotile and motile spermatozoa. Nevertheless, mitochondrial respiration occurring during motility is not sufficient to meet the high energy demands, and the energy required for sustained motility of Siberian sturgeon spermatozoa is derived from adenosine triphosphate accumulated during the quiescent state.     

Role of sperm motility and acrosome integrity in the filtration of bovine semen

In this study, the role of sperm motility and acrosome integrity in filtration of bovine semen was investigated. In Experiment 1, the treatment of semen with formaldehyde, hyperosmotic buffer, heating and direct freezing immobilized the spermatozoa completely but their acrosomal status varied significantly (P < 0.01). The immotile spermatozoa, of any kind, did not pass through the Sephadex ion-exchange column at room temperature. In Experiment 2, semen samples possessing different percentages of immobilized spermatozoa (0, 50, 75 and 100%) were filtered through the Sephadex ion-exchange column. The immotile/dead spermatozoa were removed proportionately to their number in the semen by Sephadex ion-exchange column. The type and number of immotile spermatozoa in semen had no effect (P > 0.05) on the post-filtration recovery rate of motile spermatozoa. Filtered spermatozoa exhibited higher (P < 0.01) motility (> 90%), progressive motility (> 70%) and normal acrosomes (> 95%) than non-filtered spermatozoa. In conclusion, sperm motility seems to be more important than acrosome integrity for semen filtration, and the Sephadex ion-exchange column can remove the known quantities of different kinds dead/immotile spermatozoa.     

The onset and maintenance of hyperactivated motility of spermatozoa from the mouse

Estimates were made of the proportion of freely motile mouse spermatozoa displaying hyperactivated motility by an objective photographic method employing stroboscopic illumination under dark-field conditions and examining displacements of the sperm head and bend angles of the sperm tail. In media known to support in vitro fertilisation hyperactivation gradually appeared reaching about 40% by 6 hr incubation, and it was not promoted by 2 mM caffeine or 0.1 mM Bt₂ cAMP or washing the cells free of epididymal fluid. Raising the osmolarity of the medium to 400 mOSM with electrolytes, but not nonelectrolytes, did promote hyperactivation (60% by 2 hr) suggesting that the ionic strength of the medium was important. Hyperactivation in high ionic strength media could be prevented by removing or chelating Ca²⁺, or replacing Ca²⁺ with Ba²⁺ or Mg²⁺, when nonhyperactivated motility was maintained, but Sr²⁺, like Ca²⁺, permitted hyperactivated motility. Hyperactivation in low ionic strength medium could be promoted by the ionophore A23187, suggesting that Ca²⁺ movement into the cells is important. Of a range of glycolytic substrates tested supporting nonhyperactivated motility in the presence of lactate, only glucose supported hyperactivation. Addition to glucose— or Ca²⁺ — free, high ionic strength media after 2 hr increased hyperactivation immediately (glucose) or after a lag of 2 hr (Ca²⁺) suggesting that glucose acts on a Ca²⁺ — primed system. Removal from high ionic strength medium, chelation of Ca²⁺ or inhibition of glucose metabolism did not prevent hyperactivation continuing once it had been initiated, indicating different requirements for initiation and maintenance of this form of motility.     

Sperm motility of the marine teleosts Boops boops, Diplodus sargus, Mullus barbatus and Trachurus mediterraneus

The spermatozoa of Boops boops, Diplodus sargus, Mullus barbatus, and Trachurus mediterraneus were motile in sea water, and in electrolyte solutions (NaCl) and non-electrolyte solutions (glucose) with an osmolality of 600–1000 mosmol kg^?1. Their mean motility rate 10 s after initiation was about 80%, while about 10% of the motile spermatozoa moved non-linearly, 45% linearly, and 45% circularly. The average path swimming velocity was significantly higher in M. barbatus (about 90 μm s^?1) than in the other species (70 μm s^?1). The number of motile spermatozoa decreased to 0% within 50 s after initiation of motility in T. mediterraneus, within 90 s in M. barbatus . In B. boops and D. sargus about 90% of the spermatozoa stopped movement during the first 90 s of the motility period, while the rest remained motile for 2–3 h. Motility of B. boops and D. sargus spermatozoa was reversibly suppressed in the seminal plasma, and in electrolyte and non-electrolyte solutions of 100–200 mosmol kg^?1. The trigger for motility activation was hyperosmolality (700–1000 mosmol kg^?1). Motility of M. barbatus and T. mediterraneus sperm was only partly suppressed in the seminal plasma since freshly collected semen contained about 25–50% locally motile spermatozoa. When sperm was activated immediately after collection with electrolyte and non-electrolyte solutions of 700–1000 mosmol kg^?1 spermatozoa moved progressively. The motility of those spermatozoa which had not yet been motile after collection was completely and reversibly suppressed in M. barbatus at osmolalities of 1200 mosmol kg^?1, and at osmolalities of 100–200 mosmol kg^?1 in T. mediterraneus . Therefore two triggers were necessary for initiation of motility. The nature of the first trigger was uncertain, the second trigger was a switch to hypoosmolality in M. barbatus and to hyperosmolality in T. mediterraneus . The sperm organisation of B. boops, D. sargus, M. barbatus and T. mediterraneus revealed species-specific parameters which could not be related with the sperm motility behaviour.     

Spermatozoon structure and motility in the anuran Lepidobatrachus laevis

Synthetic human gonadotropin releasing hormone (GnRH) injections were used for induction of spermatozoon release followed by cloacal lavage or mechanical stimulation of sperm release in Lepidobatrachus laevis . Light microscopic observations of Lepidobatrachus laevis spermatozoa indicated an acrosomal segment with a length of 4.1 μm delineated by an indentation, a nuclear region of 12.6 μm in length and a midpiece of 0.87 μm in length. The tail was 54.9 μm long by 1.35 μm wide with two lateral axial fibers and a central undulating membrane. At the electron microscopic level, the unusual tail had two complete axonemes that emanated from the distal centriole. The tail also contained two axial fibers 77 nm in diameter medial to the axonemes and was connected by an undulating membrane. An unusual accessory cell adherent to the head of the spermatozoon was noted in freshly obtained suspensions of spermatozoa. Spermatozoa with the accessory cell were motile and a subsequent loss of motility was correlated with the shedding of the accessory cell.     

VII. Decrease in the Rate of Respiration in the Spermatozoa of the Sea Urchin, Hemicentrotus Pulcherrimus, Caused by Long Chain Fatty Acyl-CoA-induced Inhibition of the Movement

Spermatozoa of the sea urchin, Hemicentrotus pulcherrimus , showed marked decrease in respiration, and arrested movement after interaction with the fixed eggs. Immotile spermatozoa that had reacted with fixed eggs contained higher levels of long chain fatty acyl-CoAs than normal motile spermatozoa. On treatment with carnitine, the immotile spermatozoa became motile again and their intracellular concentrations of long chain fatty acyl-CoAs decreased. On incubation with anti-mycin A or CN⁻ for 20 min, the motility of normal spermatozoa decreased gradually but their long chain fatty acyl-CoA content changed only slightly. The decrease in sperm motility in the latter case was probably due to decrease in the level of ATP, resulting from inhibition of respiration by antimycin A or CN⁻. The motility of spermatozoa extracted with Triton X-100 was restored by ATP and their movement was inhibited by long chain fatty acyl-CoAs, such as myristoly CoA and palmitoyl-CoA, but was not by short chain fatty acyl-CoAs, such as acetyl-CoA, propionyl CoA and butyryl-CoA. Na-palmitate, Na-myristate and CoA did not inhibit the reactivation of extracted spermatozoa by ATP.     

SPATC1L maintains the integrity of the sperm head‐tail junction

Spermatogenesis is a tightly regulated process involving germ cell‐specific and germ cell‐predominant genes. Here we investigate a novel germ cell‐specific gene, Spatc1l (spermatogenesis and centriole associated 1 like). Expression analyses show that SPATC1L is expressed in mouse and human testes. We find that mouse SPATC1L localizes to the neck region in testicular sperm. Moreover, SPATC1L associates with the regulatory subunit of protein kinase A (PKA). Using CRISPR/Cas9‐mediated genome engineering, we generate mice lacking SPATC1L. Disruption of Spatc1l in mice leads to male sterility owing to separation of sperm heads from tails. The lack of SPATC1L is associated with a reduction in PKA activity in testicular sperm, and we identify capping protein muscle Z‐line beta as a candidate target of phosphorylation by PKA in testis. Taken together, our results implicate the SPATC1L‐PKA complex in maintaining the stability of the sperm head‐tail junction, thereby revealing a new molecular basis for sperm head‐tail integrity.     

Relationship between sperm density, spermatocrit, sperm motility and spawning date in wild and cultured haddock

Semen was collected repeatedly from captive haddock Melanogrammus aeglefinus and the effect of seasonality on various sperm parameters was investigated. No differences in sperm traits were observed for wild and cultured haddock. A highly significant positive relationship existed between spermatocrit and spermatozoa density. A significant increase in mean spermatocrit occurred throughout the spawning season but the amount of variability explained by collection date was low (35·1%) due to variability between males. Each of 10 males sampled repeatedly throughout the spawning season demonstrated an increase in spermatocrit. No relationship existed between spermatocrit and proportion of motile spermatozoa when spermatocrit was ≤70%. Motility was reduced in semen samples with spermatocrits >70%. The proportion of spermatozoa that were motile decreased with time since activation. Some motility was still observed after 60 min in sea water (0·1–15·2%) for sperm collected at all times within the spawning season. Of those spermatozoa that were motile, the proportion that exhibited forward swimming motion decreased and the proportion that had only vibratory movement increased with time post‐activation. The speed of forward swimming spermatozoa showed no significant relationship with spermatocrit at any time between 0 and 60 min after activation. Swimming speed was negatively related to time since activation, decreasing from 174–240 μm s⁻¹ at 0 min to 80–128 μm s⁻¹ at 60 min after activation.     

Calcium ion supplementation increases brook trout Salvelinus fontinalis spermatozoa activation at the end of the spawning season

The role of environmental ion composition and osmolality in calcium ion (Ca²⁺) signalling of spermatozoa activation over the course of the spawning period of brook trout Salvelinus fontinalis was investigated. Motility at the end of spawning was low (mean ± s.d. of 5 ± 2% motile spermatozoa with curvilinear velocity of 25 ± 8 µm s^?1). Addition of 10 mM Ca²⁺ to the activation medium resulted in values similar to those recorded during the middle of the spawning period (mean ± s.d. of 95 ± 6% motile spermatozoa with curvilinear velocity of 130 ± 15 µm s^?1).     

Single‐nucleotide polymorphism c.474G>A in the SEPT12 gene is a predisposing factor in male infertility

SEPT12 is a testis‐specific gene involved in the terminal differentiation of male germ cells. SEPT12 protein is required for sperm head‐tail formation and acts as a fundamental constituent of sperm tail annulus. In this study, we screened genetic variations in exons 5, 6, 7 of the SEPT12 and assessed the annulus status in teratozoospermic, globozoospermic, and patients with immotile short tail sperm. DNA sequencing was performed for 90 teratozoospermic and 30 normozoospermic individuals. Immunocytochemistry, transmission electron microscopy and western blotting were conducted to evaluate annulus status and the expression level of SEPT12 in patients with a distinct exonic variation (c.474G>A), respectively. Five polymorphisms identified within the desired regions of the SEPT12, among them c.474G>A had the potential to induce aberrant splicing results in the expression of a truncated protein. The annulus was detected in most of the spermatozoa from teratozoospermic and normozoospermic men with c.474G>A. In contrast, in the patient with short tail sperm defect carrying c.474G>A, 99% of spermatozoa were devoid of the annulus. Based on our findings there would be no association between exons 5, 6, 7 polymorphisms of the SEPT12 gene and the occurrence of mentioned disease but c.474G>A would be a predisposing factor in male infertility.     

The spermatozoon of Eurasian murine rodents: Its morphological diversity and evolution

The murine rodents are the most speciose subfamily of mammals. Here the morphology of the spermatozoon, as determined by scanning and transmission electron microscopy of representative species from four Eurasian clades, is described. Much interspecific variability in all components of the spermatozoon was found to occur, although most species have a bilaterally flattened sperm head with a single apical hook of variable length and orientation. Ultrastructural observations indicate that this apical hook invariably contains a nuclear projection as well as a large extension of the subacrosomal cytoskeleton, as a perforatorium rostrally, and a complex asymmetrical acrosomal extension. These spermatozoa also have relatively long tails that are attached to the lower concave surface of the sperm head. Uniquely, in species in the Apodemus clade, the apical hook is orientated caudally. In a few species a highly derived sperm head morphotype that does not contain an apical hook is present. These sperm heads vary in morphology from being globular in two species of Bandicota, to bilaterally flattened and paddle-shaped in Tokudaia and Micromys. In spermatozoa of the latter two genera the subacrosomal cytoskeleton, which is less extensive than in species with a hooked sperm head, forms an apical extension, but that is not the case in Bandicota. In all species where the sperm head lacks an apical hook the acrosome is more symmetrical. The sperm tail is much shorter in these species, with attachment to the head occurring on the ventral surface in Tokudaia and basal in Micromys and the two species of Bandicota. As the sperm head morphotype with a complex apical hook is present in all the major clades of murine rodents, it is likely to be a plesiomorphic character within each of these clades, with the nonhooked sperm heads, which vary greatly in structure between species of the different lineages, probably being independently derived. The ultrastructural organization of the sperm head of Bandicota, but not those of Micromys or Tokudaia, suggest divergence in some of the morphological events associated with sperm-egg interaction at the time of fertilization.     

Ultrastructural localization of labeled acrosomal glycoproteins during in vivo fertilization in the rabbit

Rabbit spermatozoa were labeled predominantely in their acrosomal glycoproteins by 1-³H-glucosamine during spermiogenesis. Ova fertilized in vivo by spermatozoa labeled 22 days earlier were analyzed by fine-structure autoradiography for the localization of the label. The latter was found associated with 1) the fused membranes of the acrosomal cap remaining on the zona pellucida surface, 2) the material released on the zona surface after the acrosome reaction and possibly detectable after tannic acid fixation, 3) the equatorial segment of the sperm head and the preequatorial swellings, and 4) other sperm components, eg, the sperm tail. No labeling, on the other hand, was detected on the denuded leading edge of spermatozoa found either in the penetration slit or in the perivitelline space. Our observations suggest the involvement of acrosomal glycoproteins in different mechanisms of sperm/zona pellucida interaction but are not in favor of a major role of (enzymatic) glycoproteins bound to the inner acrosomal membrane during the penetration of the zona pellucida.     

Study on the role of calmodulin in sperm function through the enrichment and identification of calmodulin-binding proteins in bovine ejaculated spermatozoa

Calmodulin is a small, highly conserved acidic protein present at high levels in spermatozoa that mediates numerous intracellular Ca²⁺-dependent events. Sperm motility and fertilizing ability results from an array of biochemical pathways under Ca²⁺ control, in which the importance of calmodulin is not fully understood. The role of calmodulin in sperm function has been mostly assessed using antagonists. Nevertheless, few known calmodulin-regulated enzymes have been described in spermatozoa regarding their involvement in sperm function. To further understand the role of this important Ca²⁺ mediator in spermatozoa, different studies were also undertaken to investigate and to identify sperm calmodulin-binding proteins and determine their localization and subcellular distribution as an attempt to elucidate the role of this important Ca²⁺ mediator. In the present study, sperm calmodulin-binding proteins were identified by mass spectrometry after Ca²⁺-dependent biotinylated-calmodulin binding on sperm head proteins subjected to 2D electrophoresis and transferred on a polyvinylidene difluoride membrane. Calmodulin binding protein identification was also done on detergent extracted whole sperm proteins pulled down in a Ca²⁺-dependent manner by calmodulin-conjugated sepharose beads. In this latter group, 300 proteins were identified in at least two experiments out of three, and those identified in the three independent experiments were analyzed for overrepresented biological processes using the Bos taurus Gene Ontology database. Proteins with known function in reproductive processes, fertilization, sperm-egg recognition, sperm binding to the zona pellucida, regulation of sperm capacitation, and sperm motility were identified and further emphasize the importance of calmodulin in sperm function.     

CHEMICAL DISSECTION OF MAMMALIAN SPERMATOZOA

Spermatozoa from several mammalian species have been dissected by chemical methods to yield free heads, tails with attached midpieces, and tails from which the mitochondrial components of the midpiece were removed. Mouse and rat spermatozoa were cleaved by brief treatment with trypsin to yield free heads and tails, while human, guinea pig, and rabbit spermatozoa were cleaved by trypsin only after incubation with 2-mercaptoethanol or dithiothreitol. Spermatozoa were also cleaved at the junction of the head and the tail by treatment with acid and base. Mitochondria were removed from intact spermatozoa or isolated tails by mechanical shear after treatment with 2-mercaptoethanol or dithiothreitol. The dissected components of spermatozoa were fractionated with good yield and high purity by density gradient centrifugation. Ultrastructural analysis indicates that proteolytic cleavage to yield separated heads and tails occurs at a specific location in the neck of the spermatozoon, leaving the basal plate attached to the head of the cell. In contrast, after acid cleavage the basal plate remains with the midpiece. Proteolytic treatment has no apparent effect on any other spermatozoan structures, whereas acid or base treatment results in damage to the plasma membrane, the acrosome, and other structures. The specificity of the proteolytic cleavage suggests that a particular protein or group of proteins may be responsible for the linkage between the sperm head and tail.