Current status of sexing mammalian spermatozoa

Thousands of offspring have now been produced via artificial insemination with spermatozoa sexed by flow cytometry and cell sorting. We are unaware of any other practical approach to sexing spermatozoa that maintains fertility. Accuracy of sexing usually is 85-95% in most species, but somewhat lower with human spermatozoa. Spermatozoa are sexed in series, one at a time, at routine rates of about 3000 live spermatozoa of each sex per second for most species, and nearly twice that rate under optimal conditions for some species. Owing to various constraints and statistical considerations, there appears to be an upper theoretical limit to sexing spermatozoa of about 10,000 live spermatozoa of each sex per second with current methodology. About a quarter of the spermatozoa processed are sexed; the rest are discarded in the process or lost due to logistical constraints. Spermatozoa undergo some damage during sorting, although much less in terms of viability than with routine cryopreservation; fertility is lower with sexed than control spermatozoa. Offspring from sexed spermatozoa appear to have no more abnormalities than do controls, and both groups grow and thrive similarly. Despite high costs and complex procedures, sexing spermatozoa, usually followed by cryopreservation, is being used commercially for cattle and horse production in several countries, and is used to produce girls to avoid X-chromosome-linked genetic diseases.     

Counting mammalian spermatozoa in biological fluids containing particulate matter

Accurate counting of spermatozoa in biological fluids by particle counters requires elimination of competing background. This was accomplished by dissolving cell organelles and other lipid and proteinaceous material with sodium dodecyl sulfate to leave the sperm nucleus. Progress of selective dissolution of interfering background versus sperm nuclei was monitored by phase contrast microscopy. Rabbit semen was diluted in 2.5% sodium dodecyl sulfate (wt/vol) and then 1:1 (vol/vol) with .5 M sodium hydroxide. Suspensions were incubated, diluted 1:200 in .1 M sodium citrate-.1% Triton-X, and counted after 5, 10, and 20 min with a Coulter Counter. All treatment times resulted in similar mean counts, ranging from 367 to 369 X 10(6) sperm/ml. These means were slightly higher than the 350 X 10(6) sperm/ml for hemocytometer counts, but the correlation with hemocytometer counts was r greater than or equal to .98. Bull semen was diluted in either 1) citrate-Triton-X, 2) .25 M Tris-20% egg yolk, or 3) heated whole milk and further treated with 10% sodium dodecyl sulfate and .5 M sodium hydroxide. After 15 min, treated samples were diluted 1:200 in citrate-Triton-X and counted. Following treatment, estimated sperm concentration in the three diluents was not different from that of untreated sperm in citrate-Triton-X. The procedure was successfully applied to bull sperm diluted in milk and packaged in .5-ml French straws used routinely for artificial insemination.     

Reducing agents induce microtubule extrusion in demembranated mammalian spermatozoa

To understand the mechanism regulating flagellar bending in spermatozoa, it is important to investigate the regulation of microtubule sliding in the flagellar axoneme. It has been shown that protease treatment following demembranation with Triton X-100 disrupts the connections between microtubules and induces extrusion of microtubules from the flagellar axoneme. This approach enables a direct investigation of the regulation of microtubule sliding; however, the percentage of spermatozoa with protease-induced extrusion was relatively low, probably due to protease digestion of some regulatory motility proteins, as well as proteins connecting the microtubules. In this study, we demonstrate microtubule extrusion in most hamster and mouse demembranated spermatozoa upon treatment with a high concentration of the reducing agents dithiothreitol or 2-mercaptoethanol, without the use of proteases. The extrusion of microtubules occurred when the spermatozoa were treated with concentrations of the reducing agents that were sufficient for the reduction of the disulfide bonds of IgG. These results suggest that the arrangement of the axonemal structures connecting doublet microtubules depends to an important degree on -S-S- bonds. Close observation of the extrusion process using the present method revealed that microtubules were extruded on the same side as that of the curve of the sperm head, and also on the opposite side. Furthermore, we noted that extrusion always started on one side, followed by the other side, but was never initiated on both sides simultaneously.     

Hyperactivation of mammalian spermatozoa: function and regulation

Hyperactivation is a movement pattern observed in spermatozoa at the site and time of fertilization in mammals. It may be critical to the success of fertilization, because it enhances the ability of spermatozoa to detach from the wall of the oviduct, to move around in the labyrinthine lumen of the oviduct, to penetrate mucous substances and, finally, to penetrate the zona pellucida of the oocyte. The movement of hyperactivated spermatozoa appears different under different physical conditions and in different species, but basically it involves an increase in flagellar bend amplitude and beat asymmetry. Presumably, there is a signal or signals in the oviduct to initiate hyperactivation at the appropriate time; however, none has yet been identified. There is evidence that the source of the signal is follicular fluid, yet spermatozoa are known to hyperactivate before ovulation would release the fluid into the oviduct. Although the signal transduction cascade regulating hyperactivation remains to be described completely, it is clear that calcium ions interact with the axoneme of the flagellum to switch on hyperactivation. The process may also involve increases in intracellular cAMP, which at least is required to support motility in general. Although hyperactivation usually occurs during capacitation, the two events are regulated by different pathways.     

Angiotensin II stimulates cAMP production and protein tyrosine phosphorylation in mouse spermatozoa

Angiotensin II (AII), found in seminal plasma, has been shown to stimulate capacitation in uncapacitated mammalian spermatozoa. The present study investigated the location of AII receptors on spermatozoa and AII's mechanism of action. AT1 type receptors for AII are present on the acrosomal cap region and along the whole of the flagellum of both mouse and human spermatozoa. Because combinations of low concentrations of AII and either calcitonin or fertilization-promoting peptide (FPP), both known to regulate the adenylyl cyclase (AC)/cAMP signal transduction pathway, elicited a significant response, this study investigated the hypothesis that these peptides act on the same pathway. AII was shown to significantly stimulate cAMP production in both uncapacitated and capacitated mouse spermatozoa and this was associated with increases in protein tyrosine phosphorylation. Using an anti-phosphotyrosine antibody to visualize the location of tyrosine phosphoproteins within individual cells, AII significantly stimulated phosphorylation within 20 min in both the head, especially in the acrosomal cap region, and the flagellum, especially in the principal piece, of uncapacitated mouse spermatozoa; combined AII + FPP was stimulatory within 5 min. In addition, Western blotting revealed that AII stimulation increased phosphorylation in a number of tyrosine phosphoproteins in both uncapacitated and capacitated mouse spermatozoa, with some being altered only in the latter category of cells. These results support the hypothesis that AII stimulates AC/cAMP in mammalian spermatozoa.     

Detection and location of the enzymes of de novo pyrimidine biosynthesis in mammalian spermatozoa

Enzymes of the pathway for de novo biosynthesis of pyrimidine nucleotides have been reported in spermatozoa from fruitfly and mammals. The aim of the present study was to test the hypothesis that the enzymes for biosynthesis of uridine monophosphate (UMP) are concentrated near the mitochondria, which are segregated in the mid-piece of spermatozoa. Baby hamster kidney fibroblasts were compared with spermatozoa from rams, boars, bulls and men. Antibodies raised against synthetic peptides from sequences of the multienzyme polypeptides containing glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (CAD) and UMP synthase, which catalyse reactions 1-3 and 5-6, respectively, were used, together with an affinity-purified antibody raised against dihydroorotate dehydrogenase (DHODH), the mitochondrial enzyme for step 4. Western blot analysis, immunofluorescent microscopy and immunoelectron microscopy confirmed that CAD and UMP synthase are found in the cytoplasm around and outside the mitochondria; DHODH is found exclusively inside the mitochondria. CAD was also located in the nucleus, where it has been reported in the nuclear matrix, and in the cytoplasm, apparently associated with the cytoskeleton. It is possible that CAD in the cytoplasm has a role unconnected with pyrimidine biosynthesis.     

Capacitation-associated protein tyrosine phosphorylation and membrane fluidity changes are impaired in the spermatozoa of asthenozoospermic patients

Sperm protein tyrosine phosphorylation has been associated with capacitation, motility changes, zona binding, and fertilizing ability. We previously demonstrated that gradient-isolated human sperm subpopulations differ in their plasma membrane composition, their ability to phosphorylate proteins in tyrosine residues, and their capacity to undergo hyperactivation. In this study, we have characterized capacitation-associated changes in protein tyrosine phosphorylation and membrane fluidity in spermatozoa of asthenozoospermic and normozoospermic patients consulting for infertility. Semen samples were studied at baseline and after a capacitating incubation with or without the addition of a permeable cAMP analog and a phosphodiesterase inhibitor. Basic sperm and computer-assisted motion parameters, hyperactivation, protein tyrosine phosphorylation (immunofluorescence and Western blot), and membrane fluidity (fluorescent Laurdan probe) were the main study parameters. In comparison with normozoospermic and proven-fertile donor semen, asthenozoospermic samples showed lower motility, velocity, and amplitude of lateral head displacement, both originally and after a 6-h capacitating incubation. Unlike those in normal samples, asthenozoospermic spermatozoa were unable to increase protein tyrosine phosphorylation during capacitation. Such impairment, however, was overcome when they were incubated with a membrane-permeable cAMP analog and a phosphodiesterase inhibitor, indicating a possible membrane defect. Confirming this hypothesis, plasma membranes of asthenozoospermic sperm showed decreased fluidity (increased Laurdan polarization), even after a capacitating incubation. In conclusion, spermatozoa from functional asthenozoospermic samples may owe their poor motility, and their inability to properly capacitate and develop hyperactivation, to an impairment in the tyrosine phosphorylation of critical proteins caused by decreased membrane fluidity. These findings suggest a molecular pathogenetic mechanism for a common seminal pathology associated with male infertility.     

Phospholipase C isoforms in mammalian spermatozoa: potential components of the sperm factor that causes Ca2+ release in eggs

Injection of a soluble protein factor from mammalian spermatozoa triggers Ca2+ oscillations in mammalian eggs similar to those seen at fertilization. This sperm factor also generates inositol 1,4,5-trisphosphate and causes Ca2+ release in sea urchin egg homogenates and frog eggs. Recent studies have indicated that the sperm factor may be an inositol-specific phospholipase C (PLC) activity. This study investigated whether any of the commonly known PLC isoforms are components of the sperm factor. PLCbeta, PLCgamma and PLCdelta isoforms were shown to be present in boar sperm extracts. However, upon column fractionation of sperm extracts, none of the PLC isoforms detected correlated with the ability to cause Ca2+ release in eggs. In addition to our previous work on recombinant PLCs, it was also shown that PLCdelta3, PLCdelta4 and its splice variant PLCdelta4 Alt1 fail to cause Ca2+ release. The recently discovered 255 kDa PLCepsilon isoform also appears unlikely to be a component of the sperm factor, as fractionation of sperm extracts on a gel filtration column demonstrated that the peak of Ca2+-releasing activity was associated with fractions of 30-70 kDa. These findings indicate that the sperm factor that triggers Ca2+ release in eggs does not appear to have a known PLC isoform as one of its components.     

Protein phosphorylation in yeast mitochondria: cAMP-dependence, submitochondrial localization and substrates of mitochondrial protein kinases

We describe the identification and submitochondrial localization of four protein kinases and of their target proteins in derepressed yeast mitochondria. The activity of one of the kinases depends on the presence of cyclic AMP (cAMP). It is soluble and localized in the mitochondrial intermembrane space. Its natural target is a polypeptide of 40 kDa molecular mass, which is bound to the inner membrane. Besides this natural target this kinase phosphorylates acidic heterologous proteins, like casein, with high efficiency. The other protein kinases identified so far are cAMP-independent. At least one is localized in the matrix having its natural substrates (49 and 24 kDa) in the same compartment. Two others are firmly bound to the inner membrane phosphorylating target proteins in the inner membrane (52.5 kDa) and in the intermembrane space (17.5 kDa), respectively.     

Protein phosphatase-type 2B is involved in the regulation of the acrosome reaction but not in the temperature-dependent flagellar movement of fowl spermatozoa

The motility and acrosomal integrity of fowl spermatozoa in TES/NaCl buffer, with or without homogenized inner perivitelline layers (IPVL) prepared from laid fowl eggs, was almost negligible at 40 degrees C. However, motility became vigorous even at 40 degrees C when 2 mmol CaCl2/l was added, and the acrosome reaction was also stimulated in the presence, but not in the absence, of IPVL. The presence of deltamethrin or fenvalerate, specific inhibitors of protein phosphatase-type 2B (PP2B), did not permit the restoration of motility at 40 degrees C but, in the presence of IPVL, these compounds stimulated the acrosome reaction in a dose-dependent manner in the range of 1-1000 nmol/l. These results suggest that IPVL is necessary for the activation of the acrosome reaction in fowl spermatozoa and that Ca2+ plays an important role in the stimulation of motility and acrosomal exocytosis. Furthermore, it appears that the intracellular molecular mechanisms for the regulation of the acrosome reaction of fowl spermatozoa are different from those for the restoration of motility, i.e. protein dephosphorylation by PP2B in the former but not in the latter case.     

Energy metabolism and intracellular pH in boar spermatozoa

The effect of energy metabolism on intracellular pH was studied in boar spermatozoa using nuclear magnetic resonance (NMR) spectroscopy and confocal microscopy with the pH-sensitive dye seminaphthorhodafluor (SNARF-1). Freshly ejaculated spermatozoa had a high adenylate energy charge (AEC=0.8), which decreased to 0.6 under aerobic conditions and to 0.2 under anaerobic conditions. Correspondingly, no ATP resonances but high AMP resonance were visible in (31)P-NMR-spectra of the spermatozoa. When an artificial oxygen buffer (Fluosol) and a purpose-built air supply system were used during (31)P-NMR data acquisition, ATP resonances reappeared whereas the AMP resonance disappeared. Boar spermatozoa kept under aerobic conditions have intracellular compartments that differ markedly in pH, as demonstrated by both (31)P-NMR spectroscopy and confocal microscopy. Using confocal microscopy, the midpiece of the flagellum in which all mitochondria are located was identified as an acidic compartment (pH(i-mp) 6.7). The intracellular pH of both the head (pH(i-h)) and the long principal piece of the flagellum (pH(i-pp)) were 7.2 and, thus, only slightly below the extracellular pH (pH(e) 7.3). Storage of spermatozoa in a glucose-free medium at 15 degrees C when they are immotile slowly shifted the pH(i-mp) from 6.7 to 6.9 within 20 h, whereas pH(i-h) and pH(i-pp) remained unchanged (pH 7.1-7.2). When glucose was present in the medium, all visible compartments of the spermatozoa as well as the medium were acidified to pH 6.2 within 20 h. Under these conditions a resonance at 4.8 mg kg(-1) appeared representing glycerol 3-phosphate.     

Turbidity and Protein Aggregation in Whey Protein Beverages

ABSTRACT:  During storage of heat-treated acidic (pH ≤ 4.6) whey protein beverages, formation of protein aggregates can create undesirable turbidity and sedimentation. In this study, we found that a slow protein aggregation process controlled the rate of formation of sediment and turbidity. A heat-treated model beverage containing 12.5 g/L whey protein at a pH of 4 was stored for 6 wk at 3 different storage temperatures and analyzed for turbidity, soluble protein, and protein aggregates. One sample was stored without further treatment and the other sample was filtered to remove protein aggregates formed during heating. This was done to test the hypothesis that aggregates formed during heat treatment served as nuclei for deposition of soluble protein. Turbidity increased and soluble protein decreased as protein aggregates formed during storage. Increasing the storage temperature accelerated this process. The loss of soluble protein during storage was fit to first- and second-order kinetic equations, allowing the prediction of the effect of protein concentration, storage time, and storage temperature on the formation of protein aggregates.     

beta-Microseminoprotein in human spermatozoa and its potential role in male fertility

beta-Microseminoprotein (MSMB) is one of the most abundant proteins in human seminal plasma. The objectives of this study were: (1) to purify MSMB from seminal plasma (SP) and generate antibodies against the pure protein; (2) to investigate the interaction of MSMB with ejaculated spermatozoa and its possible effect on the spontaneous acrosome reaction (AR); and (3) to quantify MSMB content in SP and examine its relationship with the clinical sperm parameters. MSMB was purified from SP and its presence on the sperm surface was examined by indirect immunofluorescence using a specific polyclonal antibody. The effect of MSMB on the AR was evaluated using guinea pig epididymal spermatozoa as a model. MSMB quantification assay was performed with a two-site binding ELISA using two polyclonal antibodies against MSMB. MSMB was assessed in semen samples from fertile donors (controls) and subfertile patients according to World Health Organization criteria. MSMB was detected on the sperm surface and mainly localized to the acrosomal region of the head and neck. A significant spontaneous AR inhibition was observed when guinea pig epididymal spermatozoa were preincubated with MSMB. Finally, MSMB was significantly increased in subfertile patients when compared with fertile controls (P<0.02). The association of MSMB to the sperm surface, the inhibitor effect on the spontaneous AR and the increased MSMB levels found in SP in subfertile men suggests a relationship between this protein and semen quality and a possible role in the process of fertilization.