Adenine nucleotide metabolism and a role for AMP in modulating flagellar waveforms in mouse sperm.

While most ATP, the main energy source driving sperm motility, is derived from glycolysis and oxidative phosphorylation, the metabolic demands of the cell require the efficient use of power stored in high-energy phosphate bonds. In times of high energy consumption, adenylate kinase (AK) scavenges one ATP molecule by transphosphorylation of two molecules of ADP, simultaneously yielding one molecule of AMP as a by-product. Either ATP or ADP supported motility of detergent-modeled cauda epididymal mouse sperm, indicating that flagellar AKs are functional. However, the ensuing flagellar waveforms fueled by ATP or ADP were qualitatively different. Motility driven by ATP was rapid but restricted to the distal region of the sperm tail, whereas ADP produced slower and more fluid waves that propagated down the full flagellum. Characterization of wave patterns by tracing and superimposing the images of the flagella, quantifying the differences using digital image analysis, and computer-assisted sperm analysis revealed differences in the amplitude, periodicity, and propagation of the waves between detergent-modeled sperm treated with either ATP or ADP. Surprisingly, addition of AMP to the incubation medium containing ATP recapitulated the pattern of sperm motility seen with ADP alone. In addition to AK1 and AK2, which we previously demonstrated are present in outer dense fibers and mitochondrial sheath of the mouse sperm tail, we show that another AK, AK8, is present in a third flagellar compartment, the axoneme. These results extend the known regulators of sperm motility to include AMP, which may be operating through an AMP-activated protein kinase.

Signaling in sperm: toward a molecular understanding of the acquisition of sperm motility in the mouse epididymis.

Sperm motility encompasses a wide range of events involving epididymal maturation and activation of biochemical pathways, most notably cyclic AMP (cAMP)-protein kinase A (PKA) activation. Following the discovery of guanine-nucleotide exchange factors (RAPGEFs), also known as exchange proteins activated by cAMP, we investigated the separate roles of PKA and RAPGEFs in sperm motility. RT-PCR showed the presence of Rapgef3, Rapgef4, and Rapgef5, as well as several known RAPGEF partner mRNAs, in spermatogenic cells. However, Rapgef3 and Rapgef4 appeared to be less abundant in condensing spermatids versus pachytene spermatocytes. Similarly, many of these proteins were detected by immunoblotting. RAPGEF5 was detected in germ cells and murine epididymal sperm. Indirect immunofluorescence localized SGK1, SGK3, AKT1 pT(308), and RAPGEF5 to the acrosome, while PDPK1 was found in the postacrosomal region. SGK3 was present throughout the tail, while PDPK1 and AKT1 pT(308) were in the midpiece. When motility was assessed in demembranated cauda epididymal sperm, addition of ATP and the selective ligand for RAPGEFs, 8-pCPT-2'-O-Me-cAMP, resulted in motility, but the sperm were unable to undergo hyperactivated-like motility. In contrast, when demembranated cauda epididymal sperm were incubated with ATP plus dibutyryl cAMP, sperm became motile and progressed to hyperactivated-like motility. However, no significant difference was observed when intact sperm were examined. GSK3 phosphorylation was altered in the presence of H89, a PKA inhibitor. Significantly, intact caput epididymal sperm became motile when incubated in the presence of extracellular ATP. These results provide evidence for a new pathway involved in endowing sperm with the capacity to swim.

Male mice express spermatogenic cell-specific triosephosphate isomerase isozymes.

Triosephosphate isomerase 1 (TPI1) is a member of the glycolytic pathway, which is a critical source of energy for motility in mouse sperm. By immunoblotting, we detected two male, germ line-specific TPI1 bands (Mr 33,400 and 30,800) as well as the somatic-type band (Mr 27,700). Although all three bands were observed in spermatogenic cells, somatic-type TPI1 disappeared from sperm during epididymal maturation. In vitro dephosphorylation analysis suggested that the two male, germ line-specific TPI1 bands were not the result of phosphorylation of the 27,700 Mr TPI1 band. The Mr 33,400; 30,800; and 27,700 TPI1 bands corresponded to the respective sizes of the proteins predicted to use the first, second, and third possible initiation codons of the Tpi1 cDNA. We performed immunofluorescence on epididymal sperm and determined that TPI1 specifically localized in the principal piece. The antibody staining was stronger in cauda epididymal sperm than in caput epididymal sperm, a finding consistent with the identification of TPI1 as a cauda epididymal sperm-enriched protein. Immunofluorescence with sodium dodecyl sulfate (SDS)-insoluble flagellar accessory structures showed a strong TPI1 signal only in the principal piece, indicating that TPI1 is a component of the fibrous sheath. Northern blot hybridization detected longer Tpi1 transcripts (1.56 kb) in mouse testis, whereas somatic tissues had shorter transcripts (1.32 kb). As there is only one triosephosphate isomerase gene in the mouse genome, we conclude that the three variants we see in sperm result from the use of alternative translation start codons in spermatogenic cells.

Seminal plasma proteins inhibit in vitro- and cooling-induced capacitation in boar spermatozoa.

Dilute boar seminal plasma (SP) has been shown to inhibit in vitro capacitation and cooling-induced capacitation-like changes in boar spermatozoa, as assessed by the ability of the spermatozoa to undergo an ionophore-induced acrosome reaction. We hypothesised that the protein component of SP is responsible for this effect. To test this hypothesis, varying concentrations of total SP protein or SP proteins fractionated by heparin binding were assayed for their ability to inhibit in vitro capacitation, as well as cooling- and cryopreservation-induced capacitation-like changes. In vitro capacitation and cooling-induced capacitation-like changes were prevented by 10% whole SP, as well as by total proteins extracted from SP at concentrations greater than 500 microg mL(-1). No amount of SP protein was able to prevent cryopreservation-induced capacitation-like changes. Total SP proteins were fractionated based on their heparin-binding properties and the heparin-binding fraction was shown to possess capacitation inhibitory activity at concentrations as low as 250 microg mL(-1). The proteins in the heparin-binding fraction were subjected to mass spectrometry and identified. The predominant proteins were three members of the spermadhesin families, namely AQN-3, AQN-1 and AWN, and SP protein pB1. We conclude that one or more of these heparin-binding SP proteins is able to inhibit in vitro capacitation and cooling-induced capacitation-like changes, but not cryopreservation-induced capacitation-like changes, in boar spermatozoa.

Effects of seminal plasma on cooling-induced capacitative changes in boar sperm.

Porcine seminal plasma (SP) has been shown to contain factors that have a decapacitative or capacitation-inhibiting effect on sperm. The objectives of the present study were to compare the capacitative changes observed in cooled sperm with those seen in sperm after in vitro capacitation and to determine whether SP could prevent these changes. Sperm were subjected to incubation or to slow cooling under noncapacitating or capacitating conditions. The effect of SP on protein tyrosine phosphorylation and the ability of the sperm to undergo an acrosome reaction (AR) were determined. Cooled sperm displayed an increased level of tyrosine phosphorylation and a higher percentage of induced AR sperm compared to incubated sperm. The addition of SP inhibited the number of ARs that occurred during incubation and cooling. These results suggest that cooling of sperm augments the capacitative changes in sperm, and that SP contains a factor(s) that effectively prevents these changes.

Bos taurus ultralong CDR H3 antibodies.

Cow antibodies are unusual in having an exceptionally long third complementarity determining region of the heavy chain (CDR H3). These CDR H3s have a multitude of cysteines and form a distinct domain characterized by a ß-ribbon 'stalk' and disulfide bonded 'knob'. Cows appear to utilize somatic hypermutation of a single VDJ rearrangement to produce an astounding variety of distinct CDR H3 sequences with different disulfide bonding patterns within the knob. Thus, cows may be unique amongst vertebrates in evolving an antibody system with both a different scaffold for binding antigen as well as an unusual diversity creating process.

Effects of extender, incubation temperature, and added seminal plasma on capacitation of cryopreserved, thawed boar sperm as determined by chlortetracycline staining.

The effect on apparent capacitation status of frozen-thawed (FT), washed boar sperm was examined in capacitation-supporting medium at 39 degrees C without added seminal plasma (SP) or supplemented with either 10 or 20% (v/v) boar SP. The thawed sperm from three boars were washed to remove the egg yolk-based freezing medium (EY) and then incubated for 1-8h after addition of SP. Capacitation status of the sperm was determined microscopically using chlortetracycline staining patterns. At 1h after the addition of 10 or 20% (v/v) SP, capacitated sperm decreased from 59.7 to 30.3% and from 59.5 to 26.8%, respectively (P<0.001). Subsequent studies examined the effect of 10% SP on capacitation status of FT sperm extended in either phosphate buffered saline or commercial thawing extender with or without prior washing of sperm to remove EY and incubated at 17 or 39 degrees C. No effect of SP resulted from addition to sperm when EY remained or when the temperature was maintained at 17 degrees C (P>0.1). These results indicate that SP appears able to reverse capacitation of FT boar sperm, but that this effect is dependent on both temperature and composition of the thawing extender.

Effect of cooling and seminal plasma on the capacitation status of fresh boar sperm as determined using chlortetracycline assay.

Insemination of sows with frozen-thawed spermatozoa results in lower fertility, in part due to spermatozoa having undergone a capacitation-like reaction. The present study employed chlortetracycline (CTC) staining analysis to investigate the effect of adding 20% (v/v) boar seminal plasma (SP) to boar spermatozoa on the temporal progress of capacitation and the acrosome reaction in spermatozoa cooled to 5 degrees C or incubated at 39 degrees C. Based on CTC staining patterns, seminal plasma appeared to reverse capacitation in spermatozoa that had undergone capacitation while incubated at 39 degrees C in a capacitation-supporting medium from 59.7 to 36.6% capacitated (P<0.001). Similarly, the addition of SP to boar spermatozoa cooled to 5 degrees C resulted in both the prevention of the capacitation-like reaction, and the reversal of an established capacitation-like reaction from 63.3 to 34.2% capacitated (P<0.001). These observations indicated that some constituent(s) of boar SP both prevent spermatozoa from undergoing capacitation as well as reverse capacitation in spermatozoa that have already undergone the process.

From PAWP to "Pop": opening up new pathways to fatherhood.

Infertility remains a significant problem for many couples. Approximately one in seven couples who attempt to conceive will fail to do so within 1 year. In about 65% of these cases, there is a male component of infertility.1 Despite normal semen parameters, the etiology of infertility remains uncertain in more than 50% of couples.2 Defects in sperm proteins and/ or structures may underlie certain cases of male infertility. Although many men would like to be called "Pop", "Dad", or "Papa", those who are classified with idiopathic male infertility have few options for becoming fathers. Recent studies by Aarabi et al.3 may open the door to new therapies.

Mitochondrial fusion protein MFN2 interacts with the mitostatin-related protein MNS1 required for mouse sperm flagellar structure and function.

BACKGROUND: Cilia and the sperm flagellum share many structural properties. Meiosis-specific nuclear structural 1 (MNS1) is a recently characterized protein that is abundantly expressed in post-meiotic spermatids and is required for proper flagellar and motile cilia formation. To explore the possible functions of MNS1, we performed a BLAST search and determined it is homologous to the conserved domain pfam13868, exemplified by mitostatin. This protein interacts with mitofusin 2 (MFN2), a protein that participates in regulating mitochondrial associations to subcellular organelles. We hypothesized that an association between MFN2 and MNS1 in the sperm is involved in flagellar biogenesis and function. RESULTS: In the studies reported here, MFN2 was found in murine reproductive and somatic tissues high in ciliary content while MNS1 was present as two closely migrating bands in reproductive tissues. Interestingly, mitostatin was also present in reproductive tissues. Similar to Mns1 and mitostatin, Mfn2 was expressed in the testis as detected by RT-PCR. In addition, Mfn2 and Mns1 decreased in expression from pachytene spermatocytes to condensing spermatids as assessed by quantitative RT-PCR. Co-immunoprecipitation demonstrated an association between MFN2 and MNS1 in spermatogenic cells. Indirect immunofluorescence indicated that MFN2 and MNS1 co-localized to the sperm flagellum in freshly collected cauda epididymal sperm. MFN2 associated with the midpiece while MNS1 was present throughout the sperm tail in caput and cauda epididymal sperm. In spermatogenic cells, MFN2 was seen in the mitochondria, and MNS1 was present throughout the cell cytoplasm. MFN2 and MNS1 were present in detergent-resistant flagellar structures of the sperm. CONCLUSIONS: These results demonstrate that MFN2 and MNS1 are present in spermatogenic cells and are an integral part of the sperm flagellum, indicating they play a role in flagellar biogenesis and/or function.

Characterization of capacitation, cryoinjury, and the role of seminal plasma in porcine sperm.

Capacitation is a biochemical pathway sperm must undergo to be able to fertilize an oocyte, whereas cryoinjury is cryopreservation-induced biophysical damage which renders sperm immediately capable of fertilization. Similarities between capacitation and cryoinjury have not been fully elucidated. The present study attempted to characterize both processes, including the role of seminal plasma (SP). Merocyanine-540 staining detected an increase (P < 0.01) in plasma membrane disorder from 60.5% in in vitro capacitated sperm to 91.4% in cryopreserved sperm, with no effect of SP. After cryopreservation, 42.8% of sperm displayed phosphatidylserine on the outer leaflet compared to 13.6% of in vitro capacitated sperm (P < 0.01), as assessed by annexin-V staining (SP decreased phosphatidylserine inversion in both populations). Lipid raft-associated glycolipid GM(1) movement increased throughout the entire sperm membrane in cryopreserved sperm, although SP did not affect lipid raft movement in these sperm. Cryopreserved and in vitro capacitated sperm had a similar intensity of tyrosine phosphorylation (although SP reduced this intensity). In in vitro capacitated sperm, 67.5% underwent an ionophore induced acrosome reaction with 91.3% reacting in cryopreserved sperm. In both cases, SP reduced (P < 0.01) the percentage of acrosome-reacted sperm to 1.0 and 7.8%, respectively. Cryopreservation appeared to damage sperm, resulting in marked increases in membrane disorder, cholesterol efflux, and percent of capacitated sperm. In both capacitated and cryoinjured sperm, the addition of SP appeared to attenuate some of these events.

Molecular cloning and expression of the CRISP family of proteins in the boar.

The family of mammalian cysteine-rich secretory proteins (CRISP) have been well characterized in the rat, mouse, and human. Here we report the molecular cloning and expression analysis of CRISP1, CRISP2, and CRISP3 in the boar. A partial sequence published in the National Center for Biotechnology Information (NCBI) database was used to derive the full-length sequences for CRISP1 and CRISP2 using rapid amplification of cDNA ends. RT-PCR confirmed the expression of these mRNAs in the boar reproductive tract, and real time RT-PCR showed CRISP1 to be highly expressed throughout the epididymis, with CRISP2 highly expressed in the testis. A search of the porcine genomic sequence in the NCBI database identified a BAC (CH242-199E6) encoding the CRISP1 gene. This BAC is derived from porcine Chromosome 7 and is syntenic with the regions of the mouse, rat, and human genomes encoding the CRISP gene family. This BAC was found to encode a third CRISP protein with a predicted amino acid sequence of high similarity to human CRISP3. Using RT-PCR we show that CRISP3 expression in the boar reproductive tract is confined to the prostate. Recombinant porcine (rp) CRISP2 protein was produced and purified. When incubated with capacitated boar sperm, rpCRISP2 induced an acrosome reaction, consistent with its demonstrated ability to alter the activity of calcium channels.

Current concepts of molecular events during bovine and porcine spermatozoa capacitation.

Spermatozoa are required to undergo the processes of capacitation before they obtain fertilizing ability. The molecular changes of capacitation are still not fully understood. However, it is accepted that capacitation is a sequential process involving numerous physiological changes including destabilization of the plasma membrane, alterations of intracellular ion concentrations and membrane potential, and protein phosphorylation. There are no known morphological changes that occur to the spermatozoon during capacitation. The purpose of this review is to summarize current evidence on the molecular aspects of capacitation both in vivo and in vitro in bovine and porcine spermatozoa. For the purpose of this review, the process of sperm capacitation will encompass maturational events that occur following ejaculation up to binding to the zona pellucida, that triggers acrosomal exocytosis and initiates fertilization.