Significance of egg-jelly substances in the internal fertilization of the newt, Cynops pyrrhogaster

Japanese newt, Cynops pyrrhogaster, undergoes internal fertilization as do most urodeles. In this study, we focused on the roles of egg-jelly in fertilization of C. pyrrhogaster and characterized the substances associated with those roles. When dry sperm were directly inseminated onto the egg, normal fertilization occurred without the presence of water. Egg-jelly extract (JE) prepared with Steinberg's salt solution contained the activity for the initiation of sperm motility. A substance of about 50 kDa in JE was significant for this activity; an inactive form of the substance probably exists in JE. Strong activity to induce acrosome reaction was detected in JE. It was inhibited by the treatment of JE with WGA, suggesting that carbohydrate in JE may be important for the induction of the acrosome reaction. This study suggests that two significant processes of fertilization are regulated by substances in the egg-jelly of the newt, C. pyrrhogaster.     

Sperm motility-initiating activity in the egg jelly of the externally-fertilizing urodele amphibian, Hynobius lichenatus

Low osmolality initiates sperm motility during the external fertilization of aquatic anuran amphibians. It is thought that this process occurs also in urodeles, but this has not been fully examined in these species. We report here that fertilization was achieved in the externally fertilizing hynobiid, Hynobius lichenatus, by direct insemination onto the egg jelly surface without initial exposure of the sperm to a hypoosmotic solution. To identify the factors in addition to low osmolality that initiate sperm motility in Hynobius, we suspended the sperm of this amphibian in egg jelly extract (JE), and about 90% began to move within 1 min. This indicated the presence of a substance in JE that promotes motility initiation, as is also the case in the newt, Cynops pyrrhogaster. To examine whether this JE factor is homologous to the sperm motility-initiating substance (SMIS) in the newt, we tested for possible inter-species cross-reactivity of the JE. The percentage of moving Cynops sperm was increased to 67% in Hynobius JE at 5 min, and 65% of the Hynobius sperm began to move in Cynops JE within 1 min, indicating that JE is indeed cross-reactive between these species of salamander and newt. Concomitantly, pretreatment of Hynobius JE with Fab fragments of a Cynops SMIS monoclonal antibody resulted in a decreased number of moving Hynobius sperm. Immunoblotting further suggested that the substance in Hynobius JE responsible for motility initiation has an 18 kDa molecular mass, with an isoelectric point at 7.5.     

Seminal plasma proteins prolong the viability of rainbow trout (Oncorynchus mykiss) spermatozoa

Rainbow trout (Oncorhynchus mykiss) spermatozoa were incubated in artificial sperm motility inhibiting saline solution (SMIS), in SMIS containing seminal plasma proteins or in pure seminal plasma. In SMIS containing the total seminal plasma protein fraction or the <50 kDa protein fraction or in pure seminal plasma, significantly higher motility rates and swimming velocities could be activated than in SMIS without seminal plasma proteins and in SMIS containing the >50 kDa protein fraction. These preliminary results indicated that seminal plasma proteins have physiological functions in prolongation and stabilization of sperm viability when using sperm motility as viability index.     

Characteristics of sperm motility induced on the egg-jelly in the internal fertilization of the newt,Cynops pyrrhogaster

Most urodeles undergo internal fertilization and sperm are directly inseminated onto the surface of egg-jelly. Feature of sperm motility induced on the egg-jelly was examined in the newt, Cynops pyrrhogaster. When sperm were directly inseminated onto an egg-jelly, sperm motility was immediately induced on its surface. The egg-jelly of C. pyrrhogaster was composed of six sublayers that were added by turns in oviduct. When the eggs with various sets of the sublayers were obtained and sperm were inseminated onto the egg-jelly, the immediate activity for the initiation of sperm motility was observed only on the outermost sublayer. Similarly, the immediate initiation of sperm motility was induced in the sperm suspended in the extract of the egg-jelly (JE). The initiation of sperm motility was affected by the external pH, and the motility was activated in the moving sperm. A K(+)-channel antagonist, charybdotoxin (CTX), or a Ca(2+)-channel antagonist, gallopamil inhibited the initiation of sperm motility in a dose dependent manner. These results demonstrated the feature of the mechanism regulating sperm motility under stable surroundings in the internal fertilization of amphibians.     

NMDA‐type glutamate receptors mediate the acrosome reaction and motility initiation in newt sperm

The N‐methyl d ‐aspartate type glutamate receptor (NMDAR) is a ligand‐gated cation channel that causes Ca²⁺ influx in nerve cells. An NMDAR agonist is effective to the sperm motility in fowls, although the actual role of NMDAR in sperm function is unknown. In the present study, RNA‐seq of the spermatogenic testes suggested the presence of NMDAR in the sperm of the newt Cynops pyrrhogaster. Glutamate of at least 0.7 ± 0.5 mM was detected in the egg‐jelly substances along with acrosome reaction‐inducing substance (ARIS) and sperm motility‐initiating substance (SMIS). In the egg‐jelly extract (JE) that included the ARIS and SMIS, the acrosome reaction was inhibited by a NMDAR antagonists, memantine and MK801. MK801 also inhibited the spontaneous acrosome reaction in Steinberg's salt solution (ST). Furthermore, memantine and MK801 suppressed the progressive motility of the sperm in JE and spontaneous waving of the undulating membrane, which is the tail structure giving thrust for forward motility, in ST. The spontaneous waving of the undulating membrane was promoted when Mg²⁺, which blocks Ca²⁺ influx through gated NMDARs, was removed from the ST. In addition, the ARIS‐induced acrosome reaction was inhibited by a selective antagonist of the transient receptor potential vanilloid 4, whose activation might result in the membrane depolarization to release Mg²⁺ from the NMDAR. These results suggest that NMDAR acts together with other cation channels in the induction of the acrosome reaction and motility of the sperm during the fertilization process of C. pyrrhogaster.     

The activity for the induction of the sperm acrosome reaction localises in the outer layers and exists in the high-molecular-weight components of the egg-jelly of the newt, Cynops pyrrhogaster

Localisation of the acrosome reaction inducing activity in egg-jelly was examined in the newt, Cynops pyrrhogaster. The jelly has six layers: the J0, J1, J2, J3, J4 and st layers. Jelly was mechanically dissected and placed on a Millipore filter. When sperm were added from the outer surface side of the jelly, most of them exhibited the acrosome reaction after passing through the jelly. When egg-jelly was divided into four layers, strong activity for the induction of acrosome reaction was detected in the outer layers, J4+st. These findings suggest that the acrosome reaction is induced by a substance in the outer layers of the egg-jelly. Among jelly components separated by SDS-PAGE, a fraction of more than 500 kDa in molecular weight induced the acrosome reaction. Wheat germ agglutinin (WGA), Griffonia simplicifoliar agglutinin 1 (GS-1), Maclura pomifera agglutinin (MPA) and Arachis hypogaea agglutinin (PNA) inhibited the induction of the acrosome reaction by jelly extract, and WGA did so in a dose-dependent manner. Those lectins precipitated some molecules of over 500 kDa. These results suggest that the acrosome reaction is induced by the high molecular-weight components of egg-jelly in C. pyrrhogaster.     

Sperm motility-initiating substance in newt egg-jelly induces differential initiation of sperm motility based on sperm intracellular calcium levels

Sperm motility-initiating substance (SMIS), a novel motility inducer from newt egg-jelly, is activated by the release from associated jelly substances at the beginning of internal fertilization and affects female-stored sperm. We examined motility initiation kinetics of newt sperm in response to SMIS by monitoring the changes of sperm intracellular calcium ([Ca2(+)](i)). In quiescent non-motile sperm loaded with the Ca2(+) indicator Fluo-4, intracellular free Ca2(+) was observed around mitochondria using confocal scanning laser microscopy. A slight increase in [Ca2(+)](i) occurred simultaneously and transiently at motility initiation in sperm treated with either heated jelly extract (hJE) containing activated SMIS, or a low osmotic solution, which naturally initiates motility in externally-fertilizing amphibians and can initiate motility in urodele sperm. When the increase of [Ca2(+)](i) at motility-initiation was monitored using spectrofluorometry, large increases in [Ca2(+)](i) occurred immediately in the low osmotic solution and within 1.5 min in the hJE. In the intact jelly extract (no heating), small increases of [Ca2(+)](i) irregularly occurred from around 1 min and for about 4 min, during which motility was differentially initiated among sperm. These results indicate that the SMIS induces differential initiation of sperm motility depending on the activational states of the SMIS and its overall activity. The motility initiation in the jelly extract was delayed in sperm whose intracellular Ca2(+) had been chelated with BAPTA-AM. The relative levels of [Ca2(+)](i) were variable with a mean of 414 ± 256 nmol/L among resting sperm, suggesting that the level of [Ca2(+)](i) in the resting sperm modulates the responsiveness to the SMIS.     

Characterization and identification of epididymal factors that protect ejaculated bovine sperm during in vitro storage.

The role of secretory epididymal factors on sperm survival and storage in bovine cauda epididymides is poorly understood. Thus, the effects of bovine epididymal epithelium fluid (BEEF) on frozen-thawed bovine sperm motility have been evaluated in vitro. Sperm motion parameters were assessed by computer-assisted sperm analysis. Compared with serum bovine proteins, BEEF efficiently sustained bovine sperm motility after a 6-h incubation period. The positive effect of BEEF on sperm motility was even more apparent using a fractionated BEEF extract (>10 kDa, 2 mg/ml). This beneficial effect was abolished when the BEEF active fraction was heat treated before incubation. A minimal 2-h BEEF preincubation period was necessary to maintain sperm motility activity and to protect sperm against oxidative injury caused by 150 microM hydrogen peroxide. The proteins from the BEEF >10-kDa fractions were biotinylated to identify the proteins that bind to the sperm surface. Five specific sperm-surface-binding proteins were revealed by Western blot analysis probed with avidin-horseradish peroxidase conjugate. These proteins were digested with trypsin for identification by matrix-assisted laser desorption ionization time-of-flight peptide mass spectrometric analyzer. Under reducing conditions, 5 bovine proteins were identified: the beta (36-kDa spot) and alpha (38-kDa spot) chains of clusterin, the beta-adrenergic receptor kinase 2 (48-kDa spot), and the antithrombin-III and the fibrinogen gamma-B chains, both corresponding to a doublet of about 50-52 kDa. These proteins are known to be present at the sperm surface in other species and could play a role in sperm protection in vivo. These results provide new insights to explain how secretory epididymal proteins sustain sperm motility during storage in vitro.     

Snake venoms as a source of compounds modulating sperm physiology: Secreted phospholipases A2 from Oxyuranus scutellatus scutellatus impact sperm motility,acrosome reaction and in vitro fertilization in mice

The goal of this study was to identify new compounds from venoms able to modulate sperm physiology and more particularly sperm motility. For this purpose, we screened the effects of 16 snake venoms cleared of molecules higher than 15 kDa on sperm motility. Venoms rich in neurotoxins like those from Oxyuranus scutellatus scutellatus or Daboia russelii, were highly potent inhibitors of sperm motility. In contrast, venoms rich in myotoxins like those from Echis carinatus, Bothrops alternatus and Macrovipera lebetina, were inactive. From the main pharmacologically-active fraction of the Taipan snake O. scutellatus s., a proteomic approach allowed us to identify 16 different proteins, among which OS1 and OS2, two secreted phospholipases A2 (sPLA₂). Purified OS1 and OS2 mimicked the inhibitory effect on sperm motility and were likely responsible for the inhibitory effect of the active fraction. OS1 and OS2 triggered sperm acrosome reaction and induced lipid rearrangements of the plasma membrane. The catalytic activity of OS2 was required to modulate sperm physiology since catalytically inactive mutants had no effect. Finally, sperm treated with OS2 were less competent than control sperm to initiate in vitro normal embryo development. This is the first report characterizing sPLA₂ toxins that modulate in vitro sperm physiology.     

Egg-jelly structure promotes efficiency of internal fertilization in the newt, Cynops pyrrhogaster

Egg-jelly is composed of a network of fibrous components and contains substances regulating the sperm-egg interaction. Many studies on the latter have been conducted, whereas the role of the egg-jelly structure in fertilization has not yet been fully assessed. In this study, we examined the fertilization efficiency in the presence and absence of the structure around the egg of the newt, Cynops pyrrhogaster, using a gelatin gel system. Although de-jellied eggs of C. pyrrhogaster can be fertilized with an adequate number of sperm, the fertilization rate was dramatically increased through the use of the gelatin gel. Sperm showed forward motility with straight morphology in the gel, whereas they swam in circles in solution. This result indicates that the gel structure is significant for sperm guidance to the egg surface, and its presence raises the fertilization efficiency in C. pyrrhogaster. When sperm were entangled in the gel structure, they were immediately folded and never showed any forward motility. Sperm with zigzag morphology were observed in the gelatin gel as well as in the egg-jelly, indicating the elimination of sperm by the gel structure. The effect of sperm elimination on successful fertilization was estimated using gelatin gels of different thickness. Though the variation did not affect the fertilization rate, the rate of normal development gradually increased in the thicker gels. This result indicates that sperm elimination in egg-jelly can function in the fertilization system. The roles of sperm guidance and sperm elimination under the physiological condition of internal fertilization of the newt are discussed.     

Characterization of a sperm factor for egg activation at fertilization of the newt Cynops pyrrhogaster

Eggs of the newt, Cynops pyrrhogaster, arrested at the second meiotic metaphase are activated by sperm at fertilization and then complete meiosis to initiate development. We highly purified a sperm factor for egg activation from a sperm extract with several chromatographies. The purified fraction containing only a 45 kDa protein induced egg activation accompanied by an intracellular Ca2+ increase when injected into unfertilized eggs. Although injection of mouse phospholipase C (PLC) zeta-mRNA caused a Ca2+ increase and egg activation, partial amino acid sequences of the 45 kDa protein were homologous to those of Xenopus citrate synthase, but not to PLCs. An anti-porcine citrate synthase antibody recognized the 45 kDa protein both in the purified fraction and in the sperm extract. Treatment with the anti-citrate synthase antibody reduced the egg-activation activity in the sperm extract. Injection of porcine citrate synthase or mRNA of Xenopus citrate synthase induced a Ca2+ increase and caused egg activation. A large amount of the 45 kDa protein was localized in two lines elongated from the neck to the middle piece of sperm. These results indicate that the 45 kDa protein is a major component of the sperm factor for egg activation at newt fertilization.     

Sperm-derived sperm motility-initiating substance from amphioxus Branchiostoma belcheri

The sperm of amphioxus, Branchiostoma belcheri, were immotile when excised from the testis and suspended in seawater. The sperm became motile upon spawning in natural seawater, suggesting mechanisms that triggered sperm motility during spawning. When a male amphioxus that underwent spawning was transferred to a cup containing a small amount of natural seawater, and then the seawater containing the spawned sperm was centrifuged, the supernatant caused motility initiation in the immotile sperm from the testis. This sperm motility-initiating activity was also found in the supernatant of seawater in which immotile sperm from the testis were incubated overnight. These suggest that in the amphioxus, a sperm motility-initiating substance resides in the sperm, and upon spawning, the substance is transformed into a free and active form to activate the sperm. Partial purification of the substance revealed it as a small and heat-stable substance with maximum UV absorbance at 234 nm.     

Binding and inactivation of the germ cell-specific protein phosphatase PP1gamma2 by sds22 during epididymal sperm maturation

Testis- and sperm-specific protein phosphatase, PP1gamma2, is a key enzyme regulating sperm function. Its activity decreases during sperm maturation in the epididymis. Inhibition of PP1gamma2 leads to motility initiation and stimulation. Our laboratory is focused on identifying mechanisms responsible for the decline in PP1gamma2 activity during sperm motility initiation in the epididymis. Previously, using immuno-affinity chromatography, we showed that a mammalian homologue of yeast sds22 is bound to PP1gamma2 in motile caudal spermatozoa (Huang Z, et al. Biol Reprod 2002; 67:1936-1942). The objectives of this study were to determine: 1) stoichiometry of PP1gamma2-sds22 binding and 2) whether PP1gamma2 in immotile caput epididymal spermatozoa is bound to sds22. The enzyme from caudal and caput sperm extracts was purified by column chromatography. Immunoreactive PP1gamma2 and sds22 from both caudal and caput spermatozoa were found in the flow-through fraction of a DEAE-cellulose column. However, PP1gamma2 from caudal spermatozoa was inactive, whereas in caput spermatozoa it was active. The DEAE-cellulose flow-through fractions were next passed through a SP-sepharose column. Caudal sperm sds22 and PP1gamma2 coeluted in the gradient fraction. In contrast, caput sperm sds22 and PP1gamma2 were separated in the flow-through and gradient fractions, respectively. Further purification through a Superose 6 column showed that PP1gamma2-sds22 complex from caudal sperm was 88 kDa in size. Caput sperm sds22 and PP1gamma2 eluted at 60 kDa and 39 kDa, respectively. SDS-PAGE of these purified fractions revealed that in caudal sperm, the 88-kDa species is composed of sds22 (43 kDa) and PP1gamma2 (39 kDa), suggesting a 1:1 complex between these two proteins. PP1gamma2 bound to sds22 in this complex was inactive. Caput sperm sds22 eluting as a 60-kDa species was found to be associated with a 17-kDa protein (p17). This suggests that dissociation of sds22 from p17 or some other posttranslational modification of sds22 is required for its binding and inactivation of PP1gamma2. Studies are currently underway to determine the mechanisms responsible for development of sds22 binding to PP1gamma2 during epididymal sperm maturation.     

A vasopressor factor partially purified from human parathyroid glands.

Recently, a parathyroid hypertensive factor was postulated to play a role in the pathogenesis of hypertension in genetically hypertensive rats. Therefore it was examined, whether in human parathyroid glands a vasopressor substance can be detected. For this purpose, homogenates of hyperplastic parathyroid glands from 20 patients with tertiary hyperparathyroidism were deproteinized and fractionated by gel chromatography. The fractions obtained were tested for vasopressor activity in isolated perfused rat kidneys. A vasopressor fraction containing substances of 0.6-2.5 kDa was identified in the parathyroid glands. The responsible product was heat sensitive, peptidase-, trypsin- and carboxypeptidase y- sensitive and hydrophilic, as it did not bind to hydrophobic reversed-phase gel. These results suggest that parathyroid glands contain a hydrophilic peptide-like vasopressor substance different from the parathyroid hormone.     

Purification and characterization of a motility initiating protein from caprine epididymal plasma

Numerous reports have appeared on the occurrence of undefined protein factors in male reproductive fluids that promote motility of mature sperm and initiate forward motility in the immature (immotile) caput‐epididymal sperm. This study reports for the first time purification to apparent homogeneity of a motility initiating protein (MIP) from epididymal plasma and its characterization using the caprine sperm model. It is a 125 kDa (approximately) dimeric protein made up of two subunits: 70 and 54 kDa. MIP is an acidic protein with an isoelectric point of 4.75. The motility protein at 30 µg/ml (240 nM) level showed nearly maximal motility‐promoting activity. MIP is heat stable and it is maximally active at pH 8. It is a glycoprotein that binds with high affinity to concanavalin A and it contains mannose, galactose, and N‐acetyl glucosamine approximately in the ratios of 6:1:6. It is sensitive to the actions of α‐mannosidase and β‐N‐acetylglucoseaminidase thereby demonstrating that the sugar side chain of the glycoprotein is essential for its biological activity. Epididymal plasma is its richest source. It is also capable of enhancing forward motility of mature cauda‐sperm. Its antibody markedly inhibits sperm motility. MIP antibody is highly immunospecific and it recognizes both the subunits. MIP causes significant increase of the intrasperm level of cyclic AMP. MIP: the physiological motility‐activating protein has potential for use as a contraceptive vaccine and for solving some of the problems of human infertility and animal breeding. J. Cell. Physiol. 222:254–263, 2010. © 2009 Wiley‐Liss, Inc.     

Platelet activating factor activity in the phospholipids of bovine spermatozoa

Platelet activating factor (PAF) has been detected in sperm from several mammalian species and can affect sperm motility and fertilization. Because bovine sperm contain a high percentage of ether-linked phospholipid precursors required for PAF synthesis, a study was undertaken to determine the PAF activity of bovine sperm phospholipids. Total lipids of washed, ejaculated bull sperm were extracted, and phospholipids were fractionated by thin-layer chromatography. Individual phospholipid fractions were assayed for PAF activity on the basis of [3H]serotonin release from equine platelets. PAF activity was detected in the PAF fraction (1.84 pmol/mumol total phospholipid) and in serine/inositol (PS/PI), choline (CP), and ethanolamine phosphoglyceride (EP) and cardiolipin (CA) fractions. Activity was highest in the CP fraction (8.05 pmol/mumol total phospholipid). Incomplete resolution of PAF and neutral lipids may have contributed to the activity in the PS/PI and CA fractions, respectively. Phospholipids from nonsperm sources did not stimulate serotonin release. Platelet activation by purified PAF and by sperm phospholipid fractions was inhibited by the receptor antagonist SRI 63-675. These results indicate that bovine sperm contain PAF and that other sperm phospholipids, especially CP and EP, which are high in glycerylether components, are capable of receptor-mediated platelet activation.     

Detection of intracellular forms of secretory aspartic proteinase in Candida albicans.

The extracellular proteinase (EPR) of Candida albicans was induced in a medium containing bovine serum albumin as sole nitrogen source. There were two intracellular forms in cells induced to produce EPR, a 43 kDa protein (EPR) and a 45 kDa protein (cross-reacting material of EPR; CRM-EPR); these were detected by immunoblotting using anti-EPR antiserum. The 43 kDa protein (EPR) may be the same as the extracellular form judging by molecular mass, and the 45 kDa protein (CRM-EPR) may be a precursor form of EPR. Many dense granules were observed by electron microscopy near the plasma membrane of the mother cells in EPR-producing cells. Both the 43 and 45 kDa proteins were recovered in a membrane fraction and were solubilized by Triton X-100. When the membrane fraction was further fractionated by sucrose density gradient centrifugation, the 43 and 45 kDa proteins were differentially fractionated. This suggests that they were located in different membrane-bound structures and is consistent with an assumption that the 45 kDa protein is a precursor for EPR.     

Identification of apolipoprotein A1 and immunoglobulin as components of a serum complex that mediates activation of human sperm motility

Serum is superior to other body fluids in activating the progressive motility of human spermatozoa and is used in connection with sperm separation for fertilization in vitro. The major activating capacity is localized to a macromolecular fraction, purified to homogeneity by a four-step protocol with ion-exchange chromatography, chromatofocusing, exclusion FPLC (elution corresponding to a molecular mass of about 250 kDa), and Blue Sepharose chromatography (no binding but elimination of albumin). The pure protein, at a concentration of 20-70 nmol/L, activated the motility to the same extent as serum. SDS-polyacrylamide gel electrophoresis under nonreducing conditions showed one band corresponding to a molecular mass of about 180 kDa. In the presence of mercaptoethanol, two bands are obtained corresponding to 50 kDa and about 25 kDa, respectively. Without the Blue Sepharose step, the sample after reduction revealed an additional band at about 67 kDa, suggesting that the fraction is then in complex also with albumin. Amino acid sequence analysis of the Blue Sepharose eluate identified three protein chains--those of apolipoprotein A1 and immunoglobulin heavy and light chains--suggesting that the preparation is an apolipoprotein A1-immunoglobulin complex. Antiserum raised toward the pure preparation in a rabbit inhibited human sperm motility, when added directly to spermatozoa. Pretreatment of human serum with rabbit antiserum significantly reduced its ability to activate sperm motility. The sperm activating capacity of the protein complex was destroyed by heating at 100 degrees C for 5 min, suggesting that the activity was dependent on intact protein conformations. Albumin, apolipoprotein A1, and immunoglobulins by themselves had only minor effects on sperm motility.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sperm motility in the horseshoe crab,Limulus polyphemus L: II. Partial characterization of a motility initiating factor from eggs and the effects of inorganic cations on motility initiation

Egg extracts (obtained by washing intact Limulus eggs with either distilled water or artificial seawater, ASW) contain a sperm motility initiating factor (SMI). The SMI is heat stable (withstands boiling to dryness), passes through a dialysis membrane, and is retained by G-10 Sephadex (indicating a molecular weight of less than 700). Qualitative analysis (by X-ray fluorescence spectroscopy) and quantitative analysis (by atomic absorption spectroscopy) of SMI extracts revealed the presence of four divalent cations (Ca, Mg, Ni, and Cu) and one monovalent cation (K) that affect sperm motility. When assayed individually at high concentrations, all of the divalent cations initiate sperm motility and K⁺ inhibits motility initiation by the divalent cations. However, none of the divalent cations are present at concentrations high enough to produce the observed SMI activity, and since K⁺ is present when motility is initiated by SMI, K⁺ must not normally be an inhibitor. Therefore, if inorganic cations are involved in normal sperm motility initiation in Limulus, they are acting in conjunction with some other low molecular weight factor.     

Characterization of secretory proteins from cultured cauda epididymal cells that significantly sustain bovine sperm motility in vitro

Epididymis provides a safe environment in which stored-spermatozoa could survive for days before ejaculation. In vitro studies suggested that epididymal proteins seem to be implicated in sperm survival during coincubation with cultured epididymal cells. This study was basically designed to confirm if secretory proteins from bovine epididymal cell cultures provide sperm protection against rapid loss of sperm motility in vitro. Bovine spermatozoa were incubated in conditioned media (CM), which were prepared from cultured cauda epididymal cell (CEC). Motion parameters were recorded using a computer-assisted sperm analyzer. Sperm-free protein extracts from CM were fractionated by ultrafiltration through a 10-kDa cut off membrane. A significantly positive effect on sperm motility was observed when spermatozoa were incubated in CM (54 +/- 4%) and CM > 10 kDa (57 +/- 4%) compared to CM < 10-kDa fraction (30 +/- 3%) or fresh media (34 +/- 3%), after a 6-hr incubation period. This beneficial effect on sperm motility was abolished when the CM > 10-kDa fraction was heat-treated at 100 degrees C for 10 min. The CM > 10 kDa fraction provides factors that remained active even though spermatozoa were washed twice after a 2-hr preincubation period. To identify potential beneficial factors, bovine spermatozoa were incubated with radiolabeled proteins obtained using (35)S-methionine in culture medium. SDS-PAGE analysis of proteins extracted from CM-preincubated spermatozoa revealed the presence of a 42-kDa protein strongly associated to the sperm surface. This 42-kDa spot was trypsin-digested and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as a protein homologue to a 35-kDa bovine estrogen-sulfotransferase. This protein can play a role in epididymal biology and sperm function. Taken together, these results suggest that specific epididymal proteins can be implicated in the sperm protection in vitro, and can be characterized in our cell culture system.