Danhui Yang - Academia.edu (original) (raw)

Papers by Danhui Yang

Research paper thumbnail of The redox state of the plastoquinone pool controls the level of the light-harvesting chlorophyll a/b binding protein complex II (LHC II) during photoacclimation

Photosynthesis research, 2001

A cytochrome b (6) f deficient mutant of Lemna perpusilla maintains a constant and lower level of... more A cytochrome b (6) f deficient mutant of Lemna perpusilla maintains a constant and lower level of the light-harvesting chl a/b-binding protein complex II (LHC II) as compared to the wild type plants at low-light intensities. Inhibition of the plastoquinone pool reduction increases the LHC II content of the mutant at both low- and high-light intensities but only at high-light intensity in the wild type plants. Proteolytic activity against LHC II appears during high-light photoacclimation of wild type plants. However, the acclimative protease is present in the mutant at both light intensities. These and additional results suggest that the plastoquinone redox state serves as the major signal-transducing component in the photoacclimation process affecting both, synthesis and degradation of LHC II and appearance of acclimative LHC II proteolysis. The plastoquinol pool cannot be oxidized by linear electron flow in the mutant plants which are locked in a 'high light' acclimation st...

Research paper thumbnail of Regulatory Proteolysis of the Major Light-Harvesting Chlorophyll a/b Protein of Photosystem II by a Light-Induced Membrane-Associated Enzymic System

European Journal of Biochemistry, 1995

An endogenous proteolytic activity associated with spinach chloroplast thylakoid membranes has be... more An endogenous proteolytic activity associated with spinach chloroplast thylakoid membranes has been identified. This enzymic activity is involved in the degradation of the major light-harvesting chlorophyll a/b protein of photosystem II (LHCII) in response to exposure of leaves to increased irradiance. This proteolysis of LHCII requires an induction period and can only be detected 48-72 hours after transfer of the plants from low-intensity to high-intensity light. Once initiated by high-intensity light, the degradation of LHCII can readily occur in complete darkness. The proteolysis can, after induction in vivo, be experimentally followed in vitro, both in isolated intact chloroplasts and thylakoid membranes. The proteolytic process is strictly dependent on ATP and the protease involved is of the serine or cysteine type. The activity can be released from isolated thylakoid membranes by washing with high concentrations of NaCl and reconstituted by readdition of the desalted wash supernatant. It is concluded that the protease is extrinsically bound to the outer surface of the stroma-exposed regions of the stacked thylakoid membrane. The mechanism for the induction of the proteolytic process as well as its relation to previously described thylakoid proteases will be discussed.

Research paper thumbnail of The N-terminal domain of the light-harvesting chlorophyll a/b-binding protein complex (LHCII) is essential for its acclimative proteolysis

FEBS Letters, 2000

Variations in the amount of the light-harvesting chlorophyll a/b-binding protein complex (LHCII) ... more Variations in the amount of the light-harvesting chlorophyll a/b-binding protein complex (LHCII) is essential for regulation of the uptake of light into photosystem II. An endogenous proteolytic system was found to be involved in the degradation of LHCII in response to elevated light intensities and the proteolysis was shown to be under tight regulation [Yang, D.-H. et al. (1998) Plant Physiol. 118, 827^834]. In this study, the substrate specificity and recognition site towards the protease were examined using reconstituted wild-type and mutant recombinant LHCII. The results show that the LHCII apoprotein and the monomeric form of the holoprotein are targeted for proteolysis while the trimeric form is not. The Nterminal domain of LHCII was found to be essential for recognition by the regulatory protease and the involvement of the N-end rule pathway is discussed.

Research paper thumbnail of Induction of Acclimative Proteolysis of the Light-Harvesting Chlorophyll a/B Protein of Photosystem II In Response to Elevated Light Intensities

Plant …, 1998

Most plants have the ability to respond to fluctuations in light to minimize damage to the photos... more Most plants have the ability to respond to fluctuations in light to minimize damage to the photosynthetic apparatus. A proteolytic activity has been discovered that is involved in the degradation of the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCII) when the ...

Research paper thumbnail of DNA versus protein immunisation for production of monoclonal antibodies against Choristoneura fumiferana ecdysone receptor (CfEcR)

Vaccine, Jan 12, 2006

The full-length ecdysone receptor cDNA of Choristoneura fumiferana (CfEcR-B) was cloned into bact... more The full-length ecdysone receptor cDNA of Choristoneura fumiferana (CfEcR-B) was cloned into bacterial expression systems and the recombinant protein was expressed either with a His-tag (His-EcR-B) or glutathione-S-transferase (GST) fusion (GST-EcR-B). The His-EcR-B was expressed mostly as insoluble aggregates, while the GST-EcR-B was partially soluble and could be purified using affinity chromatography. Mice were then immunised with the purified GST-EcR-B protein. Due to the time-consuming protein expression and purification procedures and the solubility problem of the recombinant protein, we also inserted the full-length CfEcR-B cDNA into the mammalian DNA vaccine expression vector, pVAC1-mcs for DNA immunisation. In vitro expression of CfEcR-B in mammalian cells transfected with the pVAC-EcR-B plasmid was confirmed prior to the delivery of the DNA vaccine into mice. The anti-CfEcR-B MAbs generated from both DNA and protein vaccines were characterised and shown to recognise native...

Research paper thumbnail of Choristoneura fumiferana nucleopolyhedrovirus encodes a functional 3'-5' exonuclease

Journal of General Virology, 2004

The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III ... more The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III 39-59 exonucleases. The CfMNPV v-trex ORF was cloned into the Bac-to-Bac baculovirus expression-vector system, expressed in insect Sf21 cells with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified V-TREX confirmed that this viral protein is a functional 39-59 exonuclease that cleaves oligonucleotides from the 39 end in a stepwise, distributive manner, suggesting a role in proofreading during viral DNA replication and DNA repair. Enhanced degradation of a 59-digoxigenin-or 59-32 P-labelled oligo(dT) 30 substrate was observed at increasing incubation times or increased amounts of V-TREX. The 39-excision activity of V-TREX was maximal at alkaline pH (9?5) in the presence of 5 mM MgCl 2 , 2 mM dithiothreitol and 0?1 mg BSA ml "1 .

Research paper thumbnail of Development of an oligonucleotide-based DNA microarray for transcriptional analysis of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) genes

Journal of Virological Methods, 2007

A modified oligonucleotide-based two-channel DNA microarray was developed for characterization of... more A modified oligonucleotide-based two-channel DNA microarray was developed for characterization of temporal expression profiles of select Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) ORFs including its 7 unique ORFs. The microarray chip contained oligonucleotide probes for 23 CfMNPV ORFs and their complements as well as five host genes. Total RNA was isolated at different times post infection from Cf203 insect cells infected with CfMNPV. The cDNA was synthesized, fluorescent labelled with Cy3, and co-hybridized to the microarray chips along with Cy5-labelled viral genomic DNA, which served as equimolar reference standards for each probe. Transcription of the 7 CfMNPV unique ORFs was detected using DNA microarray analysis and their temporal expression profiles suggest that they are functional genes. The expression levels of three host genes varied throughout virus infection and therefore were unsuitable for normalization between microarrays. The DNA microarray results were compared to quantitative RT-PCR (qRT-PCR). Transcription of the non-coding (antisense) strands of some of the CfMNPV select genes including the polyhedrin gene, was also detected by array analysis and confirmed by qRT-PCR. The polyhedrin antisense transcript, based on long-range RT-PCR analysis, appeared to be a read-through product of an adjacent ORF in the same orientation as the antisense transcript.

Research paper thumbnail of Choristoneura fumiferana nucleopolyhedrovirus encodes a functional 3'-5' exonuclease

Journal of General Virology, 2004

The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III ... more The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III 39-59 exonucleases. The CfMNPV v-trex ORF was cloned into the Bac-to-Bac baculovirus expression-vector system, expressed in insect Sf21 cells with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified V-TREX confirmed that this viral protein is a functional 39-59 exonuclease that cleaves oligonucleotides from the 39 end in a stepwise, distributive manner, suggesting a role in proofreading during viral DNA replication and DNA repair. Enhanced degradation of a 59-digoxigenin-or 59-32 P-labelled oligo(dT) 30 substrate was observed at increasing incubation times or increased amounts of V-TREX. The 39-excision activity of V-TREX was maximal at alkaline pH (9?5) in the presence of 5 mM MgCl 2 , 2 mM dithiothreitol and 0?1 mg BSA ml "1 .

Research paper thumbnail of Short Communication Choristoneura fumiferana nucleopolyhedrovirus encodes a functional 39-59 exonuclease

The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III ... more The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III 39-59 exonucleases. The CfMNPV v-trex ORF was cloned into the Bac-to-Bac baculovirus expression-vector system, expressed in insect Sf21 cells with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified V-TREX confirmed that this viral protein is a functional 39-59 exonuclease that cleaves oligonucleotides from the 39 end in a stepwise, distributive manner, suggesting a role in proofreading during viral DNA replication and DNA repair. Enhanced degradation of a 59-digoxigenin-or 59-32 P-labelled oligo(dT) 30 substrate was observed at increasing incubation times or increased amounts of V-TREX. The 39-excision activity of V-TREX was maximal at alkaline pH (9?5) in the presence of 5 mM MgCl 2 , 2 mM dithiothreitol and 0?1 mg BSA ml "1 .

Research paper thumbnail of Extracellular calcium is involved in egg yolk-induced head-to-head agglutination of bull sperm

Head-to-head agglutination of bull sperm occurs when semen is highly diluted in an egg yolk-citra... more Head-to-head agglutination of bull sperm occurs when semen is highly diluted in an egg yolk-citrate diluent without streptomycin. The objectives were to investigate causes of sperm agglutination and the underlying mechanism. Aliquots of bull semen were diluted in a base diluent (BD) supplemented with various test components and the percentage of agglutinated sperm (% AggSp) was quantified at 1, 5, 24, 48, and 72 h of incubation. When sperm were incubated at 22°C, no agglutination was observed in BD for up to 72 h, whereas the % AggSp was 5.0, 41.7, 72.2, 91.1, and 92.8% in BD ϩ 5% egg yolk (BD ϩ EY) at 1, 5, 24, 48 and 72 h, respectively. However, no sperm agglutination was observed in BD ϩ EY if incubation temperature was 37°C. Addition of 5 or 10 mM ethylenebis (oxyethyleneni-trilo) tetra-acetic acid to BD ϩ EY reduced the % AggSp from 95% to Ͻ5% at 72 h (P Ͻ 0.001), but addition of 5 mM CaCl 2 to BD failed to induce sperm agglutination in the absence of egg yolk, implicating calcium and other factors in egg yolk. Addition of the citrate-soluble fraction (CSF) of egg yolk to BD induced sperm agglutination similar to whole egg yolk, whereas water-and saline-soluble fractions of egg yolk were ineffective. The spermagglutinating efficacy of CSF (the % AggSp ϭ 95% at 72 h) was reduced by dialysis (20%; P Ͻ 0.05), partially restored by addition of 5 mM CaCl 2 (70%; P Ͻ 0.05), but the calcium effect was neutralized by addition of 5 mM ethylenebis (oxyethylenenitrilo) tetra-acetic acid (1.7%; P Ͻ 0.05), again implicating calcium. Addition of 30 M of a protein kinase A inhibitor (H-89) to an agglutinating diluent failed to inhibit sperm agglutination, whereas addition of 2 mM of a cAMP analogue, dbcAMP, to a nonagglutinating diluent failed to induce sperm agglutination. Agglutination status had no effect on sperm plasma membrane/ acrosome status and mitochondrial membrane potential. In conclusion, calcium and other component(s) in the CSF of egg yolk induced head-to-head agglutination of bull sperm in a time-and temperature-dependent manner. Although the mechanism of agglutination was not determined, the cAMP-protein kinase A signaling pathway was not involved.

Research paper thumbnail of The redox state of the plastoquinone pool controls the level of the light-harvesting chlorophyll a/b binding protein complex II (LHC II) during photoacclimation

Photosynthesis research, 2001

A cytochrome b (6) f deficient mutant of Lemna perpusilla maintains a constant and lower level of... more A cytochrome b (6) f deficient mutant of Lemna perpusilla maintains a constant and lower level of the light-harvesting chl a/b-binding protein complex II (LHC II) as compared to the wild type plants at low-light intensities. Inhibition of the plastoquinone pool reduction increases the LHC II content of the mutant at both low- and high-light intensities but only at high-light intensity in the wild type plants. Proteolytic activity against LHC II appears during high-light photoacclimation of wild type plants. However, the acclimative protease is present in the mutant at both light intensities. These and additional results suggest that the plastoquinone redox state serves as the major signal-transducing component in the photoacclimation process affecting both, synthesis and degradation of LHC II and appearance of acclimative LHC II proteolysis. The plastoquinol pool cannot be oxidized by linear electron flow in the mutant plants which are locked in a 'high light' acclimation st...

Research paper thumbnail of Regulatory Proteolysis of the Major Light-Harvesting Chlorophyll a/b Protein of Photosystem II by a Light-Induced Membrane-Associated Enzymic System

European Journal of Biochemistry, 1995

An endogenous proteolytic activity associated with spinach chloroplast thylakoid membranes has be... more An endogenous proteolytic activity associated with spinach chloroplast thylakoid membranes has been identified. This enzymic activity is involved in the degradation of the major light-harvesting chlorophyll a/b protein of photosystem II (LHCII) in response to exposure of leaves to increased irradiance. This proteolysis of LHCII requires an induction period and can only be detected 48-72 hours after transfer of the plants from low-intensity to high-intensity light. Once initiated by high-intensity light, the degradation of LHCII can readily occur in complete darkness. The proteolysis can, after induction in vivo, be experimentally followed in vitro, both in isolated intact chloroplasts and thylakoid membranes. The proteolytic process is strictly dependent on ATP and the protease involved is of the serine or cysteine type. The activity can be released from isolated thylakoid membranes by washing with high concentrations of NaCl and reconstituted by readdition of the desalted wash supernatant. It is concluded that the protease is extrinsically bound to the outer surface of the stroma-exposed regions of the stacked thylakoid membrane. The mechanism for the induction of the proteolytic process as well as its relation to previously described thylakoid proteases will be discussed.

Research paper thumbnail of The N-terminal domain of the light-harvesting chlorophyll a/b-binding protein complex (LHCII) is essential for its acclimative proteolysis

FEBS Letters, 2000

Variations in the amount of the light-harvesting chlorophyll a/b-binding protein complex (LHCII) ... more Variations in the amount of the light-harvesting chlorophyll a/b-binding protein complex (LHCII) is essential for regulation of the uptake of light into photosystem II. An endogenous proteolytic system was found to be involved in the degradation of LHCII in response to elevated light intensities and the proteolysis was shown to be under tight regulation [Yang, D.-H. et al. (1998) Plant Physiol. 118, 827^834]. In this study, the substrate specificity and recognition site towards the protease were examined using reconstituted wild-type and mutant recombinant LHCII. The results show that the LHCII apoprotein and the monomeric form of the holoprotein are targeted for proteolysis while the trimeric form is not. The Nterminal domain of LHCII was found to be essential for recognition by the regulatory protease and the involvement of the N-end rule pathway is discussed.

Research paper thumbnail of Induction of Acclimative Proteolysis of the Light-Harvesting Chlorophyll a/B Protein of Photosystem II In Response to Elevated Light Intensities

Plant …, 1998

Most plants have the ability to respond to fluctuations in light to minimize damage to the photos... more Most plants have the ability to respond to fluctuations in light to minimize damage to the photosynthetic apparatus. A proteolytic activity has been discovered that is involved in the degradation of the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCII) when the ...

Research paper thumbnail of DNA versus protein immunisation for production of monoclonal antibodies against Choristoneura fumiferana ecdysone receptor (CfEcR)

Vaccine, Jan 12, 2006

The full-length ecdysone receptor cDNA of Choristoneura fumiferana (CfEcR-B) was cloned into bact... more The full-length ecdysone receptor cDNA of Choristoneura fumiferana (CfEcR-B) was cloned into bacterial expression systems and the recombinant protein was expressed either with a His-tag (His-EcR-B) or glutathione-S-transferase (GST) fusion (GST-EcR-B). The His-EcR-B was expressed mostly as insoluble aggregates, while the GST-EcR-B was partially soluble and could be purified using affinity chromatography. Mice were then immunised with the purified GST-EcR-B protein. Due to the time-consuming protein expression and purification procedures and the solubility problem of the recombinant protein, we also inserted the full-length CfEcR-B cDNA into the mammalian DNA vaccine expression vector, pVAC1-mcs for DNA immunisation. In vitro expression of CfEcR-B in mammalian cells transfected with the pVAC-EcR-B plasmid was confirmed prior to the delivery of the DNA vaccine into mice. The anti-CfEcR-B MAbs generated from both DNA and protein vaccines were characterised and shown to recognise native...

Research paper thumbnail of Choristoneura fumiferana nucleopolyhedrovirus encodes a functional 3'-5' exonuclease

Journal of General Virology, 2004

The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III ... more The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III 39-59 exonucleases. The CfMNPV v-trex ORF was cloned into the Bac-to-Bac baculovirus expression-vector system, expressed in insect Sf21 cells with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified V-TREX confirmed that this viral protein is a functional 39-59 exonuclease that cleaves oligonucleotides from the 39 end in a stepwise, distributive manner, suggesting a role in proofreading during viral DNA replication and DNA repair. Enhanced degradation of a 59-digoxigenin-or 59-32 P-labelled oligo(dT) 30 substrate was observed at increasing incubation times or increased amounts of V-TREX. The 39-excision activity of V-TREX was maximal at alkaline pH (9?5) in the presence of 5 mM MgCl 2 , 2 mM dithiothreitol and 0?1 mg BSA ml "1 .

Research paper thumbnail of Development of an oligonucleotide-based DNA microarray for transcriptional analysis of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) genes

Journal of Virological Methods, 2007

A modified oligonucleotide-based two-channel DNA microarray was developed for characterization of... more A modified oligonucleotide-based two-channel DNA microarray was developed for characterization of temporal expression profiles of select Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) ORFs including its 7 unique ORFs. The microarray chip contained oligonucleotide probes for 23 CfMNPV ORFs and their complements as well as five host genes. Total RNA was isolated at different times post infection from Cf203 insect cells infected with CfMNPV. The cDNA was synthesized, fluorescent labelled with Cy3, and co-hybridized to the microarray chips along with Cy5-labelled viral genomic DNA, which served as equimolar reference standards for each probe. Transcription of the 7 CfMNPV unique ORFs was detected using DNA microarray analysis and their temporal expression profiles suggest that they are functional genes. The expression levels of three host genes varied throughout virus infection and therefore were unsuitable for normalization between microarrays. The DNA microarray results were compared to quantitative RT-PCR (qRT-PCR). Transcription of the non-coding (antisense) strands of some of the CfMNPV select genes including the polyhedrin gene, was also detected by array analysis and confirmed by qRT-PCR. The polyhedrin antisense transcript, based on long-range RT-PCR analysis, appeared to be a read-through product of an adjacent ORF in the same orientation as the antisense transcript.

Research paper thumbnail of Choristoneura fumiferana nucleopolyhedrovirus encodes a functional 3'-5' exonuclease

Journal of General Virology, 2004

The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III ... more The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III 39-59 exonucleases. The CfMNPV v-trex ORF was cloned into the Bac-to-Bac baculovirus expression-vector system, expressed in insect Sf21 cells with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified V-TREX confirmed that this viral protein is a functional 39-59 exonuclease that cleaves oligonucleotides from the 39 end in a stepwise, distributive manner, suggesting a role in proofreading during viral DNA replication and DNA repair. Enhanced degradation of a 59-digoxigenin-or 59-32 P-labelled oligo(dT) 30 substrate was observed at increasing incubation times or increased amounts of V-TREX. The 39-excision activity of V-TREX was maximal at alkaline pH (9?5) in the presence of 5 mM MgCl 2 , 2 mM dithiothreitol and 0?1 mg BSA ml "1 .

Research paper thumbnail of Short Communication Choristoneura fumiferana nucleopolyhedrovirus encodes a functional 39-59 exonuclease

The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III ... more The Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) encodes an ORF homologous to type III 39-59 exonucleases. The CfMNPV v-trex ORF was cloned into the Bac-to-Bac baculovirus expression-vector system, expressed in insect Sf21 cells with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified V-TREX confirmed that this viral protein is a functional 39-59 exonuclease that cleaves oligonucleotides from the 39 end in a stepwise, distributive manner, suggesting a role in proofreading during viral DNA replication and DNA repair. Enhanced degradation of a 59-digoxigenin-or 59-32 P-labelled oligo(dT) 30 substrate was observed at increasing incubation times or increased amounts of V-TREX. The 39-excision activity of V-TREX was maximal at alkaline pH (9?5) in the presence of 5 mM MgCl 2 , 2 mM dithiothreitol and 0?1 mg BSA ml "1 .

Research paper thumbnail of Extracellular calcium is involved in egg yolk-induced head-to-head agglutination of bull sperm

Head-to-head agglutination of bull sperm occurs when semen is highly diluted in an egg yolk-citra... more Head-to-head agglutination of bull sperm occurs when semen is highly diluted in an egg yolk-citrate diluent without streptomycin. The objectives were to investigate causes of sperm agglutination and the underlying mechanism. Aliquots of bull semen were diluted in a base diluent (BD) supplemented with various test components and the percentage of agglutinated sperm (% AggSp) was quantified at 1, 5, 24, 48, and 72 h of incubation. When sperm were incubated at 22°C, no agglutination was observed in BD for up to 72 h, whereas the % AggSp was 5.0, 41.7, 72.2, 91.1, and 92.8% in BD ϩ 5% egg yolk (BD ϩ EY) at 1, 5, 24, 48 and 72 h, respectively. However, no sperm agglutination was observed in BD ϩ EY if incubation temperature was 37°C. Addition of 5 or 10 mM ethylenebis (oxyethyleneni-trilo) tetra-acetic acid to BD ϩ EY reduced the % AggSp from 95% to Ͻ5% at 72 h (P Ͻ 0.001), but addition of 5 mM CaCl 2 to BD failed to induce sperm agglutination in the absence of egg yolk, implicating calcium and other factors in egg yolk. Addition of the citrate-soluble fraction (CSF) of egg yolk to BD induced sperm agglutination similar to whole egg yolk, whereas water-and saline-soluble fractions of egg yolk were ineffective. The spermagglutinating efficacy of CSF (the % AggSp ϭ 95% at 72 h) was reduced by dialysis (20%; P Ͻ 0.05), partially restored by addition of 5 mM CaCl 2 (70%; P Ͻ 0.05), but the calcium effect was neutralized by addition of 5 mM ethylenebis (oxyethylenenitrilo) tetra-acetic acid (1.7%; P Ͻ 0.05), again implicating calcium. Addition of 30 M of a protein kinase A inhibitor (H-89) to an agglutinating diluent failed to inhibit sperm agglutination, whereas addition of 2 mM of a cAMP analogue, dbcAMP, to a nonagglutinating diluent failed to induce sperm agglutination. Agglutination status had no effect on sperm plasma membrane/ acrosome status and mitochondrial membrane potential. In conclusion, calcium and other component(s) in the CSF of egg yolk induced head-to-head agglutination of bull sperm in a time-and temperature-dependent manner. Although the mechanism of agglutination was not determined, the cAMP-protein kinase A signaling pathway was not involved.