Date of Award

Fall 2001

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

Program/Concentration

Biology

Committee Director

R. James Swanson

Committee Member

Wayne Hynes

Committee Member

Mary Mahony

Call Number for Print

Special Collections LD4331.B46 H39 2001

Abstract

Mammalian sperm exhibit characteristic motility patterns, termed hyperactivated (HA) motility, associated with completion of capacitation. In cynomolgus monkey (Macaca fascicularis) sperm, this HA motility is dependent in vitro upon the addition of exogenous cyclic nucleotide mediators, caffeine and dibutyryl-cyclic adenosine monophosphate (dbcAMP). Previous reports have shown protein tyrosine phosphorylation to be an integral component of this caffeine- and cAMP-stimulated HA motility. This study investigated the involvement of the mitogen-activated protein (MAP) kinase-signaling cascade. Semen specimens were collected in Talp-HEPES medium from proven breeders via electroejaculation. After washing, sperm were incubated in the presence and absence of the MAP kinase kinase (MAPKK) inhibitor, PD-98059 for 90 minutes at RT. Sperm were transferred to Talp-bicarbonate medium and incubated with and without the sperm activators, caffeine (lmM) and dbcAMP (lmM) for 0.5h at 37°C and 5% CO2. Proportion of sperm exhibiting hyperactivated motility was determined by computer assisted motion analysis (HTM-IVOS) using sorting criteria previously established in our laboratory. Tyrosine phosphorylation of sperm tail proteins was determined with the antiphosphotyrosine antibody, PY-20, by immunocytochemistry (ICC) and by immunoblotting to examine total proteins. Inhibition of the phosphorylation of MAP kinase was determined with ICC by assessing the proportion of sperm exhibiting phosphorylated MAP kinase immunoreactivity and by immunoblotting. Treatment of macaque sperm with PD-98059 resulted in a significant decrease (p50) of 0.1 μM. Similarly, PY-20 immunoreactivity decreased in a dose dependent manner with an IC50 of 0.1 μM and hyperactivated motility decreased in a dose dependent manner with an IC50 of 2 μM. However, while PD-98059 decreased MAP kinase phosphorylation from 69% ± 10 to baseline levels (14% ± 1), complete inhibition of tyrosine phosphorylation of sperm tails as evidenced by PY-20 ICC was not observed at those doses. Similarly, the proportion of sperm exhibiting hyperactivated motility did not decrease to baseline levels at the doses of PD-98059 tested. Immunoblotting detected a decrease in tyrosine phosphorylation of total proteins in a range of 40-120 kDa with PD-98059 treatment. This suggests MAP kinase is one, but not the exclusive upstream component in the signaling cascade of caffeine- and cAMP stimulated hyperactivated motility in macaque sperm.

Rights

In Copyright. URI: http://rightsstatements.org/vocab/InC/1.0/ This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).

DOI

10.25777/zmw3-g675

Download

Share

COinS