Cyclic AMP-dependent changes in phosphorylation of epididymal mouse sperm suspensions were examined in media designed to manipulate capacitation and the expression of parameters associated with full fertilizing ability, i.e. hyperactivated motility and the acrosome reaction. After initial assessment of cAMP-dependent protein kinase activity in frozen–thawed and lyophilized sperm suspensions using exogenous substrate, phosphorylation of endogenous sperm phosphoproteins was examined using sodium dodecyl sulfate poly-acrylamide gel electrophoresis followed by autoradiography or immunoblotting. Numerous phosphoproteins were detected in both uncapacitated and capacitated suspensions, the majority of which were probably concerned with motility; full expression of fertilizing ability appeared to involve an increase in the amount of endogenous phosphorylation as deduced from the decreased amount of 32P incorporation in these suspensions. The addition of the cAMP-dependent protein kinase inhibitors, H8 and PKI (6–22) amide, demonstrated that most of the phosphoproteins detected were phosphorylated in a cAMP-dependent manner. Of particular interest was a phosphoprotein with an M r of about 95 000 which was consistently observed in capacitated suspensions. Evidence suggests that this may be phosphorylated on tyrosine residues, since the inclusion of orthovanadate, a phosphoryltyrosine phosphatase inhibitor, altered phosphorylation of this protein. Furthermore, immunodetection using the antiphosphotyrosine antibody, PY-20, identified five proteins with approximate M r 116 000, 105 000, 95 000, 86 000, and 76 000, and possibly a sixth at 54 000. The 95 000 protein was consistently diminished in ionophore-treated spermatozoa, indicating that the protein was located in the acrosomal cap region. These results suggest that the protein may be the same phosphotyrosine-containing protein as that described by Leyton and Saling (1989) which has been proposed to play a role in acrosomal exocytosis.
L. R. Fraser and A. E. Duncan
Adenosine and its analogues, known to stimulate adenylate cyclase activity in somatic cells via A2 receptors, can accelerate capacitation in mouse spermatozoa and thereby enhance fertilizing ability in vitro. Indirect evidence has suggested that adenosine can modulate mouse sperm adenylate cyclase, implicating this enzyme and cAMP in the observed functional responses. In the present study we provide evidence that [3H]5′-N-ethylcarboxamidoadenosine (NECA), an adenosine analogue with specificity for stimulatory A2 adenosine receptors, can bind to mouse spermatozoa. This binding can be displaced by both unlabelled NECA and 2-chloroadenosine, another A2 receptor agonist, but not by cyclopentyladenosine, an inhibitory A1 receptor agonist, suggesting that the NECA binding is specific for A2 receptors. The presence of S-(p-nitrobenzyl)-6-thioinosine, an adenosine transport inhibitor, did not affect binding, indicating an external site for interaction with sperm cells. Saturable specific binding of [3H]NECA to mouse spermatozoa incubated at 37°C was observed, with a B max of 5.17 pmol mg−1 protein and a K d value of 930 nmol l−1. Binding data were consistent with the presence of a single major class of receptor. In addition to demonstrable binding of [3H]NECA, both NECA and 2-chloroadenosine significantly stimulated adenylate cyclase activity in a concentration-dependent manner, with NECA being effective at a lower concentration. Furthermore, the hydrolysis-resistant GTP analogue Gpp(NH)p, alone and in the presence of either NECA or 2-chloroadenosine, also significantly stimulated enzyme activity. In somatic cells, expression of responses to adenosine usually requires GTP and G proteins. These results are consistent, therefore, with the hypothesis that adenosine-induced enhancement of sperm functional ability is due to stimulation of adenylate cyclase, via A2 adenosine receptors, and hence increasing availability of intracellular cAMP. They also suggest that G proteins play a role in the receptor-mediated response.
ARLINGTON A. FORIST, JAMES E. STAFFORD and GORDON W. DUNCAN
5-(α,α,α-Trifluoro-m-tolyloxymethyl)-2-oxazolidinethione, referred to as U-11634 (Youngdale, Duncan, Emmert & Lednicer, 1966), is orally active as an antifertility agent in the rat (Duncan, Johnston & Lyster, 1966) but not in the monkey (Meyer & Wolf, personal communication). This difference could reflect differences in drug availability following oral administration or could be due to the mechanism of action of U-11634. Species-specific responses have been reported for the antifertility diphenylindenes (Duncan, Stucki, Lyster & Lednicer, 1962; Duncan & Lyster, 1962) and diphenyldihydronaphthalenes (Duncan, Lyster, Clark & Lednicer, 1963). Accordingly, a comparison of drug availability based on serum drug levels has been made following oral administration of U-11634 to the rat and monkey.
Eight groups of five female Sprague-Dawley rats, each weighing 219 to 231 g, were employed. One group served as a control. Each member of the other
Rachel E Dickinson, Lynn Hryhorskyj, Hannah Tremewan, Kirsten Hogg, Axel A Thomson, Alan S McNeilly and W Colin Duncan
In humans and domestic mammals, pivotal processes in ovary development, including primordial follicle assembly, occur prenatally. These events are essential for determining fertility in adult life; however, they remain poorly understood at the mechanistic level. In mammals, the SLITs (SLIT1, SLIT2 and SLIT3) and their ROBO (ROBO1, ROBO2, ROBO3/RIG-1 and ROBO4/MAGIC ROBO) receptors regulate neural, leukocyte, vascular smooth muscle cell and endothelial cell migration. In addition, the SLIT/ROBO pathway has functional roles in embryonic development and in the adult ovary by inhibiting cell migration and promoting apoptosis. We therefore characterised follicle formation and investigated the expression and localisation of the ROBO/SLIT pathway in the ovine fetal ovary. Using RT-PCR, we identified SLIT2, SLIT3, ROBO1, ROBO2 and ROBO4 in sheep ovaries harvested across gestation. The real-time quantitative PCR results implied that ROBO2 expression and ROBO4 expression were elevated during the early stages of follicle formation and stayed abundant during primordial follicle maturation (P<0.05). Immunohistochemistry examination demonstrated that ROBO1 was localised to the pre-granulosa cells, while ROBO2, ROBO4 and SLIT2 were expressed in the oocytes of the developing primordial follicle. This indicates that in the fetal ovary, SLIT–ROBO signalling may require an autocrine and paracrine interaction. Furthermore, at the time of increased SLIT–ROBO expression, there was a significant reduction in the number of proliferating oocytes in the developing ovary (P<0.0001). Overall, these results suggest, for the first time, that the SLIT–ROBO pathway is expressed at the time of follicle formation during fetal ovary development.