Robust in vitro capacitation protocols are necessary to assess the physiological mechanisms underlying the preparation of sperm for fertilisation. Here we describe the unreported phenomenon of head to head agglutination of ram sperm following extension in the capacitating media, Tyrodes plus albumin, lactate and pyruvate (TALP). Agglutination is immediate, specific, persistent and is not associated with a loss of motility or viability. Agglutination can be prevented or reversed by penacillamine (PEN). A dose of 250µM PEN reduced the percentage of motile, agglutinated sperm from 77 ±3% to 3±1%. Reversion back to the agglutinated state can be achieved by adding 5µM copper but other heavy metals including cobalt, iron, manganese were not as effective.
PEN can act as a chelator of heavy metals, an antioxidant and a reducing agent. To investigate PEN’s mechanism of action we compared it to the broad spectrum chelator ethylenediaminetetraacetic acid (EDTA; 1mM) and the copper specific chelator (bathocuproinedisulfonic acid (BCS: 1mM). BCS and EDTA significantly increased the percentage of motile, non-agglutinated sperm compared to the control but were significantly lower than PEN (1mM). The antioxidants superoxide dismutase (SOD, 800 IU mL-1) and ascorbic acid (1 mg mL-1) showed similar low level inhibition of agglutination compared to PEN (1mM) and catalase (150 IU mL-1) had no effect. At 0 hrs only the reducing agents cysteine (1mM) and DL-dithiothreitol (1mM) displayed similar levels of non-agglutinated sperm compared to PEN (1mM) but were less effective after 3hrs of incubation (37°C). Together these results indicate that PEN is an effective agent to reduce agglutination of ram sperm in capacitating media. It may be acting upon sulfhydryl bonds of a sperm membrane protein that binds copper.