PARENT SESSION
SLIDE SESSION 3: REGULATION OF SPERM FUNCTION
Chairs: Gary Killian, Marc-Andre Sirard, TanYa Gwathmey (Trainee)
Arts Hall 026
2:30 PM-4:30 PM

CALCIUM INCREASES IN THE ANTERIOR BOVINE SPERM HEAD DURING CAPACITATION .

Parrish, John¹, Susko-Parrish, Joan¹, ¹

ABSTRACT- Bovine sperm were incubated 4.5 hr under capacitating conditions with heparin (10 g/ml) or non-capacitating conditions with no additions (control). Fura2-AM was loaded into sperm at 3.5 hr to measure intracellular calcium (Cai). At 4.5 hr, sperm were imaged at 0, 2.5, 5, 10 and 15 min after addition of solubilized zona pellucida proteins (ZP, bovine, 50 ng/ml). Epifluorescent (340nm and 380 nm excitation) and phase contrast images were evaluated to determine Cai (nM) and acrosome reaction (AR) status respectively. The Cai was determined in the anterior (ANT) and posterior half (POST) of each sperm head. Since the nucleus failed to accumulate Fura2, the POST represented cytoplasmic Cai and the ANT represented the Cai in both the cytoplasm and acrosome. The experiment was repeated with semen from 6 bulls and a total of 387 sperm were evaluated. Three percent of the 224 control sperm and 37% of the 163 heparin-treated sperm underwent an AR in response to ZP. There was no difference (p>0.05) between the control sperm that did or did not undergo an AR in the mean±sem Cai within the ANT (85±8 vs. 74±5) or POST (58±5 vs. 46±6). In contrast, the Cai was higher (p<0.05) for heparin-treated sperm undergoing an AR both in the ANT (378±51 vs. 129±13) and POST (216±34 vs. 114±18). The Cai was also greater (p<0.05) in the ANT vs. the POST for heparin-treated sperm which underwent an AR but was not different (p>0.05) for those that did not undergo an AR. The Cai at time 0 in the ANT of heparin-treated sperm undergoing an AR at 2.5, 5, 10 or 15 min after ZP addition was 540±202, 408±110, 367±98, and 192±49. The higher the Cai at the time of ZP addition, the faster heparin-treated sperm acrosome reacted. The Cai (time 0) in the ANT of sperm which did not acrosome react in response to ZP was greater (p<0.05) in heparin-treated sperm (141±22) than in control sperm (74±5). Results suggest calcium is accumulated in the acrosome during capacitation and the more calcium accumulated the faster sperm acrosome react in response to ZP. Supported by NIH 29HD31120 and USDA 97-35203-4612.

KEY WORDS: acrosome, capacitation, calcium, image analysis