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Effects of bovine oviduct epithelial cells on ability of sperm binding and maintaining motility in vitro
336
Theriogenology
EFFECTS OF BOVINE OVIDUCT EPITHELIAL CELLS ON ABILITY OF SPERM BINDING AND MAINTAINING MOTILITY IN VITRO S. Roh, K.H. Cha, LX. Lee, B.C. Lee and W.S. Hwang Department of Theriogenology, College of Veterinary Medicine Seoul National University, Seoul 15 l-742, Republic of Korea The aim of these experiments was to determine if bovine oviduct epithelial cells derived from either the ampulla or isthmus bind sperm and maintain their motility in vitro. Oviduct epithelial cell monolayers (OEC) were prepared in the 4-well dishes (1.8 cm’) according to modified procedures of Chian and Sirard (Biol. Reprod; 52:156-162, 1995). Monolayers were formed within 5 days. The medium for OEC culture (TCM199 with 10% FBS) was replaced with TALP for sperm culture (sp-TALP) 3 h before each experiment. In Experiment 1, the number of sperm attached to OEC derived from isthmus and ampullary segments, and the motility of unattached sperm during co-culture were assessed. Sperm were co-cultured (lx 106cell/ml) with OEC, and motile unattached sperm were evaluated at l-h intervals until 6 h and then at 12, 24 and 48 h with a hemocytometer at 37°C. In Experiment 2, only isthmic OEC were used to determine the effect of heparin (hsp) on sperm binding. After swim-up and centrifugation, the sperm pellet was divided into two groups, and one group was treated with heparin (lOOpg/ml) for 15 min. Both groups of sperm were cultured in sp-TALP for 1 h. Sperm and OEC co-culture were performed and evaluated as in Experiment 1. All data were analyzed by Student’s T-test. Regardless of their origin, approximately 65% of the sperm were attached to OEC within 2 h, but only sperm co-cultured with isthmic epithelial cells maintained their attachment until 5 h (Table 1). Table 1. Changes in concentration and motility of unattached sperm co-cultured with OEC* Group
Time (h)
01
23
456
12
24
48
No. ofsperm(l04 cells/ml)
isthmus 100 ampulla 100
80.8 82.3
48.8 52.3
33.3 35.5
31.3 37.5
39.5 40.3a 47.8 64.5b
41.8a 59.3a 71.8b 83.5b
73.Sa 86.313
Motility of sperm (%)
isthmus 100 ampulla 100
72.3 74.8
69.6 70.3
60.7 64.2
49.5 56.0
52.4 53.8 53.5 52.8
49.8c 38.1c 42.8d 21.od
34.2c
No. of sperm (I O4 cells/ml)
hspA nspB
100 100
81.8 80.3
66.8e 46.3e 49.5e 68.3e 84Se 51.0f 34.8f 34.5f 39.0f 42.3f
87.0e 88.5e 50.5f 66.3f
88.8 74.3
Motility of sperm (%)
hsp nsp
100 100
73.8 71.5
70.8 68.3
28.0e 15.4e 49.8f 38.lf
4.3e 34.2f
62.8 60.3
51.8 50.5
52.9 49.2 53.0 54.2
* Oviduct epithelial cell monolayers. A Heparin treated sperm, B Non-Leparin Treated a-fDifferent superscripts in the same column of same cell differ significantly (abP<0.005, pc.o.05, efP
14.d
cd
The results show that bovine OEC derived from the isthmus play more important role(s) for sperm binding and maintaining motility than those from the ampulla, and heparin induced capacitation may change sperm binding ability on OEC in vitro.
Theriogenology
EFFECTS OF BOVINE OVIDUCT EPITHELIAL CELLS ON ABILITY OF SPERM BINDING AND MAINTAINING MOTILITY IN VITRO S. Roh, K.H. Cha, LX. Lee, B.C. Lee and W.S. Hwang Department of Theriogenology, College of Veterinary Medicine Seoul National University, Seoul 15 l-742, Republic of Korea The aim of these experiments was to determine if bovine oviduct epithelial cells derived from either the ampulla or isthmus bind sperm and maintain their motility in vitro. Oviduct epithelial cell monolayers (OEC) were prepared in the 4-well dishes (1.8 cm’) according to modified procedures of Chian and Sirard (Biol. Reprod; 52:156-162, 1995). Monolayers were formed within 5 days. The medium for OEC culture (TCM199 with 10% FBS) was replaced with TALP for sperm culture (sp-TALP) 3 h before each experiment. In Experiment 1, the number of sperm attached to OEC derived from isthmus and ampullary segments, and the motility of unattached sperm during co-culture were assessed. Sperm were co-cultured (lx 106cell/ml) with OEC, and motile unattached sperm were evaluated at l-h intervals until 6 h and then at 12, 24 and 48 h with a hemocytometer at 37°C. In Experiment 2, only isthmic OEC were used to determine the effect of heparin (hsp) on sperm binding. After swim-up and centrifugation, the sperm pellet was divided into two groups, and one group was treated with heparin (lOOpg/ml) for 15 min. Both groups of sperm were cultured in sp-TALP for 1 h. Sperm and OEC co-culture were performed and evaluated as in Experiment 1. All data were analyzed by Student’s T-test. Regardless of their origin, approximately 65% of the sperm were attached to OEC within 2 h, but only sperm co-cultured with isthmic epithelial cells maintained their attachment until 5 h (Table 1). Table 1. Changes in concentration and motility of unattached sperm co-cultured with OEC* Group
Time (h)
01
23
456
12
24
48
No. ofsperm(l04 cells/ml)
isthmus 100 ampulla 100
80.8 82.3
48.8 52.3
33.3 35.5
31.3 37.5
39.5 40.3a 47.8 64.5b
41.8a 59.3a 71.8b 83.5b
73.Sa 86.313
Motility of sperm (%)
isthmus 100 ampulla 100
72.3 74.8
69.6 70.3
60.7 64.2
49.5 56.0
52.4 53.8 53.5 52.8
49.8c 38.1c 42.8d 21.od
34.2c
No. of sperm (I O4 cells/ml)
hspA nspB
100 100
81.8 80.3
66.8e 46.3e 49.5e 68.3e 84Se 51.0f 34.8f 34.5f 39.0f 42.3f
87.0e 88.5e 50.5f 66.3f
88.8 74.3
Motility of sperm (%)
hsp nsp
100 100
73.8 71.5
70.8 68.3
28.0e 15.4e 49.8f 38.lf
4.3e 34.2f
62.8 60.3
51.8 50.5
52.9 49.2 53.0 54.2
* Oviduct epithelial cell monolayers. A Heparin treated sperm, B Non-Leparin Treated a-fDifferent superscripts in the same column of same cell differ significantly (abP<0.005, pc.o.05, efP
14.d
cd
The results show that bovine OEC derived from the isthmus play more important role(s) for sperm binding and maintaining motility than those from the ampulla, and heparin induced capacitation may change sperm binding ability on OEC in vitro.