Rat Oocytes Fertilized in Modified Rat 1-Cell Embryo: RESULTS(1)

RESULTS(1)

Effect of BSA in mR1ECM (Experiment 1)

The incidence of oocytes penetrated during coculture of oocyte-cumulus complexes with spermatozoa in mKRB, mKRB-PVA, mR1ECM, and mR1ECM-BSA for 10 h is shown in Table 2. Sperm penetration was observed only in mKRB (88.2 ± 2.7%). Although no sperm penetration occurred in mR1ECM-BSA, this medium was used in the subsequent experiments because we found the presence of BSA to be of importance for sperm penetration in mKRB. proventil inhaler

Effect of Phosphate, Sodium Lactate, and Osmolarity in mR1ECM-BSA (Experiment 2)

No penetrated oocytes were observed after coculture of oocyte-cumulus complexes with spermatozoa either in mR1ECM-BSA supplemented with 1.19 mM KH2PO4 or in mR1ECM-BSA with increased sodium lactate (21.58 mM). However, oocytes were penetrated (71.6 ± 6.9%) when the osmolarity of mR1ECM-BSA was increased to 310 mOsm by addition of NaCl (Table 3). The incidence was not different (p > 0.05) from that in mKRB (76.7 ± 13.7%). However, the incidence of sperm penetration was extremely low (p < 0.01; 7.0 ± 7.0%) when the osmolarity of mR1ECM-BSA was increased by adding 60 mM sorbitol (Table 4).

TABLE 1. Formulations of mKRB and mR1ECM.

Ingredient mKRB mR1ECM
NaCl 94.6 mM 76.7 mM
KCl 4.78 mM 3.2 mM
CaCl2 1.71 mM 2.0 mM
KH2PO4 1.19 mM
MgSO4 1.19 mM
MgCl2 0.5 mM
NaHCO, 25.07 mM 25.0 mM
Sodium lactate 21.58 mM 10.0 mM
Sodium pyruvate 0.5 mM 0.5 mM
Glucose 5.56 mM 7.5 mM
BSA 4.0 mg/ml
Polyvinyl alcohol 1.0 mg/ml
Glutamine 0.1 mM
EAAa 2% (v/v)
NEAAb 1% (v/v)
Streptomycin sulfate 50 ^g/ml
Potassium penicillin 75 ^g/ml
Osmolarity 310 mOsM 246 mOsM

a Minimal essential medium (MEM) amino acid solution (GIBCO BRL). b MEM nonessential amino acid solution (GIBCO BRL).

TABLE 2. Effects of BSA in mKRB and mRIECM on sperm penetration of rat oocytes inseminated with epididymal spermatozoa.

Media Oocytes
No. examined” % penetratedb % with male and female pronucleic % polyspermicc
mKRB 76 88.2 ± 2.7 100 ± 0 36.3 ± 1.3
mKRB-PVA 77 0
mR1ECM 85 0
mR1ECM-BSA 79 0

a Oocytes were examined 10 h after insemination; experiments were replicated 4 times. b Percentage (mean ± SEM) of oocytes examined. c Percentage (mean ± SEM) of oocytes penetrated.

TABLE 3. Effect of phosphate, lactate, and osmolarity in mR1ECM-BSA on sperm penetration of rat oocytes inseminated in vitro with epididymal spermatozoa.

Media Oocytes
No. examined8 % penetratedb % with male and female pronucleic % polyspermicc
mKRB 79 76.7 ± 13.7 94.5 ± 5.6 31.9 ± 10.9
mR1ECM-BSA 78 0
with phosphated 72 0
with high lactate’ 77 0
with high osmolarity’ 74 71.6 ± 6.9 96.0 ± 2.4 26.6 ± 6.6

a Oocytes were examined 10 h after insemination; experiments were replicated 4 times. b Percentage (mean ± SEM) of oocytes examined. c Percentage (mean ± SEM) of oocytes penetrated. d 1.19 mM KH2PO4 was added.
e Concentration of sodium lactate was increased from 10.0 mM to 21.58 mM. f Osmolarity was adjusted to 310 mOsM by increasing NaCl concentration to 106.7 mM.

TABLE 4. Sperm penetration of rat oocytes in mR1ECM-BSA with high osmolarity (310 mOsM) adjusted by adding NaCl or sorbitol.

Osmolarity(mOsM) Adjustedmethoda Oocytes
No. examinedb % penetrated’ % with male andfemale pronucleid % polyspermicd
246 52 0 ——
310 NaCl 57 77.2 ± 8.3 96.7 ± 3.3 16.8 ± 8.3
310 Sorbitol 62 7.0 ± 7.0 00

a Osmolarity was adjusted to 310 mOsM by increasing NaCl concentration to 106.7 mM (NaCl) or by adding 60 mM sorbitol.
b Oocytes were examined 10 h after insemination; experiments were replicated 4 times. c Percentage (mean ± SEM) of oocytes examined. d Percentage (mean ± SEM) of oocytes penetrated.

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