177 An active site of osteopontin restoring the endometrial epidermal growth factor profile and fertility in repeat breeder dairy cows

The loss of two peaks of the endometrial epidermal growth factor (EGF) concentrations on Days 2–4 and Days 13–14 of the oestrous cycle is related to reduced fertility in repeat breeder (RB) cows. The intravaginal infusion of osteopontin (OPN) restored the endometrial EGF profile and fertility in RB cows. Thrombin-cleaved N-terminal fragment of OPN has integrin binding motifs (RGD and SVAYGLK motifs located next to each other), whereas C-terminal fragment has the binding site to CD44. Objective of the present study is to determine an active site of OPN to restore the EGF profile and fertility in RB cows. Holstein lactating RB cows (parity 2–6, age 3–9 years, 120–180 days postpartum) with endometrial EGF concentrations below 4.70 ng g⁻¹ tissue weight on Day 3 that were housed in nine farms (400–1500 lactating cows) were used. The EGF concentration was measured by enzyme immunoassay using endometrial tissues obtained by biopsy. Cows were recruited by criteria of failing to conceive after three or more AI without a detectable abnormality in the oestrous cycle, clinical signs, uterine cytology, and genital organs by ultrasonography of the ovary and uterus. Cows with problems at calving and during the postpartum period requiring three or more veterinary visits were excluded from the study. The RB cows were infused with OPN-related proteins and peptides to the vagina on the day of AI (Day 0), diagnosed for the endometrial EGF profile on Day 3 for the second time and pregnancy on Days 30–35. Proportions of cows with the normalized EGF concentrations (≥4.70 ng g⁻¹ tissue weight) and pregnancy were compared by Fisher’s exact test. In a preliminary study, we found that C-terminal fragment of recombinant OPN (rOPN) alone had no effect, whereas N-terminal fragment alone showed an effect on the normalization of the EGF profile. Thus, in study 1, PBS alone (n = 77), rOPN (16–22 nmol, n = 111), peptide 1 (GRGDSVAYGLK) (320 or 1600 nmol, n = 50 each), peptide 2 (GRGDS) (320 or 1600 nmol, n = 20 each), and peptide 3 (SVAYGLK) (320 or 1600 nmol, n = 25 and 55, respectively) were infused to the vagina of RB cows. Peptide 1 at 320 (56.0%) and 1600 nmol (52.0%) and peptide 3 at 1600 nmol (56.3%) improved the normalization rate of the EGF profile to a similar extent to rOPN (61.3%) and greater than PBS (21.3%; P < 0.05). These treatments also improved pregnancy rate (41.8–50.0%) to a similar extent as rOPN (47.7%) and greater than PBS (21.6%; P < 0.05). Peptide 2 and 3 at 320 nmol failed to normalise the EGF profile. In study 2, role of RGD motif in peptide 1 was examined by replacing aspartic acid (D) replaced to glutamic acid (E). Peptide 1 (320 nmol, n = 78) and peptide 4 (GRGESVAYGLK) (320 or 1600 nmol, n = 50 and 26, respectively) were infused. Normalization rates of the EGF profile after peptide 4 infusions (16.0% and 19.2% for 320 and 1600 nmol, respectively) were lower than that after the peptide 1 infusion (44.9%; P < 0.05). Present results demonstrated that SVAYGLK motif may be an active site of OPN to restore the EGF profile and fertility in RB cows. The RGD motif may support the interaction between SVAYGLK motif and integrins by stabilising the secondary structure of the peptide.