Fig 1.
Aberdeen Angus (Bos taurus taurus, n = 8) and Nelore (Bos taurus indicus, n = 8) heifers were classified according to their ovarian AFC: low follicular count (LFC, n = 4) and high follicular (HFC, n = 4). All 16 animals were slaughtered one day after ovulation, and both oviducts were obtained. The ipsilateral and contralateral to ovulation side from each oviduct, including the infundibulum, ampulla, and isthmus segments, were submitted to relative gene expression by real time RT-PCR and protein quantification by western blotting.
Table 1.
Genes analyzed in bovine oviducts using TLDA® system (Gene symbols, gene full name, Applied Biosystems™ ID, function/reason for selection, and reference).
Fig 2.
Oviductal differences between bovine breeds: Aberdeen Angus (n = 8 animals) vs. Nelore (n = 8 animals).
(A) Total length of oviducts from each breed (mean ± SEM, centimeters). (B,C) Relative mRNA abundance (mean ± SEM) of AGTR1 in the ampulla (B) and in the isthmus (C) from each breed. The target gene was normalized by reference genes using 2(-ΔΔCt). Different letters indicate significant difference (p ≤ 0.05) between means.
Table 2.
p values for transcript abundance in each oviductal segment.
Fig 3.
Differences in transcripts levels in the infundibulum (ovulation side/breed and ovulation side/AFC).
Relative mRNA abundance (mean ± SEM) of OVGP1 (A) and FUCA1 (B) in ipsilateral and contralateral oviducts from Aberdeen Angus (n = 8 animals) and Nelore (n = 8 animals) heifers were normalized by reference genes using 2(-ΔΔCt). The analyses were performed by comparing different ovulation sides in the same breed and the same ovulation side in different breeds, as well as relative mRNA abundance of ACE (C) in ipsilateral and contralateral oviducts from LFC (n = 8 animals) and HFC (n = 8 animals) heifers. The analyses were performed by comparing different ovulation sides in the same AFC groups, and the same ovulation sides in different AFC groups. Different uppercase letters (A,B) indicate significant differences (p ≤ 0.05) between ovulation sides in the same breed/AFC group (horizontal analysis). Different lowercase letters (a,b) indicate significant difference (p ≤ 0.05) between breed/AFC of same ovulation side (vertical analysis).
Fig 4.
Relative mRNA abundance in the infundibulum (ovulation side effect).
HSPA5, FUCA2, ANXA2, VEGFA, and PTGER4 (mean ± SEM) in ipsilateral and contralateral oviducts (n = 16 animals). Expression of target genes were normalized by reference genes using 2(-ΔΔCt). Different letters indicate significant difference (p ≤ 0.05) between means.
Fig 5.
Relative mRNA abundance of FUCA1 in ipsilateral and contralateral ampulla from LFC (n = 8 animals) and HFC (n = 8 animals) heifers.
Expression of the target gene was normalized by references genes using 2(-ΔΔCt), and relative abundance is presented as mean ± SEM. The analyses were performed by comparing different ovulation sides in the same AFC groups, and the same ovulation side in different AFC groups. Different uppercase letters (A,B) indicate significant difference (p ≤ 0.05) between ovulation sides in the same AFC group (horizontal analysis). Different lowercase letters (a,b) indicate significant difference (p ≤ 0.05) between AFC groups on the same ovulation side (vertical analysis).
Fig 6.
Transcript levels in the ampulla (ovulation side effect).
Relative mRNA abundance (mean ± SEM) of OVGP1, HSPA5, ANXA4, PTGS2, and END1 in ipsilateral and contralateral oviducts (n = 16 animals) were normalized by reference genes using 2(-ΔΔCt). Different letters indicate significant difference (p ≤ 0.05) between means.
Fig 7.
Transcript levels in the isthmus were different depending on ovulation side and AFC.
Relative abundance (mean ± SEM) of VEGFA in ipsilateral and contralateral oviducts from LFC (n = 8 animals) and HFC (n = 8 animals) heifers were normalized by reference genes using 2(-ΔΔCt). The analyses were performed by comparing different ovulation sides in the same AFC group, and the same ovulation side in different AFC groups. Different uppercase letters (A,B) indicate significant difference (p ≤ 0.05) between ovulation sides in the same AFC (horizontal analysis). Different lowercase letters (a,b) indicate significant difference (p ≤ 0.05) between AFC groups on the same ovulation side (vertical analysis).
Fig 8.
Transcript levels in the isthmus (ovulation side effect).
Relative mRNA abundance (mean ± SEM) of FUCA2 and KDR in ipsilateral and contralateral oviducts (n = 16 animals). Target genes were normalized by reference genes using 2(-ΔΔCt). Different letters indicate significant difference (p ≤ 0.05) between means.
Fig 9.
Protein levels of FUCA1 in the infundibulum, ampulla, and isthmus.
Relative protein abundance (FUCA1/GAPDH, mean ± SEM) in the ipsilateral and contralateral oviducts (n = 8 animals), p > 0.05.
Fig 10.
Biological status of bovine ipsilateral oviducts, one day after ovulation, in Aberdeen Angus or Nelore heifers.
The ovulation side was the major factor that modulates the molecular profiles of bovine oviducts. There is a possibility of modification in the ipsilateral oviduct microenvironment to control gamete transportation, polyspermy gamete interaction, and embryo development even without the occurrence of mating, sperm presence, or fertilization. These oviductal functions could be controlled by ovulation products. 1. Upregulation of VEGF, PGE2, and ANGII systems in the infundibulum increase CBF, resulting in efficient ovum pick-up and oocyte transportation to the fertilization site. 2. Upregulation of PTGs and END1 systems in the ampulla possibly raises oviductal transport by stimulatory effects on smooth muscle contraction or CBF. 3. Downregulation of the VEGF system in the isthmus, caused by negative feedback, guarantees slow and safe transport of the embryo to the uterus. 4. Upregulation of annexin transcripts in the ampulla possibly results in an increase of sperm-bound sites, thereby controlling sperm release to oocyte fertilization. 5. Upregulation of OVGP1, HSPA5, and FUCA levels from the infundibulum to the ampulla may increase the activity of these factors on the oocytes before fertilization, participating in ZP hardening, control of sperm bound to ZP, and embryo development. We observed a difference between ipsi- and contralateral oviducts concerning the abundance of ACE (in the infundibulum), FUCA (in the ampulla), and VEGFA (in the isthmus) from low-AFC cows, but they were similar in both sides of the oviducts in high-AFC cows. The modulation by the AFC is still inconclusive, and more experiments should be performed to confirm if this subtle effect of AFC on oviduct functions can impact animal reproduction. AFC: antral follicle count, CBF: ciliary beat frequency, ZP: Zona Pellucida; CL: Corpus Luteum; (−) downregulated factor, (+) upregulated factor. Black large arrows represent the OVGP1, HSPA5, and FUCA coming from the infundibulum and ampulla.