The present study provides an extensive analysis of the bovine OF proteome considering three factors of variation, i.e., the anatomical oviduct region, the peri-ovulatory stage and the proximity of the ovulating ovary. The main findings of this study are that i) the OF proteome varies in abundance between ampulla and isthmus, in accordance with our hypothesis that each segment of the oviduct responds to specific requirements of the gametes and embryos; ii) the region of the oviduct is more determining than the proximity of the ovulating ovary and the peri-ovulatory stage in the regulation of the OF proteome; iii) OF proteins varying in abundance are involved in a broad variety of biological functions, including protein binding, response to stress, calcium homeostasis, and lipid and carbohydrate metabolism, which may play a key role in the early gamete/embryo interactions.
More than 3,700 proteins were identified in the bovine OF, which is the most comprehensive OF proteome published so far in mammals9,23−26. From the total of protein identified in our study, 37% were predicted as potentially secreted and 10% contained a signal peptide. These proportions of secreted proteins are comparable to those previously reported in the OF in cattle23,24 and sheep27. It is unclear how intracellular proteins could be exported in high proportions to the oviductal lumen. Considering the very low volume of OF in both ampulla (< 50 µL) and isthmus (< 10 µL) and to avoid cell damage, the samples were collected by gentle flushing and centrifugations were carried out to eliminate cell debris. The high rate of renewal of the oviduct epithelium during estrus28,29 may lead to intracellular release of proteins into the OF. Moreover, and in the light of our results, it is very likely that intracellular proteins could be derived from apocrine or non-canonical secretory pathways, as it has been shown for annexins (ANXAs)19,30. In accordance, a high proportion of intracellular proteins were previously identified in EVs derived from OF19–21.
The most abundant proteins quantified in the OF included several HSPs (HSP90B1, HSP90AA1, HSP90AB1, HSPA8, HSPA5 also known as GRP78), CD109 molecule, complement C3, myosins (MYH9, MYH14) and the oviduct-specific glycoprotein oviductin (OVGP1), as previously reported9,24,25. The majority (77%) of proteins quantified were detected in all conditions. The remaining 23% had on average low levels of detection (NWS < 5), suggesting that these proteins could have been detected with a higher sensitivity. Although, little attention has been paid to this small set of proteins, we should not rule out that they may have an important role in the oviduct. The use of mass spectrometer with higher sensitivity could determine whether these proteins were only present in a specific segment of the oviduct or stage, although in small amounts.
The region-related changes in the OF proteome are presented for the first time, providing valuable information on the specific requirements of gametes and embryos. Furthermore, the region was the greatest source of variation in the oviduct proteome. These results are in agreement with previous transcriptomic data evidencing differentially expressed genes between the ampulla and isthmus in cattle10–13. Also in accordance, the abundance of specific proteins was differently regulated in each region after insemination in rabbits31 and between pregnant and cyclic cows14. Differences in the vascularization32 and in the proportion of ciliated cells29 between isthmus and ampulla could explain this variation in OF proteome. It is well known that sex steroid hormones are major regulators of oviduct physiology and secretory activity6. To our knowledge, the region-specific concentrations of progesterone and estradiol in the oviduct have not been reported to date. However, the expression of progesterone receptors was reported to vary according to the oviduct region: transcripts for nuclear receptors (PGR) were more abundant in the isthmus while those for progesterone membrane component 2 (PGRMC2) were higher in the ampulla in cattle10. Furthermore, it was shown in gilts that each oviductal region responded differently to estradiol, leading to different regional patterns of secretion33. Taken together, the observed region-specific profiling of the OF proteome could result from differences in blood flow, proportion of secretory cells, gene expression and steroid hormone receptivity between ampulla and isthmus.
After mating or insemination, sperm cross the uterus and reach the isthmus where they interact with both OECs and oviduct secretions; these interactions are believed to create an optimal environment for sperm storage until ovulation2,34. Of the BP/MF overrepresented exclusively at Pre-ov, proteins involved in cell redox homeostasis (including PRDX3, PRDX4, PDIA3, PDIA6 among others), negative regulation of apoptotic process (incl. HSPA5, HSP90B1, RGN) and microtubule motor activity (incl. DNAH1, KLC3, KIF21A) may be considered as possible candidates to provide an optimal environment for sperm survival and motility until fertilization. Of those protein candidates, HSP90B1, HSPA5 and PDIA3 were among the most abundant proteins in the isthmus (NWS ≥ 100) and were also 2 to 3 times more abundant than in the ampulla at Pre-ov. HSP90B1, PDIA3 and HSPA5 were previously identified as sperm-binding proteins in the OF in cattle35. In humans, the inhibition of PDIA3 by specific antibodies reduced sperm ability to bind to the zona pellucida36. In addition, both HSP90B1 and HSPA5 promoted sperm-zona binding in human37 and pigs38. Recombinant HSPA5 enhanced human sperm calcium influx, one step of sperm capacitation39. Calreticulin (CALR) was another protein found 2,5 times more abundant in the isthmus compared to the ampulla at Pre-ov. Calreticulin is a calcium-binding protein required for cumulus oocyte complex development40. Moreover, addition of recombinant CALR in the fertilization medium blocked polyspermy in pigs41.
After ovulation, sperm undergo capacitation and migrate gradually toward the ampulla where fertilization takes place, then embryo development starts in the isthmus. Hence, we focused on identified proteins that could play a key role in supporting fertilization, embryo transport and development. Numerous DAPs between regions (incl. HSPA5, HSP90B1, ANXA3, ANXA6, MYO1D, MYO1B, PHKB) were involved in calcium homeostasis, including calcium ion binding and calcium-dependent protein binding. Calcium is well known to play major roles in sperm functions such as motility, capacitation and acrosome reaction42. In addition, several proteins (GNAS, GNG2, GNB1) were involved in cell response to catecholamines. Catecholamines have been implicated in the spontaneous contractions of the bovine oviduct43 and may help embryo transport towards the isthmus. Among the overabundant proteins in the ampulla at Post-ov, oviductin (OVGP1) was among the most abundant proteins (NWS > 150) and 2.7 times more abundant than in the isthmus. Similarly, OVGP1 transcripts were more abundant in the ampulla than in the isthmus at day 4 post-ovulation in cows12. OVGP1 is an oviduct-specific glycoprotein secreted around the time of ovulation that plays major roles in zona pellucida hardening and control of polyspermy in mammals44,45. Addition of recombinant OVGP1 in a fertilization medium improved sperm-zona pellucida binding in hamster46 and humans47. Moreover, purified or recombinant OVGP1 positively influenced sperm viability, motility and capacitation in buffaloes48 and humans47. Furthermore, numerous proteins overabundant in the ampulla were involved in the immune system (incl. CFH, CFD, PLAT, PLTP). Complement factor H (CFH) acts as a soluble inhibitor of complement and plays as such an essential role in maintaining a well-balanced immune response. CFH was reported in boar seminal plasma and it was shown that sperm possessing CFH in their outer acrosomal region evaded complement attack in the female tract49. The mRNAs for phospholipid transfer protein (PLTP) were reported in the oviduct epithelium of mice and increased in abundance in the presence of embryos but not in pseudo-pregnant mice, suggesting important roles during early development50.
Beyond the oviduct region, the proximity of the ovulating ovary was another factor regulating OF protein abundance. The regulatory effect of the POF/CL on OF proteome around the time of ovulation was previously described in cattle9 and horse51. Differentially expressed genes between sides were also identified in the ampulla and isthmus on day 3 post-estrus in cattle52. The proportion of secretory cells was reported to be equivalent between ipsilateral and contralateral oviducts53. However, highly asymmetrical concentrations of sex steroid hormones were reported in the bovine oviduct, with up to 3,5x more estradiol before ovulation54 and up to 16 times more progesterone after ovulation7 in the ipsilateral oviduct than in the contralateral one. An asymmetry in the local vascularization and anatomy of the oviduct were also reported, with a thicker, more edematous and more transparent wall in the ipsilateral side55. Overall, those differences in monovular species could probably be involved in the asymmetrical regulation of oviduct secretions.
The POF/CL effect was expected to be an important regulatory factor, preparing the suitable environment for early embryo development. In accordance, the Post-ov isthmus was the most affected by the proximity of the POF/CL, with 250 DAPs compared with 83-177 DAPs in other pair-wise comparisons between sides of ovulation. In line with the present data, transcriptomic analysis of bovine OECs identified 13 times more differentially expressed genes between sides of ovulation in the isthmus than in the ampulla on Day 3 post-estrus52. In a previous proteomic study in cattle, the proportion of DAPs in the entire oviduct was also higher at Post-ov than at Pre-ov9. The signaling pathways most significantly altered by the proximity of ovulating ovary included the EGFR, ERBB2 and PI3-kinase (PI3K) pathways after ovulation. Recent results in dogs show that exosomes secreted by oviduct cells mediate the EGFR/mitogen-activated protein kinase (MAPK) signaling pathway, which is one of the prerequisite pathways for further development, in cumulus cells56. The PI3K pathway plays also pivotal roles in numerous reproductive processes57. The PI3K-Akt pathway was one of the most affected pathways in the oviduct after mating, as shown in a transcriptomic study in pigs58.
Studies on the regulation of the OF proteome close to the ovulation time are still scarce because most studies compared the estrus/follicular phase to the luteal phase, with a time interval of around 5-12 days between conditions11,27,59. The time relative to ovulation (Pre-ov vs. Post-ov) was the factor with least variation between samples with only 2% of DAPs identified in the ampulla and isthmus. Higher rates of DAPs (13-17%) were previously evidenced in the bovine OF between stages but with no region separation9. Recently, differences before and after ovulation were reported in the equine OF proteome26. These authors reported 15 proteins downregulated in post-ovulatory OF compared to the pre-ovulatory counterpart, while 156 upregulated in post-ovulatory OF compared to pre-ovulatory OF. It is to note that the Pre-ov and Post-ov stages covered approximately 3-4 days around ovulation time and represented a limited time window relative to the 21 to 25-day length of the estrous cycle, which may explain the limited number of DAPs. Functions such as regulation of cilium assembly, intraciliary transport and negative regulation of cell proliferation were affected by the peri-ovulatory stage in the ampulla. The proportions of ciliated cells and of proliferative cells in the oviduct epithelium was reported to change during the estrous cycle in cows29 with an increase during the follicular phase and a decrease in the luteal phase, which may account for this stage-specific enrichment.
One limitation of the present study is the lack of information on cows’ breed, age and metabolic status at the time of OF collection since the samples were collected at a slaughterhouse. Although, transport and OF collection were carried out on ice with limited time after animal death, however, we cannot exclude some protein modifications due to post-mortem collection. The OF proteome was obtained from a total of 32 animals, providing data representative for this species, yet, the samples were analyzed in pools of 4 animals, precluding any evaluation of individual variations.
In conclusion, this study provides a comprehensive characterization of the bovine OF proteome, revealing a fine-tune regulation of the OF according to the anatomical region of the oviduct, the proximity to the ovary and the stage of the cycle. The strong modulatory effect of the oviduct regions points out the anatomical region as the major regulator of OF proteome for the first time. Moreover, the in deep detail of the spatiotemporal proteomic changes of the OF around the ovulation window, enhance our understanding of the oviductal milieu supporting the early reproductive events. The proteins identified here paved the way for future functional experiments towards improving sperm and embryo biotechnologies.