Researchers in Spain and the UK have used mass spectrometric analysis to demonstrate in vivo that the uterine environment can change in the presence of gametes.
While such changes had previously been shown in vitro, the work, performed in the porcine reproductive system, is the first to report and demonstrate changes in oviductal protein secretion in living mammals in response to the presence of sperm and oocytes in the oviduct at the time of ovulation, the researchers said.
“The takeaway message is that we have been able to at least show the communication between two different kinds of cells that are from two different origins” – sperm from the male and the reproductive tract of the female — according to study supervisor Alireza Fazeli, who is a senior lecturer at the University of Sheffield.
“This is one of the rare examples of communication between two completely different cell types, and the interaction of these two has resulted in a proteomic response,” he said.
The study, which appears in the current issue of the Journal of Proteome Research, also suggests that current knowledge of the oviductal environment is much more complex than previously thought, and could have implications for in vitro fertilization and other methods of artificial insemination, Fazeli said.
For example, it is not understood how sperm is able to survive in the reproductive tract — as foreign cells, they should be destroyed by the female’s immune system.
In addition, the mechanisms of sperm selection in promiscuous species — the oviduct’s ability to select so-called “good” genes and reject “bad” ones — is not understood.
In the study, the researchers report that they identified a number of proteins that were altered by the presence of sperm or oocytes in the oviduct providing “evidence for gamete modulation of the oviductal environment.”
With a “relatively big” but undisclosed grant from European funders, Fazeli and his colleagues will try to “define the whole interactome of the maternal communication [occurring] inside the embryo,” he said.
Gamete Sensor, On
For their work, the researchers began with the hypothesis that “the presence of gametes in the oviduct in vivo leads to an alteration of the oviductal secretory profile.” Furthermore, they hypothesized that the alterations in the oviductal microenvironment would be different for sperm than for oocytes.
The experiment was designed to examine the effect of the presence of sperm in the oviduct, the presence of oocyte in the oviduct, and as a control, surgical interventions on the oviduct. The experiment was performed on 15 pigs, and to avoid analytical complications due to the biological differences of oviductal fluid from different individuals, the oviductal fluid from a gamete-stimulated oviduct was compared to the oviductal fluid from a non-gamete oviduct from the same pig.
To examine how sperm affects the oviduct, pigs were artificially inseminated with sperm diluted with Beltsville thawing solution, while for surgical interventions they were inseminated with BTS-diluent only. To examine oocyte presence in the oviduct, one oviduct was clipped to prevent ooctyes from entering the oviduct.
After collecting oviductal fluid, the researchers gathered highly enriched samples of oviductal epithelial cells by opening the pigs’ oviducts longitudinally and scraping the mucosal epithelial layer with a sterile glass slide.
They then performed albumin and IgG depletion using Sigma-Aldrich’s ProteoPrep Blue Albumin-depletion kit. The samples were then purified and desalted with GE Healthcare’s PlusOne 2D clean-up kit, and a bicinchoninic acid assay was performed to determine the protein concentration of each oviductal fluid sample.
The sperm-oviduct and control-oviduct proteins were labeled using Applied Biosystems’ ICAT reagent and dried via vacuum centrifuge. After being reconstituted, they were separated by SDS-PAGE and in-gel trypsin digestion and then affinity-purified.
The team used ABI/MDS’s QStar XL ESI-qQ-TOF mass spec to analyze the samples together with the company’s ProICAT software to identify and quantitate proteins “based on the peptides analyzed during the LC-MS/MS experiment.”
“We can speculate that if they were able to sense the presence of a sperm, maybe they can sense the presence of different kinds of sperm from different individuals, and they can alter the environment of the female reproductive tract to suit some and not suit the others.”
Additional analysis was done by Western blotting and quantitative, real-time PCR to examine the gene-expression profiles of select differentially expressed proteins.
Fazeli and his team were able to identify and quantify 32 oviductal fluid proteins. In the diluent versus no-diluent ovudict experiment, almost none of the proteins showed any alteration, the exception being hemoglobin beta chain, which showed a less than two-fold increase in abundance in the oviductal fluid of an oviduct attached to the uterine horn that had been cut during surgery.
By comparison, proteins witnessed as regulated in response to the presence of sperm or oocytes showed dramatic alterations, the researchers reported. Nineteen sperm-oviduct proteins displayed an alteration of greater than two-fold, while three oocyte-oviduct proteins displayed an alteration greater than two-fold.
Western blot and real-time PCR confirmed alterations in fibrinogen, complement C3, retinol binding protein, and oviduct-specific glycoprotein.
“The apparent correlation between gene and protein expression provided further evidence for gamete modulation of the oviductal environment,” the researchers reported, adding that the observed protein changes thus cannot be attributed to proteins originating from either the sperm or oocytes.
They concluded that the data “clearly demonstrated that, in vivo, the arrival of either gamete activates a cell-type-specific signaling pathway within the oviduct, which leads directly to specific alterations in oviductal fluid composition.”
This suggests that the oviduct can recognize the presence of gametes in the reproductive tract, which “extends the concept of regulatory mechanisms within the female reproductive tract beyond hormonal regulatory systems. It presents a mechanism by which gametes have immediate and local control of their environment.”
According to Fazeli, the findings could have major implications in the area of reproductive medicine, in particular in vitro fertilization. IVF is successful only about 20 to 30 percent of the time, Fazeli said, and his team’s findings could shed light on how to raise that figure.
Further on, with data indicating that what happens during early embryonic development has life-long health consequences for an individual — for example, children born to women suffering during famine conditions have a higher tendency to develop heart disease as adults, Fazeli said — the findings of the study could have public health implications. With the data now suggesting that the female reproductive tract undergoes massive changes in the presence of sperm, the role of the oviduct in reproduction will have to be reconsidered.
“With the work that we have done, we are showing that there is recognition of the gametes there,” Fazeli said. “That [suggests] that there is a … fine tuning happening [in the reproductive tract]. Somehow we have to rethink this.”
In addition, it has been accepted convention that sperm “decides” who gets to fertilize the egg: the prize goes to the fastest swimmer. But the work by Fazeli’s group suggests that, in fact, it is the female who makes that decision when her oviduct “picks” the healthiest sperm to fertilize an egg while rejecting others.
In species where females mate with several males during one reproductive cycle, this would explain why offspring tend to belong to one male instead of being spread among several males.
“With this finding, we can show that actually the female [reproductive tract] can sense the sperm,” Fazeli said. “We can speculate that if they were able to sense the presence of a sperm, maybe they can sense the presence of different kinds of sperm from different individuals, and they can alter the environment of the female reproductive tract to suit some and not suit the others.”