Hello there, blog reader! You may be interested to know (or not) that this is my first blog post as Dr. Brooke. Unfortunately, this has not given me any apparent superpowers, but I can now get people to call me doctor, so that’s a plus.
Some time during the foggy haze that was the build-up to and let-down from my thesis defense, I came across this gem of a paper: “The bitch uterine response to semen deposition and its modification by male accessory gland secretions” (England, Russo, and Freeman at the University of Nottingham). Right from the title, you know this will be a good one. Bitches, semen, and my personal favorite: male accessory gland secretions.
This paper does two things: first, it lets me write a blog post in which I use the word ‘bitch’ 20 times (including that time). Second, and arguably more importantly, the results of this paper confirm what many other studies have shown over the years, in many different animals (including people): all that extra “stuff” in the semen is really important for optimal sperm performance.
The introduction of semen into the uterus causes an inflammatory response in mammalian females. White blood cells involved in the immune response rush to the uterus to deal with all the foreign material (aka: man juice). As far as the female’s body is concerned, sperm are foreign cells that need to be destroyed. Attacking the sperm is a bad thing if the goal is pregnancy, and attacking the embryo, which is also “foreign” is even worse. But shutting down the immune response isn’t a great idea, either. That’s because sex can also introduce bacteria that really do need to be destroyed. But how can the female’s body discriminate between the good (ie: sperm and the eventual embryo) and the bad (bacteria)?
A recent paper from Sarah Robertson’s lab in Adelaide, Australia showed that after unprotected sex, there is a rush of pro-inflammatory cells and molecules to the woman’s cervix. Of course, this wasn’t a total surprise: the inflammatory response to sex has been shown in mice, pigs, horses, rabbits, sheep, and even primates. And the induction of immunity-related proteins has been shown in many insects. But there hasn’t been much focus on this phenomenon in women.
Dr. Robertson and colleagues collected biopsies of the ectocervix before and after sex. They also compared the changes in the cervix between unprotected sex and sex with a condom or abstinence. They then measured the levels of white blood cells and molecules associated with the immune response. They found that semen is required to get the post-sex increase in these immune-related measurements, confirming that we’re not so different from a mouse after all.
The one thing they didn’t test, though, was whether you would still get the same response with only the sperm–hopefully that will be a follow-up.
The induction of an immune response by semen could help “condition” the female to her partner’s proteins so that she doesn’t attack the embryo after implantation, while at the same time allowing her to flush out bad stuff like bacteria. But how do the sperm survive the onslaught?
This brings us to the bitch paper.
The first part of the bitch paper demonstrates the same result as the human paper (though much less thoroughly)–that sex leads to an immune response. Researchers England and colleagues measured the number of polymorphonuclear neutrophils (PMNs) in the bitch uterus before and 48 hours after insemination. PMNs are sort of the “first responders”–they’re a type of white blood cell that is most important in the early stages of inflammation and immune response.
Want to know what a PMN in action looks like? Check out this video of a PMN attacking pathogenic fungus (the bad stuff is in orange).
At 48 hours after artificial insemination, the bitch uterus contained about 10 million PMNs per mL, whereas there were only about 5.9 million/mL before insemination. (As the authors themselves point out, the increase might have been more dramatic if they checked earlier, but they had to work within the bitches’ schedules, you know.) That means about 10 million of those crawling cells are on the prowl for bacteria–and for sperm.
The authors also made some measurements on blood velocity and other things that confuse me. But the most exciting part of the story is what they did with bits of bitch uterus that they cut out and tested in a petri dish. The uteri they used were taken from bitches getting spayed. The authors also collected ejaculates from male Labradors (the bitches were either Labrador retrievers or Golden retrievers).
The bits of uterus were incubated with either control buffers, seminal plasma (ie: semen minus the sperm), fluid from the prostate, or PMNs. Seminal plasma contains prostatic fluid, but also has secretions from other male glands.
Separately, sperm were treated with a fluorescent dye (so they could be easily counted at the end of the experiment). They were then neatly diluted in buffer so that each uterus sample would receive 10 million glowy sperms.
30 minutes after letting the sperm hang out with the uterus samples, the samples were vigorously washed to remove any sperm that weren’t tightly attached. If those sperm were in a live bitch, and couldn’t attach to the to the uterine lining, they would have no chance of fertilizing an egg. After washing, the researchers counted how many sperm were still hanging on.
In the control treatment (buffer only), an average of 4300 sperm stayed attached after washing (out of the original 10 million). The numbers were a little better, but not in a statistical sense, when seminal plasma or fluid from the prostate was present.
But those treatments didn’t include the PMNs–the immune cells that would normally be present after sex in the bitch (and in other mammalian females). When the samples were incubated with PMNs, but not seminal plasma, the number of sperm that could stay put went down–from 4300 to 3500. Seminal plasma and prostatic fluid brought the numbers back up. With seminal plasma, more than 3900 sperm could hold on. The prostatic fluid did a little better: about 4100 sperm stayed attached.
This wasn’t a dramatic increase, but every little sperm helps when you’re trying to get a bitch pregnant. The more dramatic results came from looking directly at how the bitch’s immune cells interacted with the sperm.
In a separate experiment, the authors incubated sperm with PMNs from the bitches. They incubated about 10 million PMNs with 50 million sperm, went to grab a coffee (I assume) and then calculated the percentage of PMNs with at least one sperm attached–meaning those sperm are out of the running for fertilizing eggs.
Sperm that were incubated with PMNs and just the control buffer were more likely to be attacked–nearly 70% of the PMNs in the control samples had attacked at least one sperm. The addition of seminal plasma and/or prostatic fluid saved the lives of many a sperm: only about 20% of the PMNs in those samples were able to snag a sperm.
To tie the results back into the final outcome–the production of new canine units–the authors note that when semen was diluted in extra seminal plasma or prostatic fluid during artificial insemination, the resulting litter size was about the same. Keep in mind–the diluted samples only have about half as many sperm. This agrees with other papers that have found a positive fertility effect of seminal plasma when performing artificial insemination.
The study falls a little short of proving that semen leads to the increase in PMNs, since there was no real negative control used–this should definitely be done–but the fact that so many other studies have shown this in other mammals makes it pretty likely. For negative controls, measurements should be taken from bitches that don’t get inseminated, as well as from bitches that get inseminated with sperm that’s been separated from the other fluids. This would help determine which part of the semen cause the immune response.
So just to recap: females attack sperm after mating, but the fluids from male accessory glands help defend sperm from attack, letting them hang around and compete to fertilize eggs. Isn’t canine love a beautiful thing?
England GC, Russo M, & Freeman SL (2012). The bitch uterine response to semen deposition and its modification by male accessory gland secretions. Veterinary journal (London, England : 1997) PMID: 22652115
Sharkey DJ, Tremellen KP, Jasper MJ, Gemzell-Danielsson K, & Robertson SA (2012). Seminal fluid induces leukocyte recruitment and cytokine and chemokine mRNA expression in the human cervix after coitus. Journal of immunology (Baltimore, Md. : 1950), 188 (5), 2445-54 PMID: 22271649