Sperm Cryopreservation in Egyptian Spiny Mice

My very first publication, and the first study from my PhD completed!

This paper describes protocols for successfully cryopreserving and thawing mature sperm from Egyptian spiny mice.

I looked at sperm vitality, motility, mitochondrial health, DNA and acrosome (the part that binds to the egg) integrity. Thankfully, the results were great! The spermatozoa survived the process quite well, and a high number of spermatozoa were highly viable post-thawing – meaning a large portion have the capacity to fertilise an egg and pass on healthy DNA.

This is not only important for future studies involving assisted reproductive techniques (ART; IVF, for example), but it also provides a way to reliably store the genetics of the world’s only known menstruating rodent.

Fluorescent image showing different sperm staining patterns post cryopreservation. 1) Living spermatozoa with intact acrosomes and poor mitochondrial health; 2) living spermatozoon with intact acrosome and good mitochondrial health; 3) living spermatozoon with a damaged acrosome and good mitochondrial health; 4) damaged spermatozoon with a damaged acrosome with good mitochondrial health.

Superovulation in Egyptian Spiny Mice

The collection of mature eggs (superovulation) is a very common ART, and is a critical step in infertility treatment. As spiny mice have a menstrual cycle, and a human-like hormone pattern, we believed a human-like superovulation protocol would be the most appropriate method for this species and decided to define exactly that.

Based on what we knew about the spiny mouse menstrual cycle and human superovulation protocols, we defined a set of protocols for the collection of oocytes and embryos from mice stimulated with a deslorelin implant – an implant that harnesses the body’s natural pathway from the brain to the ovary that stimulates egg development.

Hopefully, the next generation of reproductive biologists can test and use these protocols to set up the system of egg collection, IVF, and embryo transfers, to effectively use spiny mice as models of human reproduction.

Our protocol for superovulation and collection of spiny mouse eggs.
Female mice are monitored until they reach the menstrual phase (~ day 1–3 of the cycle) before a hormonal implant (Deslorelin; A GnRH agonist) is placed under the skin of each mouse. All mice are given 3 cycle lengths (27 days total) to recover from surgery and for the implant to have taken effect. Mice are then injected once daily for 4 days with egg-growth hormones (gonadotrophins) followed by a single injection on day 5 to stimulate ovulation (hCG) before eggs are collected and either used for IVF or frozen for future studies and experiments.

Early Pregnancy in Egyptian Spiny Mice

The most difficult study from my PhD, and the publication I’m most proud of.

Spiny mice have several unique reproductive traits, and the combination of menstruation and postpartum ovulation (ovulation immediately after giving birth) have never been seen before in mammals. We decided to explore how this could be, as it questions a lot of what we know about menstruation and the hormones regulating menstruation and parturition.

We looked at the uterine and ovarian tissues as well as sex hormones (estrogen, progesterone) from mice that recently gave birth (day of birth – 10 days later) to monitor the changes in reproductive tissues and hormones.

Amazingly, we found that this is a natural process for spiny mice, and they are able to be simultaneously nursing young/lactating, and pregnant at the same time – something unheard of in menstruating species due to a process called lactational emenorrhea where breastfeeding naturally inhibits ovulation.

Spiny mice continue to get weirder, and more amazing the more we research them. Results like this make us question what we think ‘true menstruation’ is, what controls it, how it works, and how we can treat some of the issues of menstruation.

Fluorescent image of the spiny mouse uterus 5 after parturition.
The large green area is the myometrium or uterine musculature, the blue area is the endometrium (where the embryo will implant into and the placenta will grow from), and the wave of red/orange/yellow is the luminal epithelium (where the embryo attaches to during implantation). The green circles within the endometrium are arteries, and the arteries found closer to the luminal epithelium are called spiral arteries – vasculature critical for supporting the placenta and baby during pregnancy.

Spiny Mice: The bridge from Bench to Bedside

Hopefully, by now, you can tell my PhD revolved around spiny mice and their incredible reproduction. This paper is a collaborative review that myself, Dr Nadia Bellofiore (the researcher who discovered menstruation in spiny mice), and our other collaborators wrote to highlight the potential use of spiny mice as a translational animal model for female reproductive health.

It’s very hard to find cheap, effective, and reliable animal models of female reproduction outside of gorillas and chimpanzees. Of course, they have plenty of issues and limitations with their use in research, which is why we use rodents as proxy species; they’re far smaller, cheaper, and easier to deal with.

We concluded that spiny mice have an incredible amount of potential to model or help find treatments for several disorders of female reproduction including implantation failure, preeclampsia, menstrual cycle variation, and heavy menstrual bleeding, so watch this space!

The three main forms of embryo implantation.
1) Centric implanting embryos do not fully invade the endometrium, but, rather, superficially attach to the luminal epithelium (e.g cows and ewes). 2) Eccentric implantation is one step further, where the embryo moderately invades the endometrium but is not completely engulfed by the tissues (e.g most rodents). 3) Interstitial implantation is the most invasive form where the embryo is completely embedded in the uterine stroma (e.g humans and spiny mice).

Good Things Come to Those Who Mate: Analysis of the Mating Behaviour in the Menstruating Rodent, Acomys cahirinus

Definitely not what I thought I would be studying during my PhD, but something that I’m really proud of for completing – although I’m not sure which I’m happier about: This paper finally being published, or the fact that I was allowed a pun in the title.

Most rodents, form what’s called a ‘copulatory plug’ (coagulated semen) after they’ve mated, and it’s an easily visible way of confirming if mice had mated or not and is reliably used by researchers to pinpoint exact days in gestation. Of course, spiny mice being the unique rodents that they are, do not plug. So, in this study, I compared the mating behaviour of spiny mice across the menstrual cycle to determine if there is any effect on copulation and if we could define a new system to reliably estimate gestational age. Thankfully, we did!

Mice in the later stages of the cycle (late luteal) gave birth 46–48 days after pairing compared to 40–43 days during the earlier stages, and animals paired in menses didn’t mate at all. This means there is a direct effect of the menstrual cycle on mating behaviour and therefore gestation!

Any spiny mouse researchers can now use this understanding to more efficiently pair animals and more reliably estimate gestational age – yay!

Mounting behaviour in spiny mice during the lights-on (AB) and lights-off (CD) periods. Male spiny mice approach females from behind and place their front paws on the middle of the female’s back. Females will either allow mating to occur or move away to prevent unwanted mating. Frames 2B and 2D are zoomed-in images (2X) of 2A and 2C respectively