Know Your Sperm

Part of the "Frontiers in Reproduction" series

With a little knowledge of biology, and some basic lab equipment, you can do some very useful things with sperm. This post explains the basics of how to isolate sperm, evaluate their quality, and preserve them for shipment or long-term cryostorage.

If you’d like to know about anything in more detail, please ask in the comments below.

Sperm Development

Although this is mainly a practical guide, it’s good to have some background knowledge about what sperm are and where they come from.

The word “sperm” refers to male gametes; in mammals these are spermatozoa¹. Sperm develop within the testes inside seminiferous tubules. Spermatogonial stem cells around the edge of the tubules differentiate into spermatocytes, which undergo meiosis and form spermatids. The spermatids futher mature into spermatozoa; this process involves replacing histones with protamines to tightly package their DNA, as well as growing a flagellum to provide motility. Spermatozoa are not fully mature until after they leave the testis and travel through the epididymis, which is a 6 meter long,² tightly coiled tube. It’s so long because it needs to match the transit time to the sperm maturation time, which is typically about 4 days in humans. Overall, the entire process takes about 70 days to go from spermatogonia to mature spermatozoa.

Diagram of spermatogenesis (source), with some annotations added. “2n” and “1n” refer to diploid and haploid cells.

A mature spermatozoon contains three main segments:

1. the head, containing the nucleus as well as machinery to fuse with the egg

2. the midpiece, containing mitochondria which power the sperm

3. the flagellum, which provides motility

To be released during ejaculation, sperm travel from the epididymis through the vas deferens, and mix with fluids from the seminal vesicles and prostate. The overall mixture is known as semen. Removing the vas deferens (a vasectomy) is a form of male sterilization since this prevents the sperm from leaving the testes.

Sperm Isolation

The first step of using sperm is obtaining them. Fortunately this isn’t too difficult.

In mouse research, most sperm is collected surgically since it’s the easiest way to get high concentrations of mouse sperm³. We learned this technique in the course, and you can watch a demonstration here:

Sperm maturation happens as they move through the epididymis, so mature sperm are found only in the vas deferens as well as the portion of the epididymis farthest from the testis, known as the cauda. These are the parts that are dissected. This can also be done in humans (although usually it is performed only if there are no sperm present in semen). It also is possible to surgically collect viable sperm from recently deceased men, and this has been the subject of a few interesting court cases.

Still, most men would strongly prefer a non-invasive⁴ method of sperm collection. Sperm can be obtained from semen, the collection method of which is left as an exercise to the reader. Semen contains proteins from the seminal vesicles which cause it to coagulate shortly after ejaculation, as well as proteases from the prostate which slowly liquefy the semen. Therefore, semen must be allowed to liquefy before subsequent isolation of sperm. Typically this takes about 20 minutes at 37 °C.⁵

Many uses of sperm (for example, in vitro fertilization or intrauterine insemination) require that the sperm be separated from the seminal fluid. This is commonly accomplished by a “swim up” method where liquefied semen is overlaid with a culture medium (components listed below) into which the sperm can swim. After 30–60 minutes the upper layer is retrieved. This works quite well to isolate a pure population of motile sperm, although the efficiency of isolation is rather low. Another method to separate sperm from seminal fluid is density gradient centrifugation. This recovers a higher fraction of the sperm, but requires more equipment and is harsher on the sperm.

Sperm Evaluation

There are two main parameters that determine sperm quality. First is the sperm count. For counting sperm with a microscope and Neubauer chamber⁶, it is useful to dilute the sample in water first. The hypotonic solution immobilizes the sperm, making them easier to count. A typical sperm count for a healthy man is 50 – 100 million/mL although this is rather variable,⁷ and depends on how long it was since the previous ejaculation. A count of <15 million/mL is associated with infertility.

This video shows sperm on a Neubauer chamber; these sperm were not immobilized.

Second, the sperm must be motile in order to reach the egg. Clinically, sperm motility is measured with an unnecessarily expensive “computer-assisted sperm analysis” machine which is basically a microscope, a video camera, and image recognition software. Various parameters such as velocity and path curvature are combined to create an overall score. Practically speaking, you can also just estimate it by eye. When measuring motility it’s important to keep the sperm as close to 37 °C as possible, because motility will decrease if they get too cold.

Sperm Sorting

It is sometimes desired to separate X and Y sperm to favor one sex⁸. This can be done by two methods, which both exploit the fact that the X chromosome is larger. First, density gradient centrifugation can separate sperm by mass. This doesn’t require specialized equipment, but the degree of enrichment achievable is limited. Second, a fluorescent dye can be added to stain DNA, followed by cell sorting. This can generate highly pure (>95%) X and Y sperm populations, but the downsides are that the fluorescence-activated cell sorter is expensive, the procedure can damage sperm, and the throughput is limited⁹. Therefore, this method is best combined with fertilization methods not requiring large numbers of sperm, such as IVF or ICSI.

Sperm Capacitation

Freshly ejaculated sperm lack the ability to fertilize the egg until they “capacitate” in the female reproductive tract. I won’t get into the biochemical details of capacitation here¹⁰, but this process can also be performed in vitro by incubating sperm in a medium containing bicarbonate and a protein, such as albumin, that can remove cholesterol from sperm membranes. A formulation for such medium is given below. Capacitation takes about 1 hour in mice and 4 hours in humans. After capacitation, sperm become “hyperactivated” and have increased motility (although linear motility does not increase much because the hyperactivated sperm travel in curved paths).¹¹

Sperm Shipment and Storage

Sperm can be stored refrigerated for short periods of time (up to 96 hours) in various media known as “semen extenders”. There are various different formulations, optimized for different species. One formulation commonly used with human sperm is given below. The extender is mixed with semen, typically in a 3:1 ratio of extender to semen. Results are somewhat better if the sperm are separated from the seminal fluid before mixing with the extender, but for most applications this is not required. Semen extenders allow the at-home collection of sperm, followed by shipment in a refrigerated container to a recipient.¹²

For longer-term storage, sperm can be mixed with cryopreservation medium and frozen in straws, which are stored in liquid nitrogen. The overall process is as follows:

1. Mix sperm with cryopreservation medium.

2. Using a syringe, suck the sperm suspension into the middle of a thin plastic straw, leaving air at both ends. If the sperm sample volume is small, you can also suck some medium in first so that the straw doesn’t float in liquid nitrogen.

3. Heat-seal the ends of the straw.

4. Lower the straw and hold it just above liquid nitrogen (in the cold nitrogen vapor) for 10 minutes¹³, then quickly immerse it. Using an aluminum freezing canister, multiple straws can be frozen at once.

5. For thawing, immerse the straw in a 37 °C water bath for 10 minutes, making sure the part containing the sperm is fully immersed. Then cut one side of the straw and attach it to a syringe. Finally, cut the other side and expel the sperm (typically into capacitation medium for subsequent IVF).

   

   A sperm straw. The cotton plug at the top helps avoid contamination.

Frozen and thawed sperm isn’t as good as fresh sperm for artificial insemination, but it’s fine for in vitro fertilization.

Appendix: Media compositions

Note: some alternate extender formulations substitute citric acid for TES.

1. As opposed to things like pollen in plants. For the rest of this post, “sperm” means spermatozoa.

2. 6m is the length in humans. For more info see the paper amusingly titled, “How do you get six meters of epididymis inside a human scrotum?” Also, interestingly sperm spend less time in the human epididymis than the mouse epididymis, even though the human one is longer.

3. And also because mice are generally considered as disposable in the research world.

4. And more enjoyable.

5. Coagulation and liquefaction acts as a “slow release” mechanism to maximize the number of sperm retained in the female reproductive tract. In mice and many other animals, semen forms a solid plug which lasts for hours.

6. For a guide see here.

7. Average sperm counts have been decreasing considerably over the past few decades. This worrying trend might be the subject of a future blog post.

8. For example, dairy cow breeders prefer females . . . or certain parents prefer males.

9. At a relatively fast sorting rate of 10,000 per second it would still take 3 hours to process a typical sample containing ~108 million sperm.

10. For further reading: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6078053/

11. Additionally, in order to fertilize the egg the sperm must undergo acrosomal exocytosis, and capacitation is a prerequisite for this.

12. Obviously, this could spread diseases, so don’t do it with anyone you wouldn’t have sex with.

13. This can be done using a Styrofoam float.