Sea urchin sperm: Appearance, collection and measurement of sperm motility – the ideal CASA solution!

Sea urchins belong to the phylum Echinodermata meaning “spiny skinned”. There are five classes of Echinodermata and three of these are indicated below in Fig. 1 (A to C): The sea stars, the sea urchins and the sea cucumbers.

Fig. 1: A: Sea stars; B: Sea urchins; C: Sea cucumbers (Modified from Branch and Branch (1985)).

Why are sea urchins important? They have ecologically and economically important roles and above all have served as the best models for understanding the basis of sperm motility and the acrosome reaction. Sea urchins typically live in wave exposed rocky pools and are broadcast spawners like most Echinoderms. They accordingly release their gametes in the sea-water and furthermore serve as an important “reference broadcasting species”.

But how is sperm collected from sea urchins? The classical method is by injecting 1mL of 0.55 mM KCl on the oral side with spawning taking place from the 5 gonads via the 5 gonoducts after a few minutes to the 5 genital pores on the aboral side (Fig. 2A). As much as 2mL semen of highly concentrated semen can be collected (Fig. 2B). More recent information suggests that much lower concentrations of KCl (0.20 mM) can be injected which ensure survival of most of the animals.

Fig. 2: A: semen ejected via 5 gonopores on the aboral side; B: After 10 min as much as 2 mL semen may be ejected for collection using a Pasteur pipette or similar from Parechinus angulosus.

The structure of the sperm is very simple and consists of a conical head, with a small acrosome, a small midpiece and a thin tail. A special sea-water SpermBlue stain also differentiates the various components of the sperm clearly.

Fig. 3: A: Scanning electron micrograph (van der Horst and Cross, 1978) B: Transmission electron micrograph showing simple acrosome, conical head, very small midpiece containing a single mitochondrion and a tail about (42 µm) (Bennett, 2020) of the sea-urchin Parechinus angulosus.

The two best methods to study sperm motility in the sea urchin and most external sea-water spawners is by either the flush technique or the swim-up technique (Bennett, 2020) (Fig. 4).

Fig. 4: Diagrammatic presentation of the flush and swim-up technique for SCA motility analysis (Bennett 2020).

Fig. 5 is a typical SCA motility analysis field showing the individual sperm tracks of Parechinus in red, green and blue. Superficially it may seem that the sperm are swimming in a circle but on closer inspection they swim in a “tight” helix. Fig. 6 shows the two dimensional track of sperm 351 and then its 3D reconstruction using the method of van der Horst and Sanchez (2016, 2018). This model is based on many supportive studies and assumes that the sperm swims in spherical helix. In the 3D reconstruction three kinematic parameters VCL, ALH and BCF were used. We have furthermore established that the faster the sperm swims the greater the diameter of the helix. This has important implications in terms of sperm competition. It appears that sperm that swims in a helix instead of a straight line has a better chance of meeting an oocyte and, moreover, the faster they swim in a helix the greater the helix diameter and consequently the greater the chance of meeting an oocyte (van der Horst et al. 2018).

Fig. 5: A typical SCA motility analysis field. The white dots are the sperm heads that have been tracked by SCA motility module swimming in a tight helix. Red, rapid swimming sperm; green medium, swimming sperm and blue slow swimming sperm.

Fig. 6: SCA analysis motility field, showing the two dimensional track in the middle and the 3D reconstruction after van der Horst and Sanchez (2016, 2018). While only 3 kinematic parameters are indicated here, SCA measures 8 kinematic parameters and then a whole range of percentage distributions.

In conclusion, one is limited in a blog due to space limitations to provide more detailed information. However, the above shows the basis for CASA analysis of broadcast spawners using a sea-urchin species as a classical example. In previous studies we have shown that the above analysis and theoretical basis pave the way for further investigations of other broadcast spawners such as mussels, oysters and abalone.

Finally, there are numerous research fields to apply these quantitative approaches in the sphere of sperm competition, pollution, toxicology, drug development and cryopreservation studies.

Prof Gerhard van der Horst (PhD, PhD), Senior Consultant, MICROPTIC S.L.

Ms Monique Bennett (MSc, PhD will be awarded end of year), Comparative Spermatology Laboratory, Department of MBS, University of the Western Cape, Bellville, South Africa.

Prof Maryna de Kock (PhD), Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa.

• By MICROPTIC S.L.
• 16/10/2020
• cryopreservation, urchin sperm, CASA, sperm motility, SpermBlue, toxicology, drug development