A schematic of the cumulus-oocyte complex.

Legend

CC= Cumulus Cells
CM= Cumulus Matrix
PV= Perivitilline Space
EPM= Egg Plasma Membrane
EGG= Oocyte
ZP= Zona Pellucida

A. Hyaluronan (HA): A high molecular weight mucopolysaccharide formed from alternating N-acetylglucosamine and 3-glucuronic acid.

• HA plays a central role in the formation of the extracellular matrix of tissue, especially skin, vitreous humor of the eye, joints, cartilage, etc.

• HA solutions are viscoelastic. HA contributes to the lubricity and “shock absorption” of articulating joints.

• HA receptors, such as CD44 and RHAMM, are expressed on the surface of a number of both normal and malignant cells and are involved in cell motility and tissue repair.

B. Normal Conception

• Normal semen is comprised of a mixture of fully functional, motile, mature sperm and up to 50% immature, motile or non-motile, abnormal and infertile sperm.

• Normal conception includes a series of steps that select mature, fertile sperm and only allows the selected sperm to contact the egg plasma membrane and to fertilize the egg.

• Sperm-HA interactions play key roles in this selection process.

C. HA-Sperm Interactions

• Mature sperm express specific surface receptors that bind HA. These receptors include PH-20 and RHAMM^1,2, but there may be others.

Two-second time-lapse fluroresence micrographs of sperm in the control chamber (A, lift side) and the HA-coated chamber (B, right side). The sperm were treated with a dye that illuminates only their heads. Wavy lines show progressive swimming by motile sperm. In the HA-coated chamber, the majority of sperm have bound to the hyaluronan and appear immotile.

• When mature sperm bind HA in solution they strongly increase their swimming vigor and velocity³. HA-stimulated swimming gives these sperm an advantage in the selection process.

• HA is a component of cervical mucus. Mature sperm with HA- stimulated swimming have an advantage exiting the hostile environment of the vagina passing through the cervical mucus and entering the uterus.

• HA is a major component of the cumulus matrix, the viscous layer surrounding the oocyte. Passage through the cumulus is facilitated by HA-stimulated swimming.

• HA-sperm interactions are also involved in other essential steps in normal conception, including capacitation and the acrosome reaction^1,5.

D. When mature sperm encounter immobilized hyaluronan, they display a remarkable behavior— they bind themselves to the layer.

• Binding changes the visual appearance of the sperm from motile to immotile

• The heads of HA-bound sperm show no progressive movement

• The sperm tail, however, beats vigorously

• True immotile sperm show no motion in head or tail

Thus HA-bound sperms are easily distinguished from non-bound sperm.
A microdroplet of hyaluronan was bound to a Petri dish and sperm contacted to it.

Kinetics of Sperm Binding
Semen samples were videotaped for two-minute intervals over a 20-minute period. The mean percent un-bound sperm are shown versus incubation time. From these results, a minimum of ten minutes were required for complete binding to be established.

HYDAK^® HBA Sperm-Hyaluronan Binding Assay

• Double-chamber microscope slides (Cell-Vu^®) were converted to HBA devices by coating both chambers with hyaluronan.
  
  

• The proportion of mature sperm, competent to bind to immobilized HA was obtained by counting the unbound motile sperm cells (U) and the HA-bound sperms cells (B) in one of the chambers.
  
  

  The percent bound = 100 x B / (U + B)< /p >

  
  

• HA depth in the coated chambers was determined by dyeing with Toluidine Blue O and quantifying the bound dye⁴. The depth was less than 0.1 microns.
  
  

• Sperm binding was specific to hyaluronan: Sperm failed to bind to heparin, chondroitin sulfate, carboxymethyl cellulose, methyl cellulose, and poly (hydroxyethyl methacrylate).

Range of Percent Binding
Thirty sperm samples were assayed for binding before and after washing with human tubal fluid (HTF).