TABLE OF CONTENTS
Acrosome Integrity of Sperms: Importance, Evaluation and Giemsa Staining Method
Acrosome integrity of sperms refers to the structural and functional soundness of the acrosome, a cap-like organelle that covers the anterior portion of a sperm’s head. The acrosome contains digestive enzymes that are crucial for penetrating the zona pellucida (outer membrane) of the ovum during fertilization.
Acrosome a cap like structure on the head of the spermatozoa covers 60% of the anterior portion of the sperm head.
The morphology of the acrosome should be maintained for the sperm to undergo capacitation and acrosome reaction in the female reproductive tract for attaining fertilizing ability.
Appreciable modifications to the structure of the plasma membrane and the outer acrosomal membrane follow after capacitation has run its course, in the form of acrosome reaction.
Acrosome reaction consists of fusion at multiple points between the two membranes and formation of vesicles made up of fragments of two membranes.
The sperm must be able to undergo these changes in the female reproductive tract to attain fertilizing ability by release of specific enzymes.
The subcellular enzymes present in the acrosome facilitates dissolution and penetration of the zona by the spermatozoa which will leads to the union of male and female nucleus. For this to happen, the acrosome should be intact.
Acrosome can be detached from sperm under the influence of different physical and chemical factors.
Freezing and thawing can also bring about damage to the acrosome. Hence the acrosomal cap has received considerable attention in sperm morphology due to its importance during fertilization.
Any damage or loss of the acrosome leads to infertility or sterility problem. Hence the evaluation of the acrosomal status gets importance. The acrosome is evaluated by the Giemsa stain.
Materials Required
- Stock Giemsa stain
- Semen (fresh/frozen)
- Glass slides
- 5% formaldehyde
Stain Preparation
Preparation of Stock Giemsa Stain
| Chemical | Quantity |
|---|---|
| Giemsa powder | 1 gm |
| Glycerol | 60 ml |
| Methanol | 66 ml |
Weigh the Giemsa powder, put in a pestle and mortar. The glycerol is added slowly and grind it well. Then add methanol slowly and grind it. Finally the solution is filtered and stored in an amber coloured bottle.
Preparation of Working Giemsa Solution
| Chemical | Quantity |
|---|---|
| Stock Giemsa stain | 3 ml |
| Sorenson’s buffer | 2 ml |
| Distilled water | 45 ml |
Working Giemsa solution is prepared immediately prior to use.
Preparation of Sorenson’s Buffer
| Chemical | Quantity |
|---|---|
| Na2HPo4.2H2O (21.682 gm/500 ml) | 200 ml |
| KHPo4H2O (22.254 gm/500 ml) | 80 ml |
| Stock buffer | 280 ml |
Preparation of 5% formaldehyde
| Chemical | Quantity |
|---|---|
| Formalin (37-41% W/V) | 12.5 ml |
| Distilled water | 87.5 ml |
Procedure
- A drop of diluted semen (1:5 to 1:10 in 2.9 percent sodium citrate) is kept on a clean, grease free, pre warmed glass slide and is dried in air. The frozen thawed semen needs no dilution.
- The slide is immersed in 5% formaldehyde for 30 minutes for fixing semen smear.
- The slide is washed in running tap water and dry it.
- The slide is immersed in working Giemsa solution for 3 hours at 37:C.Finally wash the slide in running tap water and dry it.
- Examine the slide under oil immersion of phase contrast microscope and count 200 sperms.
The acrosome based on the morphology is classified as follows:
- Normal acrosome
- Partially lost acrosome
- Completely lost acrosome
- Ruffled/wrinkled acrosome
- Knobbed acrosome
- Diadem defect
- Detached galea capitis
- Apical notch/ridge
The fresh semen should have 80 percent and above acrosome integrity and the frozen semen should have minimum of 65 percent intact acrosome.
The hereditary defects like knobbed acrosome and diadem defect should not exceed 5 percent.
