Acrosome integrity

ACROSOME INTEGRITY

Introduction

  • 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 disolution 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. Maintenance of optimum fertility depends on the acrosome being structurally and biochemically 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.

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Materials required

  • Stock Giemsa stain
  • Semen (fresh/frozen)
  • Glass slides
  • 5% formaldehyde

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 colored bottle.

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Preparation of working Giemsa solution Preparation of Sorenson’s Buffer Preparation of 5% formaldehyde

  • It is prepared immediately prior to use

Chemical

Quantity

Stock Giemsa stain

3 ml

Sorenson’s buffer

2 ml

Distilled water

45 ml

Chemical

Quantity

Na2HPo4.2H2O (21.682 gm/500 ml)

200 ml

KHPo4H2O (22.254 gm/500 ml)

80 ml

Stock buffer

280 ml

Chemical

Quantity

Formalin (37-41% W/V)

12.5 ml

Distilled water

87.5 ml

Staining procedure and result interpretaion

Procedure (View videoVideo demonstration: Giemsa staing procedure Part 1 & Giemsa staing procedure Part 2)

  • A drop of diluted semen (1:5 to 1:10 in 2.9 per cent 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 f or 30 minutes for fixing semen smear.
  • The slide is washed in running tap water and dries it.
  • The slide is immersed in working Giemsa solution for 3 hours at 37 ⁰ 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

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S.No.

Defect

Picture

1

Normal acrosome

2

Partially lost acrosome

3

Completely lost acrosome

4

Ruffled/wrinkled acrosome

5

Knobbed acrosome

6

Diadem defect

7

Detached galea capitis

8

Apical notch/ridge

  • The fresh semen should have 80 per cent and above acrosome integrity and the frozen semen should have minimum of 65 per cent intact acrosome.
  • The hereditary defects like knobbed acrosome and diadem defect should not exceed 5 per cent.

Various acrosomal defects are given below.

Click to enlarge Click to enlarge Click to enlarge Click to enlarge

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Last modified: Monday, 4 June 2012, 10:05 AM