Figure 3

Immunocytochemistry demonstrates that Tg8/+ males have significantly increased numbers of sperm retaining enlarged cytoplasmic droplets (CDs) with overexpressed Spam1 or Hyal5, and a concomitant absence of the proteins on the heads. a) Histograms showing the proportion of sperm, from populations of 200, with CDs in wild-type and Tg8/+ males. The greater than 10-fold increase in caudal sperm compared to wild-type is highly significant (χ² = 10.8; P < 0.01), as is the >30-fold increase in caput sperm (χ² = 28.5; P < 0.001). b) Retention of CDs with overexpressed Spam1 (green staining) in sperm taken from an aliquot used for flow cytometry in Fig. 2a. A sperm with the normal amount and normal location of Spam1 is shown for comparison with the overexpressed protein in the CD. Note that there is non-specific background staining on the tails. c) Sperm with overexpressed Hyal5 (green staining) in enlarged CDs near the neck and the absence of the protein on the heads are seen in B and C, while A shows a normal CD without Hyal5. FISH signals on flow sorted sperm showing double signals in transgenic cells d) and a single signal in wild-type cells e).