Estimation error of Leydig cell numbers in atrophied rat testes due to the assumption of spherical nuclei

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Abstract
In this study, Leydig cell numbers in control and atrophied testes (induced via subcutaneous implants of testosterone plus 17 beta estradiol for 16 weeks; TE-implanted) of rats, estimated via the fractionator method (independent of any assumptions) were compared to those estimated via the disector (unbiased, but dependent on shrinkage) and Floderus (assumes spherical particles, dependent on shrinkage) methods. Estimates of Leydig cell numbers in control rats produced by all three stereological methods were similar. In rats with atrophied testes, both the fractionator and the disector methods produced significantly lower (P < 0.01; 47% and 41% with fractionator and disector, respectively) Leydig cell number estimates per testis than in the controls. By contrast, the estimates of Leydig cell number in atrophied testes derived via the Floderus equation were not significantly different from those of controls, but larger than those obtained via the fractionator and the disector methods. These results suggested that the assumptions of the Floderus method were violated in the atrophied rat testes. Why was the Floderus method of estimating Leydig cell number applicable to control rats but not to the TE-implanted rats? In an attempt to answer this question the diameter measurement together with its correction factor used in the Floderus equation (i.e. D+t - 2H) was also derived from the data collected for the disector method. The values for D+t - 2H used in the Floderus method and also calculated via the disector method were found to be identical in controls, but for the TE-implanted rats a 32% lower value was obtained with the Floderus equation when compared to the disector. These findings suggested that this estimation error caused an overestimation of Leydig cell numbers in the TE-implanted rat testes.