Theory and practice of stereological techniques applied to the estimation of cell number and nuclear volume in the testis

Abstract

The historical background to contemporary approaches to the estimation of cell/nuclear number and volume in the testes is reviewed. The limitations of older geometric model-based approaches to the estimation of cell/nuclear number are discussed, and the need for absolute estimates of cell number rather than ratio estimates is examined. The physical and optical disector approaches to the direct estimation of numerical density and, hence, absolute cell number are presented together with data illustrating their operational efficiency in the testis. New approaches to the direct estimation of nuclear/cell volume, using the point-sampled intercept family of methods, are presented and illustrated, using the example of the Sertoli cell nucleus. The use of both classical transverse and the newer vertical section approaches is explored. Estimation of Sertoli cell/nuclear volume in the volume (point-sampled intercept procedure) and number (nucleator and rotator methods) distributions on both conventional transverse and vertical sections is discussed. The use of transverse sections of the testis is shown to produce a consistent bias in the estimation of Sertoli cell nuclear volume in 120-day-old animals, with all the estimators. Comparison of the Sertoli cell nuclear volume (measured on vertical sections) in the volume and number-weighted distribution suggests a coefficient of variation of volume in the number distribution of 0.4-0.5, suggesting either a random or stage-dependent variation in Sertoli cell nuclear size which requires further exploration.

Quantification of stem cell factor mRNA levels in the rat testis: usefulness of clusterin mRNA as a marker of the amount of mRNA of sertoli cell origin in post pubertal rats

“…It must be underlined, that the ratio of the number of Sertoli cells to the total number of cells counted on histological sections was in no way aiming to be an estimation of the number of Sertoli cells in the testis; this requires much more sophisticated methodologies to be determined (Wreford 1995). However, the ratio provides evidence that the proportion of Sertolian tissue in testis samples may vary greatly according to the different experimental/pathological conditions, even when the number of Sertoli cells remains unchanged.…”

Alterations of Sertoli cell activity in the long-term testicular germ cell death process induced by fetal androgen disruption

“…After full polymerization, 25 mm sections were cut on an Ultracut microtome (Reichert-Jung Inc., Wien, Austria) using glass knives, mounted onto adhesive glass slides, and stained with Harris hematoxylin. Sertoli and Leydig cells were then counted using the optical dissector method as described by Wreford (1995) and Sharpe et al (1998Sharpe et al ( ), (2000. The thick sections were viewed at high magnification and optically sectioned with a microscope equipped with a microcator to measure stage movement in the z-axis.…”

Quantification of healthy follicles in the neonatal and adult mouse ovary: evidence for maintenance of primordial follicle supply

“…Estimates of primordial and primary follicle number were made using the Olympus CAST 2 stereological software v2.1.4 (Olympus Denmark A/S, Albertslund, Denmark) on a PC in conjunction with an Olympus BX-51 microscope fitted with !100 oil immersion objective with high numerical aperture (N A Z1$4). Microscopic fields were selected using a motorised stage (Autoscan Systems, Brighton, Australia) and systematic uniform random sampling (Gundersen & Jensen 1987, Wreford 1995. A microcator (D8225, J Heidenhain GmbH, Traunreut, Germany) fitted to the microscope was used to measure the movement of the stage in the z-axis.…”

“…A fractionator/optical disector design (Gunderson et al 1988, Wreford 1995) was used to sample tissue in conjunction with an unbiased sampling frame (Gundersen & Jensen 1987) as described previously (Myers et al 2004). The number of sections selected from the 2585 making up the serial set (sampling fraction 1 (f1)), the area of the sampling frame and the step length (sampling fraction 2 (f2)Zframe area/(x-step!y-step)) was optimized to give the most efficient counting protocol.…”