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

Department of Anatomy, Monash University, Clayton, Victoria, Australia.
Microscopy Research and Technique (Impact Factor: 1.15). 12/1995; 32(5):423-36. DOI: 10.1002/jemt.1070320505
Source: PubMed


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.

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    • "Mean nucleus volume was calculated using the formula for a prolate sphere (4/3 pab 2 , where a ¼ longest radius and b ¼ shortest radius; McCoard et al. 2001). The total number of Sertoli cells per testis was estimated by dividing the absolute volume of Sertoli cell nuclei per testis by the mean volume of Sertoli cell nucleus (Wreford 1995). "
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    ABSTRACT: We tested whether the reversible effects of nutrition on spermatogenesis in sexually mature sheep were mediated by Sertoli cells. Rams were fed with diets designed to achieve a 10% increase (High), no change (Maintenance) or a 10% decrease (Low) in body mass after 65 days. At the end of treatment, testes were lighter in the Low than the High group (P<0.01). The Maintenance group had intermediate values that were not significantly different from those of the other two groups. Spermatogenesis (Johnsen score) was impaired in the Low group, but normal in both other groups. There was no effect of treatment on Sertoli cell numbers, although 1% of Sertoli cells appeared to retain their ability to proliferate. By contrast, Sertoli cell function was affected by dietary treatment, as evidenced by differences between the High and Low groups (P<0.05) in the expression of seven Sertoli cell-specific genes. Under-nutrition appeared to reverse cellular differentiation leading to disruption of tight-junction morphology. In conclusion, in sexually mature sheep, reversible reductions in testis mass and spermatogenesis caused by under-nutrition were associated with impairment of basic aspects of Sertoli cell function but not with changes in the number of Sertoli cells.
    Reproduction Fertility and Development 01/2015; Published online. DOI:10.1071/RD14368 · 2.40 Impact Factor
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    • "The total testis volume was estimated using the Cavalieri principle [35]. The optical disector technique [36] was used to count the number of Leydig cells in each testis. The cells were recognised by their position, round nucleus and relatively abundant cytoplasm as described previously [18], [37]–[39]. "
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    ABSTRACT: The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.
    PLoS ONE 08/2014; 9(8):e105687. DOI:10.1371/journal.pone.0105687 · 3.23 Impact Factor
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    • "For the testicular cells count, twenty stage VII seminiferous tubules for each testicular sample were randomly selected and the crude counts (nuclear count) of the germ cells (spermatogonia , pachytene spermatocytes, and round spermatids) and Sertoli cells in the tubules were recorded according to a previous report (Leblond and Clermont, 1952). Subsequently, the true count was determined after the crude count values were corrected with the respective nuclear diameter of each cell type and the section thickness using a corrected formula, true count = crude count × section thickness/(section thickness + nuclear diameter of cells) according to the method described before (Abercrombie, 1946; Wreford, 1995). The numbers of Sertoli cells were expressed as true counts of Sertoli cells/per seminiferous tubule, and the numbers of germ cells were expressed as true counts of each germ cell/per Sertoli cell (germ cell/Sertoli cell ratios). "
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    ABSTRACT: To investigate the effects of a low bisphenol A (BPA) concentration on male reproduction, adult rats were administered a concentration of BPA that was less than the no observable adverse effect level (0.0005–5 mg/kg/bw) for 8 weeks. General toxicity, reproductive hormones, and spermatogenesis were then determined. The expression of genes related to hormone synthesis and spermatogenesis was also analyzed. These BPA concentrations generated no general toxicity and no significant changes on serum hormones. However, the testicular testosterone, hormone synthesis-related genes StAR and Cyp450scc increased, whereas 3β-HSD, 17β-HSD, and Cyp450arom decreased. Additionally, BPA significantly decreased the epithelial height and round spermatids in seminiferous tubules, sperm count, androgen receptor expression, and the expression of the spermatogenesis-related genes outer dense fiber protein 1 (ODF1) and transition protein 1. Our results indicate that a low BPA concentration can induce spermatogenesis disorders mainly through decreasing androgen receptor expression. The present results may bring attention to the risk of environmental BPA exposure.
    Toxicology Letters 05/2013; 219(2):116–124. DOI:10.1016/j.toxlet.2013.03.011 · 3.26 Impact Factor
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