Methods for quantifying follicular numbers within the mouse ovary

Abstract

Accurate estimation of the number of ovarian follicles at various stages of development is an important indicator of the process of folliculogenesis in relation to the endocrine signals and paracrine/autocrine mechanisms that control the growth and maturation of the oocytes and their supporting follicular cells. There are 10-fold or greater differences in follicular numbers per ovary at similar ages and/or strains reported in earlier studies using various methods, leading to difficulties with interpretation of ovarian function in control vs experimental conditions. This study describes unbiased, assumption-free stereological methods for quantification of early and growing follicular numbers in the mouse ovary. A fractionator approach was used to sample a defined fraction of histological sections of adult wild-type ovaries. Primordial and primary follicles were counted independently with the optical and physical disector methods. The fractionator/disector methods, which are independent of follicular size or shape, gave estimations of 1930 6 286 (S.E.M.) and 2227 6 101 primordial follicles, and 137 6 25 and 265 6 32 primary follicles per ovary at 70 and 100 days of age respectively. From exact counts on serial sections, secondary and later follicular numbers at 100 days of age were estimated at 135 per ovary. Remnants of zona pellucidae (a marker of previous follicular atresia) were estimated using a fractionator/physical disector approach and were approximately 500 per ovary. The application of the quantitative methods described will facilitate an improved understanding of follicular dynamics and the factors that mediate their growth and maturation and allow for a better comparison between different studies.

The genetics of human infertility by functional interrogation of SNPs in mice

“…Testes and ovaries were fixed for 24 h at 23°C in Bouin's solution, paraffin-embedded, sectioned at 5 μm, and then stained with H&E. Ovarian follicle quantification was done by counting the every fifth section, as described previously (41). For immunofluorescence, testes were fixed in 4% paraformaldehyde for ∼24 h, paraffin-embedded, sectioned at 7 μm, deparaffinized, and then boiled for 30 min in 10 mM citric acid (pH 6.0) for antigen retrieval.…”

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

“…All types of healthy follicles were classified according to the same criteria as detailed in Myers et al (2004). 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).…”

“…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.…”

“…This information is critical to evaluation of claims of germline stem cells in the mouse that can give rise to new primordial follicles. Whilst many studies have assessed follicle numbers in the mouse ovary, the numbers varied significantly according to the stage of folliculogenesis, the methods used, and the strain and age of the mice (reviewed by Myers et al 2004). Although Faddy et al (1987) have studied the kinetics of follicle development from birth to day 100, they used the uncommon CBA/Ca strain and their calculations relied upon several unspecified correction factors.…”

“…These techniques have recently been validated in our laboratory (Myers et al 2004) and by others using C3H wild-type and hpg mice (Wang et al 2005). In addition, we used immunocytochemistry and ultrastructure to further characterize the healthy follicles in the ovary.…”