Date Published: August 30, 2012
Publisher: Hindawi Publishing Corporation
Author(s): Yanfei Yang, Ali Honaramooz.
Significant intrinsic fluorescence in tissues and in disassociated cells can interfere with fluorescence identification of target cells. The objectives of the present study were (1) to examine an intrinsic fluorescence we observed in both the piglet testis tissue and cells and (2) to test an effective method to block the autofluorescence. We observed that a number of granules within the testis interstitial cells were inherently fluorescent, detectable using epifluorescence microscopy, confocal laser scanning microscopy, and flow cytometry. The emission wavelength of the autofluorescent substance ranged from 425 to 700 nm, a range sufficiently broad that could potentially interfere with fluorescence techniques. When we treated the samples with Sudan Black B for different incubation times, the intrinsic fluorescence was completely masked after treatment for 10–15 min of the testis tissue sections or for 8 min of the testis cells, without compromising specific fluorescence labeling of gonocytes with lectin Dolichos biflorus agglutinin (DBA). We speculate that the lipofuscin or lipofuscin-like pigments within Leydig cell granules were mainly responsible for the observed intrinsic fluorescence in piglet testes. The method described in the present study can facilitate the identification and characterization of piglet gonocytes using fluorescence microscopy.
The mammalian testis is composed of seminiferous tubules, primarily containing germ and Sertoli cells, and interstitial tissues containing Leydig cells. As the earliest identifiable germ cell progenitors, primordial germ cells (PGCs) proliferate and differentiate in the fetal testis gonad into gonocytes [1–3]. After birth, gonocytes proliferate in the testis and develop into spermatogonial stem cells (SSCs) prior to puberty [4, 5]. In the mature testis, SSCs initiate and maintain the continuity of spermatogenesis through self-renewal, proliferation, and differentiation to produce daughter germ cells eventually leading up to spermatozoa [4, 6]. In the neonatal testis, gonocytes are the only germ cells present [7–11], and although they give rise to SSCs and are considered germline stem cells, there is controversy as to whether gonocytes have the capability to initiate spermatogenesis on their own, that is, without first developing into SSCs [12–16]. Compared with PGCs and SSCs, gonocytes are the least investigated germline progenitor cells ; therefore, obtaining new knowledge about gonocytes may also shed light on the germline stem cells as a whole.
Autofluorescence within the target tissue or cells could interfere with detection of specific signals from the labeling fluorophores, leading to inaccurate or even false-positive results. In preliminary observations, we noticed that because of the strong innate autofluorescence in isolated testis cells, identifying piglet gonocytes using fluorescence staining was difficult. In the present study, we observed that the autofluorescence was limited to the interstitial tissue/cells of the testis (Figures 1–3), and the source was primarily the intrinsically fluorescent granules within the cytoplasm of Leydig cells. Gonocytes did not emit fluorescence. The autofluorescence had a wide excitation and emission spectrum, strong enough to potentially mimic the appearance of fluorescence labeling. When testis cells were cultured, this intrinsic fluorescence decreased in intensity (Figures 6 and 7). Treatment of the testis tissue and cells with the lysochrome SBB completely masked the intrinsic fluorescence while not compromising with the identification of gonocytes through detection of specific fluorescent signal (Figures 8 and 9).
In conclusion, we characterized an intrinsic fluorescence in piglet testes and showed that the use of SBB can completely quench this autofluorescence, without interfering with identification of specific testis cells by fluorescence microscopy.