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 –), 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 and ). 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 and ).
The sources of intrinsic fluorescence in tissue and cell samples can be divided into natural and fixative-induced fluorescence causes. In the present study, the autofluorescence was equally detectable in the testis tissue in both fresh and fixed samples (Figures –). This may imply that the observed autofluorescence was indeed intrinsic to the cells and not acquired during the processing. Flavins and porphyrins are among the most common natural fluorescence substances in fresh tissues/cells, but they are generally extracted during fixation and dehydration processes. Natural sources of the remaining autofluorescence in fixed and processed tissues include elastin, collagen, and lipofuscin. While elastin and collagens comprise major sources of natural autofluorescence in the extracellular matrices, lipofuscin is the main intracellular natural source of autofluorescence in both fresh and fixed samples [27
]. Throughout our observations, the intrinsic fluorescence was consistently detected within interstitial cells in both in vitro
and in situ
(i.e., in freshly disassociated testis cells, tissue whole mounts, tissue sections, and testis cell culture), indicating that the source of autofluorescence in the neonatal piglet testes may likely be lipofuscin or lipofuscin-like pigments.
Lipofuscin is a nondegradable autofluorescent pigment (composed mostly of lipid and protein) which could not be exported from the cells, therefore, accumulates in an almost linear manner with increasing age in the cells, and is stored in the lysosomes as waste materials [30
]. In addition to aging, stress has also been cited as a potential reason for accumulation of lipofuscin. In mice under chronic stress treatment (noise exposure), similar granules were reported to form and accumulate particularly in Leydig cells, and lipofuscin was suggested as the fluorescent substance in the granules [32
]. While aging or chronic stress is unlikely causes of lipofuscin accumulation in the cells of newborn animals, perhaps other factors may also regulate the accumulation of lipofuscin or lipofuscin-like pigments in the neonatal testes. An intrinsic fluorescence has also been reported in neonatal mouse testis cells, with intensities comparable to the labeling fluorophores [15
] and in neonatal bovine testis cells, interfering with the purification of spermatogonia using fluorescence-activated cell sorting [34
]. Fluorescent granules were also observed in Leydig cells in testes of mature crossbred boars [35
]; however, to our knowledge autofluorescence has not been reported in neonatal piglet testes. It is important to note that autofluorescence with broad excitation and emission wavelengths is considered typical of lipofuscin and lipofuscin-like pigments [26
], and quenching of such autofluorescence by lipid staining (e.g., SBB) was suggested to further indicate the lipofuscin origin of the autofluorescence [26
]. Therefore, the fact that the autofluorescence in the present study had a broad spectrum (425 to 700
nm with solid signal from 480 to 620
nm and was observable with widely used filters) and was quenchable with SBB supports the speculation that lipofuscin or lipofuscin-like pigments are mainly responsible for the autofluorescence in the piglet testes.
Staining of different neural tissues from multiple species with several dyes leads to the conclusion that SBB was the most in reducing the lipofuscin-like autofluorescence, without causing the loss of the specific fluorescence signal in immune staining [25
]. In the present study, SBB also completely masked the intrinsic fluorescence of pig testis cells both in situ
and in vitro
but allowed the identification of gonocytes with fluorescein-labeled DBA. While degradation and exocytosis do not remove lipofuscin from the cells, mitotic division is the only reported mechanism that can reduce the lipofuscin concentration within cells [30
]. In the present study, decreasing autofluorescence was observed over the duration of the testis cells culture. We speculate that the lipofuscin content of the cells was divided into the newly formed cells.