Abstract
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.
Highlights
The mammalian testis is composed of seminiferous tubules, primarily containing germ and Sertoli cells, and interstitial tissues containing Leydig cells
An intense fluorescence was consistently detected by both epifluorescence and confocal laser scanning microscopes in testes from neonatal, prepubertal, and mature pigs (Figures 1, 2, and 3)
The autofluorescence was exclusively observed in the testis interstitial cells in situ, mainly in the cytoplasm of most Leydig cells that contained strongly fluorescent intracellular granules, some Leydig cells did not seem to contain the autofluorescent granules (Figures 1–3)
Summary
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 [1,2,3]. 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]. Gonocytes are the only germ cells present [7,8,9,10,11], and 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 [12,13,14,15,16]. Compared with PGCs and SSCs, gonocytes are the least investigated germline progenitor cells [17]; obtaining new knowledge about gonocytes may shed light on the germline stem cells as a whole
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
