|Suppressed testicular development or suppressed spermatogenesis||Ram and Sathyanesan (1983); Wester (1991); Kirubagaran and Joy (1992); Friedmann., et al. (1996); Vergílio., et al. (2014); Aziz., et al. (2017)|
|Testicular degeneration, hypertrophy, or atrophy||Wester and Canton (1992); Friedmann., et al. (1996): Miles-Richardson., et al.(1999); Weis (2009); Ebrahimi and Taherianfard (2009); Rajan and Kuzhivelil (2013); Vergílio., et al. (2014)|
|Disorganization of testicular tissue or disorganization of cyst structure||Wester and Canton (1992); Hammerschmidt., et al. (2002); Liao., et al. (2006); Ebrahimi and Taherianfard (2009); Vergílio., et al. (2014); Aziz., et al. (2017)|
|Congestion in testicular circulatory vessels||Vergílio., et al. (2014)|
|Proliferation of interstitial tissue||Rajan and Kuzhivelil (2013); Vergílio., et al. (2014)|
|Increased prevalence of ovotestes||Wester (1991); Stentiford., et al. (2003)|
|Testicular tumors (Sertoli cell adenoma, fibroplasia and fibrosis)||Granado-Lorencio., et al. (1987); Řehulka (2013)|
|Germ cell atrophy||Vergílio., et al. (2014)|
|Leydig cell hypertrophy and changes in pycnosis||Kirubagaran and Joy (1992)|
|Sertoli cell atrophy||Wester and Canton (1992)|
|Aggregation||Vergílio., et al. (2014)|
|Altered morphology||Vergílio., et al. (2014)|
|Arrested sperm development or reduced abundance||Wester 1991; Wester and Canton (1992); Miles-Richardson., et al. (1999); Rajan and Kuzhivelil (2013); Vergílio., et al. (2014)|
|Necrosis (pycnosis and karyorhexus) or apoptosis||Wester and Canton (1992); Miles-Richardson., et al.(1999); Blazer (2002); Drevnick., et al. (2006)|
Methods for the treatment of adult fish were those of Govoni., et al. (2017). Groups of adult L. xanthurus, were caught in the wild, held in the laboratory for 90d, and fed: (1) a control diet of Finfish Hi-Performance feed pellets; and (2) a treatment diet of ground axial muscle of Makaira nigricans with naturally high concentrations of Hg (Barber and Whaling, 1983), mixed with ground pellets. Muscleof M. nigricans provided a natural dietary vector for tracing Hg into testes. Hg concentrations in muscle of M. nigricans and in ovaries of L. xanthurus were estimated in solids and in solution by thermal decomposition, amalgamation, and atomic absorption spectrophotometryusing a Milestone DMA-80 Hg analyzer. The concentration of total Hg in muscle of M. nigricans was 3.37 μg/g wet weight (WW); the concentration of extracted MeHg was 0.48 μg/g (WW); and by difference, the concentration of HgII was 2.89 μg/g WW. Fish were fed ad libitum for 90 d. Gametogenesis, including spermatogenesis was induced by shortening photoperiod and increasing temperature.
Sections (5 µm) were cut from treatment and control livers, ovaries, and batches of spawned eggs (Govoni., et al. 2017), and from the testes of treatment fish. Sections were treated with AMG following Danscher and Møller-Madsen (1985), with developing solutions and protocols of Danscher., et al. (2000), and counter stained with Mayer’s ̶ Harris’s hematoxylin and eosin-y-phloxine. A preliminary study to assess the efficacy of AMG in demonstrating Hg in liver and ovaries with densitometry indicated that Hg-sulfide granules, the reaction products of AMG, were significantly related to total Hg concentration in ovaries (Govoni., et al. 2017, Supplement 1). Total Hg in treated ovaries ranged from 0.018 to 1.0 µg/g wet weight. In Govoni., et al. (2017), Hg-sulfide granules were demonstrated in hepatic and ovarian tissues and cells of treatment fish, with some granules were evident in fish from control fish, not fed muscle of M. nigricans, but the prevalence of granules was greater in treatment fish; the presence of some granules in control fish was attributed to environmental exposure before capture (Govoni., et al. 2017).
Spermatogenesis is complex in fishes and consists of multiple phases and stages with alternate nomenclature (Schultz., et al. 2010; Uribe., et al. 2014). Here, we apply the simple, standardized nomenclature for phases of Brown-Peterson., et al. (2011), with the tissue and cellular definitions of Shaw., et al. (2012).
All 20 slides prepared from testes (from separate fish) exhibited Hg-sulphide granules, although the extent and distribution in tissues and cells varied among slides, and therefore among specimens. Ten slides exhibited slight distribution with granules scattered in developing germ cells and support tissue around internal lobular sperm ducts, not the main sperm duct. Testicular tissue was in various phases of spermatogenesis, with developing and spawning-capable phases prevalent; one slide was from a testis in immature stage.
Hg-sulphide granules were evident in spermatogenic germ cells, epithelial cells and connective tissue surrounding sperm ducts, Sertoli cells surrounding spermatocytes, and in spermatozoa. Granules were widespread in spermatogenic cells and in the epithelial cells that surround sperm ducts (Figure 1). In testes that were in developing phase, Sertoli cells in the germinal epithelium of spermatocytes exhibited granules (Figure 2). Sertoli cells associated with either collapsed spermatocytes or collapsed, small, sperm ducts had a high prevalence of granules. Granules were evident in melano-macrophages in interstitial tissue and in association with Sertoli cells (Figure 3). In testes that were in spawning capable phase, spermatozoa within the main sperm duct (Figure 4) and in smaller, radiating sperm ducts within lobular tissue were heavily invested with granules. These spermatozoa appeared necrotic and cellular debris was present within the ducts (Figure 5).
This paper is written in memory of D. W. Evans. Auto metallography was prepared by the North Carolina State University, College of Veterinary Medicine, Raleigh, NC; S. Horton and M. Mattmuller. P. H. Crumley assisted in experiments. We thank C. A. Harms (N.C. College of Veterinary Medicine) and M. Vandersea (National Oceanic and Atmospheric Administration, National Ocean Service, National Centers for Coastal Ocean Science, Marine Spatial Ecology Division) for their review of this manuscript. Protocols of fish experimentation were in accord with the standards recommended by the Guide for the Care and Use of Laboratory Animals and Directive 63/2010. Mention of trade names does not imply endorsement by NOAA. The U.S. Government has the right to retain a non-exclusive, royalty-free license in and to any copyright covering this paper.
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