Temporal germ cell development strategy during continuous spermatogenesis within the montane lizard, Sceloporus bicanthalis (Squamata; Phrynosomatidae

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Temporal germ cell development strategy during continuous spermatogenesis within the montane lizard, Sceloporus bicanthalis (Squamata; Phrynosomatidae
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  This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:http://www.elsevier.com/copyright  Temporal germ cell development strategy duringspermatogenesis within the testis of the GroundSkink,  Scincella lateralis  (Sauria: Scincidae) J.L. Rheubert a,b , H.H. McHugh a , M.H. Collier a , D.M. Sever b , K.M. Gribbins a, * a  Department of Biology, Wittenberg University, PO Box 720, Springfield, OH 45501-0720, USA b  Department of Biological Sciences, Southeastern Louisiana University, Hammond, Louisiana 70402, USA Received 26 December 2008; accepted 18 January 2009 Abstract Ground Skink ( Scincella lateralis ) testes were examined histologically to determine the testicular organization and germ celldevelopment strategy employed during spermatogenesis. Testicular tissues were collected from 19 ground skinks from AikenCounty, South Carolina during the months of March-June, August, and October. The testes consisted of seminiferous tubules linedwith germinal epithelia in which germ cells matured in close association with Sertoli cells. As germ cells matured, they migratedaway from the basal lamina of the epithelia towards the lumina of the seminiferous tubules. The testes were spermatogenicallyactiveduringthemonthsofMarch,April,May,June,andOctober(largestseminiferoustubulediametersandepithelialheights),butenteredaquiescentperiodinAugust(smallestseminiferoustubulediameterandepithelialheight)whereonlyspermatogoniatypeAand B and early spermatocytes were present in low numbers within the seminiferous epithelium. Although the testicularorganization was similar to other amniotes, a temporal germ cell development strategy was employed during spermatogenesiswithinGroundSkinks,similartothatofanamniotes.Thus,thisskink’sgermcelldevelopmentstrategy,whichalsohasbeenrecentlyreported in all other major reptilian clades, may represent an evolutionary intermediate in terms of testicular organization betweenanamniotes and birds and mammals. # 2009 Published by Elsevier Inc. Keywords:  Testis; Spermatogenesis; Skink; Lizard; Histology 1. Introduction Recently, within the European Wall Lizard,  Podarcismuralis , a new germ cell development strategy has beendescribed for lizards and other squamates that is muchdifferent than the typical spatial arrangement of germcells within the amniotic (bird and mammal) testes[1,2]. Germ cells within the testis of   P. muralis developed through the stages of spermatogenesis as asingle population and produced mature spermatozoa ina single spermiation event at the end of thespermatogenic cycle. This episodic germ cell develop-ment strategy, in which the entire seminiferous tubule/ epithelium participated in spermiation, was morereminiscent of that seen within anamniotic (amphibian)testes and was distinct from the continuous spatial germcell development that results in waves of sperm releaseat certain portions along the length of the seminiferoustubules within the amniotic testes of avian andmammalian taxa during their breeding seasons [3–7]. www.theriojournal.com  Available online at www.sciencedirect.com Theriogenology 72 (2009) 54–61* Corresponding author. Tel.: +1937 327 6478. E-mail address:  kgribbins@wittenberg.edu (K.M. Gribbins).0093-691X/$ – see front matter # 2009 Published by Elsevier Inc.doi:10.1016/j.theriogenology.2009.01.021  Furthermore, this new temporal germ cell developmentstrategy during spermatogenesis also has beendescribed in all major taxa within Reptilia (Sauria:[1]; Chelonia: [8]; Serpentes: [2,9]; Crocodylia: [10]). Historically, spermatogenesis has been explainedusing various stages (I-VIII), which describe thepresence or absence of groups of cells within theseminiferous epithelium, presence/absence of thelumen of the seminiferous tubule, and spermatozoa inthe epididymis in reptiles [11]. These studies did notconcentrate on the individual cell types and omitteddetails pertaining to germ cell morphological changesduring mitosis, meiosis, and spermiogenesis. For thecurrent study, these stages were not included; sperma-togenic activity was based onthe presence orabsenceof specific cell types, and whether consistent associationsof groups of cells were present within the seminiferousepithelium.The purpose of this study was to expand ourunderstanding of the method of germ cell maturationand developmental strategy employed during sperma-togenesis within the temperate Ground Skink, and todetermine if the temporal germ cell developmentstrategy observed in other reptiles also existed withinthe family Scincidae. Ground Skinks are medium-sized‘‘brown-backed’’ lizards (ranging from 75–146 mm)that are common in the moist woodlands of thesoutheastern United States [12]. In Florida populations,male Ground Skinks have the greatest enlargement andactivity within the testis from October through Januaryand the testes reach minimal size in August [13].Populations of Ground Skinks in South Carolina are  J.L. Rheubert et al./Theriogenology 72 (2009) 54 – 61  55Fig. 1. Germ cell types found within the seminiferous epithelium of the Ground Skink. Bar = 20  m m. SpA, spermatogonia A; SpB, spermatogoniaB; PL, pre-leptotene spermatocyte; LP, leptotene spermatocyte; ZY, zygotene spermatocyte; PA, pachytene spermatocyte; DI, diplotenespermatocyte; M1, meiosis I; SS, secondary spermatocyte; M2, meiosis II; S1, step 1 spermatid; S2, step 2 spermatid; S3, step 3 spermatid;S4, step 4 spermatid; S5, step 5 spermatid; S6, step 6 spermatid; S7, step 7 spermatid; MS, mature spermatozoa.  spermatogenically active March through July, withquiescence in August [14]. The Ground Skink is foundin the largest and most diverse family of lizards,Scincidae, which contains more than 1200 species, is acosmopolitan taxon, with representative species onevery continent except Antarctica [15]. Furthermore,skinks have recently become popular in the pet trade.To date,  Podarcis muralis  of the family Lacertidae, Seminatrix pygaea  of the family Colubridae, and  Agkistrodon piscivorous  of the family Viperidae aretheonlyfamilieswithinSquamata(  40families)withacomplete description of the germ cell developmentstrategy exhibited during active spermatogenesis. Thehistological data obtained on spermatogenesis within Scincella lateralis  will be compared to the temporalgerm cell development of   P. muralis , to the knownhistology of the testes in other skinks, and to what isknown of the life history and reproductive characterswithin the Ground Skink. Also, data from this studymay eventually help to elucidate or strengthen thephylogenetic relationships (by adding nontraditionalcharacters such as germ cell development morphologiesto molecular phylogenetic information) of skinks andother squamates, which is highly controversial atpresent [18,19]. 2. Materials and methods Adult male Ground Skinks were collected within theUnited States Department of Energy’s Savannah RiverSite in Aiken County, SC, during the months of April(n = 3), May (n = 3), and October (n = 3) 2001 andMarch (n = 3), June (n = 3), August (n = 4) 2002.Although a complete series of testicular tissues fromevery month of the year were not collected, individualswere collected during what is believed to be thereproductively active and inactive seasons. Skinks werekilled by exposure to ether (technique approved byAnimal Care and Use Committee of Saint Mary’sCollege, Notre Dame, IN, USAwhere the animals werekilled), and the testes were excised and fixed in Trump’sfixative (Electron Microscopy Sciences, Hatfield, PA,USA).Testicular tissues were dehydrated in a graded seriesof ethanol, infiltrated with a 1:2 solution of Spurr’splastic (Electron Microscopy Sciences)/100% ethanol,and again in 1:1 Spurr’s plastic/100% ethanol, beforebeing infiltrated in 100% Spurr’s plastic overnight.Fresh plastic then was used to embed tissues andpolymerized blocks were allowed to cure for 2 d in aFisher Isotemperature Vacuum Oven (Fisher Scientific,Pittsburg, PA, USA). Thin sections (2-3  m m) were cutfrom blocks using an LKB-Ultramicrotome III (LKBProdukter AB, Bromma, Sweden) and a dry glass knife.The sections were stained using a basic fuchsin/ toluidine blue composite stain, as described by Hayat[20].Sections of each testis were examined using a Zeisscompound light microscope (Carl Zeiss Microimaging,Inc. Thornwood, NY, USA) at various magnifications todetermine the morphology of germ cells and germ celldevelopment strategy. Photographs were taken using aSPOT digital camera (Diagnostic System Laboratories,Webster, TX, USA) and composite plates wereconstructed using Adobe Photoshop CS (AdobeSystems, San Jose, CA, USA). Thirty cross sectionsof seminiferous tubules for each represented monthwere chosen at random and the tubular diameter andgerminal epithelial heights were measured using anocular micrometer. Data analysis was performed usingMinitab 15.0 (Minitab Inc., State College, PA, USA) forWindows. Results were deemed significant if  a  0.05.  J.L. Rheubert et al./Theriogenology 72 (2009) 54 – 61 56Fig. 2.  Top : Variation in seminiferous tubule diameter and  Bottom :Variation in germinal epithelial height during the months of March-June,August,andOctoberwithinthetestisoftheGroundSkink.Valuesare means  1 SEM. Different superscripts indicate differencesbetween monthly means (P  0.05; Dunn-Sidak multiple range test).  Tubule diameter and germinal epithelial height datawere tested for normality and homogeneity of variancesusing the Kolmogorov-Smirnov and Bartlett’s tests,respectively, before statistical analyses were performed[21,22]. These data did not meet assumptions of normality; thus, nonparametric Kruskal-Wallis analysiswas used to assess seasonal variation in seminiferoustubulediameterandgerminalepithelialheight.Post-hocnonparametric multiple comparison tests using Dunn-Sidak procedures were then used to detect significantdifferences among pairs of means [2,23]. 3. Results Testes of Ground Skinks were composed of seminiferous tubules that are lined with a germinalepithelium consisting of Sertoli cells and developinggerm cells. Spermatogonia A and B (Fig. 1, SpA andSpB) were found in the basal compartments of theepithelia during all months analyzed. Both sperma-togonia types were located near the basementmembrane of the seminiferous epithelium, withSpA being ovoid in shape and SpB being slightlylarger and more round. The number of spermatogoniaand the mitotic divisions of these gonial cellsdecreased during August (one layer of gonial cells)compared to all other months sampled (two or threelayers of gonial cells). Little difference occurredbetween the abundance of spermatogonia and mitoticdivisions in testes of specimens collected fromMarch-June. There was a slight increase (  5%) inthe number of layers of spermatogonia (  3) andmitotic activity in testes of animals collected inOctober, compared to all other months sampled.Meiotic cells were characterized by a sequentialincreasing in size of the nucleus and a condensation of chromatin into chromosomes. The cell types represent-ing prophase I of meiosis were found during all monthsof the year, including August (usually only pre-leptotene cells) when the testis was reduced in size.Pre-leptotene cells (Fig. 1, PL) were the immediateresult of mitotic divisions of Spermatogonia B. Pre-leptotene cells contained well-defined nuclear envel-opes with dark staining nucleoli and were the smallestof all meiocytes.Leptotene spermatocytes (Fig. 1, LP) were bigger insize compared to pre-leptotene cells and weredistinguished by their dense filamentous chromatin.Zygotene spermatocytes (Fig. 1, ZY) were roughly thesame size as leptotene cells, but had large clumps of condensed filamentous chromatin that stained moreintensely than previous germ cells. Pachytene sperma-tocytes (Fig. 1, PA) were the largest of all developingspermatocytes, had more open nucleoplasm, and con-tainedthickerchromatinfibers.Pachytenespermatocyteswere found during March, May, June, and October.Diplotene spermatocytes (Fig. 1, DI), metaphase 1(Fig. 1, M1), secondary spermatocytes (Fig. 1, SS), and metaphase 2 (Fig. 1, M2) cells were found duringspermatogenically active months (March, April, May,June, and October). The nuclear membranes of diplotene cells began to degenerate and condensedchromatin fibers formed a tight circle just under thesedegenerating membranes. Metaphase 1 cells had fullycondensed chromosomes that aligned at the metaphaseplate. The results of meiosis 1 were the secondaryspermatocytes. The chromatin fibers of the secondaryspermatocytes were randomly dispersed throughouttheir nucleoplasms. During metaphase 2, chromosomesaligned again at the metaphase plate. The germ cell sizeand amount of chromatin present in metaphase 2 wasapproximately halfthatseen inmetaphase1. Theresultsof metaphase 2 were step 1 spermatids.Spermiogenesis within the Ground Skink could bedivided into seven steps, based on the terminology of Russell et al. [6] for mammals. Step 1 spermatids(Fig. 1, S1) were found during every month analyzed,exceptAugust.Thesespermatidsweresmallinsize,hadwell-defined nuclear membranes, and two conspicuousacrosome vesicles in contact with the apex of eachnuclear membrane. These two acrosome vesicles of each spermatid fused to produce a single acrosome instep 2 spermatids (Fig. 1, S2). Acrosome granules weremost commonly seen in the acrosome of Step 2spermatids. Step 3 spermatids (Fig. 1, S3) had definedacrosome vesicles that began to increase in size andenvelop the nuclear heads. As the development of theacrosomes continued, a deep depression within the apexof the nuclear head formed, a feature that characterizedstep 4 spermatids (Fig. 1, S4). Nuclear elongationcharacterized step 5 spermatids, which began oppositeof the acrosome (Fig. 1, S5) and initiated the stretchingof the spermatids’ dorsoventral planes. As elongatingspermatids developed, they migrated towards the apicalportions of Sertoli cells, with the heads of elongatesfacing the basement membrane and the flagella facingthe lumen.Elongation continued and condensation dominatedthe nuclei of steps 6 and 7 spermatids (Fig. 1, S6 andS7). As condensation of the DNA progressed, thethickness of the nuclear heads decreased, resulting invery thin and aerodynamic nuclear heads on the maturespermatozoa (Fig. 1, MS). Mature spermatozoa wereshed to the lumina of the seminiferous tubules.  J.L. Rheubert et al./Theriogenology 72 (2009) 54 – 61  57
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