hernandez y uribe 2012

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Seasonal Spermatogenic Cycle and Morphologyof Germ Cells in the Viviparous Lizard Mabuya

brachypoda (Squamata, Scincidae)

Arlette Hernandez-Franyutti1 and Mari Carmen Uribe2*

1Laboratorio: Acuicultura Tropical, Division Academica de Ciencias Biologicas, Universidad Juarez Autonoma

de Tabasco, 86039 Villahermosa, Tabasco, Mexico2Laboratorio: Biologa de la Reproduccion Animal, Departamento de Biologa Comparada, Facultad

de Ciencias, Universidad Nacional Autonoma de Mexico, 04510 Mexico D.F.

ABSTRACT We describe seasonal variations of the his-tology of the seminiferous tubules and efferent ducts ofthe tropical, viviparous skink, Mabuya brachypoda,throughout the year. The specimens were collected

monthly, in Nacajuca, Tabasco state, Mexico. The resultsrevealed strong annual variations in testicular volume,stages of the germ cells, and diameter and height of theepithelia of seminiferous tubules and efferent ducts.Recrudescence was detected from November to December,when initial mitotic activity of spermatogonia in the semi-niferous tubules were observed, coinciding with thedecrease of temperature, photoperiod and rainy season.From January to February, early spermatogenesis contin-ued and early primary and secondary spermatocytes weredeveloping within the seminiferous epithelium. FromMarch through April, numerous spermatids in metamor-phosis were observed. Spermiogenesis was completedfrom May through July, which coincided with an increasein temperature, photoperiod, and rainfall. Regressionoccurred from August through September when testicularvolume and spermatogenic activity decreased. During thistime, the seminiferous epithelium decreased in thickness,and germ cell recruitment ceased, only Sertoli cells andspermatogonia were present in the epithelium. Through-out testicular regression spermatocytes and spermatidsdisappeared and the presence of cellular debris, and scat-tered spermatozoa were observed in the lumen. Theregressed testes presented the total suspension of sperma-togenesis. During October, the seminiferous tubules con-tained only spermatogonia and Sertoli cells, and the sizeof the lumen was reduced, giving the appearance that itwas occluded. In concert with testis development, the effer-ent ducts were packed with spermatozoa from May through

August. The epididymis was devoid of spermatozoa by Sep-tember. M. brachypoda exhibited a prenuptial pattern, in

which spermatogenesis preceded the mating season. Theseasonal cycle variations of spermatogenesis in M. brachy-

podaare the result of a single extended spermiation event,which is characteristic of reptilian species. J. Morphol.273:11991213, 2012. 2012 Wiley Periodicals, Inc.

KEY WORDS: spermatogenesis; testicular cycle;efferent ducts; viviparous lizard; Mabuya

INTRODUCTION

The identification and definition of the seasonalchanges of the male reproductive cycle, e.g., the

spermatogenesis, is as important as the femalereproductive cycle in determining the reproductivepattern of a species (Trauth, 1979). Spermatogene-sis is an essential process in the biology of repro-

duction and results in production of haploid sper-matozoa from diploid spermatogonial cells. Thestudy of the spermatogenesis requires the recogni-tion and identification of germ cell morphology dur-ing their differentiation and their occurrencethroughout the seasonal cycle. The differentiationof germ cells involves specific cellular events: mito-sis of spermatogonia, meiosis of spermatocytes, andspermiogenesis of spermatids. This process is fol-lowed by spermiation, the release of sperm fromthe seminiferous epithelium (Bakst et al., 2007;Hess and de Franca, 2008).

In reptilian sauropsids, as amniotic species, sper-matogenesis has special evolutionary interest among

vertebrates because germ cell development repre-sents a strategy between the anamniotic clades(fishes and amphibians) and the other amniotic taxa(birds and mammals; Gribbins et al., 2003, 2007,2008, 2009; Rheubert et al., 2009a,2009b). In thetestes of birds and mammals, germ cells are sequen-tially situated along the seminiferous tubules in acontinuous spatial development of spermatogenesisthat results in waves of sperm release from specificportions of the tubules during the reproductive cycle(Leblond and Clermont, 1952; Clermont, 1972; Lin

Additional Supporting Information may be found in the online

version of this article.

*Correspondence to: Mari Carmen Uribe, Departamento de Biolo-ga Comparada, Facultad de Ciencias. Circuito Exterior, CiudadUniversitaria, Universidad Nacional Autonoma de Mexico, Insur-gentes Sur 3000, Delegacion Coyoacan, 04510 Mexico, D.F. Mexico.E-mail: [email protected]

Received 22 January 2012; Revised 4 April 2012;Accepted 13 April 2012

Published online 13 July 2012 inWiley Online Library (wileyonlinelibrary.com)DOI: 10.1002/jmor.20050

JOURNAL OF MORPHOLOGY 273:11991213 (2012)

2012 WILEY PERIODICALS, INC.


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and Jones, 1990; Franca and Godinho, 2003; Hessand de Franca, 2008). In contrast, reptilian sperma-togenesis develops a single population of spermato-zoa, which is released during a single spermiationevent (Mayhew and Wright, 1970; Guillette andCasas-Andreu, 1987; Guillette and Mendez de la

Cruz, 1993; Villagran-Santa Cruz et al., 1994; Grib-bins et al., 2003, 2007, 2008, 2009; Ferreira et al.,2009; Rheubert et al., 2009a,2009b). Consequently,the analysis of morphological and physiological char-acteristics of spermatogenesis in reptiles representsa main aspect in the understanding of the evolutionof male germ cell development in vertebrates.

Species of the genus Mabuya are tropical grassskinks that are distributed in regions of America,

Asia, and Africa. These species present remarkablereproductive patterns having several unique spe-cializations among lizards: a) the genus Mabuyaincludes oviparous and viviparous species in Asiaand Africa. However, all the species of America are

viviparous (Fitch, 1970; Vitt and Blackburn, 1983,1991); b) the females may initiate gestation at asmall body size and subsequently grow duringgestation (Vitt and Blackburn, 1983); c) viviparousspecies have a long period of gestation (912months; Vitt and Blackburn, 1983, 1991); d) theovaries develop different types of eggs, some speciesovulate macrolecithal eggs (containing abundantyolk; Vitt and Blackburn, 1983) while other speciesovulate microlecithal eggs (containing little yolk;Webb, 1958; Vitt and Blackburn, 1983, 1991; Flem-ming, 1994; Hernandez-Franyutti et al., 2005); e)therefore, there are different levels of lecithotrophy(when most nutrients for the embryo are provided

by the yolk), or matrotrophy (when most nutrientsfor the embryo are transferred from maternal tis-sues during gestation; Vitt and Blackburn, 1983,1991; Blackburn, 2000, 2005; Uribe et al., 2006).

Male reproductive activity ofMabuya species hasbeen described as a continuous process by grossanalysis of the testes (weight and volume), in M.striata (Simbotwe, 1980) and M. mabouya (Sommaand Brooks, 1976; Ramirez-Pinilla et al., 2002); inother species, spermatogenesis was defined as aseasonal cycle, as in M. heathi (Vitt and Blackburn,1983), M. capensis (Flemming, 1994). In these spe-cies, the peak of spermatogenesis occurred duringspring and early summer, which is characteristic of

the prenuptial pattern (Saint Girons, 1982). Histo-logical changes in the seminiferous tubules of

Mabuya were mentioned only in the study of M.capensis by Flemming (1994), but he did not pres-ent histological images.

Mabuya brachypodais a viviparous lizard distrib-uted from the southeast of Mexico to Costa Rica inCentral America (Webb, 1958). Some reproductiveaspects have been studied in adult females of thisspecies. The ovarian cycle and oogenesis ofM. bra-chypoda were documented by Hernandez-Franyuttiet al. (2005), who identified a seasonal process dur-

ing which microlecithal eggs (0.91.3 mm in diame-ter) developed. The microlecithal eggs are consideredthe smallest described thus far in reptiles. The eggsattained maximum diameters during June-July.Macroscopic examination of the oviducts ofM. bra-chypoda, collected between late June to late July by

Webb (1958), showed embryos in the uterus. Histo-logical analysis of the uterus ofM. brachypoda dur-ing the annual cycle by Uribe et al. (2006), showedthe presence of embryos between August and May.There are no previous descriptions of male reproduc-tive system of M. brachypoda. Therefore, the goalsof this study are to characterize the morphology ofthe germ cells during spermatogenesis and deter-mine the changes of the seminiferous epithelia andefferent ducts in M. brachypoda during the annualcycle and to relate these characteristics to the longgestation period of the female reproductive cycle.

MATERIAL AND METHODSMale M. brachypoda (snout-vent length 5978 mm; N 5 36, 3

specimens per month), from Nacajuca, Tabasco, Mexico (18810N, 9381 W), were collected during 2004. Meteorological data,mean monthly maximum and minimum temperatures and pre-cipitation in Tabasco, Mexico, were obtained from the ComisionNacional del Agua, Mexico (2004) (Supporting InformationGraph 1). The specimens were euthanized using ether anddecapitation. A mid ventral incision opened the abdominal cav-ity. Maximum and minimum testicular diameters were meas-ured. Testicular volume was subsequently calculated using theequation for the volume of an ellipsoid (V5 4/3p (1/2L) (1/2W)2).

Testes, and efferent ducts were removed and fixed in alcoholicBouins (815 h). After fixation, the tissues were washed in 70%alcohol, dehydrated in a series of graded alcohols (80%, 96%and 100%), cleared in xylene, and embedded in paraffin. Tissueswere serially sectioned at 6 lm in longitudinal planes and

stained with hematoxylin-eosin (Aguilar-Morales et al., 1996).The morphological changes of the seminiferous epithelium dur-ing the annual cycle were divided into eight phases (I to VIII)according to the classification described by Mayhew and Wright(1970) based on the morphological changes of the seminiferousepithelium in lizards of the genus Uma. This classificationfocuses on the type of germ cells bordering the tubular lumenduring the annual cycle. The VIII phases are characterized by:I, mitosis of spermatogonia and no lumen; II, primary sperma-tocytes at luminal margin; III, secondary spermatocytes atluminal margin; IV, undifferentiated spermatids at luminalmargin; V, spermatids in spermiogenesis at luminal margin; VI,spermatozoa in the lumen; VII, early regression with cellulardebris in the lumen; VIII, complete regression, no mitosis ofspermatogonia and reduced lumen. The progressive changesthat occurred during the spermiogenesis were divided intoseven steps according to Gribbins et al. (2009). The terms used

for the two types of efferent ducts were ductuli efferentes andductus epididymis as described by Sever (2010). Selected mor-phometrics such as: diameter and epithelial height of the semi-niferous tubules, ductuli efferentes, and ductus epididymis weremeasured (N 5 25 per specimen) using a Carl Zeiss AxionVision Imaging System. Digital photomicrographs were takenusing an Olympus digital camera model C5050Z coupled to anOlympus CX31 microscope.

RESULTS

The testes of M. brachypoda are ellipsoid struc-tures (Fig. 1A,B), in the abdominal cavity and con-

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nected to the dorsal wall of the body by the meso-rchium. Adjacent to the testis is the epididymis asa convoluted duct (Fig. 1A,B). The efferent ductsconsisted of two segments. The proximal segmentof the epididymis is closest to the testis and isformed by the ductuli efferentes. The distal seg-ment is the ductus epididymis, which connects tothe ductus deferens. The testes of M. brachypoda

present annual variations in testicular volume, di-ameter and epithelial height of the seminiferoustubules and diameter and epithelial height of theductuli efferentes, and ductus epididymis (see sup-porting Information, Table 1).

The reproductive cycle of M. brachypoda ishighly seasonal. The phases of the spermatogeniccycle in the testis, and the presence of spermato-

Fig. 1. Testes and epididymis ofMabuya brachypoda during different periods of spermatogenesis. A. Testes and epididymis dur-ing phase II of the seminiferous epithelium cycle. The seminiferous tubules of the testes are reduced. The ductus of the epididymisdoes not contain spermatozoa. B. Testes and epididymis during phase VI of the seminiferous epithelium cycle. The seminiferoustubules are larger compared to those seen in phase II in the Fig. 1A. The ductus epididymis encloses abundant spermatozoa.C. The seminiferous epithelium during phase II contains few germinal cells and there are no spermatozoa in the lumen of the sem-

iniferous tubules. D. The seminiferous epithelium during phase VI contains abundant germinal cells and there are late spermatidsat the apical end, adjacent to the lumen of the seminiferous tubules. Epididymis (E), late spermatids (1St), lumen (*), seminiferousepithelium (sep), spermatozoa (Sz), testes (T). Bars 5 A, B: 500lm, C, D: 100lm.

SPERMATOGENESIS OF VIVIPAROUS LIZARD Mabuya brachypoda 1201



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zoa in the ductus epididymis defined the seasonalchanges (Fig. 1C,D). Between November and Feb-ruary, the size of the testis, its volume, diameterand the height of epithelium of the seminiferoustubules, as well as the diameter and epithelialheight of the efferent ducts, in early spermatogen-

esis were minimal (Fig. 1A,C) but they were maxi-mal between May and July, i.e., during late sper-matogenesis (Fig. 1B,D; Table 1, supporting Infor-mation). In early spermatogenesis, theseminiferous epithelium of the testicular tubulescontains few germinal cells and there are no sper-matozoa in the lumina of the seminiferous tubules(Fig. 1C). The ducts of the epididymis do not con-tain spermatozoa. In late spermatogenesis, theseminiferous epithelia of the testicular tubulescontain abundant germinal cells and there arespermatids at the apical end, adjacent to thelumen of the seminiferous tubules (Fig. 1D), andthe ductus epididymis contain abundant spermato-

zoa.

Testicular Components

The wall of the testis is surrounded externallyby the tunica albuginea, formed by vascularizedconnective tissue with abundant collagenousfibers. The testes contain the seminiferous tubuleslined by seminiferous epithelia. These epitheliaconsist of Sertoli cells and germ cells. Sertoli cellsform a permanent epithelium and are distributedaround the circumference of the seminiferoustubules (Fig. 2A). Sertoli cells have a basal nu-cleus, are frequently triangular in shape with one

or two nucleoli. The seminiferous epithelium islimited by a basement membrane (Fig. 2A) thatseparates the germinal compartment inside theseminiferous tubules, from the interstitial com-partment outside the seminiferous tubules. The in-terstitial compartment is formed by connective tis-sue composed of Leydig cells, fibroblasts, collagenfibers, myo-epithelial cells, nerve fibers and lym-phatic and blood vessels.

Morphology of Germ Cells During theSpermatogenesis

Spermatogonia. Spermatogonia (Fig. 2BE)

are located at the base of the seminiferous epithe-lium, bordering the basement membrane. Duringthe annual cycle they proliferate by mitosis, pro-ducing a large population of spermatogonia orinitiating meiosis and becoming primary spermato-cytes. There are two types of spermatogonia: sper-matogonia A (Fig. 2B), and spermatogonia B(Fig. 2CE). Spermatogonia A have an average di-ameter of 21lm; are ovoid in shape, and theirnuclei are spherical and contain fine granular het-erochromatin with one or two dense and roundnucleoli. The spermatogonia B are slightly larger;

have an average diameter of 23.3 lm and are morespherical than the spermatogonia A. Their nucleusand cytoplasm are similar to those seen in thespermatogonia A. During mitosis the fibers of thespindle were easily observed (Fig. 2E).

Primary spermatocytes. Primary spermato-

cytes (Fig. 2FK) are spherical cells with a roundnucleus. The nucleus undergoes morphologicalchanges during the progression of meiosis I.

According to the morphology of the chromosomes,several steps of meiotic prophase I were seen: a)during the leptotene stage (Fig. 2F), the averagecell diameter is smaller than that of the spermato-gonia, attaining a 20.2 lm average diameter. Thechromatin initiates condensation and the chromo-somes appear as thin, densely stained threads. b)During zygotene (Fig. 2G), the average cell diame-ter increases to 23.2 lm and condensation of thechromosomes advances. Next, the chromosomesbecome more filamentous as the homologous chro-

mosomes pair form. c) During the pachytene stage(Fig. 2H), the spermatocytes have an average di-ameter of 25.6 lm. The paired chromosomes areeven thicker and shorter than those seen in zygo-tene and maintain their intense stained affinity.These spermatocytes were the most abundantamong the primary spermatocytes. d) During thediplotene stage (Fig. 2I), the spermatocytes are atthe maximum diameter for primary spermatocytes,they attained an average diameter of 26.8 lm. Thepaired and dense chromosomes become graduallyseparated but remain united in several sites, irreg-ularly distributed all along the chromosomes,forming chiasmata. These cells were the rarest of

primary spermatocytes. The primary spermato-cytes divide forming the spindle and the chromo-somes arranged into metaphase I (Fig. 2J), con-tinue through anaphase I (Fig. 2K), and telophaseI, and complete cytokinesis to produce the second-ary spermatocytes.

Secondary spermatocytes. Secondary sperma-tocytes (Fig. 2L) are the result of the first meioticdivision. Since secondary spermatocytes are rarelyobserved it is assumed that rapidly enter the sec-ond division of meiosis. Secondary spermatocytesare spherical in shape and smaller in averagediameter (18.9 lm) compared to the primary sper-matocytes. The nucleus is spherical and the chro-

matin consists of fine filaments, indicating the con-tinuation of meiosis. After the second meiotic divi-sion, the secondary spermatocytes form step 1spermatids.

Spermatids. Spermatids (Fig. 2MS), in theirearly stages (Fig. 2M), are spherical cells andsmaller, compared to secondary spermatocytes,with an average diameter of 14.8 lm. The sperma-tids go through metamorphosis during spermiogen-esis. The morphological changes of the spermatidincluded formation of the acrosomal system, chro-matin condensation, elongation of the nucleus and




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tion of the nucleus; and step seven (Fig. 2S), ischaracterized by easy visualization of the flagel-lum protruding into the lumen which suggests thegerm cell is near to be released into the lumen.

Spermatozoa. Once spermatids complete sper-miogenesis (Fig. 2T), they are released into the

lumen as spermatozoa. Spermatozoa are elongated,and the flagellum is long and thin. Mature sper-matozoa are transported to the efferent ducts.

Annual Changes During the SeminiferousEpithelium Cycle

The presence/absence of specific germ cellsaltered the morphology of the seminiferous epithe-lium during the annual cycle of Mabuya brachy-

poda through eight phases (I-VIII). Spermatogene-sis was synchronous in all the seminiferoustubules.

Phase I. The testes were in this phase during

November and December (Fig. 3A,B). The majorityof the seminiferous tubules contained only sperma-togonia and Sertoli cells. The volume of the testeswas 8.7 6 1.2 mm3. The diameter of seminiferoustubules was 61.8 6 2.2 lm. The seminiferous epi-thelium height was 28.3 6 1.7 lm. The lumen isoccluded in most of tubules, and only a few ofthem had a visible small lumen (Fig. 3A). At thisphase, recrudescence of spermatogenesis wasdetected undergoing spermatogonia in mitosis(Fig. 3B). As a consequence of the mitotic activityof spermatogonia, the number of germ cells in theseminiferous epithelium appeared to increase.

Phase II. The testes were in phase II during

January and February at which time the volumeof the testes was 8.9 6 3.1 mm3(Fig. 3C). The di-ameter of the seminiferous tubules was 107.2 64.9 lm, and the seminiferous epithelium heightwas 31.4 6 1.0 lm. Spermatogonia continued pro-liferating entering into meiosis and formed abun-dant primary spermatocytes that were located atthe margin of the lumen.

Phase III. The testes were in phase III duringMarch at which time the volume of the testes was12.9 6 3.4 mm3 (Fig. 3D). The diameter of thetubules increased to 182.6 6 5.0 lm. The seminif-erous epithelium heights also increased to 61.3 61.6 lm. Primary spermatocytes were numerous.

Meanwhile, secondary spermatocytes were occa-sionally seen at the apical end of the seminiferousepithelium. Primary spermatocytes in pachytene ofprophase I of meiosis were the most abundanttype of germ cells identified in this phase. Occa-sionally, spermatids were also located at the mar-gin of the tubular lumen.

Phase IV. The testes were in phase IV duringApril at which time the volume of the testes was47.7 6 5.5 mm3 (Fig. 4AD). The diameter of theseminiferous tubules was 271.7 6 6.7 lm, and theheight of the seminiferous epithelium was 94.5 6

2.4 lm.The seminiferous epithelium contained sev-eral layers of spermatids situated along the mar-gin of the lumen. Most of the spermatids showedthe initial morphological changes that occur dur-ing spermiogenesis, including acrosomal develop-ment and the initial elongation of the nucleus, as

evidence of early spermiogenesis.Phase V. The testes were in phase V duringMay at which time the volume of the testes was75.9 6 8.5 mm3 (Fig. 5AD). The diameter of theseminiferous tubules attained 306.2 6 7.4 lm,slightly larger than those seen in the previousstages. The height of the seminiferous epitheliumattained 112.6 6 2.6 lm. The germinal epitheliumformed large columns in which spermatids werelocated along the lumen. The number of primaryspermatocytes decreased; meanwhile, the sperma-tids were very abundant. The spermatids advancedinto late spermiogenesis presenting further elonga-tion of the spermatid nucleus.

Phase VI. The testes were in phase VI duringJune and July at which time the volume of thetestes was 89.2 6 10.5 mm3 (Fig. 6AD). The semi-niferous tubules attained their maximum diameter(356.6 6 17.4 lm), similar to that seen in theheight of the seminiferous epithelia 122.1 6 5.9lm. Spermatogenesis was completed during thisphase. Numerous spermatids lined the margins ofthe seminiferous epithelium and spermatozoa werefree within the lumen of the tubules (Fig. 6C,D).

Phase VII. The testes were in phase VII duringAugust at which time the volume of the testes was52.6 6 12.0 mm3 (Fig. 7A,C). The diameter of theseminiferous tubules was reduced to 312.5 6 16.8

lm. During early regression (Fig. 7A,B), the semi-niferous epithelium decreased in thickness to 89.26 5.4 lm; but, there were smaller regions withonly three or four germ cell layers, containingspermatogonia, and some spermatids in spermio-genesis. During late regression (Fig. 7C), most ofspermatocytes and spermatids disappeared, onlyremaining spermatogonia and Sertoli cells in theseminiferous epithelium. Abundant cellular debris,and some scattered spermatozoa were seen in thelumen of the seminiferous tubules.

Phase VIII. The testes were in phase VIII dur-ing September and October at which time the vol-ume of the testes was 20.6 6 9.0 mm3 (Fig. 7D).

The seminiferous tubules attained the minimumdiameter (77.7 6 1.8 lm), as the height of the sem-iniferous epithelium (11.6 6 1.4 lm). The lumen ofthe tubules was mostly occluded. The seminiferousepithelium contained, exclusively, spermatogoniaand Sertoli cells. No mitotic activity of the sperma-togonia was seen.

Efferent Ducts

The efferent ducts of M. brachypoda (Fig. 8AC)are ductuli efferentes and ductus epididymis. Near




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the efferent duct is observed the sexual segment ofthe kidney.

The ductuli efferentes (Fig. 8AD) were thesmallest efferent ducts. They were lined by cuboi-dal epithelium that had large stereocilia borderingthe lumen (Fig. 8D) that we need to verify theirpresence at the electron microscope level. Theseducts had a similar size throughout the reproduc-tive cycle (Table 1, supporting Information). Their

minimal and maximal diameters were 17.5 6 3.4lm from January through February, and 24.6 62.9 lm from June through July. Their epithelialheight varied from 8.1 6 2.1 lm to 10.5 6 3 lmrespectively. Only during the stage VII, at the endof the spermiation and during the early regressionof the reproductive cycle in the seminiferoustubules, the ductuli efferentes contained a fewspermatozoa.

Fig. 3. Mabuya brachypoda, seminiferous tubules during phases I, II and III of the cycle. A. The seminiferous epithelium duringphase I contains only Sertoli cells and spermatogonia. The lumen is very small or it is occluded. B. Spermatogonia in metaphase ofmitosis are seen, where the fibers of the spindle are evident. C. Seminiferous tubules during phase II of the cycle show spermatogo-nia A and B at the base of the seminiferous epithelium. Primary spermatocytes are abundant at the center of the tubules. Thelumen is lightly larger than in the previous phase. D. Seminiferous tubules during phase III of the cycle. Secondary spermatocytesare seen at the luminal margin of the seminiferous epithelium. The lumen is clearly larger than in the previous phase. Lumen ( *),Sertoli cells (Se), spermatogonia A (SgA) spermatogonia B (SgB), spermatogonia in metaphase of mitosis (arrowheads), primaryspermatocytes (1Sc). Bars 5 12lm.




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The ductus epididymis (Fig. 8AC,E) was distalto the ductuli efferentes, and was larger than theductuli efferentes. The diameter of the ductus epi-didymis and the height of the epithelial cellschanged during the reproductive cycle. Minimumlumen size was measured as 67.8 6 8.1 lm in Jan-uary-February; maximum lumen was 226.3 6 15.2

lm between June through August, during thephases V to VII when masses of spermatozoa werein the lumen. The ductus epididymis was lined bycolumnar epithelium (Fig. 8E), and it hypertro-phies during June-July. The height of the epithe-lium varied from 9.1 6 1.2 lm from January-February to 33.0 6 3.0 lm during June-July. From

Fig. 4. Mabuya brachypoda, seminiferous tubules during phase IV of the cycle. A, B. Spermatids before metamorphosis at the

luminal margin of the seminiferous epithelium. Some of them present early metamorphosis. The lumen is seen. C, D. Abundantspermatids during early metamorphosis are seen at the luminal margin of the seminiferous epithelium. Spermatids (St), sperma-tids in early metamorphosis (eSt), lumen (*). Bars 5 A: 20lm. B: 10lm. C: 30lm. D: 20lm.




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May through August, the cytoplasm of the epithe-lial cells contained abundant secretory granules(Fig. 8E).

DISCUSSION

The results of our study are in agreement withearlier studies of reptilian sauropsids by Trauth

(1979). Histological analysis of Mabuya brachy-poda shows the precise time and duration of repro-ductive activity and reflects upon the environmen-tal factors that may affect the cyclical changes ofthe reproductive system.

Mabuya brachypoda males exhibit a strongseasonal cycle of spermatogenesis. Spermatogenicrecrudescence was detected from November to

Fig. 5. Mabuya brachypoda, seminiferous tubules during phase V of the cycle. AD. Spermatids during middle metamorphosisand late metamorphosis are seen at the luminal margin of the seminiferous epithelium. The seminiferous epithelium presents col-

umns extended to the lumen. Spermatids in middle metamorphosis (mSt) and late metamorphosis (lSt), columns (co), lumen (*

).Bars 5 A: 40lm. B: 10lm. C: 20lm. D: 10lm.




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December, when mitotic activity of spermatogoniaand development of a central lumina in the semi-niferous tubules were observed, coinciding withthe decrease of temperature, photoperiod, and thebeginning of the rainy season. From January toFebruary, primary and secondary spermatocytesdeveloped. From March through April, numerous

spermatids in various stages of spermiogenesiswere apparent in the apical layers of the seminif-erous tubules. Spermiogenesis was completed fromJune through July, when the increases in tempera-ture, photoperiod and rainy season occurred.During these months, the seminiferous tubulescontained abundant spermatozoa, and the maxi-

Fig. 6. Mabuya brachypoda, seminiferous tubules during phase VI of the cycle. A, B. Late spermatids are seen at the luminalmargin of the seminiferous epithelium. The seminiferous epithelium presents columns extended to the lumen. C, D. Abundantspermatozoa during spermiation are observed in the lumen of the seminiferous tubules. Late spermatids (lSt), lumen (*), spermato-zoa (Sz). Bars 5 10lm.




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mal testicular volume was detected. Testicularregression occurred from August through Septem-ber when testicular volume and spermatogenicactivity decreased; the seminiferous tubules con-tained only a few spermatocytes, the spermatids in

metamorphosis had diminished and the seminifer-ous epithelium decreased in thickness, having onlythree or four cell layers composed of spermatogo-nia, occasionally some spermatocytes and someremaining spermatids and spermatozoa. Progres-

Fig. 7. Mabuya brachypoda, seminiferous tubules during phases VII and VIII of the cycle. A, B. During early regression, inphase VII of the cycle, the reduction of the seminiferous epithelium is evident. Spermatogonia, spermatids and spermatozoa areseen. Few spermatozoa are seen in the lumen of the seminiferous tubule. C. During middle regression, in phase VII of the cycle,the seminiferous epithelium is reduced to spermatogonia and Sertoli cells and abundant cellular debris (cd) in the lumen of thetubule are seen. D. Seminiferous tubules of Mabuya brachypoda in late regression, in phase VIII of the cycle. During completeregression, only spermatogonia and Sertoli cells are seen in the seminiferous epithelium. The lumen is very small or it is occluded.Cellular debris (cd), few spermatozoa (arrow head), lumen (*), reduction of the seminiferous epithelium (thick arrows), Sertoli cells(Se), spermatids (St), spermatogonia (Sg), spermatozoa (Sz), A: Bar 5 20lm. B: Bar 5 10lm. Bar 5 12lm. Bar 5 12lm.




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sively, with the advance of the regression, thespermatocytes and spermatids disappeared andthe presence of hyaline material, abundant cellulardebris, and some scattered spermatozoa were seen

in the lumen of the seminiferous tubules. Theregressed testes defined the total suspension ofspermatogenesis. During October, the seminiferoustubules contained only spermatogonia and Sertoli

Fig. 8. Epididymis of Mabuya brachypoda. A, B. Epididymis, testis and sexual segment of the kidney, during early spermato-

genesis, in phase II of the seminiferous epithelium cycle. The ductuli efferentes of the efferent ducts have not spermatozoa. Stromaof connective tissue surrounds the efferent ducts. C. Epididymis and sexual segment of the kidney, during late spermatogenesis inphase VI of the seminiferous epithelium cycle. The ductuli efferentes have not spermatozoa, and the ductus epididymis containsabundant spermatozoa. The testis is observed near the epididymis. D. Detail of Fig. 8C. The ductuli efferentes have a cuboidalepithelium with large stereocilia in the apical end. The stroma surrounds the ductuli efferentes. E. Detail of Fig. 8C. The ductusepididymis has a large columnar epithelium and contains abundant spermatozoa. The stroma (S) surrounds the ductus epididymis.Ductuli efferentes (dep), ductus epididymis (Dep), epididymis (E), sexual segment of the kidney (SK), spermatozoa (Sz), stereocilia(arrowhead), stroma (S), testis (T). Bars 5 A: 500lm. B: 100lm. C: 500lm. D: 10lm. E: 20lm.




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cells, and the lumen became completely occluded.Similar reptilian spermatogenic seasonality wasdocumented primarily, in temperate species, and itwas indicated that spermiation occurred duringthe warmer months of the year (Licht, 1984;

Aldridge et al., 1990; Gribbins et al., 2009). Mean-

while, spermatogenesis in some tropical specieshas been described both as a continuous (Licht,1984; Hernandez-Gallegos et al., 2002; Ferreiraet al., 2009; Gribbins et al., 2009), or as seasonal;if seasonal it was triggered either by the rainy sea-son or the photoperiod (Fitch, 1970; Licht, 1984;Guillette and Casas-Andreu, 1987; Villagran-SantaCruz et al., 1994; Ramrez-Bautista et al., 2006;Ferreira et al., 2009), a similar pattern to thatdescribed in temperate species.

The seasonal morphological changes of the testisin M. brachypoda coincided with those reported forovarian seasonality of this species (Hernandez-

Franyutti et al., 2005). According to this report,females were in early vitellogenesis during Maythrough July, and ovulation occurred during Julythrough August. The concurrence of gametogenesisbetween males and females of M. brachypoda indi-cates synchrony in the reproductive cycles of bothsexes. The synchrony of gametogenic cycles ofmales and females was seen in other species of thegenus Mabuya, as M. heathi (Vitt and Blackburn,1983), and M. capensis (Flemming, 1994).

The histological analysis of testes and epididy-mis of M. brachypoda evidences that the malesreached the maximum level of spermatogenic ac-tivity, before mating, corresponding to a prenuptial

pattern, as it was described by Saint-Girons (1982)and Licht (1984). Other lizards were documentedas having prenuptial spermatogenesis, including

Barisia imbricata (Guillette and Casas-Andreu,1987) and M. capensis (Flemming, 1994). Lizards,usually have a prenuptial pattern of reproduction,in contrast, multiples species of snakes as Tham-nophis sirtalis parietalis (Krohmer et al., 1987),Opheodrys aestivus (Aldridge et al., 1990), andSeminatrix pygaea (Gribbins, 2010), have a post-nuptial pattern, as the spermatozoa are producedafter the completion of the mating season andstored in the male ducts until the following matingseason. In the prenuptial pattern, both events,

spermatogenesis and mating, are initiated by thesame hormonal condition, called the associatedpattern (Whittier and Crews, 1987). In the post-nuptial pattern, spermatogenesis and mating aretemporally separated, occurring at different timesof the year. Separate hormonal patterns may exist,and this is known as dissociated pattern ofreproduction (Whittier and Crews, 1987).

The observation of the VIII phases of the annualspermatogenic cycle in the seminiferous epitheliumof M. brachypoda, based in the classification usedby Mayhew and Wright (1970), indicated the sea-

sonality of the testicular cycle. Spermatogenic ac-tivity in M. brachypoda, wherein all of the seminif-erous tubules have similar stages of spermatogene-sis, in a chronological uniformity, during theannual reproductive cycle, coincides with that ofseveral species of reptiles, such as lizards (Me ndez-

de la Cruz et al., 1988; Villagran-Santa Cruz et al.,1994; Ochotorena et al., 2005; Gribbins et al., 2007;Gribbins, 2010; Rheubert et al., 2009a,2009b), aturtle (Gribbins et al., 2003; Gribbins, 2010), alliga-tors (Gribbins et al., 2006) and a snake (Gribbinset al., 2008; Gribbins, 2010). In all of these species,there is a synchronous development of a singlepopulation of germ cells during spermatogenesisfollowed by a single spermiation event.

Gribbins (2010, 2011) compared the spermato-genesis in several reptilian sauropsids consideringthat the spermatogenic strategy in reptiles, releas-ing sperm in a single event, is reminiscent to thatdescribed in anamniotic taxa (fishes and amphib-

ians). The testis of fishes (Grier and Uribe, 2009),and amphibians (Uribe et al., 1994; Ogielska andBartmanska, 2009; Uribe, 2009), contains tubulesor lobules where the germinal cells develop in acyst or spermatocyst. The cysts are formed whenspermatogonia are enclosed by Sertoli cells. Withinthe cysts, germ cells undergo the events of mitosis,meiosis and spermiogenesis as a single cohort.When spermiation occurs, the cysts open andrelease spermatozoa into the tubules or lobules,from where they travel to the deferent ducts. Incontrast, in all amniotic taxa (sauropsids, birdsand mammals), the testes contain seminiferoustubules that are lined by permanent, seminiferous

epithelium formed by Sertoli cells and germ cells,and the germ cells are not within spermatocysts.

As described in several species of reptilian saurop-sids by Gribbins (2010, 2011), the testes ofM. bra-chypoda have Sertoli cells and spermatogonia as apermanent seminiferous epithelium, throughoutthe year. During spermatogenesis, the germ cellscommence meiosis while moving into the seminif-erous epithelium from the base to the lumen andare subsequently released into the lumen at sper-miation. During the reproductive season, in bothbirds and mammals, the seminiferous epitheliumcontains Sertoli cells associated with layers ofgerm cells. The germ cells are in different stages

of spermatogenesis (Leblond and Clermont, 1952;Hess and Franca, 2008). Unlike reptilian saurop-sids, which have synchronous spermatogenesisalong the lengths of tubules, in birds and mam-mals there is a successive order of spermatogenicphases along the seminiferous epithelium whichoccurs in a wave of cyclic cell division and spermdevelopment (Lin and Jones, 1990; Hess andFranca, 2008). Leblond and Clermont (1952) recog-nized in the rat the cellular associations that occuralong the seminiferous epithelium during the sper-matogenic phases. Later, the seminiferous epithe-




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lium cycle was studied in several species of mam-mals, such as mouse, hamster, gerbil, guinea pig,ram, monkey (Clermont, 1972), domestic cat(Franca and Godinho, 2003), grey squirrel (Taitand Johnson, 1982), and in birds as the Japanesequail (Lin and Jones, 1990; Jones and Lin, 1993).

Jones and Lin (1993) concluded that in birds thereis a well-defined cycle of the seminiferous epithe-lium, and spermatogenesis involves the activitiesof germ cells within and between successive gener-ations to produce cellular associations of the semi-niferous epithelium, which are essentially similarto those described in mammals. Therefore, as itwas reviewed by Gribbins (2011), the functionalstrategy for germ cell development in reptiliansauropsids is synchronized within the seminiferousepithelium along the lengths of the tubules, andthere is a single spermiation event. As a conse-quence, reptiles represent a dynamic of spermato-genesis between the anamniotes fishes and

amphibians, and the amniotes birds and mam-mals.

Several studies described annual changes in theepididymis of reptilian sauropsids (Mayhew andWright, 1970; Dufaure and Saint-Girons, 1984;Ravet et al., 1987; Mesure et al., 1991; Ferreiraet al., 2009; Sever, 2010; Rheubert et al., 2010),coinciding with our observations in M. brachypoda.These studies correlate the cyclic changes in thetestes with those seen in the epididymis and its se-cretory activity, in particular, in the ductus epididy-mis. Sever (2010) considers that, during the repro-ductive cycle, the functions of these ducts includereabsorption of luminal fluid and active secretion.

Rheubert et al. (2010) documented the presence ofglycoproteins in the secretions of the ductus epidi-dymis in the Gecko (Hemidactylus turcicus), andsuggested that the function of the glycoproteins inthe epididymal secretions perhaps produce an envi-ronment for sperm storage and/or maturation.Mesure et al. (1991), studying the epididymis of thelizard Lacerta vivipara, suggested that these secre-tions are discharged into the lumen of the ductusepididymis where they bind to the heads of thespermatozoa. Ferreira et al. (2009) and Sever (2010)consider the importance of analyzing the morpho-logical changes of the epididymis and its secretionsin the interpretation of its role in the maturation of

the spermatozoa. The sexual segment of the kidneyis a distal portion of the nephron of squamates, andis observed near the testis and epididymis. The sex-ual segment of the kidney is believed to provideseminal fluid that mixes in the ureter with sperm(Sever et al., 2002; Sever and Hopkins, 2005).

Thus, spermatogenesis, including the cell typesand the dynamics of the seminiferous epitheliumcycle and features of the duct system in reptiles,provide essential aspects for the understandingof the evolution of male reproduction in verte-brates.

ACKNOWLEDGMENTS

The authors thank Marcela E. Aguilar-Moralesand Adriana Garca-Alarcon for help in histologicalprocessing, Ana Isabel Bieler Antolin and GabinoDe la Rosa Cruz for the kind technical assistancewith digital photography; and Jose Antonio Her-

nandez Gomez for the excellent digital preparationof histological figures and Edgar Abraham LozanoMendoza and Ulises HernA ndez Vidal for the as-sistance in the preparation of the Supporting In-formation Graph.

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