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Spermatogenesis. 2011 Jul-Sep; 1(3): 250–269.
Published online Jul 1, 2011. doi:  10.4161/spmg.1.3.18092
PMCID: PMC3271667

Reptilian spermatogenesis

A histological and ultrastructural perspective

Abstract

Until recently, the histology and ultrastructural events of spermatogenesis in reptiles were relatively unknown. Most of the available morphological information focuses on specific stages of spermatogenesis, spermiogenesis, and/or of the mature spermatozoa. No study to date has provided complete ultrastructural information on the early events of spermatogenesis, proliferation and meiosis in class Reptilia. Furthermore, no comprehensive data set exists that describes the ultrastructure of the entire ontogenic progression of germ cells through the phases of reptilian spermatogenesis (mitosis, meiosis and spermiogenesis). The purpose of this review is to provide an ultrastructural and histological atlas of spermatogenesis in reptiles. The morphological details provided here are the first of their kind and can hopefully provide histological information on spermatogenesis that can be compared to that already known for anamniotes (fish and amphibians), birds and mammals. The data supplied in this review will provide a basic model that can be utilized for the study of sperm development in other reptiles. The use of such an atlas will hopefully stimulate more interest in collecting histological and ultrastructural data sets on spermatogenesis that may play important roles in future nontraditional phylogenetic analyses and histopathological studies in reptiles.

Key words: spermatogenesis, reptile, testis, spermiogenesis, cell cycle, seminiferous epithelium, spermatogonia, spermatocyte, spermatid, ultrastructure

Introduction

Spermatogenesis is a complex process in which progenitor cells (spermatogonia) mitotically divide, undergo meiosis, and then differentiate into one of the few motile cells, spermatozoa, within the male vertebrate body. It is not surprising that a multitude of books and reviews have been devoted to this topic.15 These reviews concern birds and mammals in terms of amniotic sperm development. Only one review has looked specifically at the major steps of spermatogenesis (light and electron microscopy) in a reptilian taxon, the Ophidia (snakes), but even here the ultrastructure is provided only for spermiogenesis and the mature spermatozoon.6 The ultrastucture of the early stages of spermatogenesis have not been explored in the Reptilia. Thus, this original review provides the first complete account of the entire process of spermatogenesis in a morphological context that includes ultrastructure of all the major generational types (stages and steps) of sperm development in reptilies. The class Reptilia is a major taxon within Amniota and in the context of this review includes: Chelonia, Squamata, Crocodylia, Rhynchocephalia, thus excluding the class Aves paraphyletically from the archosaurs.

The last decade has produced a large number of manuscripts that focus on the light and electron microscopy of the process, progression, morphology, germ cell cycle, and developmental strategies of spermatogenesis within the class Reptilia (for example: spermatogenesis in the turtle, Trachemys scripta and Ophidia6,7). Spermatogenesis is required of all vertebrate males, whose main means of passing on genetic material is via sexual reproduction. In amniotes (reptiles, birds and mammals), a shift in testicular architecture and germ cell arrangement occurred within the seminiferous tubules and epithelia that are morphologically unique to Amniota (Fig. 1) among vertebrates.6 The significance of which is most likely associated evolutionarily with the adaptations of the amniotic egg and complete reliance of amniotes on internal fertilization, which frees them completely from water for breeding.8,9 The differences morphologically and ultrastructurally within the germ cell development cycle, spermiogenesis, and the spermatozoa between both Reptilia and other amniotic clades may in time provide powerful nontraditional information that could be important in understanding the phylogenetics within reptiles, which at best is considered highly controversial at the present time.10

Figure 1
(A) Sagittal section of entire testis and anterior excurrent ducts of Carphophis vermis (Western Worm Snake). Bar = 0.2 cm. Testis (T), efferent ducts (black arrowhead), epididymis (black arrows). (B) Low power view of seminiferous tubules in cross and ...

Seminiferous Epithelium Organization and Germ Cell Development Strategy

The anamniotic (fish and amphibians) testis consists of lobules or tubules that are lined with cysts (Fig. 2) where germ cells progress as a single population through the main stages of spermatogenesis: proliferation (mitosis), meiosis and spermiogenesis.1114 Once the population of germ cells has completed maturation, they undergo spermiation and are released from the seminiferous epithelium as spermatozoa.15,16 In order for this to occur, cysts rupture and sperm are often shed as a unit, which in many cases compromises Sertoli cell integrity and the blood testis barrier that is often seasonal in temperate and some tropical anamniotic species.17,18

Figure 2
(A) Transverse section through a seminiferous tubule in Notophthalmus viridescens (Red-spotted Newt). The tubule has multiple cysts (C) resting on a prominent basal lamina (black arrow). Sertoli cell processes (black arrowheads) enclose the entire population ...

Reptiles have a permanent population of Sertoli cells and a more stable blood testis barrier than anamniotic taxa.6,17,1921 With this morphological shift in the amniotic seminiferous epithelia, the germ cells are also arranged differently than within the cysts of fish and amphibians. Within all amniotes, the testis is composed of germ cell generations that are associated with the Sertoli cells (Fig. 3) and are arranged in a consistent pattern between the basal lamina and the lumen of the seminiferous epithelium (Fig. 4). Reptiles also appear to have a regular histological arrangement of germ cell generations (Figs. 3 and and44) within their seminiferous epithelia similar to mammals. No study until the beginning of this decade had determined that reptiles lacked a consistent spatial germ cell development like the birds and mammals. Birds and mammals always have the same spermatogonial and spermatocyte generations associated with each of the step spermatids within the seminiferous epithelium.2225 The term step is adopted from the mammalian literature.26 Spermatid steps refers to each morphological transition during spermiogenesis within the testis of a species (i.e., Fig. 8, steps 1–7). This spatial arrangement only allows for 3 to sometimes 4 spermatids to be observed within a cross section of seminiferous tubule within the testes of birds and mammals.26 Studies devoted to the germ cell development strategy in reptiles representing different orders and exhibiting different seasonal patterns have now shown that reptiles no matter their seasonal cycle type exhibit a germ cell development very similar to that of amphibians and not like the consistent spatial development seen in other amniotes.7,2732

Figure 3
Light (A) and low power electron (B) micrographs depicting the seminiferous epithelia within Agkistrodon contortrix (northern Copperhead Snake) and Thamnophis sauritus (Garter Snake) testes. Sertoli cells sit on a prominent boundary layer (black arrows) ...
Figure 4
A cross section of June seminiferous epithelium within Seminatrix pygaea (Black Swamp Snake) and higher magnifications of represented cell types. Bar = 50 µm for seminiferous epithelium section and 10 µm for magnified germ cell types. ...
Figure 8
Germ cell types found within the seminiferous epithelia of testes of Seminatrix pygaea (Black Swamp Snake). Bar = 15 µm. Type A spermatogonia (SpA), type B spermatogonia (SpB), pre-leptotene spermatocytes (PL), leptotene spermatocytes (LP), zygotene ...

In reptiles, the majority of germ cell generations progress through the stages of spermatogenesis as a single population (Fig. 5). The mitotic and meiotic cells are restricted to the basal compartment (close to the basement membrane) and dividing spermatogonia are always present, except in turtles late in the summer/fall and winter when only resting spermatogonia are present and are quiescent.7 Meiosis is also consistent during active spermatogenesis and large populations or cohorts of cells progress through meiosis together and then enter spermiogenesis as large populations (example: Fig. 5: Germ cell development in the turtle, Trachemys scripta, Fig. 6 in the snake, Agkistrodon piscivorus).

Figure 5
The appearance and duration of time that germ cell types are seen within the seminiferous epithelium of the Red-eared Slider Turtle, Trachemys scripta. Resting, resting spermatogonia; Sp. A and B spermatogonia A and B; Pre-Lept., preleptotene spermatocyte; ...
Figure 6
Specific monthly samples of seminiferous epithelia within the testis of Agkistrodon contortrix (northern Copperhead Snake). Bars = 20 µm. (A) May epithelium showing high lipid (white arrowheads) content and the following cell types: spermatogonia ...

During spermiogenesis, large cohorts of cells develop together through acrosome development, elongation and maturation (Fig. 6B and C) and the seminiferous epithelium expands into large columns that are separated by deep luminal crypts (Fig. 3A and B). These columns accumulate many sequential generations of spermatids at one time within the seminiferous epithelium. Five to six different spermatids can be observed within the seminiferous epithelium at the same time, which is much different than the usual three found consistently within birds and mammals. The elongating steps of spermiogenesis accumulate apically within cellular processes of Sertoli cells. The accumulation of these elongates that are finishing spermiogenesis are then released in a large single spermiation event that leads to dilations of the lumina and increased sizes or diameters of the seminiferous tubules (Fig. 6B and C). In seasonal breeders, such as those reptiles found in temperate environments, once this single major spermiation event occurs in the summer or late fall, the testis enters a quiescent period where spermatogenesis slows or ceases altogether. The histology of the seminiferous tubules at this time shows reduced epithelia with typically only spermatogonia present (Fig. 6D). Within the lumina of the seminiferous tubules are pieces of the Sertoli cells with various generations of germ cells that have been sloughed from the epithelia and are thought to be recycled and reabsorbed along the length of the reproductive tract.

In continuous breeding reptiles, such as Sceloporus bicanthalis33 and Anolis lineatopus31 (Fig. 7), once a cycle of spermatogenesis is complete the basal compartment shows an increased rate of mitosis and meiosis, which amplifies the seminiferous epithelial height. These new generations of germ cells then enter spermiogenesis and spermiation again as a single wave or cohort. These steps are repeated over and over again leading to bimonthly sequential increases and decreases of the seminiferous epithelium and tubule diameter (Fig. 7). Notice from Figure 7 that when the diameter is the widest in this bimonthly cycle the epithelium height is the lowest. Thus, when the seminiferous tubule diameter width is high then spermiation has occurred increasing the size of the lumen and tubule overall. In contrast, the spermiation event has caused a loss of germ cells and Sertoli cell columns therefore decreasing the overall seminiferous epithelial height.

Figure 7
Top: Variation in seminiferous tubule diameter across months of the year in the testes of Sceloporus bicanthalis (Trans Volcanic Bunchgrass Lizard). Values represented on this graph are means ± 1 standard error. A and B on the graph indicate significant ...

Understanding the morphology and progression of germ cells within the seminiferous epithelium of reptiles might provide better insight on how the reptilian testis functions during seasonal cycles. For example, endocrinologists know that in mammals FSH (Follicle Stimulating Hormone) stimulates type A spermatogonial division during early spermatogenesis.34 FSH has been found in reptiles,35,36 and all present data suggest that it causes recrudescence of spermatogenesis in temperate reptiles either before or after mating.37,38 However, it is not known whether type A spermatogonia with the ability to divide are present within the seminiferous epithelium at all times of the year or whether the population of dividing spermatogonia is temporal (may be responsible for refractory period for example, see ref. 39) and only available for FSH activation during certain times of the reptilian spermatogenic cycle. Understanding the basic cytological events of spermatogenesis in reptiles may help resolve the existing controversies and inconsistencies with regard to refractory periods, temperature effects, and hormonal stimulation within the testicular cycles of reptiles. Furthermore, since a temporal germ cell development strategy is employed by all major taxa of reptiles studied to date, they may provide a feasible model for histopathological studies of the testis in response to pesticides or heavy metals. The simplified germ cell development strategy allows for easier observation of germ cell types because they all progress together through the stages of spermatogenesis. Thus, morphological abnormalities, morphometric differences, and absences or overabundance of germ cell types can potentially be discernable in reptilian models.

Germ Cell Cycle and Germ Cell Morphologies during Spermatogenesis

Spermatogenesis is the process of sperm development and begins with spermatogonia that divide, undergo meiosis, and then differentiate into spermatozoa. Sperm development is typically broken down into three phases based upon functional and morphological criteria.26

(1) The proliferative phase involves the spermatogonia, which undergo rapid successive incomplete (cytoplasmic) mitotic divisions that lead to a large clonal population of germ cells that will allow the testis to produce immense numbers of spermatozoa. (2) The meiotic phase includes the spermatocytes, which undergo the reduction divisions, segregation and recombination of genetic material. The final aspect of spermatogenesis is the maturational or spermiogenic phase, (3) where haploid undifferentiated spermatids transform into specialized hydrodynamic sperm cells equipped and capable of reaching and fertilizing eggs. Germ cells, particularly the mitotic and meiotic cell types, within these phases have very specific morphologies that are highly conserved among vertebrates.1,2 Russell et al.26 provides a thorough review of the process of spermatogenesis and the morphology of the individual germ cell types for mammals. Once all germ cell morphologies for each phase of spermatogenesis are known, then a germ cell cycle map can be produced that allows the visualization of the transformation of spermatogonia to mature spermatozoa (Fig. 8). Because of the conserved nature of especially the morphologies of the earlier germ cells of spermatogenesis (Fig. 9), within this review only one representative example will be provided for each cell type to prevent redundancy. However, within spermiogenesis, two major morphological themes play out within reptiles, which will be equally represented within the figures provided here. Also, it should be noted that all cell types were taken at the same magnification (for both light and electron microscopy) whenever possible throughout this manuscript unless stated within the figure legend. Thus, scale bars that represent magnifications for all the micrographs within a figure are often provided for just one of the sets of images within a plate. At all times, caution should be taken when interpreting the information provided in this review, although the figures represent the normal morphologies of cell types, differences within reptiles may exist, especially among species not studied to date.

Figure 9
Spermatogonia representatives from the alligator (Alligator mississippiensis), lizard (Podarcis muralis) and snake (Seminatrix pygaea) at the light and electron microscope level. Notice how similar is morphology the cell types are at both levels of microscopy. ...

Proliferative stage: spermatogonia morphology.

Regardless of seasonal cycle (i.e., prenuptial, postnuptial, mixed) type within reptiles, all species produce large numbers of cells in the beginning of each spermatogenic cycle, this is true of most amniotes studied to date.26 One of the major functions of the spermatogonia is to produce a large population of clonal germ cells that can then enter the other phases of spermatogenesis. Since large numbers of spermatozoa are typically needed for successful fertilization,1,2 the rapid and successive division of the spermatogonia population fulfils this requirement.

Three major types of spermatogonia can be recognized within the reptilian seminiferous epithelium. Spermatogonia are typically restricted to the basal compartment within the seminiferous epithelium in close association to the basement membrane of the Sertoli cells (Fig. 3, Sp). The resting spermatogonia (Fig. 10A) have only been described to date in the turtle, Trachemys scripta.7 Spermatogonia of similar morphology have been observed from other histological studies of turtles (Gribbins KM, unpublished data). All temperate turtles studied to date are considered postnuptial in the seasonal pattern of spermatogenesis.39 This means that turtles start spermatogenesis after spring mating and spermiation occurs in the late summer or early fall. Most temperate snakes are also postnuptial and only one postnuptial snake has complete spermatogenic data (light microscopy) that details the germ cell cycle.29 In this study, no resting spermatogonia were reported. However, upon re-examination for the present review, resting spermatogonia are actually observed in October testes within the snake, Seminatrix pygaea, similar in morphology to that described here for Trachemys scripta (Fig. 10A). Thus, to date, resting spermatogonia have only been observed within postnuptial breeding reptiles, where an extended quiescent period may be necessary during late summer to prevent another round of spermatogenesis from progressing into the late fall or winter months.7

Figure 10
(A) Resting spermatogonia from the seminiferous epithelium of the Red-eared Slider Turtle (Trachemys scripta). Note tight dark staining heterochromatin and lack of organelles cytoplasmically. (B) Spermatogonia A from the seminiferous epithelium of the ...

Resting spermatogonia have dark staining chromatin that are tightly packed within the nuclei and lack visible nucleoli (Fig. 10A). They also have few to no major organelles present within their cytoplasms except for mitochondria. The function of these spermatogonial cells is unknown, as little is understood of the mechanics behind division and differentiation of spermatogonia in reptilian testes. We hypothesize that they are stem cells with similar function to those types of spermatogonia described in mammals. Stem cell spermatogonia in mammals have been reported to be resistant to stress, damage, and are often non-dividing.26,40,41 Resting spermatogonia in reptiles have tightly packed DNA and thus might be more resistant to damage because the availability of cleavage sites of the DNA in this condensed form is restricted. Furthermore, resting spermatogonia are rarely seen dividing in the testes of turtles and snakes. Reptilian resting spermatogonia most likely provide a resilient population of stem cells that can survive insult and hibernation to be utilized in the following early/late spring, early summer months to repopulate the spermatogonia populations within the testes of temperate snakes and turtles.

The second type of spermatogonia seen within the testis of reptiles is the type A spermatogonia (Fig. 10B). Spermatogonia A in reptiles are very similar in function and morphology to those described in mammals (Apaired and Aaligned type spermatogonia).26 These spermatogonia are known as proliferative spermatogonia in mammals and within reptiles provide much of the mitotic power of the seminiferous epithelium. They also are typically found juxtapositioned to the basement membrane within the basal compartment of the reptilian seminiferous epithelium. However, type A and B (described next) spermatogonia can produce several layers of spermatogonia that extend out into the interior of the seminiferous epithelium during the early divisions of spermatogenesis in reptiles. Type A spermatogonia are ovoid in shape with typically one cell membrane surface resting on the basement membrane of the seminiferous epithelium. Their chromatin is diffuse except next to the inner leaflet of the nuclear envelope and nucleoli are present. The organelles responsible for protein production are also present within the cytoplasm, which suggests these cells are anabolically active. Spermatogonia A are the most abundant spermatogonia seen within the reptiles studied to date.6

Type B spermatogonia are the last type of spermatogonia observed within the seminiferous epithelium of reptiles (Fig. 11A). They are similar to the differentiating spermatogonia (A1,2,3,4, B and Intermediate types) illustrated within the mammalian testis.26,42 Spermatogonia B have more heterochromatin within the nucleoplasm and associated with the inner nuclear envelope compared to spermatogonia A. They are also more round compared to the ovoid-shaped spermatogonia A. Spermatogonia B still possess the nucleoli and organelle machinery responsible for protein production and are thus anabolically active cells. They in some case have much more rough ER and larger Golgi complexes to that of other spermatogonia in reptiles.

Figure 11
Spermatogonia B (A) and preleptotene spermatocyte (B) from the seminiferous epithelium of the Black Swamp Snake (Seminatrix pygaea). Notice that both cell types' nuclei are more round that speramtogonia A and they have very similar morphology except for ...

Meiotic stage: spermatocyte morphology.

The spermatogonia B divide to form the first cell type of meiosis. Prophase 1 of meiosis is exceptionally long in reptiles (roughly 4 weeks) like that of other vertebrates.26 There are gradual morphological transitions that are easily recognizable with histological practice during this prolonged phase of meiosis (Fig. 8: PL-DI). A common theme among all reptiles and other amniotes is that spermatocyte size increases dramatically during prophase 1. Thus, the largest and most recognizable germ cells within the seminiferous epithelium are the terminating spermatocytes of prophase 1. This elongated prophase allows plenty of time for recombination of paired chromosomes to occur before the first nuclear division of meiosis. The morphology of pre-leptotene spermatocytes is very similar to spermatogonia B. The diagnostic character that separates the two cell types is size and location. Pre-leptotene spermatocytes (Fig. 11B) typically have less heterchromatin and are smaller than B type spermatogonia. Pre-leptotene cells are not only roughly 30% smaller than spermatogonia B but are also found further away from the basement membrane. Rarely do pre-leptotene cells make contact with the basement membrane of the seminiferous epithelium in reptiles.

Leptotene spermatocytes represent the true beginning of prophase. Heterochromatin common in the pre-leptotene phase has now disappeared and when underexposed the “wispy” threads of the condensing chromosomes can be visualized (Fig. 12A and black arrows). The chromosomes however are not yet paired into homologs. Zygotene spermatocytes are often hard to differentiate from leptotene cells especially via the light microscope. The only real differences are a slight size increase in zygotene cells and a thickening of chromosomal fibers. The continued condensation and repackaging of the chromosomes during the zygotene phase of meiosis leads to the initial pairing of homologous chromosomes into tetrads and the formation of synaptonemal complexes seen only by the electron microscope (Fig. 12B and black arrows). Since the homologous chromosomes are now paired along most of their length, the condensing chromosomes appear thicker in light microscope sections of zygotene spermatocytes.

Figure 12
(A) Leptotene spermatocyte within the testis of the Black Swamp Snake (Seminatrix pygaea). The nucleus and the entire cell is now round as it has traveled away from the basal lamina. Linear densities (beginning of chromosome condensation) (black arrows) ...

Pachytene spermatocytes have fully paired homologous chromosomes with very thick chromosomal fibers and large areas of open nucleoplasm separating the chromosome clumps when observed via the light microscope (Fig. 13A). Pachytene cells are also the most abundant and longest phase of prophase 1 in reptiles and most amniotes studied to date. Synaptonemal complexes are abundant and robust within the thinning nuclear membrane of pachytene cells (Fig. 13A and black arrows). These structures are where the actual crossing over events occur within regions called chiasmata between the homologs of the tetrads. This pairing apparatus appears as an elongated tripartite (two thick lateral elements and one thin central element) structure that is only recognizable by the electron microscope (Fig. 13A and inset). The arms of the chromosomes can also be easily distinguished radiating out from the synapses. The time germ cells spend in the pachytene phase lends itself to the importance of genetic recombination and its significance to genetic diversity and reproductive success within species of vertebrates. Pachytene spermatocytes within the reptilian testis, like that of mammals,43,44 become highly synthetic toward the middle and end of their tenure, which lends to their great size. From the ultrastructure, one can see the enlarged nucleoli and tremendous amount of rough endoplasmic reticula. However, unlike mammals, a prominent sex vesicle26 (represents the sex chromatin) that is juxtapositioned to the inner leaflet of the nuclear envelope does not arise in reptilian pachytene cells in the species studied to date.

Figure 13
(A) A pachytene spermatocyte in the Cottonmouth Snake (Agkistrodon piscivorus) testis. Completed synaptonemal complexes (black arrows) can be seen in cross sections of the nucleus. These complexes are a tripartite structure with two lateral thick components ...

The diplotene phase (Fig. 13B) of meiosis 1 in reptiles is very brief as are metaphase 1 and the rest of the division stages of the 1st and 2nd parts of meiosis. Typically cross-sections of seminiferous tubules will show the diplotene, metaphase 1 (Fig. 14A), metaphase 2 (Fig. 14A), and secondary spermatocytes (Figs. 8 and 14B) all in the same area of seminiferous epithelium. In this phase, synaptonemal complexes dissipate and chromosomal condensation and packaging is nearly complete. Like mammals,26,45 reptilian diplotene cells possess an electron dense body with a central clear area when observed under the electron and light microscopes (Fig. 13B and black arrows). Diplotene spermatocytes are also easier to recognize in cross section in reptiles than within mammals because condensed chromosomes under the light microscope appear near the inner nuclear membrane resulting in a spoke-like pattern within the diplotene nucleus. Diplotene cells progress into metaphase, anaphase and telophase very rapidly. Usually all of these phases except metaphase (Fig. 14A) are hard to differentiate between one another and in most cases are absent in cross-sections of seminiferous tubules in reptiles. The completion of the reduction division of meiosis results in the formation of secondary spermatocytes (Fig. 14B).

Figure 14
(A) Electron and light micrographs of meiosis metaphase 1 in the testis of the Northern Copperhead Snake (Agkistrodon contortrix). Note the thick spindle fiber microtubules (white arrows) and the aligned chromosomes (black arrow). The light micrographs ...

Secondary spermatocytes (Fig. 14B) are also very short lived during spermatogenesis in reptiles. Like mammals, they are hard to discern from the first step of spermiogenesis and are very rare within the seminiferous epithelium. Secondary spermatocytes are about 30% to 40% larger than step 1 spermatids (Fig. 14B and inset) and have a bit more heterochromatin and very prominent nucleoli. A feature that both cell types share is a prominent form of nuage called the chromatoid body, a feature they also share with mammals.26 Nuage is defined as a prominent inclusion that is amorphic and found within the cytoplasm of spermatids during spermiogenesis.4547 The form of this material can be variable. In many cases it is electron dense and granular (Fig. 14B and black arrows) or can be more translucent and less dense (Fig. 14B and inset: white arrowhead). Nuage should not be confused with structures that resemble intracellular residual bodies26 (Fig. 14B and inset; white arrow; Fig. 23, turtle, black arrowhead) or deteriorating membrane bound organelles. Another ultrastructural characteristic of secondary spermatocytes are their vacuolated mitochondria (Fig. 14B and black arrowheads). Metaphase 2 cells can be differentiated from metaphase 1 cells based on size and amount of heterochromatic material located within the cytoplasm. Metaphase 2 cells are smaller and have about half the amount of heterochromatin (Fig. 14A) compared to metaphase 1 germ cells. This size difference in cytoplasm and genetic material portrays nicely the reduction division and the production of haploid cells.

Figure 23
Turtle (Trachemys scripta): Step 7 spermatid flagellar morphology. The mitochondria (mi) of the midpiece have conspicuous outer concentric layers of membrane (cristae) and are large and round compared to the elongated mitochondria of the snake and lizard. ...

Spermiogenic stage: spermatid morphology.

The resulting haploid and undifferentiated round step 1 spermatid within the reptilian testes undergoes one of the most dramatic changes of any cell type within the adult vertebrate body. This process takes about 5 to 8 weeks in reptiles (Fig. 5), which is longer than the typical 4 weeks observed in most mammals studied to date.26 This transformation occurs without cell division and several cytological events occur at the same time during spermiogenesis; it is easier to concentrate on one histological process at a time. In order to simplify what is occurring within spermatids, three major morphogenic themes will be presented in this review for spermiogenesis: acrosome development, elongation of the nucleus/condensation of DNA, and development of the flagellum. The end result of these three morphological courses is a highly hydrodynamic motile cell, the spermatozoon, which has three major structures: the acrosome complex, the nucleus, and the flagellum (Fig. 15).

Figure 15
A sagittal section through the head and proximal flagellum of a spermatozoon from the lumen of a seminiferous tubule of the American Alligator (Alligator mississippiensis). The major parts of the spermatozoa in reptiles and other amniotes include the ...

Even though spermiogenesis is historically broken down into distinct morphological steps,1,26 it is best to think of the process as a continual progression or transition and that spermatids can easily be observed between stages or what some have called early and late steps of spermiogenesis.26 Thus, cycle maps and spermatid step morphological descriptions for a given species are not set in stone. The number of spermatid steps varies among mammalian species, for example: 19 in the rat,5,46 16 in the mouse,5,48 12 in the dog.42 In contrast, reptiles are more consistent in the number of ontogenic steps required to complete spermiogenesis. All the species studied to date have 7 or 8 steps (Fig. 8, s1–s7) and include reptiles representing multiple orders within class Reptilia.7,2731,33

Detailed ultrastuctural studies of the complete process of spermiogenesis within the Reptilia are rare. Many studies have incomplete descriptions of different aspects of spermiogenesis within the testis of reptiles. They are too numerous to list here and the concentration of this review is on complete descriptions of spermiogenesis in order to better understand spermatogenesis as a whole morphological mechanism within reptiles. Comprehensive studies of spermiogenesis, as well as the morphology of the spermatozoa have been shown to be important data as non-traditional characters used in phylogenetic research of Reptilia over the last decade (for example, see references 4951). In Lepidosauria (snakes, lizards and tuataras), only 8 studies have entire ultrastructural descriptions of spermiogenesis: the skink, Chalcides ocellatus;52 the tuatara, Sphenodon punctatus;53 the lizard, Tropidurus torquatus;54 the iguana, Iguana iguana;55 the skink, Scincella lateralis;56 the snake, Agkistrodon piscivorus;57 the anole, Anolis lineatopus;58 and the Gecko, Hemidactylus turcicus.59 Even less information on spermiogenesis exists in turtles and crocodilians. Four studies that provide entire ultrastructure details of spermatid development occur in the turtles Trachemys scripta60 and Pelodiscus sinensis,61 and in the crocodilians Caiman crocodylus62 and Alligator mississippiensis.63

The round spermatids (Fig. 8, S1–S4) are uniformly consistent in morphology and development across Reptilia. They along with all other steps of spermiogenesis develop within the columns of the apical regions of the seminiferous epithelia (Figs. 3 and and44) closest to the lumina of the seminiferous tubules. The major events of acrosome formation occur during the tenure of the round spermatid stage. Step 1 spermatids (Fig. 16) are the result of the 2nd division of meiosis. They are not only smaller than secondary spermatocytes but also have an enlarged Golgi complex (Fig. 16 and black arrows) and typically a small developing acrosome vesicle (called the proacrosome in mammals64) (Fig. 16A and B, white arrows) that does not made contact with the nucleus. As Step 1 spermatids progress through their development this vesicle grows in size and makes contact with the apex of the nucleus (Fig. 16A and C). The size of the developing acrosome increases due to membrane and vesicular materials (Fig. 16B and black arrowhead) gained from transport vesicles (Fig. 16C and white arrowheads) that originate from the Golgi complex. In all reptiles studied to date (reviewed in ref. 6) only one Golgi complex is associated with the developing acrosome, except in the snake, Agkistrodon controtrix, which has two complexes aiding the development of the acrosome (Fig. 16D and black arrows). The significance of possessing two Golgi complexes is not known but this morphological setup does allow for early formation and observation of the acrosome granule (Fig. 16D and white*). Of course Scincella lateralis56 has the earliest developing acrosome granule in the reptiles studied to date. This lizard's granule within the acrosome vesicle develops before the vesicle even contacts the nucleus, and this species only has one Golgi complex. Thus, a species of reptile does not need two Golgi complexes to acquire an early developing acrosome granule. Also note, as stated earlier, that because of incomplete mitotic divisions during proliferation all developing cohorts of germ cells within the reptilian testis are connected via cytoplasmic bridges (Fig. 16A and black*). These bridges are thought to allow for cytoplasmic sharing and are most likely involved with coordinated communication that controls the simultaneous development of the reptilian temporal germ cell development strategy.6

Figure 16
(A) Light and electron micrographs of step 1 spermatids from the testes of the American Alligator (Alligator mississippiensis) and the Cottonmouth Snake (Agkistrodon piscivorus). Note the characters that define this step, chromatoid body (cd), the large ...

During the remainder of the round spermatid stage, the acrosome vesicle continues to develop and enlarge (Fig. 17). Within this vesicle (Fig. 17, S1, ac) an electron dense acrosome granule (Fig. 17, S2, black arrowhead) forms most likely from the materials delivered from the transport vesicles of the Gogli complex. The cytoplasm at this time is packed with mitochondria and a large amount of rough and smooth endoplasmic reticula. As the acrosome grows in size, it first indents the apex of the nucleus so that the newly forming acrosome complex resides in a deep nuclear fossa. Then late in the development of the round spermatid, a cytoplasmic shift occurs, forcing most the organelles and cytoplasm caudally within the round spermatid (Fig. 17, S3, black*). This results in the outer acrosome membrane becoming directly juxtapositioned to the cellular membrane (Fig. 18 and inset, black arrowheads) and causes the acrosome to collapse and flatten on to the apical surface of the round spermatid nucleus. The force produced from the meeting of acrosome and cellular membrane eventually causes the acrosome to completely flatten the apical nucleus, which also has thin electron dense subacrosome space (Fig. 17, S4, black arrow) adhering the inner acrosome membrane to the apical nuclear envelope. This shift from a centrally located nucleus to an eccentrically located nucleus becomes even more pronounced during the elongation phase of spermiogenesis (Fig. 18). The exact mechanism that controls the cytoplasmic shift and the beginning stages of elongation are not completely understood in amniotes. It has been suggested that actin components of the cytoskeleton play a major role in the cytoplasmic shift and early events of elongation.65,66 Later events of elongation and head formation are most likely controlled by the manchette,67 a cytoskeletal scaffolding of microtubules associated with the body of the elongating nucleus.

Figure 17
The major steps of the round stage of development during spermiogenesis within the seminiferous epithelium of the American Alligator (Alligator mississippiensis). The acrosome (ac) not only grows in size but a prominent granule (black arrowhead) is observed ...
Figure 18
Comparison of a step 1 spermatid (American Alligator, Alligator mississippiensis) nuclear location to that of a step 5 spermatid location (Ground Skink, Scincella lateralis; Inset: Red-eared Slider Turtle, Trachemys scripta) location. The step 1 nucleus ...

The continued elongation (Fig. 17, S4) of the nucleus caudally forces the distal cytoplasm to push the apex of the nucleus further against the cell membrane, which most likely results in the enveloping of the acrosome complex around the now rounded nuclear head (Figs. 18 and and1919). In many instances during reptilian spermiogenesis the squeezing of the enveloping cell membrane, acrosome membranes and nuclear membranes causes prominent nuclear shoulders on the caudal lateral boards of the nuclear apex (Figs. 18 and and1919). The complete progression and transition of the acrosome vesicle and the nuclear head is illustrated in Figure 19.

Figure 19
Progression of acrosome development across reptile taxa showing the similarities and conserved ontogenic morphology of the acrosome during its development. (A) S1, American Alligator (Alligator mississippiensis); (B) S2, Transvolcanic Bunchgrass Lizard ...

When combining the known complete morphological data on the process of elongation during spermiogenesis, two major ultrastructural themes separate the squamates (lizard, snakes) from the rest of extant reptiles (turtles, crocodilians and tuataras). Thus for simplicity and ease of understanding, most the of morphology on elongating spermatids presented here will show a squamate representative (several different lizards and snakes) and Alligator mississippiensis or Trachemys scripta for the non-squamate examples. As elongation continues, the nuclear body lengthens leading to a more cylindrical appearance to the nucleus (Fig. 20). Elongation during both themes results in similarly shaped spermatid nuclei. Also, the manchette (Fig. 20 and Scincella lateralis, Inset, ma) is found in all species of reptile studied to date during elongation except in Anolis lineatopus, which lacks a manchette all together.58 The manchette is thought to aid in the elongation of the nucleus when present and is also represented in almost every mammal and bird.2,26,56,64,68 The manchette has long been thought to be a major player in the elongation and constriction of the nuclear width during amniote spermiogenesis,64,66 but the exact mechanism is not known. It also seems that a functioning manchette is not necessarily needed for elongation to occur, as it does not exist in A. lineatopus although normal elongation does take place.

Figure 20
Light (Ground Skink step 5) and electron microscopy of mid-elongation in the American Alligator (Alligator mississippiensis) and the Ground Skink (Scincella lateralis) from the seminiferous epithelium. Acrosome, black arrows; acrosome granule, black arrowhead; ...

As the nucleus elongates, it also becomes restricted in width. As you compare the size of the nucleus of a late elongating spermatid to the larger spermatocytes one can see the disproportionate size difference in favor of the spermatocytes. The chromatin of these germ cells must be tightly packed together in order for that amount of DNA to fit within the nucleus of the hydrodynamic spermatozoa. Condensation is the process by which the DNA is packaged within the body of the nucleus. It occurs in two ways and roughly follows the two themes of elongation between squamate and non-squamate reptiles. Non-squamate reptiles have a granular type of condensation pattern. Chromatin is packed into larger and ever tightening granules as the nucleoplasm shrinks within the elongating spermatid nucleus (Fig. 21A and B). In contrast, the squamates typically have a filamentous type of condensation of the their chromatin (Fig. 21C). The filamentous pattern can condense into more or less straight fibers (Fig. 21E and F) as it does in Scincella lateralis56 spermatids or actually concentric fibers (Fig. 21C) that wrap in ever tightening coils within cross sections of elongating spermatids (Fig. 21D) as in Agkistrodon piscivorus.57 Also open areas of nucleoplasm can occur, often termed lacunae (Fig. 21C and black arrow),49,56 within the body of the nucleus during and after elongation have been completed. Many squamate elongating spermatids have two prominent translucent areas (Fig. 21C and white arrows) within the nucleoplasm on either side of the caudal nuclear fossa (Fig. 21E and white arrowhead) where the flagellum (proximal and distal centrioles, Fig. 21C, pc and dc) attaches to the caudal nuclear body. Reptilian elongating spermatids not only have the manchette (Fig. 21F, ma) along the length of the body of the nucleus but also in some cases possess a large amount of smooth endoplastic reticula (Fig. 21B and black arrowheads) running in juxtaposition to the body of the nucleus during middle elongation.

Figure 21
Electron micrographs of Step 5 and Step 6 spermatids in (A and B) S5, American Alligator, Alligator mississippiensis; (C and D) S5, Cottonmouth Snake, Agkistrodon piscivorus; (E and F) S6, Ground Skink, Scincella lateralis. Within the Alligator (A and ...

The acrosome and flagellar development is complicated in reptiles and many of the differences between species within and among orders of reptiles occur in the compartmentalized acrosome complex or within such parts of the flagellum as the midpiece. Therefore, these variations within these parts of the spermatozoa and/or spermatids are the focus of recent nontraditional phylogenetic studies in reptiles.6,49,50,53 The plates that are represented within this review are at the climax of acrosome and flagellar development as seen within the anatomy of the step 7 or 8 spermatids, which are the stages right before spermiation. This high complexity of the acrosome complex in reptiles53,57,69,71 is often absent in mammals and birds,69,70 and has been suggested to aid in the sequential release of hydrolytic enzymes during fertilization.72

In turtles, tuataras and crocodilians53,63 the major differences within their acrosome complexes are the presence of deep endonuclear canals (1 to 3) (Fig. 22A, en) that house the perforatoria. These nuclear canals are absent in squamates (Fig. 22B) and their perforatoria are completely epinuclear and reside in the subacrosome spaces right under the proximal acrosome vesicles (Fig. 22B, pf). The acrosome complex (Fig. 22A and B, ac) consists of the acrosome superficially, which envelops the entire nuclear head. This enzymatic sac is typically rounded or slightly pointed at its most anterior end and has thin elongated shoulders (Fig. 22A and black arrowheads; B, as) that run at least a third of the way down the lateral aspects of the apical nucleus (Fig. 22). In both non-squamate and squamate reptiles the acrosome lumen can be striated, with a darker cortex and a light medulla as in Alligator mississippiensis73 and Hemidactylus turcicus.59 The acrosome is glued to the nuclear head by the paracrystalline subacrosome space that is typically made up of electron dense granules. The subacrosome space (Fig. 22A and B, sa) can be striated also as in the lizard, Barisia imbricata (Fig. 22B) (Gribbins KM, unpublished data). The basal part of the subacrosome space, with the epinuclear translucent zone (Fig. 22B, ep), is separated from the rostrally located basal plate (Fig. 22B and white arrowhead) and perforatorium (Fig. 22B, pf), which is right under the most proximal part of the acrosome head (Fig. 22A and B), by a thin white translucent line (Fig. 22B and white arrowheads) in most squamates. The nucleus penetrates the subacrosome space as a narrow pointed rostrum in almost all reptiles studied to date (Fig. 22A and B, nr). Surrounding the nuclear head and acrosome are several layers of Sertoli cell membrane (Fig. 22A and black arrows) that most likely aid in the continual attachment of the developing spermatid to the Sertoli cells of the seminiferous epithelium via desmosome (anchoring) junctions (Fig. 21E, ds).

Figure 22
(A) The nuclear head with acrosome complex of the American Alligator (Alligator mississippiensis) in sagittal and cross sections. The letters A–G of the sagittal section correspond to the same letters of each cross section. Acrosome vesicle, ac; ...

In the Reptilia, the flagellum becomes evident early in the round spermatid stage as a small principle piece growing from the distal centriole (Fig. 17, s3, white arrowhead). The 9 + 2 arrangement of the microtubules growing from the centrioles in cross section is termed the axoneme (Fig. 23, ax). The centrioles have a 9 + 3 arrangement of concentric peripheral tubules and a centrally located microtubule pair in cross section for the distal centriole (Fig. 23). The flagellum attaches to the caudal end of the nucleus within an implantation fossa (Fig. 21E and white arrowhead). Most non-squamate reptiles, like Alligator mississippiensis (Fig. 23), lack a prominent neck region of the flagella and the midpiece abuts directly to the caudal nucleus. In contrast, in many snakes and lizards (Fig. 23) there is a neck region between the implantation (nuclear) fossa and the beginning of the midpiece. The laminar projects (Fig. 23, lp), pericentrioler matieral (Fig. 21E, pe), dense collar (Fig. 23, dl), and the central doublet of the distal centriole all aid in the support, robustness of the neck, and to the future propulsional function of the reptilian sperm tail.

The midpiece of the flagellum is defined by the concentric rings of mitochondria surrounding the axoneme (Fig. 23). In non-squamate reptiles these mitochondria are large uniformly round with many peripheral concentrically layered cristae (Fig. 23, turtle). The number of mitochondria appears from the few studies done to date to be fairly consistent along the length of the midpiece in turtles,60,61 crocodilians,62,63 and the tuatara53 and ranges from 8 to 10 in sagittal section. In squamates, the sperm tend to maintain slightly higher and more variable numbers of elongated mitochondria (on average around 12 in lizards) (Fig. 23, lizard) along the length of the midpiece (except some geckos, 2) with snakes having much higher numbers than that of non-squamate reptiles with 14 or more mitochondria in sagittal sections of the midpiece.69 Snakes also tend to have many rows of dense bodies (presumably deteriorating mitochondria), which leads to very long midpieces (Fig. 23, snake). Non-squamate reptiles tend to lack dense bodies altogether within their midpieces (Fig. 23, turtle). The other major difference within the midpiece of non-squamate reptiles is the distal centriole runs along the entire length of the midpiece axoneme (Fig. 23, turtle, dc), whereas squamates have a short distal centriole that is typically restricted to the neck region only. Therefore, the principle piece penetrates up into the midpiece region (Fig. 23, snake and lizard, pp) of the flagellum in Squamata. Associated with microtubule triplets within the distal centriole are peripheral axonemal fibers (Fig. 23, lizard, pf) within most reptiles studied to date. These fibers again are hypothesized to aid in the strengthening of the proximal attachment point of the flagella to the body of the nucleus. These peripheral fibers continue into the principle piece but tend to dissipate further from the terminating point of the midpiece, which is marked by the annulus (Fig. 23 and white arrowheads) in reptiles and other amniotes. Of these fibers numbers 3 and 8 tend to stay enlarged in turtles, some crocodilians, and in most squamates (Fig. 23, lizard, lizard,3f3f).69

The principle piece (Fig. 23) has an axomene that is surrounded by a fibrous sheath, which tends to be thicker proximally and thins as you move distally along the principle piece's length (Fig. 23, fs). The fibrous sheath is made up of uniform annulated fibrous rings that are stacked sequentially along the length of the principle piece in reptiles similar to that of all amniotes.69 The endpiece (Fig. 23) in reptiles is easily discernable from the principle piece as it has lost the fibrous sheath and all that remains is the axoneme and the cell membrane (Fig. 23, lizard, cm and ax).

Conclusions

The purpose of this review is to provide basic information on the histology and ultrastucture of spermatogenesis in reptiles. This qualitative study may encourage other scientists to begin to more thoroughly explore sperm development in reptiles. The studies that have been done to date provide relatively large but incomplete data sets that could be important or useful in phylogenetic analyses if and only if more ultrastructural information is collected for reptilian representatives across this class. Studying both spermatogenesis and spermatozoon ultrastructure within many new species representing families across Reptilia will only add to the robustness of such data. Ultrastructural spermatogenic characters collected from such studies will provide for better phylogenetic analysis overall and enable a better understanding of the evolution of sperm development and morphology within reptiles and across amniotes.

These data also provide a basic model that could be used for histopathological studies of the testis in amniotes. In order to understand how toxins or heavy metals affect sperm development, a thorough understanding of the histology of spermatogenesis has to be understood.26 Reptiles provide the perfect model for these types of studies because the temporal development of germ cells as they progress through spermatogenesis provides a simplistic and straightforward series of germ cell morphologies that can be easily delineated during the major phases of spermatogenesis. This should make it easier to discern abnormalities among different generations of germ cells within the testis and the absence or disruptions of particular stages of sperm development. Also, many reptiles are semi-aquatic and relatively abundant and thus could be sentinel species for the studies of aquatic pesticides and their toxicological impacts on germ cell development. Chemicals such as atrazine have been shown to alter sperm development in amphibians and fishes.74 Reptiles could impact these studies even further as they have a testis more similar to those of other amniotes, including humans, than to anamniote fish and amphibians.

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