Cytological evaluation of the germ cell development strategy within the testis of the American alligator, Alligator mississippiensis

The cytological changes to germ cells were investigated within the seminiferous epithelium of the American alligator (Alligator mississippiensis). Testicular tissues were collected, embedded in plastic, sectioned on an ultramicrotome, and stained with the periodic acid–Schiff+ procedure followed by a haematoxylin counterstain. Alligators have a prenuptial pattern of germ cell development, where spermatogenesis begins in early spring and sperm is mature by the time mating begins in May. Consistent spatial relationships between germ cells are absent within the seminiferous epithelium of the alligator. Their germ cells progress through the phases of spermatogenesis as a single cohort, leading to one continuous spermiation event that occurs during their mating season (May–June). This temporal germ cell development is different from the consistent spatial development seen within seasonally breeding birds and mammals but is similar to the recently described germ cell development strategies of two other temperate breeding reptiles, the slider turtle and the European wall lizard. The germ cell development strategy shared by these three temperate reptiles representing three different taxa within the class Reptilia is reminiscent of the temporal strategy seen within the anamniotic testis. Thus, alligators and at least two other temperate reptiles exhibit primitive spermatogenic cycles within derived amniotic testes and may be consider intermediates in terms of testicular organization, which may have significance phylogenetically.     

Ultrastructure of the reproductive system of the black swamp snake (Seminatrix pygaea). V. The temporal germ cell development strategy of the testis

The germ cell development strategy during spermatogenesis was investigated in the black swamp snake (Seminatrix pygaea). Testicular tissues were collected, embedded in plastic, sectioned by ultramicrotome, and stained with methylene blue and basic fuchsin. Black swamp snakes have a postnuptial pattern of development, where spermatogenesis occurs from May to July and spermiation is completed by October. Though spatial relationships are seen between germ cells within the seminiferous epithelium during specific months, accumulation of spermatogonia and spermatocytes early in spermatogenesis and the depletion of spermatocytes and accumulation of spermatids late in spermatogenesis prevent consistent cellular associations. This temporal germ cell development within an amniotic testis is consistent with that seen in other recently studied temperate reptiles (slider turtle and wall lizard). These reptiles’ temporal development is more similar to the developmental strategy found in anamniotes than the spatial germ cell development that characterizes birds and mammals. Our findings also imply that a third germ cell development strategy may exist in temperate breeding reptiles. Because of the phylogenetic position of reptiles between anamniotes and other terrestrial amniotes, this common germ cell development strategy shared by temperate reptiles representing different orders may have significant implications as far as the evolution of sperm development within vertebrates.     

The germ cell development strategy and seasonal changes in spermatogenesis and Leydig cell morphologies of the spiny lizard Sceloporus mucronatus (Squamata: Phrynosomatidae)

The viviparous lizards of the Sceloporus genus exhibit both seasonal and continuous spermatogenesis. The viviparous lizard Sceloporus mucronatus from Tecocomulco, Hidalgo, México, exhibits seasonal spermatogenesis. This study demonstrates the relationship between changes in testis volume, spermatogenesis activity, and Leydig cells during the male reproductive cycle of S. mucronatus. A recrudescence period is evident, which starts in the winter when testicular volume is reduced and climaxes in February, when the greatest mitotic activity of spermatogonia occurs. The testicular volume and Leydig cell index increase gradually during the spring with primary spermatocytes being the most abundant cell type observed within the germinal epithelium. In the summer, the secondary spermatocytes and undifferentiated round spermatids are the most abundant germinal cells. The breeding season coincides with spermiogenesis and spermiation; testicular volume also increases significantly as does the Leydig cell index where these cells increase in both cytoplasmic and nuclear volume. During fall, testicular regression begins with a significant decrease in testicular volume and germinal epithelium height, although there are remnant spermatozoa left within the lumen of the seminiferous tubules. During this time, the Leydig cell index is also reduced, and there is a decrease in cellular and nuclear volumes within these interstitial cells. Finally, during quiescence in late fall, there is reduced testicular volume smaller than during regression, and only spermatogonia and Sertoli cells are present within the seminiferous tubules. Leydig cells exhibit a low index number, their cellular and nuclear volumes are reduced, and there is a depletion in lipid inclusion cytoplasmically.     

Germ cells of male mice express genes for peroxisomal metabolic pathways implicated in the regulation of spermatogenesis and the protection against oxidative stress

Peroxisomes are organelles with main functions in the metabolism of lipids and of reactive oxygen species. Within the testis, they have different functional profiles depending on the cell types. A dysfunction of peroxisomes interferes with regular spermatogenesis and can lead to infertility due to spermatogenic arrest. However, so far only very little is known about the functions of peroxisomes in germ cells. We have therefore analyzed the peroxisomal compartment in germ cells and its alterations during spermatogenesis by fluorescence and electron microscopy as well as by expression profiling of peroxisome-related genes in purified cell populations isolated from mouse testis. We could show that peroxisomes are present in all germ cells of the germinal epithelium. During late spermiogenesis, the peroxisomes form large clusters that are segregated from the spermatozoa into the residual bodies upon release from the germinal epithelium. Germ cells express genes for proteins involved in numerous metabolic pathways of peroxisomes. Based on the expression profile, we conclude that newly identified functions of germ cell peroxisomes are the synthesis of plasmalogens as well as the metabolism of retinoids, polyunsaturated fatty acids and polyamines. Thus, germ cell peroxisomes are involved in the regulation of the homeostasis of signaling molecules regulating spermatogenesis and they contribute to the protection of germ cells against oxidative stress.     

Impaired fertility and spermiogenetic disorders with loss of cell adhesion in male mice expressing an interfering Rap1 mutant

The guanosine trisphosphatase Rap1 serves as a critical player in signal transduction, somatic cell proliferation and differentiation, and cell-cell adhesion by acting through distinct mechanisms. During mouse spermiogenesis, Rap1 is activated and forms a signaling complex with its effector, the serine-threonine kinase B-Raf. To investigate the functional role of Rap1 in male germ cell differentiation, we generated transgenic mice expressing an inactive Rap1 mutant selectively in differentiating spermatids. This expression resulted in a derailment of spermiogenesis due to an anomalous release of immature round spermatids from the seminiferous epithelium within the tubule lumen and in low sperm counts. These spermiogenetic disorders correlated with impaired fertility, with the transgenic males being severely subfertile. Because mutant testis exhibited perturbations in ectoplasmic specializations (ESs), a Sertoli-germ cell-specific adherens junction, we searched for expression of vascular endothelial cadherin (VE-cadherin), an adhesion molecule regulated by Rap1, in spermatogenic cells of wild-type and mutant mice. We found that germ cells express VE-cadherin with a timing strictly related to apical ES formation and function; immature, VE-cadherin-positive spermatids were, however, prematurely released in the transgenic testis. In conclusion, interfering with Rap1 function during spermiogenesis leads to reduced fertility by impairment of germ-Sertoli cell contacts; our transgenic mouse provides an in vivo model to study the regulation of ES dynamics.     

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

Sceloporus bicanthalis is a viviparous lizard that lives at higher elevations in Mexico. Adult male S. bicanthalis were collected (n = 36) from the Nevado de Toluca, Mexico (elevation is 4200 m) during August to December, 2007 and January to July, 2008. Testes were extracted, fixed in Trumps, and dehydrated in a graded series of ethanol. Tissues were embedded, sectioned (2 μm), stained, and examined via a light microscope to determine the spermatogenic developmental strategy of S. bicanthalis. In all months examined, the testes were spermiogenically active; based on this, plus the presence of sperm in the lumina of seminiferous tubules, we inferred that S. bicanthalis had year-round or continuous spermatogenesis, unlike most reptiles that occupy a temperate or montane habitat. It was recently reported that seasonally breeding reptiles had a temporal germ cell development strategy similar to amphibians, where germ cells progress through spermatogenesis as a single population, which leads to a single spermiation event. This was much different than spatial development within the testis of other derived amniotes. We hypothesized that germ cell development was temporal in S. bicanthalis. Therefore, we wanted to determine whether reptiles that practice continuous spermatogenesis have a mammalian-like spatial germ cell development, which is different than the typical temperate reptile exhibiting a temporal development. In the present study, S. bicanthalis had a temporal development strategy, despite its continuous spermatogenic cycle, making them similar to tropical anoles.     

Cyclic changes in the testis of the brook stickleback Eucalia inconstans (Kirtland)

The cyclic changes in the testis of the five-spined stickleback Eucalia inconstans (Kirtland) were studied histologically. Specimens were trapped between July 1965 and July 1967 in a shallow pond near London, Ontario. A three-dimensional microscopic study showed a main vas deferens and a system of primary, secondary and tertiary tubules. The testis cycle was divided into seven arbitrary stages. Spawning takes place from mid-April to mid-July. This is followed by the division of primary spermatogonia which are located along the walls of the tubules, producing cysts of spermatogonia enclosed in connective tissue which is surrounded by a thin epithelium. Both primary and secondary spermatocytes develop within these cysts. Breakdown of the cysts occurs with the development of spermatids and spermiogenesis occurs while spermatids are free in the tubules. Over-wintering of mature sperm takes place. Development of mature sperm from primary spermatogonia takes about 156 days. Germinal epithelium is absent but primary germ cells are believed to be those cells occupying the spaces between the tubules of the testis. No tissue which might be implicated in hormone production was observed. Phagocytic invasion of the testis has been studied. Massive infiltration by phagocytes is believed to be responsible for the sudden increase in testis weight observed during spawning. These cells ingest sperm nuclei and groups of them have been observed in the lumen of the tubules and the vas deferens, probably on their way out of the body.     

Putative steroid-binding receptors and nonreceptor components and testicular activity in the lizard Podarcis sicula sicula.

Labelled testosterone- and oestradiol-binding molecules have been found in the cytosol and nuclei of lizard testes. DNA-cellulose affinity chromatography was used to separate putative sex-steroid-binding receptors (adhering molecules) and nonreceptor components (nonadhering molecules). A putative androgen receptor (Kd: 10(-10) mol l-1; 3-9 fmol g-1 tissue) was found mainly in the nuclei of testicular cells when actively undergoing spermatogenesis. This suggests that, as in higher vertebrates, testosterone is implicated in spermatogenetic step regulation (meiosis and spermiogenesis) in lizard testis. In the cytosol, testosterone-binding molecules (Kd: 10(-9) mol l-1; 384-784 fmol g-1 tissue) with several properties of androgen-binding proteins are present from autumn to spring. The behaviour of these molecules is consistent with the role assigned to androgen-binding proteins as androgen reservoir. A putative oestrogen receptor is present throughout the sexual cycle, except during the culmination phase (breeding). The putative oestrogen receptor may be involved in the regulation of the first spermatogenetic step (spermatogonia multiplication) and in the induction of post-reproductive refractoriness. This phase is present in temperate-zone lizards. These studies show that the evaluation of sex-steroid-binding molecules is useful in considering the relationships between sex hormones and spermatogenetic activity in the testes of lizards.     

Interrelationship among testicular cells in wall lizard Hemidactylus flaviviridis (Rüppell): an ultrastructural seasonal and experimental study

The present study was aimed at investigating ultrastructure of different testicular cells and their interactions through various junctional specializations during different phases of reproductive cycle in wall lizard H. flaviviridis to develop an integrated approach of cell-cell interaction in control of testicular functions. Specialized steroid synthesizing cell organelles such as smooth endoplasmic reticulum (SER) and long slender mitochondria with tubulo-vesicular cristae were predominantly seen in Leydig as well as Sertoli cells during spermatogenically active phase, suggesting their active involvement in steroid biosynthesis. Peritubular cells also exhibited marked seasonal variations. Multi-layered fibroblast-like peritubular cells during regressed phase became single layered myoid-like during spermatogenically active phase. The presence of various types of junctions, including gap and tight junctions (occluding junctions) and adhering junctions such as desmosomes, septate-like junction, ectoplasmic specializations and tubulo-bulbar complexes, were demonstrated among testicular cells in wall lizard H. flaviviridis. However, the nature and degree of junctional (environmental) interaction varied with the reproductive state of the wall lizard. Further, administration of dihydrotestosterone in wall lizards during regressed phase resulted in increase of lipid droplets in Leydig cells and accumulation of germ cell debris in seminiferous tubules. Some of the Sertoli cells were seen darker in response to testosterone treatment probably due to its inhibitory effect on lipid metabolism. These results suggest that testosterone either directly or via inhibiting pituitary basal gonadotropin secretion has suppressive effect on testicular cells.     

Initial observations on the organization of the testis of Trisopterus minutus capelanus]

Testis of the Teleostean fish Trisopterus minutus capelanus has been examined to study the organization of the seminiferous tubules and the ultrastructural features of the germ cells. The testis is shown to be composed of seminiferous tubules full of cells: only few of them have just a very narrow lumen. Each tubule is divided by thin septa of connective tissue in zones containing homogeneous cells; such an organization is confirmed by ultrastructural images showing groups of synchronously developing germ cells. By morphological characterization of the germ cells found in each zone, 6 maturation stages have been identified. During spermiogenesis, a progressive shrinkage of germ cells and a nuclear chromatin condensation have been observed. Intercellular bridges, homogeneously dispersed granules of glycogen and groups of mitochondria associated with dense granular material have been described. Such features are present in the earlier stages of spermiogenesis and are retained until the later stages of spermatid differentiation. The spermatozoon shows a lack of acrosome as in many other teleosts previously studied.     

Annual changes in germinal epithelium determine male reproductive classes of the cobia

Five reproductive classes of cobia Rachycentron canadum , caught along the Gulf of Mexico and the south-east Atlantic coast of the U.S.A., are described during the annual reproductive cycle. These are based upon changes in the testicular germinal epithelium and the stages of germ cells that are present: early maturation, mid maturation, late maturation, regression and regressed. During early maturation, the germinal epithelium is continuous from the testicular ducts to the periphery of the testis and active spermatogenesis occurs throughout the testis. In mid maturation, the germinal epithelium near the ducts becomes discontinuous, but it remains continuous distally. In late maturation, a discontinuous germinal epithelium extends all along the lobules to the testicular periphery; lobules are swollen with sperm and there is minimal spermatogenesis. The regression class is characterized by a discontinuous epithelium throughout the testis, sperm storage and widely scattered spermatocysts. Spermatogonial proliferation also occurs along the lobule walls and at the periphery of the testis. In regressed testes, spermatogonia exist only in a continuous or discontinuous germinal epithelium, although residual sperm are nearly always present in the lobules and ducts. The presence or absence of sperm is not an accurate indicator of reproductive classes. At the periphery of the testis in the regression and regressed classes, the distal portions of lobules elongate as cords of cells containing spermatogonia and Sertoli cells. All reproductive classes can be identified in paraffin sections, although plastic sections provide better resolution. Using maturation classes defined by changes in the germinal epithelium to describe testicular development and spermatogenesis gives a more accurate picture than does using the traditional terminology.     

Impairment of spermatogenesis and enhancement of testicular germ cell apoptosis induced by exogenous all-trans-retinoic acid in adult lizard Podarcis sicula

In mammals, retinoic acid is involved in the regulation of testicular function by interaction with two families of nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR). Among RAR isoforms, the testicular cells of the lizard were found to express only RARalpha (3.7 kb) and RARbeta (3.4 kb) mRNAs, as reported here. In this study, the effects of exogenous all-trans-retinoic acid (atRA) on spermatogenesis of a non-mammalian seasonal reproducer were investigated. Daily intraperitoneal injections of atRA or atRA plus testosterone (atRA+T) were given for 2 weeks to adult males of the lizard Podarcis sicula. In animals treated with atRA, the seminiferous tubules were markedly reduced in cross-area. The seminiferous epithelium collapse was responsible for a sensible reduction in the number of germ cells and disruption in normal epithelial organization. In comparison, in atRA+T-treated lizards the loss of germinal cells was significantly less. The loss of germ cells observed in both experimental groups results from an induction of apoptotic process, as revealed by TUNEL analysis. Although low in number, apoptotic germ cells were also observed in the control groups (saline- and T-treated lizard), where the main germ cells undergoing apoptosis are primary spermatocytes (most frequently) and some spermatogonia.In conclusion, it is shown here that retinoic acid has deleterious effects on lizard spermatogenesis, causing a severe depletion of seminiferous epithelium, probably via induction of apoptotic processes. These effects are not completely inhibited by simultaneous administration of testosterone, although this hormone, once injected, is able to stimulate spermatogenesis and protect germinal cells from apoptotic cell death.     

The ultrastructure of germinal beds in the ovary of Gerrhonotus coeruleus (Reptilia: Anguidae)

A study of ovarian structure in adult Alligator Lizards (Gerrhonotus coeruleus) was conducted by light microscopy and transmission electron microscopy. Particular attention was directed to characterizing the ultrastructure of germ-line cells, prior to follicle formation. General ovarian structure in this lizard is similar to that of other lizards. The paired organs are hollow, thin-walled sacs containing follicles in roughly 3 to 4 size classes. Ovarian germinal tissue consists of oogonia (diploid cells which divide mitotically) and oocytes (meiotic cells), intermixed with ovarian surface epithelial cells. Germ cells reside in two dorsal patches of epithelium per ovary (germinal beds), as is common in lizards. Oogonia in interphase show a highly dispersed chromatin pattern. Within oogonia cytoplasm, Golgi complexes are scarce, rough endoplasmic reticulum is absent, and lipid droplets are rare. Ribosomes are scattered in small clusters. Small, round vesicles are common in all oogonia; glycogen-like granules are present in some. Mitochondria form a juxtanuclear mass within which groups of several mitochondria surround a dense granule. “Nuage” granules also are found unassociated with mitochondria. Oocytes are present in stages of meiotic prophase up to diplotene. Synaptinemal complexes are seen in several (pachytene) cells. The cytoplasm of oocytes differs from that of oogonia in that mitochondria do not form groups, and nuage and glycogen are absent, whereas small round vesicles and large irregular vesicles are common. The ultrastructural similarities in germ cells of a reptile as compared to those of other vertebrates strengthens the notion that germ-line cells possess (or lack) qualities related to the undifferentiated state of these cells.     

Spermatogenesis in Atlantic cod (Gadus morhua): a novel model of cystic germ cell development

Precocious male puberty significantly compromises sustainability aspects of aquaculture in a number of finfish species. As part of a program aiming to understand and eventually control testis maturation in farmed Atlantic cod, we studied the first reproductive cycle. The gonadosomatic index shows a 41-fold increase from immature (August) to mature (March) stages, reaching almost 10% of the total body weight. The paired cod testes are composed of several lobes arranged around a central collecting duct. In each individual lobe, spermatogenesis occurs in a marked gradient of development, with undifferentiated spermatogonia in the periphery of the lobe and the most advanced germ cells in the vicinity of the collecting duct, suggesting a tight spatiotemporal organization of spermatogenesis in the testis lobes of this species. Spermatogonial proliferation starts in August and continues for about 6 mo. Meiosis and spermiogenesis are first observed in October and are completed in all cysts by February, when a 2-mo-long spawning season starts. Spermatogonia go through 11 mitotic divisions before differentiating to primary spermatocytes. Apoptosis is rare, but when observed it occurs mainly during the last spermatogonial generations. Our observations suggest a model in which a maturational wave progresses through each growing lobe that is first driven by appositional growth from the lobe's periphery, reflecting spermatogonial proliferation and cyst formation which, when ceasing, is terminated by completing spermiogenesis and spermiation that progress toward the lobe's periphery.     

Testis morphology and spermatogenesis in two species of Elenchus (Strepsiptera : Elenchidae)

Testes ultrastructure and spermatogenesis were studied in two species of Strepsiptera (Insecta), namely, Elenchus tenuicornis and E. japonicus, using light and electron microscopy. In both species, the testis is paired and consists of several large irregularly shaped follicles. Each follicle consists of a single clone of germ cells surrounded by a thin epithelium. During the larval and pupal stages, all the germ cells of each testis develop synchronously, and at eclosion, the gonads contain solely mature sperm. One of the most interesting findings is the morphogenesis of a large nuclear vesicle bounded by the fenestrate part of the nuclear envelope. This vesicle contains an electron-dense spherical structure, the chromatoid body. At the end of spermiogenesis, both the nuclear vesicle and its chromatoid body are eliminated with the excess cytoplasm. Large drops of residual cytoplasm containing several nuclear vesicles are present in the lumen of the testis and inside the cytoplasm of phagocytic cells.     

The interstitial origin of germinal cells in the testis of the stickleback

The brook stickleback, Culaea inconstans (Kirtland), in common with other bony fishes, lacks a germinal epithelium in the tubules of the testis, and the tubule wall is composed of a thin, discontinuous layer of myoid cells and collagenous fibers. Labelling of germ cells with tritiated thymidine has shown that the germ cells are derived from clumps of spermatogonia in the interstitial area. Large companion cells within the lumina of the tubules extend their processes to engulf spermatogonia from the interstitium which then enter the lumen of the tubule. Subsequent development of the germ cells takes place within individual compartments formed by folds of the plasma membrane of a companion cell. The companion cell, together with its complement of germ cells, constitutes a cyst. A companion cell may surround spermatogonia in the interstitium and at the same time encompass residual sperm of the previous season within the lumen. The plasma membranes of the germ cells and the companion cells remain discrete. Mature sperm are released into the lumen of the tubule and the companion cell again extends its processes into the interstitium and engulfs more spermatogonia for the following year. Companion cells may be homologous to the Sertoli cells of higher vertebrates although their processes penetrate the interstitium during the initial stages of spermatogenesis and they do not contain a permanent stock of spermatogonia.