An ultrastructural study of the effect of a diabetogenic dose of alloxan on the secretory ameloblasts of the rat incisor

Sertoli cells of the ground squirrel (Spermophilus lateralis), a seasonal breeder, were examined by light and electron microscopy and their structure, particularly the organization of the cytoskeleton, was related to events that occur in the seminiferous epithelium during spermatogenesis. Among the events considered and described are the apical movement of elongate spermatids, withdrawal of residual cytoplasm from germ cells, transport of smooth endoplasmic reticulum (SER) between the base and apex of the Sertoli cells, and sperm release. These events are dramatically evident in this species because the seminiferous epithelium is thin, i.e., there are few germ cells, and both the germ cells and Sertoli cells are large. Sertoli cells of the ground squirrel have a remarkably well developed cytoskeleton. Microfilaments occur throughout the cell but are most evident in ectoplasmic specializations associated with junctions. Intermediate filaments occur around the nucleus, as a layer at the base of the cell, and adjacent to desmosome-like junctions with germ cells. Intermediate filaments, together with microtubules, are also abundant in regions of the cell involved with the transport of SER, in cytoplasm associated with elongate spermatids, and in processes that extend into the residual cytoplasm of germ cells. Our observations of ultrastructure are consistent with the hypothesis that Sertoli cell microtubules are involved with the movement of germ cells within the seminiferous epithelium, and further implicate these structures as possibly playing a role in the retraction of residual cytoplasm from germ cells and the intracellular transport of SER. The abundance and organization of intermediate filaments suggest that these cytoskeletal elements may also be involved with events that occur during spermatogenesis.     

Fine structural changes in Sertoli and Leydig cells during the reproductive cycle of the ground squirrel, Citellus lateralis

During spermatogenesis in sexually mature ground squirrels Leydig and Sertoli cells were morphologically well differentiated. For Leydig cells the most prominent organelles were lipid droplets, mitochondria with tubulo-vesicular cristae and abundant agranular reticulum organized as a mass of anastomosing tubules. These morphological criteria suggest that the Leydig cells were steroidogenically active. Sertoli cells exhibited a topographical distribution of certain organelles with basal regions containing stacks of granular reticulum, and large areas of agranular reticulum. The cytoplasm surrounding maturing germ cells contained numerous microtubules, and an adluminal layer of spermatids at a certain stage of spermiogenesis became enveloped by Sertoli cytoplasm containing an enormous proliferation of agranular reticulum. The presence of these organelles in Sertoli cells suggests that during spermatogenesis they are active in the synthesis of proteins and steroids. In particular the mass of agranular reticulum surrounding late stage spermatids indicates that steroids may be required for spermatid maturation and/or spermiation. By contrast Leydig and Sertoli cells observed during testicular regression, when only spermatogonia remain in the seminiferous tubules, had undergone structural changes. Leydig cells were still numerous and large with abundant agranular reticulum that was now organized as a loose assemblage of single unbranched tubules. Sertoli cells were drastically reduced in both cytoplasmic volume and content of organelles.     

FORMATION OF GERM CELL-LIKE CELLS IN THE PERITONEAL EPITHELIUM OF THE TESTES OF ESTROGEN-TREATED ADULT MALES OF THE JAPANESE RED-BELLIED NEWT, CYNOPS PYRRHOGASTER PYRRHOGASTER

Columnar cells of the peritoneal epithelium in slender cords of the testes were examined in normal and estradiol benzoate-treated Japanese red-bellied newt, Cynops pyrrhogaster pyrrhogaster, by light and electron microscopy. In normal newts, the peritoneal epithelium covering the slender cord consists of columnar cells, which contain extraordinarily large, oval or spindle-shaped nuclei with conspicuous indentations. The nucleus contains chromatin granules and the cytoplasm is filled with numerous tonofilaments. The primordial germ cells are scattered throughout the slender cord, and each cell is surrounded by a few follicle cells. Between the germ cells and follicle cells there are microvilli-like processes. The nucleus of primordial germ cells is multilobate and has electron lucent areas, dispersed chromatin and several electron-dense nucleoli. In the lighter cytoplasm, the nuage material is found very near to nuclear pores, and is frequently seen among the mitochondria. The nucleolus-like body is not associated with other organelles. The primary spermatogonia have bilobate nuclei. It is remarkable that most of the cytoplasmic organelles are found in the deep nuclear indentations. The nuage material and nucleolus-like body are well developed in the cytoplasm. After treatment of newts with estradiol benzoate for one year, four types of cells can be distinguished in the peritoneal epithelium. One type is quite different from the columnar cells. These newly appeared cells are large and light in appearance. Their nucleus is highly lobate, and contains dispersed chromatin and several nucleoli with compact electron dense material in its periphery. The cells are characterized by the presence of nuage material and nucleolus-like bodies in the cytoplasm. There are microvilli-like processes between these cells and adjacent elongated cells. These ultrastructural characteristics of the light cells are very similar to those of primordial germ cells and/or primary spermatogonia in normal testes. These findings suggest that the light cells which appear in the peritoneal epithelium of the testes on administration of estrogen may be germ line cells.     

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.     

Cytological study on Sertoli cells and their interactions with germ cells during annual reproductive cycle in turtle

Sertoli cells (SCs) play a central role in the development of germ cells within functional testes and exhibit varying morphology during spermatogenesis. This present study investigated the seasonal morphological changes in SCs in the reproductive cycle of Pelodiscus sinensis by light microscopy, transmission electron microscopy (TEM), and immunohistochemistry. During hibernation period with the quiescent of spermatogenesis, several autophagosomes were observed inside the SCs, the processes of which retracted. In early spermatogenesis, when the germ cells started to proliferate, the SCs contained numerous lipid droplets instead of autophagosomes. In late spermatogenesis, the SCs processes became very thin and contacted several round/elongated spermatids in pockets. At this time, abundant endoplasmic reticulum and numerous mitochondria were present in the SCs. The organization of the tight junctions and the adherens junctions between the SCs and germ cells also changed during the reproductive cycle. Moreover, SCs were involved in the formation of cytoplasmic bridges, phagophores, and exosome secretions during spermatogenesis. Tubulobulbar complexes (TBC) were also developed by SCs around the nucleus of the spermatid at the time of spermiation. Strong, positive expression of vimentin was noted on the SCs during late spermatogenesis compared with the hibernation stage and the early stage of spermatogenesis. These data provide clear cytological evidence about the seasonal changes in SCs, corresponding with their different roles in germ cells within the Chinese soft‐shelled turtle Pelodiscus sinensis.     

Short-time effects of taxol on the seminiferous epithelium of the rat

Summary The effect of taxol, an inhibitor of microtubule degradation, on the seminiferous epithelium was studied. Taxol arrested spermatogenesis at metaphase in both mitotic and meiotic germ cell division. Microtubules were seen to accumulate, especially in the cytoplasm of the spermatogonia, and also in the early spermatids and Sertoli cells. No microtubule accumulation was observed in germ cells during meiotic prophase. Formation of the flagellum was affected in developing spermatids. Peculiar lamellar structures, probably derived from degenerating mitochondria, were seen in the cytoplasm of late spermatids and Sertoli cells.The results are compared with the effects of other mitotic inhibitors such as colchicine and vinca alcaloids.     

The ultrastructure of Sorubim lima (Teleostei, Siluriformes, Pimelodidae) spermatogenesis: premeiotic and meiotic periods

The ultrastructure of Sorubim lima spermatogenesis during the premeiotic and meiotic periods was studied. Our observations showed that the germ cells in the cysts are connected by cytoplasmic bridges and the mitotic and meiotic divisions are slightly asynchronous. The first and the last spermatogonial generations differ in the cellular and nuclear volume, nucleolus, chromatin condensation, distribution, size, density, and shape of the mitochondria, presence of 'lamellae anulata', amount and dimension of the 'nuages', and movement of the centrioles. In addition to the nuclear prophase structures, the spermatocyte I shows changes in all other cellular organelles and elongated vesicles appear in the cytoplasm. The accentuated cytoplasmic density and thickened walled vesicles are morphological characteristics that differentiate spermatocytes II from the other germ cells in the cysts of Sorubim lima testis.     

Rho GTPases and spermatogenesis

Rho GTPases, such as Rho, Rac and Cdc42, are known to regulate many cellular processes including cell movement and cell adhesion. While the cellular events of germ cell movement are crucial to spermatogenesis since developing germ cells must migrate progressively from the basal to the adluminal compartment but remain attached to the seminiferous epithelium, the physiological significance of Rho GTPases in spermatogenesis remains largely unexplored. This paper reviews some recent findings on Rho GTPases in the field with emphasis on the studies in the testis, upon which future studies can be designed to delineate the role of Rho GTPases in spermatogenesis.     

Morphology of the bovine epididymis

The epididymis of the bull was divided into six regions, and morphological differences between regions were studied. The epithelium of all regions contained four cell types: principal and basal epithelial cells, and intraepithelial lymphocytes and macrophages. The epithelium of regions II-V also contained a few apical cells. Principal cells of all regions possessed an endocytotic apparatus including stereocilia underlain by canaliculi, coated vesicles, and subapical vacuoles (up to 1 micron in diameter); however, large vacuoles with a flocculent content and multivesicular bodies (up to 5 microns in diameter) were most numerous in regions II, III, and IV. The unique features of principal cells of region I were the presence of well-developed Golgi bodies, few lipid droplets, and whorls of smooth endoplasmic reticulum in the supranuclear cytoplasm. Numerous mitochondria, distended cisternae of rough endoplasmic reticulum, and dense granules characterized the infranuclear cytoplasm of the principal cells of regions II-VI; however, these features were more developed in region V. Apical cells were characterized by the apical location of the nucleus, many mitochondria in the apical cytoplasm, and few microvilli at the luminal border. Basal cells with few cytoplasmic lipid droplets were present throughout the length of the epididymis but appeared more numerous in region V. Intraepithelial lymphocytes were present at all levels of the epithelium but were never seen in the lumen. Intraepithelial macrophages containing heterogeneous granules, eccentric nuclei, and pseudopods were invariably seen near the basal area of the epithelium in all regions. These observations are discussed in an effort to define the role of each cell type in the epididymal epithelium.     

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.     

Distribution of sulfhydryloxidase (SOx) immunoreactivity in the testis of the axolotl (Ambystoma mexicanum) (amphibia, urodela)

Immunohistochemical localization of sulfhydryloxidase (SOx) has been examined in the testis of the Axolotl (Ambystoma mexicanum). The urodelan testis contains germ cells in various phases of differentiation from primordial germ cells to mature spermatozoa. SOx immunoreactivity is present in mitochondria of primordial germ cells and primary spermatogonia and declines within the population of secondary spermatogonia, suggesting, that the antibody used to localize SOx may serve to estimate the developmental stage of spermatogonia towards meiosis, since more undifferentiated cells react positively. Intensity of immunostaining increases again in spermatocytes and becomes most intense in early round spermatids correlating on ultrastructural level with an accumulation of numerous mitochondria in that part of the cytoplasm, where the acrosome vesicle is formed. Mature sperm are immunonegative. Additionally, Leydig cells within the glandular tissue are stained by the antibody. Thus the distribution pattern of SOx immunoreactivity principally resembles that in the mammalian testis found during ontogenesis or in the adult seminiferous epithelium. The possible functional significance of mitochondrial SOx in germ cells and Leydig cells is discussed. These results suggest, that the amphibian testis is a model for experimental problems dealing with the investigation of germ cells in various developmental phases including very undifferentiated premeiotic germ cells. The cystic testis may be of value in studying influences of various experimental conditions on varied homogeneous populations of germ cells.     

The ultrastructure of the salt gland of Spartina foliosa

Summary The salt gland in Spartina foliosa is composed of two cells, a large basal cell and a smaller, dome-shaped cap cell which is located on a neck-like protrusion of the basal cell. There is no cuticular layer separating the salt gland from the mesophyll tissue. The basal cell has dense cytoplasm which contains numerous mitochondria, rod-like wall protuberances, and infoldings of the plasmalemma which extend into the basal cell and partition the basal cell cytoplasm. The protuberances originate on the wall between the basal and the cap cells and are isolated from the basal-cell cytoplasm by the infoldings of the plasmalemma. While the cap cell has no partitioning membrane system or wall protuberances, it resembles the basal cell by having dense cytoplasm and numerous mitochondria.The basal cell seems to be designed for efficient movement of ions toward the cap cell. The long, dead-end extracellular channels in the basal cell of Spartina appear comparable to surface specializations seen in the secreting epithelium of animal cells which carry out solute-linked water transport. The number of mitochondria and their close association with the plasmalemma extensions suggest that they have an important role in the transfer of ions through the basal cell.The accumulated ions would move into the extracellular spaces along an osomotic gradient where the accompanying passive flow of water would move the ions into the cap-cell wall and from there the solution would pass out through the pores in the cuticle.     

Ultrastructural study of the boar seminiferous epithelium: changes in cryptorchidism

The present study compares the ultrastructural features of Sertoli cells and germ cells between scrotal testes of healthy boars and abdominal testes of unilateral and bilateral cryptorchid boars. In healthy boars, spermatogonia are flat cells lying in close association with the basal lamina. As differentiation progresses, spermatogonia acquire an oval profile and lose their contact with the basal lamina. Spermatocytes are round cells moving from the basal compartment of the seminiferous epithelium to the luminal compartment. Spermatids exhibit complex morphological changes leading to the formation of spermatozoa. Sertoli cells extend from the basal lamina to the tubular lumen. The nucleus encloses fine euchromatin and one or two nucleoli; the nuclear envelope has a few deep infoldings. The lateral cell membranes form junctional specializations that constitute the blood-testis barrier. The cytoplasm encloses smooth endoplasmic reticulum, vesicles, aggregates, and scattered mitochondria. The seminiferous epithelium of abdominal testes from unilateral and bilateral cryptorchid boars contains few spermatogonia with an abnormal appearance; the alteration in germ cell number is more severe in the bilateral disease. In unilateral cryptorchid boars, spermatogonia appear as either large pyramidal cells or roundish cells; in bilateral cryptorchid boars, spermatogonia show roundish profiles and degenerative patterns. Abdominal testes of both unilateral and bilateral cryptorchid boars are constituted by immature Sertoli cells that show abnormal cytoplasmic content, defective development of the blood-testis barrier, and atypical nuclear appearance; in bilateral cryptorchid boars, immature Sertoli cells exhibit degenerative signs. At postpubertal age, unilateral and bilateral cryptorchidism induce total arrest of spermatogenesis at spermatogonial stage as a result of an abnormal differentiation of the Sertoli cells. Moreover, the degeneration of abdominal testes initiates earlier in bilateral cryptorchidism than in unilateral cryptorchidism.     

Testicular involution following optic enucleation

Summary The testes of adult male Syrian hamsters underwent involution within six weeks after optic enucleation. The diameter of the seminiferous tubules was 39% less than controls. Sertoli cells, spermatogonia, and primary spermatocytes were still present, but all steps of spermatids were completely absent from the involuted testes. Lipid droplets filled the Sertoli cell cytoplasm and often encroached upon the nucleus. Sertoli cells had sparse mitochondria and smooth endoplasmic reticulum, but Golgi cisternae were abundant. Typical SertoliSertoli junctions attached contiguous Sertoli cells. With lanthanum tracers it was demonstrated that these junctions were impenetrable; therefore, the bloodtestis barrier was deemed intact. Irregularly shaped protrusions often arose from the peritubular tissue and extended inward toward the seminiferous epithelium, often displacing the cytoplasm of the Sertoli cells and spermatogonia. The core of these protrusions consisted of irregular extensions of myoid cell cytoplasm surrounded by the myoid cells' basal lamina. External to the myoid cell basal lamina were bundles of collagen filaments with the basal lamina of the seminiferous epithelium forming the outermost layer of these protrusions. The apices of the Sertoli cells gave rise to numerous leaf-like processes that extended into and obliterated the lumen of the tubules. The Sertoli cell basal cytoplasm often contained phagocytized degenerating germ cells that appeared to give rise to the lipid droplets that filled the Sertoli cell cytoplasm. Acid phosphatase rich lysosome-like organelles were seen fusing with the degenerating germ cells and lipid droplets. The degenerating germ cells also were shown to contain acid phosphatase activity.     

Maternal inheritance of mitochondrial DNA (mtDNA) in the Pacific oyster (Crassostrea gigas): a preliminary study using mtDNA sequence analysis with evidence of random distribution of MitoTracker-stained sperm mitochondria in fertilized eggs

In many bivalve species, paternal and maternal mitochondrial DNA (mtDNA) from sperm and eggs is transmitted to the offspring. This phenomenon is known as doubly uniparental inheritance (DUI). In these species, sperm mtDNA (M type) is inherited by the male gonad of the offspring. Egg mtDNA (F type) is inherited by both male and female somatic cells and female gonadal cells. In Mytilidae, sperm mitochondria are distributed in the cytoplasm of differentiating male germ cells because they are transmitted to the male gonad. In the present study, we investigated maternal inheritance of mtDNA in the Pacific oyster, Crassostrea gigas. Sequence analysis of two mitochondrial non-coding regions revealed an identical sequence pattern in the gametes and adductor muscle samples taken from six males and five females. To observe whether sperm mitochondria were specifically located in the cytoplasm of differentiating germ cells, their distribution was recorded in C. gigas fertilized eggs by vital staining with MitoTracker Green. Although the 1D blastomere was identified in the cytoplasm of differentiating germ cells, sperm mitochondria were located at the 1D blastomere in only 32% of eggs during the 8-cell stage. Thus, in C. gigas, sperm mitochondria do not specifically locate in the germ cell region at the 1D blastomere. We suggest that the distribution of sperm mitochondria is not associated with germ cell formation in C. gigas. Furthermore, as evidenced by the mtDNA sequences of two non-coding regions, we conclude that mitochondrial DNA is maternally inherited in this species.     

Transmission electron microscopy of impaired spermatogenesis in an avian hybrid

The mechanisms involved in the impaired spermatogenesis of male goldfinch x canary hybrids were investigated by transmission electron microscopy and compared with spermatogenesis in the testes of the parent species. In the parent species the testes were of normal structure, with the only unusual observation being that the Sertoli cells were variable in cytoplasmic electron density. In hybrid birds the Sertoli cells were either electron dense or electron lucent with respect to both nucleus and cytoplasm. In the hybrids examined in this study, no spermatozoa were produced. Spermatogenic stages were arrested without formation of synaptonemal complexes. Centrioles were abnormally arranged in both somatic and germ cells. When they moved away from the basement lamina the germ cells degenerated and were phagocytosed. No focal tight junctions were present between Sertoli cells overlying what would normally have been the basal compartment of the tubule. The basement lamina was unusually thickened, peritubular cells were abnormal in structure, and numerous plasma cells were present in the interstitial tissue. The observations reported here suggest that there was an immunological basis for degeneration of germ cells in the hybrid testis.     

Fine structure of the dorsal epithelium of the tongue of the freshwater turtle,Geoclemys reevesii (Chelonia,Emydinae)

Scanning electron microscopy shows that lingual papillae occur all over the dorsal surface of the tongue of the freshwater turtle, Geoclemys reevesii. The surface of each papilla is composed of compactly distributed hemispherical bulges, each composed of a single cell. Microvilli are widely distributed over the surface of cells. Histological examination reveals that the connective tissue penetrates deep into the center of papillae and that the epithelium is stratified columnar. Under the transmission electron microscope, the cells of the basal and the deep intermediate layers of the epithelium appear rounded. A large nucleus lies in the central area of each cell. The cytoplasm contains mitochondria, endoplasmic reticulum and free ribosomes. The cell membrane form numerous processes. The shallow intermediate layer contains two types of cell. The cytoplasm of the first has numerous fine granules, in addition to mitochondria, ribosomes, and endoplasmic reticulum. The other type of cell contains highly electron-dense granules. The surface layer shows two cell types. One type consists of typical mucous cells. The other type of cell contains fine, electron-lucent granules. The latter cells lie on the free-surface side, covering the mucous cells, and have microvilli on their free surfaces.     

The cycle of follicular and interstitial cells (Leydig cells) in the testis of the marbled newt, Triturus marmoratus (Caudata, Salamandridae)

Ultrastructural examination of the marbled newt (Triturus marmoratus) testis throughout the annual cycle revealed that during the period of testicular quiescence (November-February), primordial germ cells proliferate within cords of filament-rich epithelial cells that will become follicular cells (FCs). Fibroblast-like cells surround the FCs and form the lobule-boundary interstitial cells (ICs). During the period of germ cell development from primordial germ cells to round spermatids (March-June), the FCs surrounding the developing germ cells contain scanty cytoplasm with abundant rough endoplasmic reticulum and scarce filaments. With spermatid elongation (July-August), the FC size grows, its nucleus becomes irregularly outlined, and its cytoplasm displays abundant smooth endoplasmic reticulum, residual bodies, lipid droplets, and large vacuoles. After spermatozoon release by the FCs (August-September), the adjacent ICs increase their size and transform into Leydig cells with abundant smooth endoplasmic reticulum, mitochondria with tubular cristae, and lipid droplets. During the period of testicular quiescence (November-February), the Leydig cells undergo involution, eventually developing the morphological attributes of mesenchymal cells. Intermingled among these cells, cords of filament-rich cells are observed. During this period of the cycle, spermatozoon cysts supported by FCs are present. At the beginning of the germ cell proliferation period (March), these spermatozoa are released, and the adjacent ICs undergo a transformation into Leydig cells similar to those observed in August-September. Maturation and involution of ICs occur when testosterone levels are known to be rising and falling, respectively.     

Occurrence of unusual heterogeneous lipid-containing granule-storing cells in the main excretory duct epithelium of the male mouse submandibular gland

Summary The fine structure of the main excretory duct epithelium of the male mouse submandibular glands was investigated by scanning and transmission electron microscopy. Three principal cell-types were observed: type I and II, and basal cells. This epithelium was characterized by the presence of intercellular canaliculi. Type-I cells were the most numerous. They had an abundance of mitochondria, well-developed Golgi apparatus, a few electron-lucent lipid-containing granules and poorly developed basal infoldings. These cells were also characterized by many glycogen granules throughout the cytoplasm and abundant smooth endoplasmic reticulum in the apical cytoplasm. Type-II cells were the second most numerous. Their most characteristic feature was the presence of abundant heterogeneous lipid-containing granules having acid phosphatase activity at the periphery. They were concentrated in the infra- and supranuclear cytoplasm. The granules may be derived from mitochondrial transformation and seem to be a special kind of secondary autolysosome. Type-II cells also contained abundant mitochondria throughout the cytoplasm, much smooth endoplasmic reticulum in the apical cytoplasm, a well developed Golgi apparatus adjacent to the heterogeneous lipid-containing granules and no basal infoldings. Basal cells were situated adjacent to the basal lamina. They had a large nucleus and the cytoplasm was filled with glycogen granules.     

Caspase 2 activity contributes to the initial wave of germ cell apoptosis during the first round of spermatogenesis

Early in postnatal life the first phase of spermatogenesis is accompanied by an initial wave of germ cell apoptosis. This wave of germ cell death is thought to reflect an adjustment of germ cell numbers that can be adequately maintained by Sertoli cells. Caspase 2 is an initiator caspase whose activation has been found to stimulate apoptosis through the mitochondria. The present study investigates if germ cell apoptosis during the first phase of spermatogenesis involves activation of caspase 2. Germ cell apoptosis was found to peak at Postnatal Days (pnds) 15 and 16 in male C57BL/6 mice. Western blot analysis revealed that caspase 2 also increased in the testes at pnd 16. Immunolocalization of total caspase 2 showed staining of germ cells in the periphery of the seminiferous tubules as well as germ cells more centrally located in an area where apoptotic germ cells were observed. Cytoplasmic as well as nuclear staining was observed. Western blot analysis of cytoplasmic and nuclear proteins from pnd 16 testis revealed pro-caspase 2 in both fractions. Further Western blot analysis for caspase 2 detected an increase in the activation of caspase 2 at pnd 16 in proteins isolated from the cytoplasm but not from the nucleus. Proteins isolated from mitochondria from pnd 16 testes revealed an increase in pro-caspase 2 as well as activated caspase 2 corresponding with an increase in cytochrome c in cytoplasmic fractions. Injection of the caspase 2-specific inhibitor z-VDVAD-fmk directly into the testis significantly reduced the observed germ cell apoptosis at pnds 15 and 16. These results suggest that caspase 2 is present in germ cells in the murine testis in early postnatal life and increases in expression in correspondence to the initial wave of germ cell apoptosis. Caspase 2 also localizes to mitochondria, where it is correlated with a release of cytochrome c and germ cell apoptosis. Blockade of caspase 2 activation reduced the number of apoptotic germ cells in the initial wave of germ cell apoptosis, indicating that caspase 2 plays an important role upstream of the mitochondria in germ cell apoptosis during the first phase of spermatogenesis.