The amphibian testis as model to study germ cell progression during spermatogenesis

Testicular morphology of vertebrate testis indicates requirement of local control. In urodeles, the testis is organized in lobes of increasing maturity throughout the cephalocaudal axis. The anuran testis is organized in tubules. Spermatogenesis occurs in cysts composed by Sertoli cells enveloping germ cells at synchronous stages. Moreover, in numerous species germ cell progression lasts a year which defines the sexual cycle. Due to the above quoted features, research on factors regulating germ cell progression in amphibians may reach greater insight as compared with mammalian animal models. In particular, studies on endocrine and paracrine/autocrine factors involved in the regulation of germ cell functions reveal that fos activation and a J protein, previously specifically found in mouse testis, exert an important role in spermatogonial proliferation and maturation of post-meiotic stages, respectively.     

Murine Asb-17 expression during mouse testis development and spermatogenesis

In this study we isolated a murine mAsb-17 from mouse testis by RT-PCR using primers designed based on the sequences from the GenBank database. The sequence analysis showed that mAsb-17 encodes a 295 amino acid polypeptide with a molecular weight of approximately 34 kDa containing two ankyrin repeats and one SOCS box. The amino acid sequence of mASB-17 showed 87.5%, 98.3% and 92.9% identity with that of human, rat and dog, respectively. Interestingly, northern blot analysis showed that mAsb-17 was expressed only in the testis. The expression analysis by RT-PCR for mAsb-17 in mouse indicates that mAsb-17 is expressed from the fourth week after birth to adult, with the highest expression in round spermatids. Both northern blot and RT-PCR analyses suggest that mASB-17 may play essential roles in testis development and spermatogenesis.     

Deciphering the pathways of germ cell apoptosis in the testis

A growing body of evidence demonstrates that germ cell death both spontaneous (during normal spermatogenesis) and that induced by suppression of hormonal support or increased scrotal temperature occurs via apoptosis. The mechanisms by which these proapoptotic stimuli activate germ cell apoptosis are not well understood. In order to provide some insight, here we report the key molecular components of the effector pathways leading to caspase activation and increased germ cells apoptosis triggered by mildly increased scrotal temperature. Short-term exposure (43 °C for 15 min) of the testis to mild heat results, within 6 h, in stage- and cell-specific activation of germ cell apoptosis in rats. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to paranuclear localization in heat-susceptible germ cells. Such relocation of Bax is further accompanied by sequestration of mitochondria and endoplasmic reticulum (ER) into paranuclear areas, cytosolic translocation of cytochrome c and is associated with activation of the initiator caspase 9 and the executioner caspases 3, 6, and 7, and cleavage of PARP. Furthermore, Bax is co-localized with ER in the susceptible germ cells as assessed by combined two-photon and confocal microscopy and Western blot analyses of fractionated testicular lysates. In additional studies, using gld and lpr^cg mice, which harbor loss-of-function mutations in Fas-ligand (FasL) and Fas, respectively, we demonstrated that heat-induced germ cell apoptosis is not blocked, thus providing further evidence that the Fas signaling system is dispensable for heat-induced germ cell apoptosis in the testis. Taken together, these results demonstrate that the mitochondria- and possibly also ER-dependent pathways are the key apoptotic pathways for heat induced germ cell death in the testis.     

Chemical anoxia delays germ cell apoptosis in the human testis

An understanding of testicular physiology and pathology requires knowledge of the regulation of cell death. Previous observation of suppression of apoptosis by hypoxia suggested a role for ATP in germ cell death. However, the exact effects of ATP production on germ cell death and of apoptosis on the levels of ATP and other adenine nucleotides (ANs) have remained unclear. We investigated the levels of ANs during human testicular apoptosis (analyzed by HPLC) and the role of chemical anoxia in germ cell death (detected by Southern blot analysis of DNA fragmentation, in situ end labeling of DNA, and electron microscopy). Incubation of seminiferous tubule segments under serum-free conditions induced apoptosis and concomitantly decreased the levels of ANs. Chemical anoxia, induced with potassium cyanide (KCN), an inhibitor of mitochondrial respiration, dropped ATP levels further and suppressed apoptosis at 4 h. After 24 h, many of the testicular cells underwent delayed apoptosis despite ATP depletion. Some cells showed signs of necrosis or toxicity. The addition of 2-deoxyglucose, an antimetabolite of glycolysis, did not alter the results obtained with KCN alone, whereas a toxic concentration of hydrogen peroxide switched apoptosis to necrosis. In most of the testicular cells, mitochondrial respiration appears to play a crucial role in controlling primary cell death cascades. In the human testis, there seem to be secondary apoptotic pathways that do not require functional respiration (or ATP).     

Ultrastructural changes in the male germ cells of Bulinus truncatus during spermatogenesis

The structure of the spermatogonia, spermatocytes, spermatids and Sertoli cells of the hermaphroditic snail Bulinus truncatus was studied by electron microscopy. The spermatogonia are small, with relatively large nuclei. The acrosome develops from a small proacrosomal granule which is probably derived from the Golgi apparatus in the spermatocyte stage. Condensation and elongation of the nuclei were found in the spermatids. The shape and components of the Sertoli cells did not change during the spermatogonium and spermatocyte stages. Before spermiation the Sertoli cells have the morphological features of steroid-producing cells. The study showed that the Sertoli cells are involved in the nutrition and transportation of the spermatogenic cells, in spermiation and in hormone production.     

Overexpression of Bcl-W in the testis disrupts spermatogenesis: revelation of a role of BCL-W in male germ cell cycle control

To explore physiological roles of BCL-W, a prosurvival member of the BCL-2 protein family, we generated transgenic (TG) mice overexpressing Bcl-w driven by a chicken beta-actin promoter. Male Bcl-w TG mice developed normally but were infertile. The adult TG testes displayed disrupted spermatogenesis with various severities ranging from thin seminiferous epithelium containing less germ cells to Sertoli cell-only appearance. No overpopulation of any type of germ cells was observed during testicular development. In contrast, the developing TG testes displayed decreased number of spermatogonia, degeneration, and detachment of spermatocytes and Sertoli cell vacuolization. The proliferative activity of germ cells was significantly reduced during testicular development and spermatogenesis, as determined by in vivo and in vitro 5'-bromo-2'deoxyuridine incorporation assays. Sertoli cells were structurally and functionally normal. The degenerating germ cells were TUNEL-negative and no typical apoptotic DNA ladder was detected. Our data suggest that regulated spatial and temporal expression of BCL-W is required for normal testicular development and spermatogenesis, and overexpression of BCL-W inhibits germ cell cycle entry and/or cell cycle progression leading to disrupted spermatogenesis.     

Temporal germ cell development strategy during spermatogenesis within the testis of the Ground Skink, Scincella lateralis (Sauria: Scincidae)

Ground Skink (Scincella lateralis) testes were examined histologically to determine the testicular organization and germ cell development strategy employed during spermatogenesis. Testicular tissues were collected from 19 ground skinks from Aiken County, South Carolina during the months of March-June, August, and October. The testes consisted of seminiferous tubules lined with germinal epithelia in which germ cells matured in close association with Sertoli cells. As germ cells matured, they migrated away from the basal lamina of the epithelia towards the lumina of the seminiferous tubules. The testes were spermatogenically active during the months of March, April, May, June, and October (largest seminiferous tubule diameters and epithelial heights), but entered a quiescent period in August (smallest seminiferous tubule diameter and epithelial height) where only spermatogonia type A and B and early spermatocytes were present in low numbers within the seminiferous epithelium. Although the testicular organization was similar to other amniotes, a temporal germ cell development strategy was employed during spermatogenesis within Ground Skinks, similar to that of anamniotes. Thus, this skink's germ cell development strategy, which also has been recently reported in all other major reptilian clades, may represent an evolutionary intermediate in terms of testicular organization between anamniotes and birds and mammals.     

Spontaneous germ cell death in the testis of the adult rat takes the form of apoptosis: re-evaluation of cell types that exhibit the ability to die during spermatogenesis

Spontaneous germ cell degeneration occurs in the testis of the adult rat. Accumulating data supports the idea that this degeneration takes place via apoptosis. We have determined that morphology, acridine orange staining and ultrastructural features of these cell deaths clearly take the form of apoptosis. Furthermore, with acridine orange staining it was possible to detect a cell population showing early signs of death. The characterization of the main morphological features of these cells allowed us to identify several steps of maturing germ cells undergoing degeneration that have not previously been described. We have reevaluated in toluidine blue stained semithin sections the germ cell types that undergo cell death at every stage of the spermatogenic cycle in the adult rat and concluded that, spermatogonia undergo cell death coinciding with their mitotic peaks, spermatocytes during preleptotene, leptotene, zygotene, pachytene and during metaphase I and spermatids during all their maturation steps. The biological significance of these cell deaths, at these steps of germ cell development, in relation to apoptosis, is discussed.     

Continuous spermatogenesis and the germ cell development strategy within the testis of the Jamaican Gray Anole, Anolis lineatopus

Testicular tissues from Anolis lineatopus were examined histologically to determine testicular structure, germ cell morphologies, and the germ cell development strategy employed during spermatogenesis. Anoles (N = 36) were collected from southern Jamaica from October 2004 to September 2005. Testes were extracted and fixed in Trump's fixative, dehydrated, embedded in Spurr's plastic, sectioned, and stained with basic fuchsin/toluidine blue. The testes of Jamaican Anoles were composed of seminiferous tubules lined with seminiferous epithelia, similar to birds and mammals, and were spermatogenically active during every month of the year. However, spermatogenic activity fluctuated based on morphometric data for February, May and June, and September-December. Sequential increases for these months and decreases in between months in tubular diameters and epithelial heights were due to fluctuations in number of elongating spermatids and spermiation events. Cellular associations were not observed during spermatogenesis in A. lineatopus, and three or more spermatids coincided with mitotic and meiotic cells within the seminiferous epithelium. Although the germ cell generations were layered within the seminiferous epithelium, similar to birds and mammals, the actual temporal development of germ cells and bursts of sperm release more closely resembled that reported recently for other reptilian taxa. All of these reptiles were temperate species that showed considerable seasonality in terms of testis morphology and spermatogenesis. The Jamaican Gray Anole has continuous spermatogenesis yet maintains this temporal germ cell development pattern. Thus, a lack of seasonal spermatogenesis in this anole seems to have no influence on the germ cell development strategy employed during sperm development.     

Ultrastructure of germ cell development in the human fetal testis

Summary Electron-microscopic examination of the human fetal testis between 10 and 20 weeks gestation reveals the presence of two distinct cell types within the tubules: Sertoli cells and germ cells. The latter are distinguished by their spherical shape, smooth nuclear membranes, globular mitochondria and paucity of cytoplasmic organelles. The gonocytes, or primitive germ cells, occur as single cells in the central portions of the tubules. Their chromatin is finely granular and evenly dispersed. Nucleoli are centrally placed and of uniform electron density. Various stages in the migration of gonocytes to the tubular periphery are indicated by the extension of cytoplasmic processes toward the basal lamina. Bands of microtubules are present within the processes. Spermatogonia are arranged in pairs and groups at the tubular periphery. They lack the nucleolar and mitochondrial characteristics of adult spermatogonia. Except for slight changes in chromatin density and nucleolar structure, the fetal spermatogonia retain the ultrastructural characteristics of gonocytes. Intercellular bridges connect adjacent spermatogonia. Degeneration affecting large numbers of germ cells, but primarily gonocytes, begins with nuclear infolding and chromatin condensation and eventually involves both nuclear and cytoplasmic structures. The degenerated cells are removed by phagocytosis by adjacent Sertoli cells. Large phagosomes are present in the cytoplasm of many of the Sertoli cells.Supported by a grant from the Ford Foundation and by General Research Support Grant RR055511 from the National Institutes of Health. Technical assistance was provided by Mrs. Lucy A. Conner.     

Critical period of nonpromoter DNA methylation acquisition during prenatal male germ cell development

The prenatal period of germ cell development is a key time of epigenetic programming in the male, a window of development that has been shown to be influenced by maternal factors such as dietary methyl donor supply. DNA methylation occurring outside of promoter regions differs significantly between sperm and somatic tissues and has recently been linked with the regulation of gene expression during development as well as successful germline development. We examined DNA methylation at nonpromoter, intergenic sequences in purified prenatal and postnatal germ cells isolated from wildtype mice and mice deficient in the DNA methyltransferase cofactor DNMT3L. Erasure of the parental DNA methylation pattern occurred by 13.5 days post coitum (dpc) with the exception of approximately 8% of loci demonstrating incomplete erasure. For most loci, DNA methylation acquisition occurred between embryonic day 13.5 to 16.5 indicating that the key phase of epigenetic pattern establishment for intergenic sequences in male germ cells occurs prior to birth. In DNMT3L-deficient germ cells at 16.5 dpc, average DNA methylation levels were low, about 30% of wildtype levels; however, by postnatal day 6, about half of the DNMT3L deficiency-specific hypomethylated loci had acquired normal methylation levels. Those loci normally methylated earliest in the prenatal period were the least affected in the DNMT3L-deficient mice, suggesting that some loci may be more susceptible than others to perturbations occurring prenatally. These results indicate that the critical period of DNA methylation programming of nonpromoter, intergenic sequences occurs in male germline progenitor cells in the prenatal period, a time when external perturbations of epigenetic patterns could result in diminished fertility.     

Stereological studies on germ cell mitochondria of rat during spermatogenesis and spermiogenesis

The following stereological parameters of mitochondria were calculated in rat germ cells during the spermatogenesis: volume density of matrix, outer compartment, outer membrane and inner membrane, surface density of outer membrane and inner membrane. They were the basis to calculate the partition coefficient of matrix and partition coefficient of outer compartment. The matrix volume demonstrated a decreasing in mitochondria of germ cells during spermatogenesis. The relative volume of outer compartment was calculated with the intracristal spaces and revealed increasing from spermatogonia to spermatids. The partition coefficient for the matrix significantly decreased. Our observations suggest that transformation of mitochondrial configuration during spermatogenesis and spermiogenesis is the expression of intensive metabolic processes and activity of membrane transport in germ cells.     

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.     

Temporal and spatial expression of neurotrophins and their receptors during male germ cell development.

Spermatogenesis is a stepwise cellular differentiation process involving proliferation and commitment to differentiate in spermatogonia, meiosis in spermatocytes, and morphological changes in round spermatids. The whole process is regulated by intercellular communication between the germ cells and the supporting cells. In order to investigate whether neurotrophin family and their receptors contribute to the intercellular communication, we examined the expression of neurotrophins and their receptors in testis during spermatogenesis. One of neurotrophin family, NT-3 was expressed in spermatocytes and spermatogonia while its high affinity receptor, TrkC was found mainly in late spermatids and their low affinity receptor, TrkA in spermatocytes and round spermatids. On the other hand, BDNF immunoreactivity was found in Sertoli cells while its high affinity receptor, TrkB was found in spermatogonia. The temporally and spatially regulated expression of neurotrophins, NT-3 and BDNF, and their receptors, TrkC and TrkB, during male germ cell development suggests that neurotrophins play as the paracrine factors in the intercellular communication between the germ cells and the supporting somatic cells to control germ cell development.