Annual cyclical changes in the testicular activity of a freshwater teleost, Barbus luteus (Heckel) from Shatt-Al-Arab, Iraq

The testes of Barbus luteus are a pair of elongated structures composed of an anastomosing system of seminiferous lobules. Six spermatogenetic stages of germ cells have been described in the gonad. The endocrine factor lies in the interstitial cells. Spermiation occurs during the months of May, June and July. Fertilization appears to be external. Un-expelled spermatozoa undergo pycnotic degeneration. The testes show a regular annual cycle which has been described in five arbitrary stages.     

Schistosoma mansoni: reversible destruction of testes by procarbazine

1. The anticancer drug procarbazine is profoundly damaging to the testes of Schistosoma mansoni when administered at 200 mg/kg or more to the mouse host. Somatic tissues appear entirely unaffected. 2. Within 2 days the meiotic process is disrupted, and primary and secondary spermatocytes and spermatids are destroyed and replaced by amorphous granular material. 3. The testes regenerate within about 15 days, apparently from surviving spermatogonial resting cells near the germinal epithelium of the testis. 4. Livers of mice treated once 7 weeks earlier have numerous egg granulomas and give rise to many miracidia, suggesting that full testis function is regained. 5. Male worms given the drug at 200 mg/kg at 19 days of age and fixed 7 weeks later have on average one testis less than control worms, indicating that about 15% of immature testes are unable to regenerate, whereas mature worms given the drug regenerate the normal number of testes. 6. The drug did not have significant antispermatogenic effects when male worms were incubated for a week with various concentrations in vitro, suggesting that a host metabolite is the active agent.     

Presentation of antigen by B cells: functional dependence on radiation dose, interleukins, cellular activation, and differential glycosylation

Whether resting B cells can present antigen to T cells is controversial. Several factors can influence the outcome of an assessment of the presenting function of resting B cells: the method of purifying resting B cells and maintaining them in culture without altering their resting state, the sensitivity of resting B cells to gamma-irradiation, the activation state of the T cells used to assess presenting function, and the requirement for exogenous interleukin 1. We have examined all of these variables and find that one adherent antigen-presenting cell is functionally equivalent to four LPS-activated B cells and to 1000 resting B cells. In addition, we have examined the potential functional relevance of the differential glycosylation of Ia molecules on resting B cells compared with adherent antigen-presenting cells. Altering the surface glycosylation of resting B cells by neuraminidase treatment results in a 25-fold increase in B cell antigen presentation without altering their resting state. More important, among antigen-presenting cells the effect of neuraminidase is limited to resting B cells. It also appears to involve a restricting element such as the Ia molecule rather than total cell surface charge, because neuraminidase treatment has no effect on the capacity of resting B cells to serve as accessory cells in the Con A response.     

Intermediate-sized filaments present in Sertoli cells are of the vimentin type.

The cytoplasmic structure of Sertoli cells of rat testes has been studied by electron microscopy of ultrathin sections. Sertoli cells contain numerous intermediate-sized (7-11 nm) filaments which form a meshwork extending throughout the whole cytoplasm. Often the frequency of such filaments appears especially high in juxtanuclear and cortical regions, including the apical recesses containing the spermatids. Examination of frozen sections of testes by indirect immunofluorescence microscopy using guinea pig antibodies to prekeratin and vimentin has shown the absence of intermediate-sized filaments of the cytokeratin type in all cells of the testes but the presence of filaments of the vimentin type in Sertoli cells as well as in cells of the interstitial space. These results show that the intermediate-sized filaments, abundant in Sertoli cells, are of the vimentin type. In addition we conclude that the "germ epithelium" differs from others true epithelia by the absence of cytokeratin filaments and typical desmosomes and, in Sertoli cells, the presence of vimentin filaments, suggestive of a mesenchymal character or derivation.     

Histological and ultrastructural investigation of early gonad development and sex differentiation in Adriatic sturgeon (Acipenser naccarii,Acipenseriformes, Chondrostei)

Gonad development and sex differentiation from embryos to 594‐day‐old individuals were investigated in farmed Acipenser naccarii using light and transmission electron microscopy. The migrating primordial germ cells first appear along the dorsal wall of the body cavity in embryos 1.5 days before hatching. The gonadal ridge, containing a few primary primordial germ cells (PGC‐1) surrounded by enveloping cells, appears in 16‐day‐old larvae. At 60 days, the undifferentiated gonad is lamellar and PGC‐1 multiply, producing PGC‐2. In 105‐day‐old juveniles, a distinct germinal area with advanced PGC‐2 appears on the lateral side near the mesogonium and the first blood vessels are visible. At 180 days, putative ovaries with a notched gonadal epithelium and putative testes with a smooth one appear, together with adipose tissue on the distal side. In 210‐day‐old juveniles, active proliferation of germ cells begins in the putative ovaries, whereas putative testes still contain only a few germ cells. The onset of meiosis and reorganization of stromal tissue occurs in ovaries of 292‐day‐old individuals. Ovaries with developed lamellae enclosing early oocyte clusters and follicles with perinucleolar oocytes occur at 594 days. Meiotic stages are never found, even in anastomozing tubular testes of 594‐day‐old individuals. Steroid producing cells are detected in the undifferentiated gonad and in the differentiated ones of both sexes. Anatomical differentiation of the gonad precedes cytological differentiation and female differentiation largely precedes that of the male. Gonad development and differentiation are also associated with structural changes of connective tissue, viz. collagen‐rich areas are massive in developing testes and reduced in ovaries. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Comparative study of enzymes in testes and ovaries from adult Dipetalogaster maximus (Uhler) and triatoma infestans (Klug) (Hemiptera: Reduviidae). correlation with fine structural organization.

Activities of hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PDH), fructose-6-phosphate kinase (F6PK), glutamate dehydrogenase (GlutDH), aspartate aminotransferase (AAT), malate dehydrogenase (MDH) and glycerol-3-phosphate dehydrogenase (GPDH) were determined in tissue extracts of testes and ovaries of adult Dipetalogaster maximus (Uhler) and Triatoma infestans (Klug) (Hemiptera: Reduviidae), insect vectors of Chagas disease. The fine structure organization of the same organs were studied by electron microscopy. Results allow the following inferences: in testes from both species, most of the glucose would be utilized through the glycolytic pathway. Amino acid catabolism for energy purposes appears to be unimportant. The number of mitochondria and the development of the rough endoplasmic reticulum in cells of the spermatogenic line indicate the occurrence of active oxidative metabolism and protein synthesis; in ovaries, levels of G6PDH indicate the existence of an active pentose pathway which would supply the NADPH required for fat and ecdysteroid synthesis. Amino acid catabolism appears to be relatively more important in ovary than in testis. Fat and glycogen are stored in follicular cells of D. maximus; oocytes of both species contain numerous fat droplets. Abundant mitocondria are present in follicular cells and oocytes. A well developed rough endoplasmic reticulum and free ribosomes are also conspicuous in these cells. The malate/aspartate H-transfer system seemed to be relatively more important than the glycerophosphate shuttle in ovaries as well in testes.     

Asynchronous expression of the homeodomain protein CUX1 in Sertoli cells and spermatids during spermatogenesis in mice

The homeodomain CUX1 protein exists as multiple isoforms that arise from proteolytic processing of a 200-kDa protein or an alternate splicing or from the use of an alternate promoter. The 200-kDa CUX1 protein is highly expressed in the developing kidney, where it functions to regulate cell proliferation. Transgenic mice ectopically expressing the 200-kDa CUX1 protein develop renal hyperplasia associated with reduced expression of the cyclin kinase inhibitor p27. A 55-kDa CUX1 isoform is expressed exclusively in the testes. We determined the pattern and timing of CUX1 protein expression in developing testes. CUX1 expression was continuous in Sertoli cells from prepubertal testes but became cyclic when spermatids appeared. In testes from mature mice, CUX1 was highly expressed only in round spermatids at stages IV-V of spermatogenesis, in both spermatids and Sertoli cells at stages VI-X of spermatogenesis, and only in Sertoli cells at stage XI of spermatogenesis. While most of the seminiferous tubules in wild-type mice were between stages VI and X of spermatogenesis, there was a significant reduction in the percentage of seminiferous tubules between stages VI and X in Cux1 transgenic mice and a significant increase in the percentage of seminiferous tubules in stages IV-V and XI. Moreover, CUX1 was not expressed in proliferating cells in testes from either wild-type or transgenic mice. Thus, unlike the somatic form of CUX1, which has a role in cell proliferation, the testis-specific form of CUX1 is not involved in cell division and appears to play a role in signaling between Sertoli cells and spermatids.     

Reproductive anatomy of the Australian grayling, Prototroctes maraena Gunther

Gross anatomy of Australian grayling testes showed progressive enlargement from March to the peak of the spawning season in late April–early May. The single, left testis changed from a flat, translucent wisp of tissue consisting of an anterior and posterior lobe connected by a thin filament to a three-dimensional mass of milky-white tissue which filled the coelomic cavity. Spawning lasted 2 weeks and testicular regression was apparent by 8 May when the bilobed condition of the testis was noted. The testis cleared rapidly and resembled pre-spawning conditions by mid-June. Histological examination revealed that Australian grayling testes follow the pattern of teleost spermatogenesis whereby clusters of spermatogonia arise from resting germ cells and all of the cells within a given cluster mature at approximately the same time.
The single left ovary of Australian grayling females showed a similar pattern of development, filling the body cavity with an average of 47 000 eggs of 0.9 mm diameter by late April–early May. Females exhibited swollen genital vents near spawning and males developed breeding tubercles which are illustrated by scanning electron micrograph.     

Stem cell therapy for the treatment of Leydig cell dysfunction in primary hypogonadism

The production of testosterone occurs within the Leydig cells of the testes. When production fails at this level from either congenital, acquired, or systemic disorders,the result is primary hypogonadism. While numerous testosterone formulations have been developed, none are yet fully capable of replicating the physiological patterns of testosterone secretion. Multiple stem cell therapies to restore androgenic function of the testes are under investigation. Leydig cells derived from bone marrow, adipose tissue, umbilical cord, and the testes have shown promise for future therapy for primary hypogonadism. In particular, the discovery and utilization of a group of progenitor stem cells within the testes, known as stem Leydig cells(SLCs), has led not only to a better understanding of testicular development, but of treatment as well. When combining this with an understanding of the mechanisms that lead to Leydig cell dysfunction, researchers and physicians will be able to develop stem cell therapies that target the specific step in the steroidogenic process that is deficient. The current preclinical studies highlight the complex nature of regenerating this steroidogenic process and the problems remain unresolved. In summary, there appears to be two current directions for stem cell therapy in male primary hypogonadism. The first method involves differentiating adult Leydig cells from stem cells of various origins from bone marrow, adipose, or embryonic sources. The second method involves isolating, identifying, and transplanting stem Leydig cells into testicular tissue. Theoretically, in-vivo re-activation of SLCs in men with primary hypogonadism due to age would be another alternative method to treat hypogonadism while eliminating the need for transplantation.     

Phospholipid hydroperoxide glutathione peroxidase of rat testis. Gonadotropin dependence and immunocytochemical identification.

A high glutathione peroxidase activity toward phospholipid hydroperoxides is present in rat testis. The attribution of this activity to the selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPX) was supported by cross-reactivity with antibodies raised against pig heart PHGPX which had been purified and characterized. Rat testis PHGPX is partially cytosolic and partially linked to nuclei and mitochondria. The soluble and organelle-bound enzymes appear identical by Western blot analysis. PHGPX, but neither selenium-dependent nor non-selenium-dependent glutathione peroxidase activity, is expressed in testes only after puberty, disappears after hypophysectomy, and is partially restored by gonadotropin treatment. Specific immunostaining of testes by antiserum against PHGPX appears as a fine granular brown pattern localized throughout the cytoplasm in more immature cells but is confined to the peripheral part of the cytoplasm, the nuclear membrane, and mitochondria in maturating spermatogenic cells. As expected, immunostaining of spermatogenic cells in hypophysectomized animals was negative, but gonadotropin treatment only marginally increased the immunoreactivity. The expression of PHGPX in testes is consistent with the previously described specific requirement for selenium for synthesis of a 15-20-kDa selenoprotein which is related to the production of functional spermatozoa.     

Expression of antigen interaction structures on resting and activated cytotoxic lymphocytes

The expression of T-cell receptor protein and Lyt-2 was examined on resting and activated antigen-specific cytotoxic lymphocytes. Receptor expression was largely independent of the activation state of the cells. In contrast Lyt-2 expression was approximately threefold higher on activated than on resting cells. Since lytic activity varied by as much as 100-fold, it appears that lytic activity is primarily regulated by processes independent of quantitative changes in antigen interaction structures.     

A histological study of maturity in male goldlined seabream Rhabdosargus sarba (Forskål, 1775) in the Oman Sea

The goldlined seabream Rhabdosargus sarba (Forskål, 1775) is farmed in several countries of the Western Pacific Rim and is a candidate species for aquaculture in the Gulf States of the Middle East. This study was undertaken to examine the onset and patterns of maturity in male goldlined seabream in support of the development of practical hatchery techniques. The testes of 337 male fish (0 + to 7 + , average FL 183 ± 20 to 315 ± 12 mm) collected from the Oman trap fishery (August 2001–April 2002; September 2002–2003) were classified into four stages, based on cell type (immature, developing, active or resting). Four spermatogenetic cell types were identified in the testes spermatogonia (types a and b), spermatocytes, spermatids and spermatozoa. Active testes appeared more regularly during the peak of the reproductive season in January and February. In March, resting testes were observed indicating the end of the reproductive season. There was clear evidence of protandry. Intersex gonads were observed in male fish from age 0 + to 7 + (33% at age 0 + to 75% at age 7 + with maximum 82% intersex frequency observed at age 5 + ). From the evidence presented, male fish age 1 + produced free‐flowing sperm under slight abdominal pressure during the period November–March.     

The messenger RNA sequences in growing and resting mouse fibroblasts.

The sequences present in messenger RNA in resting and growing 3T6 cells have been examined. First, the abundance and complexity classes of mRNA in growing 3T6 were compared to those in other established cell lines. The overall complexities measured for mRNA from HeLa cells and the three mouse fibroblast lines, 3T6, SV-PY-3T3, and L, are qualitatively similar and correspond to approximately 10,000 sequences. The relative amount of the two major abundance classes and their complexities appear identical in the three mouse fibroblast lines despite their different histories. HeLa cell mRNA is significantly different both in the amount and the complexity of the two major classes. The complexity of the two mRNA classes appears the same in resting and growing 3T6, although there is a small difference in relative amounts. Cross hybridizing cDNA and mRNA from resting and growing cells shows that the majority of mRNA sequences are the same in the two states. However, cross hybridization after the common sequences are removed shows that about 3% of the mRNA in resting cells is not present in the growing state, while the opposite cross shows 3% of the mRNA in growing cells is not present in resting cells. These differences may result from alterations in gene expression which are related to the growth state of the cell.     

Morphology and kinetics of spermatogenesis in Xenopus laevis.

The light microscopic characteristics of spermatogenic stages of the germ cell line in the anuran, Xenopus laevis, have been described as they appear in both nuclear squash preparations and plastic embedded thick sections of intact testes. Tritiated thymidine autoradiography was employed to unequivocally identify stages. Using this methodology, it was determined that the premeiotic DNA synthetic period occurs in a cell which is morphologically indistinguishable from a late secondary spermatogonial cell. In addition, kinetic studies with tritiated thymidine were employed to determine the duration of meiotic prophase and spermiogenesis in Xenopus. Results indicate that at 18 degrees C, the most rapidly matururing cells in the testis spend four days in leptotene, six days in zygotene, twelve days in pachytene, one day in diplotene, one day in meiotic division, and twelve days in spermiogenesis. An estimate for the duration of the premeiotic S stage was indirectly calculated from combined data to yield a value of six to seven days. Pooled spermatocyte counts measuring the frequency of occurrence of individual stages produced results which correlated closely with estimates obtained by tritiated thymidine labelling. Individual counts, however, show wide variations between testes from single animals and between testes from different animals, whether sacrificed together or at different times of the year. Nevertheless, in all cases, both morphology and labelling patterns indicate that spermatogenesis is continuously active. The variation observed in individual testes appears to be the result of waves of non-random entry of spermatogonial stem cells into the population of cells irreversibly committed to differentiation.     

Clinical aspects of male germ cell apoptosis during testis development and spermatogenesis

Apoptosis appears to have an essential role in the control of germ cell number in testes. During spermatogenesis germ cell deletion has been estimated to result in the loss of up to 75% of the potential number of mature sperm cells. At least three factors seem to determine the onset of apoptosis in male germ cells: (1) lack of hormones, especially gonadotropins and androgens; (2) the specific stage in the spermatogenic cycle; (3) and the developmental stage of the animal. Although male germ cell apoptosis has been well characterized in various animal models, few studies are presently available regarding germ cell apoptosis in the human testis. The first part of this review is focused on germ cell apoptosis in testes of prepubertal boys, with special emphasis on apoptosis in normal and cryptorchid testes. A higher percentage of apoptotic spermatogonia was seen in the cryptorchid testes than in the scrotal testes. The hCG-treatment increased the number of apoptotic spermatogonia. The hCG-treatment-induced apoptosis in spermatogonia had severe long-term consequences in reproductive functions in adulthood. Increased apoptosis after hCG-treatment was associated with subnormal testis volumes, subnormal sperm density and pathologically elevated serum FSH. This finding indicates that increased apoptosis in spermatogonia in prepuberty leads to disruption of testis development. To evaluate the role of apoptosis in human adult testes, apoptosis was induced in seminiferous tubules that were incubated under serum-free conditions in the absence or presence of testosterone. Most frequently apoptosis was identified in spermatocytes. Occasionally some spermatids also showed signs of apoptosis. In short term incubations apoptosis was suppressed by testosterone. Our findings lead to the conclusion that apoptosis is a normal, hormonally controlled phenomenon in the human testis. The role of apoptosis in disorders of spermatogenesis remains to be established.     

Comparison of testosterone and insulin-like peptide 3 secretions in response to human chorionic gonadotropin in cultured interstitial cells from scrotal and retained testes in dogs

Levels of testosterone and insulin-like peptide 3 (INSL3) secretions in response to different doses of human chorionic gonadotropin (hCG) in cultured interstitial cells were compared between retained and scrotal testes in dogs. Retained (n=10) and scrotal (n=9) testes were obtained from small-breed dogs. The testicular tissues were dispersed in Dulbecco's Modified Eagle Medium with Ham's nutrient mixture containing 2000 PU/ml dispase II and 10% fetal bovine serum. The cells were plated with differing concentrations (0-10 IU/ml) of hCG for 18 h in multiwell-plates. Testosterone and INSL3 in the same spent medium were measured by enzyme-immunoassays (EIA). A new EIA with a reliable detection range of 0.025-5 ng/ml was developed in order to measure canine INSL3 in culture medium. Dose-dependent stimulation of testosterone by hCG was observed in the cells of both retained and scrotal testes. The incremental rate of testosterone secretion was significantly lower at 0.1, 1 and 10 IU/ml hCG in the cells of retained testes than in scrotal testes, however. INSL3 secretion was significantly stimulated at 10 IU/ml hCG relative to unstimulated controls comprising cells of scrotal testes; no such stimulation was observed in the cells of retained testes. At 10 IU/ml hCG, the incremental rate of INSL3 was significantly lower in the cells of retained testes than scrotal testes. These results suggest that LH-induced secretory testosterone and INSL3 responses are lower in the interstitial cells of retained testes than of scrotal testes. Furthermore, the high concentrations of LH may acutely stimulate INSL3 release in scrotal testes of dogs, but not in retained testes.     

Enzyme levels in testis and other tissues of genetically sex-reversed mice

The specific activity of G6PD and PGK were measured in the testes, seminal vesicles, and livers of Sxr/+,XX mice, their Sxr/+,XY littermates and normal mice. While G6PD activity was high in the testes of young normal mice and declined as the testes matured, in the testes of Sxr/+,XX mice activity remained high, suggesting a failure of the Sertoli cells to mature normally. The activity of PGK was low in the testes of young normal mice, and increased as the testes matured. The testes of young Sxr/+,XX mice had high activity of this enzyme which remained high into adulthood. The high activity in young mice suggests an abnormality in the somatic cells. The seminal vesicle and liver measurements of G6PD and PGK confirmed that the Sxr/+,XX mice were phenotypically normal males except with respect to the testis. The developmental patterns of both enzymes in testes lacking germinal cells indicate that the maturation of the somatic cells of the normal testis is influenced by the presence of germinal cells.     

Ultrastructural and biochemical characteristics of leydig cells from newborn androgen-insensitive rats

Summary Leydig cells of the testis of newborn pseudohermaphrodite (tfm) rats have an ultrastructure similar to that of the normal, containing well developed organelles and inclusions. The cytoplasm is filled with smooth endoplasmic reticulum forming a network of interconnected tubules. Lipid droplets are surrounded by cisternae of smooth endoplasmic reticulum and are in close association with pleomorphic mitochondria. Many of the latter are cup-shaped and have tubular cristae and intramitochondrial dense bodies.Essentially, these are characteristics of normal Leydig cells. Accordingly, the production of testosterone by testes from newborn tfm rats is the same as that by testes from normal newborns and adults. However, it is significantly higher than that by testes of tfm adults. Also, the plasma testosterone levels of newborn tfm rats are the same as in the normal newborn, but lower than in normal adults and much lower than in adult tfm animals.Thus, since in the tfm rat the morphology of Leydig cells, androgen production, and maintenance of plasma levels of testosterone are normal in the newborn, but become abnormal with advancing age, it appears that defective androgen action rather than insufficient androgen production is the cause of male pseudohermaphroditism.