Temporal synthesis of mitochondrial proteins from mouse epididymal spermatozoa

Mitochondria from ejaculated bovine spermatozoa contain a group of polypeptides ranging in molecular weights from 13,000 to 35,000 not found in other bovine or murine testicular mitochondria [Hecht and Bradley, 1981]. These proteins are present in the mitochondria isolated from both epididymal and ejaculated spermatozoa. To establish when during epididymal transport, spermiogenesis, and/or meiosis these proteins are synthesized, the synthesis intervals for the mitochondrial proteins from cauda epididymal spermatozoa were established following intratesticular injection of (³⁵S)methionine. Mice were killed every third day over a 33-day period and cauda epididymal spermatozoa were fractionated into mitochondrial and head components. Radioactivity in each fraction was monitored by liquid scintillation counting. Maximal incorporation was observed during spermiogenesis, although substantial amounts of protein were synthesized during meiosis. Analysis of the mitochondrial polypeptides by gel electrophoresis revealed that many polypeptides such as the cysteine-rich structural protein of the mitochondrial capsule were synthesized over prolonged intervals of spermiogenesis and meiosis rather than in a brief specific time period. These results suggest that spermatozoal mitochondria are produced by a sequential substitution of new proteins into the differentiating mitochondria rather than the abrupt appearance of a new class of mitochondria during spermatogenesis.     

Over-expression of IL-18, ICE and IL-18 R in testicular tissue from sexually immature as compared to mature mice

In this study we examined the cellular origin and the expression levels of interleukin-18 (IL-18), IL-18 receptor (IL-18R) and IL-1beta-converting enzyme (ICE), which activates pro-IL-18, during normal maturation of murine testis. The levels of IL-18, IL-18R and ICE were significantly higher in testicular tissues and homogenates (but not in the spleen or liver) from sexually immature than mature mice. Immunohistochemical staining of testicular tissues from sexually immature and mature mice shows that testicular germ cells and Leydig cells/interstitial cells express higher levels of IL-18, as compared to other testicular cells. Peritubular cells of sexually immature and mature mice also expressed IL-18. Our results demonstrate, for the first time, over-expression of the IL-18 family in testicular tissues of sexually immature mice, as compared to mature mice, as well as the expression of IL-18 in the different stages of differentiation of testicular germ cells. Thus, our results may indicate involvement of the endocrine system (gonadotropins and testosterone) in the regulation of the testicular IL-18 family, which could be involved in the regulation of testicular functions, development and spermatogenesis under physiological conditions.     

Characterization of cAMP-dependent protein kinase and its endogenous substrate proteins in ram testicular,cauda epididymal,and ejaculated spermatozoa

Mammalian spermatozoa have been shown to possess cAMP-dependent protein kinase (A-PK) and endogenous substrate proteins for this enzyme. A study of the kinase system was undertaken to determine changes that may be associated with sperm maturation by comparing immature testicular with mature cauda epididymal and ejaculated spermatozoa. Absolute activity levels of A-PK, stimulated over a concentration range of 10^?9 to 10^?5 M, was significantly greater in testicular than ejaculated spermatozoa. At an optimal cAMP concentration (10^?6M), testicular spermatozoa had significantly greater amounts of cAMP-dependent protein kinase activity than did cauda or ejaculated spermatozoa. Electrophoretic analysis and autoradiography of NP-40-soluble protein extracts revealed the presence of two substrate proteins (M_r = 62,000 and 44,000) in all three types of spermatozoa. In addition, a phosphoprotein (M_r = 20,000) was detected in mature cauda and ejaculated but not immature testicular spermatozoa. The phosphorylation of these substrate proteins was both dose and time dependent. Examination of cyclic AMP phosphodiesterase activity revealed significantly higher levels in testicular than ejaculated spermatozoa. These results indicate marked alterations in cAMP-modulated protein phosphorylation and dephosphorylation systems in ram spermatozoa during epididymal maturation.     

Ceramides and sphingomyelins with high proportions of very long-chain polyunsaturated fatty acids in mammalian germ cells

Very long-chain polyunsaturated fatty acids (VLCPUFA) have previously been shown to be components of sphingomyelin (SM) of mammalian testis and spermatozoa. Here we examined the fatty acids of testicular ceramide (Cer) in comparison with those of SM in some mammals with a special focus on the rat testis. In bull, cat, dog, rabbit, mouse, and rat, VLCPUFA were found in both testicular lipids, Cer having a higher percentage of VLCPUFA than SM. Rat testis had the highest percentage of VLCPUFA in both lipids, the major ones being 28:4n-6 and 30:5n-6. VLCPUFA-containing SM and Cer occurred in cells located in the seminiferous tubules, where germ cells had a higher percentage of these species than Sertoli cells. Seminiferous tubule fractionation showed that SM and Cer of mitochondria and lysosomes had mostly saturates and negligible VLCPUFA, the latter being important in the SM and Cer of microsomes and other membrane fractions. VLCPUFA were absent from the SM and Cer of rat prepuberal testis, increased with the onset of spermatogenesis to account for nearly 15 and 40% of the total fatty acids of testicular SM and Cer, respectively, remained at those levels throughout the adult life of fertile rats and tended to decrease at advanced ages. Four conditions that lead to selective death of germ cells in vivo, namely experimental cryptorchidism, post-ischemic reperfusion, focalized x-ray irradiation and treatments with the antineoplasic drug doxorubicin, caused the VLCPUFA to disappear from the testicular SM and Cer of adult fertile rats, showing that these lipids are specific traits of spermatogenic cells.     

Cryoconservation du tissue testiculaire chez l’enfant: comment préserver la fertilité chez le jeune garçon?

Survival rates for almost all types of childhood cancer have improved over the last 30 years. Estimates suggest that, in 2010, 1 out of 715 adolescents and young adults will be a long-term survivor of childhood cancer. With current therapy, 70% of children are cured. The increased number of survivors has focused attention on the many long-term or late sequelae of treatment. Most of the effects cannot be detected at the end of therapy, but only become apparent with puberty, growth and the normal aging process. Among the various sequelae, gonadal dysfunction is observed and the degree of gonadal damage depends on the type and total doses of chemotherapy and/or radiotherapy. The male gonald is also more sensitive to cancer therapy than the female gonad. Cryopreservation of ejaculated spermatozoa should be proposed for sexually mature boys. However, when ejaculated semen samples cannot be collected, or in the case of prepubertal boys who are not yet able to produce spermatozoa, another strategy must be used: testicular biopsy associated with cryopreservation of (i) testicular tissue, or (ii) isolated testicular spermatozoa or (iii) immature germ cells. The future use of immature testicular tissue will depend on the development of novel technologies in humans such as germ cellin vitro maturation, or autologous or xenogeneic germ cell transplantation.     

Monoclonal antibodies to bull sperm surface antigens

Four monoclonal antibodies were generated during the present investigation. Mice were immunized with washed ejaculated bull spermatozoa. Spleen cells from the immunized mice were fused with myeloma cells (SP2/0) and four different hybridoma clones were obtained, producing specific monoclonal antibodies. These antibodies were designated as SP₂A₅, SP₁A₂, SP₁C₄ and SP₁C₆ respectively. All belonged to the IgG sub-class 1. The specificity of these monoclonal antibodies was tested using both ELISA (enzyme-linked immunosorbent assay) and indirect immunofluorescence staining techniques. Quantitative estimation of antibody-antigen reaction was done by optical density measurements. Ejaculated bull spermatozoa were always reactive for each ELISA procedure. Other test cells including spleen, testicular, ovarian, uterine and pancreatic cells from both bull and rabbit were non-reactive. Bull testicular spermatozoa were also non-reactive. However, seminal fluid (without sperm) was reactive. All four monoclonal antibodies were reactive to the midpiece of the ejaculated bull spermatozoa. In addition, SP₂A₅ was also reactive to the acrosomal area and SP₁A₂ was reactive to the acrosomal and the post-acrosomal area. Cytoplasmic droplets of ejaculated spermatozoa from bull and human were also possibly reactive to one antibody (SP₁C₆). The results clearly suggest the heterogeneous nature of the sperm cell plasma membrane and precise molecular alteration in the plasma membrane components (antigens) as the sperm cells differentiate and mature during their transit through the epididymis.     

Interleukin-6 expression during normal maturation of the mouse testis

In this study, we examined the cellular origin and the expression levels of interleukin-6 (IL-6) during normal maturation of mouse testis. The levels of IL-6 (protein and mRNA) were higher in testicular homogenates of sexually immature than mature mice. Immunohistochemical staining of testicular tissues of sexually immature and adult mice show that testicular germ cells, at different stages of differentiation, Leydig cells/interstitial cells and peritubular cells express IL-6. Our results demonstrate, for the first time, overexpression of IL-6 in testicular tissues of immature mice, as compared to mature mice, as well as the expression of IL-6 in germ cells of testicular tissues of adult and sexually immature mice. Thus, our results may indicate the involvement of the endocrine system (gonadotropins and testosterone) in the regulation of IL-6, which is involved in the regulation of testicular development, functions and spermatogenesis.     

Specific expression of VCY2 in human male germ cells and its involvement in the pathogenesis of male infertility

Abnormal spermatogenesis in men with Y-chromosome microdeletions suggests that genes important for spermatogenesis have been removed from these individuals. VCY2 is a testis-specific gene that locates in the most frequently deleted azoospermia factor c region in the Y chromosome. We have raised an antiserum to VCY2 and used it to characterize the localization of VCY2 in human testis. Using Western blot analysis, the affinity-purified polyclonal VCY2 antibody gave a single specific band of approximately 14 kDa in size, corresponding to the expected size of VCY2 in all the collected human testicular biopsy specimens with normal spermatogenesis. Immunohistochemical analyses showed that VCY2 localized to the nuclei of spermatogonia, spermatocytes, and round spermatids, except elongated spermatids. At the ultrastructural level, VCY2 expression was found in the nucleus of human ejaculated spermatozoa. To determine the possible relationship of VCY2 with the pathogenesis of male infertility, we examined a group of infertile men with and without Y-chromosome microdeletions and with known testicular pathology using VCY2 antibody. VCY2 was weakly expressed at the spermatogonia and immunonegative in spermatocytes and round spermatids in testicular biopsy specimens with maturation arrest or hypospermatogenesis. The specific localization of the protein in germ cell nuclei indicates that VCY2 is likely to function in male germ cell development. The impaired expression of VCY2 in infertile men suggests its involvement in the pathogenesis of male infertility.     

Changes in circulating hormone concentrations, testes histology and testes ultrasonography during sexual maturation in beef bulls

Nine groups of bull calves (n = 5 to 6 per group) were castrated every 5 wk from 5 to 45 wk of age, and the stages of spermatogenesis were identified histologically. Prior to castration, the testes of each calf were examined by ultrasonography, and the pixel intensities of the parenchyma were quantitated. Testis ultrasonograms were also recorded every 2 wk from 10 bull calves between 2 and 40 wk of age. Blood samples were collected at weekly intervals until castration. There was an early transient rise in circulating LH concentrations between 4 and 25 wk of age, while circulating FSH concentrations were high initially but decreased between 14 and 30 wk of age. Circulating testosterone concentrations increased gradually from 6 to 35 wk of age and then rapidly to 42 wk of age. There was a progressive increase in the more mature cell types during spermatogenesis as the animals aged, with the most dramatic changes occurring between 15 and 45 wk of age. Outer seminiferous tubule diameter increased between 10 and 45 wk of age, with the most rapid increase occurring from 30 wk of age. Inner tubule diameter increased between 30 and 35 wk of age. The echogenicity of the testes (as determined by ultrasonography) increased between 20 and 40 wk of age. From these data we conclude that testis echogenicity increased during the most active phase of growth of the seminiferous tubules as more mature germ cells were produced. Cessation of the early rise in gonadotrophin secretion immediately preceded this active phase of testicular development. Testosterone secretion rose markedly with the production of mature spermatozoa.     

Protein carboxyl methyltransferase activity specific for age-modified aspartyl residues in mouse testes and ovaries: Evidence for translation during spermiogenesis

An antiserum prepared against the purified protein carboxyl methltransferase (PCMT) from bovine brain has been used to compare testicular and ovarian levels of the enzyme and to study the regulation of PCMT concentrations during spermatogenesis. The PCMT, which specifically modifies age-damaged aspartyl residues, is present at a significantly higher concentration in mature mouse testis than in ovary. However, the PCMT is present at nearly equal concentrations in extracts of germ cell-deficient ovaries and testes obtained from mutant atrichosislatrichosis mice. In normal testis, the concentration of the PCMT increases severalfold during the first 4–5 weeks after birth, paralleling the appearance and maturation of testicular germ cells. Both immunochemical and enzymatic measurements of PCMT specific activities in purified spermatogenic cell preparations indicate that PCMT levels are twofold and 3.5-fold higher in round spermatids and residual bodies, respectively, than in pachytene spermatocytes. The results are consistent with the enhanced synthesis and/or stability of the PCMT in spermatogenic cells and with the continued translation of the PCMT during the haploid portion of spermatogenesis. The relatively high levels of PCMT in spermatogenic cells may be important for the extensive metabolism of proteins accompanying spermatid condensation or for the repair of damaged proteins in translationally inactive spermatozoa.     

Xenografting of adult mammalian testis tissue

Xenografting of testis tissue from immature males from several mammalian species to immunodeficient mouse hosts results in production of fertilization-competent sperm. However, the efficiency of testis tissue xenografting from adult donors has not been critically evaluated. Testis tissue xenografting from sexually mature animals could provide an option to preserve the genetic material from valuable males when semen for cryopreservation cannot be collected. To assess the potential use of this technique for adult individuals, testes from adult animals of six species (pig, goat, cattle, donkey, horse and rhesus monkey) were ectopically grafted to host mice. Grafts were recovered and analyzed at three time points: less than 12 weeks, between 12 and 24 weeks and more than 24 weeks after grafting. Histological analysis of the grafts revealed effects of species and donor tissue maturity: all grafts from species with greater daily sperm production (pig and goat) were found to have degenerated tubules or grafts were completely degenerated. None of the xenografts from mature adult bull and monkeys contained differentiated spermatogenic cells when examined more than 12 weeks post-grafting but tubules with Sertoli cells only remained. In grafts from a young adult bull, Sertoli cells persisted much longer than with the mature adult grafts. In grafts from a young adult horse, spermatogenesis proceeded to meiosis. In grafts from a young adult donkey and monkey, however, complete spermatogenesis was found in the grafts. These results show that testis tissue grafts from mature adult donors did not support germ cell differentiation but seminiferous tubules with Sertoli cells only survived in some species. The timing and progression of tubular degeneration after grafting of adult testis tissue appear to be related to the intensity of spermatogenesis at the time of grafting. Testis tissue from sub-adult donors survives better as xenograft than tissue from mature adult donors, and complete spermatogenesis can occur albeit with species-specific differences.     

Effects of osmolality, bicarbonate and buffer on the metabolism and motility of testicular, epididymal and ejaculated spermatozoa of boars.

Spermatozoa were collected from the rete testis of conscious boars, from the cauda epididymidis by retro-flushing, and by ejaculation. Testicular spermatozoa showed no progressive motility, and that of ejaculated was greater than that of epididymal spermatozoa. Glycolysis and respiration of testicular spermatozoa, while lower than that of the more mature cells, were only slightly affected by the incubation conditions. Epididymal spermatozoa converted 83% of the glucose they utilized to CO2 or lactate, but testicular cells converted only 35% to these metabolites. Synthesis of lipid was greatest by testicular spermatozoa. With the more mature cells hyperosmolar conditions depressed CO2 production, but increased lactate production, and these changes were greater for ejaculated than for epididymal spermatozoa. Glycolysis plus respiration of these cells was related to their motility. These results were interpreted as showing increasing motility, glycolysis and respiration with maturation, but also decreased synthetic capacity and increased sensitivity to the environment.     

Cryoconservation des tissus testiculaires: Quel avenir?

Over the last twenty years, an increased incidence of cancer has been observed in the population of adolescents and young adults. With the progress in cancer diagnosis and therapy, childhood cancer has become a curable disease. The efficacy of treatment is associated with a high degree of toxicity and gonadal function is particularly sensitive to this toxicity. Prevention of sterility in childhood cancer survivors will become a major challenge in reproductive medicine. Cryopreservation of ovarian tissue is performed in girls and women before cancer treatment. Cryopreservation of ejaculated spermatozoa is possible in sexually mature boys. However, for prepubertal boys or after failure of ejaculated sperm collection, mature or immature testicular tissue banking should be proposed. However, an optimal cryopreservation protocol is a prerequisite for clinical applications. Furthermore, the future applications of immature testicular tissue banking should be developed, not solely autologous testicular tissue grafting, but also in vitro maturation of germ cells. Cryopreservation protocols, transplantation and in vitro maturation techniques should be improved in animal models and in humans.     

The testicular cycle of Gambusia affinis holbrooki (Teleostei: Poeciliidae)

Fifteen male mosquito fish ( Gambusia affinis holbrooki ) were collected in 1989 on the 15th of each month to perform a quantitative histologic study of the annual testicular cycle including a calculation of the gonadosomatic index, testicular volume, and the total volume per testis occupied by each germ cell type. The cycle comprises two periods: spermatogenesis and quiescence. The spermatogenic period begins in April with the development of primary spermatogonia into secondary spermatogonia, spermatocytes and round spermatids. In May, the first spermatogenic wave is completed and the testicular volume begins to increase up to June when the maximum testicular volume and gonadosomatic index are reached. Germ cell proliferation with successive spermatogenetic waves continues until August. In September germ cell proliferation ceases and neither secondary spermatogonia nor spermatocytes are observed. However, spermiogenesis continues until October. In November, spermiogenesis has stopped and the testis enters the quiescent period up to April. During this period only primary spermatogonia and spermatozoa are present in the testis. In addition, a few spermatids whose spermiogenesis was arrested in November are observed. Testicular release of spermatozoa is continuous during the entire spermatogenesis period. The spermatozoa formed at the end of this period (September-October) remain in the testis during the quiescent period and are released at the beginning of the next spermatogenesis period in April. Developed Leydig cells appear all year long in the testicular interstitium, mainly around both efferent ducts and the testicular tubule sections showing S₄ spermatids.     

Phospholipid hydroperoxide glutathione peroxidase: expression pattern during testicular development in mouse and evolutionary conservation in spermatozoa

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein belonging to the family of glutathione peroxidases and has been implicated in antioxidative defense and spermatogenesis. PHGPx accounts for almost the entire selenium content of mammalian testis. In an attempt to verify the expression pattern of PHGPx, testes of mouse mutants with arrest at different stages of germ cell development and testes of mice at different ages were subjected to immunostaining with a monoclonal anti-PHGPx antibody. PHGPx was detected in Leydig cells of testes in all developmental stages. In the seminiferous tubuli, the PHGPx staining was first observed in testes of 21-day-old mice which is correlated with the appearance of the first spermatids. This result was confirmed when the testes of mutant mice with defined arrest of germ cell development were used. An immunostaining was observed in the seminiferous tubuli of olt/olt and qk/qk mice which show an arrest at spermatid differentiation. In Western blot analysis of proteins extracted from testes of mutant mice and from developing testes, two signals at 19- and 22-kDa were observed which confirm the existence of two PHGPx forms in testicular cells. In mouse spermatozoa, a subcellular localization of PHGPx and sperm mitochondria-associated cysteine-rich protein (SMCP) was demonstrated, indicating the localization of PHGPx in mitochondria of spermatozoa midpiece. For verifying the midpiece localization of PHGPx in other species, spermatozoa of Drosophila melanogaster, frog, fish, cock, mouse, rat, pig, bull, and human were used in immunostaining using anti-PHGPx antibody. A localization of PHGPx was found in the midpiece of spermatozoa in all species examined. In electronmicroscopical analysis, PHGPx signals were found in the mitochondria of midpiece. These results indicate a conserved crucial role of PHGPx during sperm function and male fertility.     

Developmental changes of DNA ligase during ram spermatogenesis

The molecular forms and activities of ram DNA ligase have been investigated during spermatogenesis from the stage of early round spermatids to ejaculated spermatozoa. Through germ cell maturation, two consecutive forms of the enzyme (6S and 7S) have been found. The 6S form (DNA ligase II) is observed in primary and secondary spermatocyte, as well as in round spermatids. The 7S form (DNA ligase I) is present in elongated spermatids and in the sole round cell population with spermatogonia and young primary spermatocytes. In ram germ cells, DNA ligase I and DNA ligase II appear to be respectively associated with DNA replication repair. The absence of DNA ligase II associated with the absence of DNA repair in testicular and ejaculated spermatozoa might be related to male infertility.     

Lipid composition and metabolism in testicular and ejaculated ram spermatozoa

1. Spermatozoa collected directly from the testis of the conscious ram contain 25% more phospholipid than ejaculated spermatozoa. The concentration of lecithin, phosphatidylethanolamine and ethanolamine plasmalogen was greater in testicular spermatozoa; little difference was observed in choline plasmalogen. Both types of spermatozoa had significant amounts of cardiolipin and alkyl ether phospholipid. 2. The fatty acids in the phospholipid extracted from testicular spermatozoa have a very high content of palmitic acid. The phospholipids of ejaculated spermatozoa contained less palmitic acid, but more myristic acid. 3. Ejaculated spermatozoa contained less acyl ester and cholesterol. It is suggested that lipids are a source of substrate for spermatozoa during their passage through the epididymis. 4. Testicular spermatozoa when incubated with [U-¹⁴C]glucose incorporated more radioactivity into the glycerol part of the phospholipid and neutral lipid fractions than did ejaculated cells. The distribution of radioactivity in the individual phospholipids and neutral lipids was similar for both cell types. No radioactivity was detected in choline plasmalogen, which accounted for approx. 40% of the total phospholipid. 5. Testicular spermatozoa incorporated more radioactivity from glucose into formate than into acetate, whereas a higher proportion of radioactivity was found in acetate in ejaculated cells. 6. The implications of these lipid changes in the process of spermatozoal maturation are discussed.