Early Stages Of Spermatogenesis Research Articles

Immunohistochemical localization of androgen receptor in mouse testicular germ cells during fetal and postnatal development.

Determination of the cellular distribution of the androgen receptor (AR) in testicular cells is necessary for understanding the mode of AR action in the testis. We here investigated immunohistochemically the localization of AR by use of anti-human AR polyclonal antibody NH27, with special reference to the AR in germ cells in the developing mouse testis. ICR mouse testes taken from day 14 post coitum (p.c.) to day 56 post partum (p.p) were used for AR immunohistochemistry by the routine immunoperoxidase method at the light microscopic level and the pre-embedding method at the electron microscopic level. On day 14 p.c., AR immunoreactivity was present in nuclei of prospermatogonia but not in those of Sertoli cells or interstitial cells. On day 14 p.p., the AR was detected in the nuclei of spermatogonia, Sertoli cells, and myoid cells. AR immunoreactivity in nuclei of Leydig cells appeared on day 21 p.p. In the mature mouse testis, the AR was present in the nuclei of spermatogonia, Sertoli cells, myoid cells, and Leydig cells. AR was present both in germ cells and in somatic cells during fetal and postnatal development of the mouse testis. In the fetal testis, AR was localized exclusively in prospermatogonia and spermatogonia, suggesting that androgen may act directly on germ cells during prespermatogenesis and the early stage of spermatogenesis. Based on the fact that AR is expressed in Sertoli cells, myoid cells, and Leydig cells around the onset of spermatogenesis, the regulation of AR expression in the germ cells seems to be different from that in the somatic cells. Furthermore, our present data suggest the ultrastructural localization in nuclei of mouse testicular cells is similar to that of some other steroid receptors, both in germ cells and somatic cells.

Physiological evidence for two sturgeon gonadotrophins in Acipenser transmontanus

Physiological evidence in white sturgeon Acipenser transmontanus suggests that two gonadotrophins, termed sturgeon gonadotropin I (stGTH I) and sturgeon gonadotropin II (stGTH II), are involved in regulating reproduction. Pituitary and plasma concentrations of stGTH I were found to be higher than concentrations of stGTH II during vitellogenesis and the early stages of spermatogenesis. Conversely, pituitary and plasma concentrations of stGTH II were greater compared with stGTH I during ovulation and spermiation. The gonadotropin releasing hormone analog D-Ala 6-des-Gly 10-GnRH ethylamide (GnRHa) was effective in stimulating the release of both gonadotrophins in mature males and preovulatory females, with a maximal response occurring in the spring. Collectively, the data supports our view that sturgeon possess a dual gonadotropin system controlling reproduction.

Expression of new KDN-gangliosides in rainbow trout testis during spermatogenesis and their structural identification.

The developmental expression of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid-containing glycosphingolipids (KDN-gangliosides) in rainbow trout testis during spermatogenesis was studied using a monoclonal antibody, mAb.kdn3G, which recognizes the KDN alpha 2-->3Gal beta 1-->epitope. A major KDN-ganglioside found in mature sperm, (KDN)GM3, KDN alpha 2-->3Gal beta 1-->4Glc beta 1-->Cer (where Cer is ceramide), was expressed in testis throughout all stages of its maturation. On the contrary, four new KDN-gangliosides which were reactive with mAb.kdn3G were not detected in mature sperm, although they were identified in immature testis and expressed during spermatogenesis. The structures of these KDN-gangliosides were established by chemical, enzymatic and immunochemical methods as: (i) (KDN)GD1a, KDN alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4(KDN alpha 2-->3)Gal beta 1-->4Glc beta 1-->Cer; (ii) (KDN, Neu5Ac)GD1a, KDN alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4(Neu5Ac alpha 2-->3)Gal beta 1-->4Glc beta 1-->Cer and Neu5Ac alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4(KDN alpha 2-->3)Gal beta 1-->4Glc beta 1-->Cer; (iii) (KDN) GD1 alpha, KDN alpha 2-->3Gal beta 1-->3(KDN alpha 2-->6)GalNAc beta 1-->4Gal beta 1-->4Glc beta 1-->Cer; and (iv) (KDN,Neu5Ac)GD1 alpha, KDN alpha 2-->3Gal beta 1-->3(Neu5Ac alpha 2-->6)GalNAc beta 1-->4Gal beta 1-->4Glc beta 1-->Cer. (KDN)GD1a and (KDN,Neu5Ac)GD1a first appeared at an early stage of spermatogenesis, but (KDN)GD1 alpha and (KDN,Neu5Ac)GD1 alpha were not expressed until 2 months prior to spermiation. While (KDN)GM3 was previously shown to contain only 4-sphingenine (d18:1) acylated with a C16:0 fatty acid, the new KDN-gangliosides discovered in this study were composed of 4-hydroxysphinganine (t18:0) or 4-sphingenine (d18:1), and were acylated with a C24:1 or C16:0 fatty acid. A possible function of these KDN-gangliosides is suggested.

Metabolism and Testicular Toxicity of 1,3-Dinitrobenzene in Rats of Different Ages

Metabolism and Testicular Toxicity of 1,3-Dinitrobenzene in Rats of Different Ages. Brown, C. D., Forman, C. L., McEuen, S. F., and Miller, M. G. (1994). Fundam. Appl. Toxicol. 23, 439-446.Susceptibility to 1,3-dinitrobenzene (1,3-DNB)-induced testitular damage is known to increase with age. The present study investigated the possibility that age-dependent differences in metabolism and disposition could account for differences in toxicity. [14C]1,3-DNB (25 mg/kg, ip) was administered to Sprague-Dawley rats which were 31, 75, or 120 days of age. Levels of 1,3-DNB and 1,3-DNB metabolites were determined in blood and urine. As animal age increased, peak blood concentrations of 1,3-DNB were lower and declined more slowly indicating an age-dependent decrease in rate of metabolism and a possible increase in volume of distribution. In younger animals, faster elimination rates were associated with higher blood levels of metabolites. Urinary metabolites were generally similar for all age groups with the exception of the diacetamidobenzene metabolite which was significantly lower in the urine of 31 day old rats. There were clear differences in the toxicokinetic profile for 1,3-DNB between the 31 day old rats and the other two age groups. However, differences between the 75 and 120 day old animals were less marked. Testicular damage induced by 1,3DNB (25 mg/kg, ip) was hardly detectable in the youngest animals, while the intermediate age group showed a moderate lesion particularly in later stages of spermatogenesis. For the oldest animals, testicular damage was more severe, particularly in the earlier stages of spermatogenesis. Overall, the rapid elimination rate could account for the lack of 1,3-DNB toxicity in very young animals. However, simple metabolic differences were less likely to adequately explain the increase in testicular damage found as animal age increased from 75 to 120 days.

An ultrastructural study of vertical transmission of Rickettsia tsutsugamushi during oogenesis and spermatogenesis in Leptotrombidium pallidum.

Rickettsia tsutsugamushi in Leptotromibidium pallidum was observed by electron microscopy and rickettsiae were found in the various tissues and organs of both larvae and adults. Budding of rickettsiae, a manner of release from the host cells, was observed only in the rudiments of the reproductive organs in larvae. Oogonia and maturing oocytes in adult females and eggs after oviposition contained the microorganisms. In adult males, rickettsiae were also found in the spermatogonia, spermatocytes, and spermatids in the early stage of spermatogenesis, but were eliminated from these cells during maturation. Only the maturing spermatids, but not the eliminated rickettsiae, migrated to another rickettsia-free area of the testis, resulting in the separation of spermatids from rickettsiae and in the production of rickettsia-free spermatophores. Based on these observations, the mechanism of vertical transmission of the rickettsiae to the progeny occurs only in the female parents. Most rickettsiae in the somatic cells of larvae and adults were coccoid, but some rickettsiae in the ovary and the testis of adult mites showed bacillary forms and were enveloped by a membrane of unknown origin.

Metabolism and Testicular Toxicity of 1,3-Dinitrobenzene in Rats of Different Ages

Susceptibility to 1,3-dinitrobenzene (l,3-DNB)-induced testicular damage is known to increase with age. The present study investigated the possibility that age-dependent differences in metabolism and disposition could account for differences in toxicity. [14C]1,3-DNB (25 mg/kg, ip) was administered to Sprague-Dawley rats which were 31, 75, or 120 days of age. Levels of 1,3-DNB and 1,3-DNB metabolites were determined in blood and urine. As animal age increased, peak blood concentrations of 1,3-DNB were lower and declined more slowly indicating an age-dependent decrease in rate of metabolism and a possible increase in volume of distribution. In younger animals, faster elimination rates were associated with higher blood levels of metabolites. Urinary metabolites were generally similar for all age groups with the exception of the diacetamidobenzene metabolite which was significantly lower in the urine of 31 day old rats. There were clear differences in the toxicokinetic profile for 1,3-DNB between the 31 day old rats and the other two age groups. However, differences between the 75 and 120 day old animals were less marked. Testicular damage induced by 1,3-DNB (25 mg/kg, ip) was hardly detectable in the youngest animals, while the intermediate age group showed a moderate lesion particularly in later stages of spermatogenesis. For the oldest animals, testicular damage was more severe, particularly in the earlier stages of spermatogenesis. Overall, the rapid elimination rate could account for the lack of 1,3-DNB toxicity in very young animals. However, simple metabolic differences were less likely to adequately explain the increase in testicular damage found as animal age increased from 75 to 120 days.

Ultrastructure of spermatogonia, spermatocytes, and sertoli cells in the testis of the crayfish, Procambarus paeninsulanus

The testes of the crayfish, Procambarus paeninsulanus, were prepared for light and transmission electron microscopy. During early stages of spermatogenesis, when the spermatogonia are dividing, processes from a single Sertoli cell extend between numerous spermatogonia. As the cells enter meiosis, many points of contact can be observed between the Sertoli cell processes and spermatocytes. These desmosome-gap junctions are maintained between the germ and Sertoli cells until the early spermatid stage.

Presence of a repressor protein for testis-specific H2B (TH2B) histone gene in early stages of spermatogenesis.

The rat testis-specific TH2B histone gene assumes a hypomethylated chromatin structure at all stages of spermatogenesis. However, the TH2B mRNA level is very low in pre-meiotic spermatogenic cells and rises sharply in meiotic pachytene spermatocytes. The low level of TH2B mRNA in pre-meiotic spermatogenic cells appears to be a result of transcriptional repression of the gene by a pre-meiotic cell-specific protein which binds to a site between the TATA element and the transcription initiation site of TH2B gene.

Transcriptional expression of a testis-specific variant of the mouse pyruvate dehydrogenase E1 alpha subunit.

An isogene of the E1 alpha subunit of the mouse pyruvate dehydrogenase complex was shown in a previous study to map to chromosome 19. Here we demonstrate using Northern blot analysis that this gene is expressed in a testis-specific fashion. Two testis-specific E1 alpha transcripts were detected: (1) a 2.0-kb transcript that is abundant in pachytene cells but also detectable during earlier stages of spermatogenesis, and (2) a shorter 1.7-kb transcript detectable only in round spermatids. Analysis of both transcripts following RNase H digestion revealed that the smaller message is not derived from the shortening of the poly(A) tail and most likely results from the alternate use of two polyadenylation signals. Finally, analysis of polysomes isolated from 20- and 80-day-old mouse testes demonstrated that the 2.0-kb transcript is associated with the polysomal fraction, suggesting that this message is transcribed and actively translated at the same time. We conclude from these results that expression of the testis-specific E1 alpha is initially expressed during the meiotic prophase stage of spermatogenesis and not under any apparent post-transcriptional regulation.

Presence of a repressor protein for testis-specific H2B (TH2B) histone gene in early stages of spermatogenesis

Presence of a repressor protein for testis-specific H2B (TH2B) histone gene in early stages of spermatogenesis

Growth and reproduction of the Antarctic nototheniid fish Pagothenia borchgrevinki

Growth and reproductive parameters were recorded from Pagothenia borchgrevinki captured from beneath the sea ice in McMurdo Sound during October and November 1987. Fish were aged on the basis of unvalidated check rings in otoliths. Fish were estimated to be 2–7 years old. There was good correlation of estimated ages with length-frequency modes for a number of year classes. Growth was linear, and did not approach an asymptote with increasing age. Most females were in the early stages of vitellogenesis, however, 2 fish showed evidence of imminent or recent spawning activity. Males were all in the early stages of spermatogenesis. Relative fecundity was low, and this was associated with large egg size. Ovarian development was group synchronous, and it appears that spawning mainly occurs during winter.

Sex difference in methylation of single-copy genes in human meiotic germ cells: Implications for X chromosome inactivation, parental imprinting, and origin of CpG mutations

To determine the methylation status of female germ cells in reference to the programmed reversal of X chromosome inactivation in these cells, we examined human fetal ovaries at developmental stages from the time germ cells initiate meiosis to when they cease to synthesize DNA (8-21 weeks gestation). Using methylation-sensitive restriction enzymes, we analyzed 57 MspI sites (32 sites in the CpG islands, and 25 nonclustered sites) from five X-linked housekeeping genes (HPRT, G6PD, P3, PGK, and GLA) and two tissue specific genes (X-linked F9 and autosomal EPO). Methylation patterns were compared to those of male germ cells, sperm, and somatic tissues of both sexes. All 32 MspI sites in CpG islands were unmethylated in germ-cell fractions of fetal ovary and adult testes, which could explain the reversibility of X inactivation in these tissues. However, whereas male meiotic germ cells were extensively methylated outside the islands (in the body of genes) and the methylation patterns resembled those of most somatic tissues, none of the 25 nonclustered CpGs was methylated in DNA contributed by the germ-cell component of fetal ovaries. The presence of faint MspI-like fragments in HpaII digests of fetal testes as well as fetal ovary prior to the onset of meiosis suggests that DNA of primordial germ cells is unmethylated in both sexes. Our observations of meiotic germ cells suggest that the female germ cells remain unmethylated, but that methylation in male germ cells occurs postnatally, prior to or during the early stages of spermatogenesis. In any event, the striking sex difference in methylation status of endogenous single-copy genes in meiotic germ cells could provide a molecular basis for parental imprinting of the mammalian genome.

The duality of teleost gonadotropins

The duality of salmon gonadotropins has been proved by biochemical, biological, and immunological characterization of two chemically distinc gonadotropins. GTH I and GTH II were equipotent in stimulating estradiol production, whereas GTH II appears to be more potent in stimulating maturational steroid synthesis. The ratio of plasma levels and pituitary contents of GTHs and the secretory control by a GnRH suggest that GTH I is the predominant GTH during vitellogenesis and early stages of spermatogenesis in salmonids, whereas GTH II is predominant at the time of spermiation and ovulation. GTH I and GTH II are found in distinctly separate cells. In trout, GTH I is expressed first in ontogeny, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis, and increase dramatically at the time of final reproductive maturation. Comparison of the amino acid sequences of polypeptides and the base sequences of cDNA revealed that salmon GTH I β is more similar to bovine FSHβ than bovine LHβ and salmon GTH II β shows higher homology to bovine LHβ than to bovine FSHβ. The existence of two pituitary gonadotropins in teleosts as well as tetrapods suggests that the divergence of the GTH gene took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods.

Changes in dehydrodolichyl diphosphate synthase during spermatogenesis in the rat

The levels of dolichyl phosphate and 2,3-dehydrodolichyl diphosphate synthase were determined in seminiferous tubules of prepuberal rats to assess any changes occurring during early stages of spermatogenesis. Dolichyl phosphate increased in concentration two- to threefold from Day 10 to Day 23 after birth. A method was optimized to measure dehydrodolichyl diphosphate synthesis from Δ 3-[ 14C]isopentenyl diphosphate and t,t-farnesyl diphosphate in homogenates of seminiferous tubules. Both dehydrodolichyl mono- and diphosphates were observed as products of the in vitro assay. The specific activity of tubular synthase increased twofold between Day 7 and Day 23 and decreased similarly between Day 23 and Day 60. Since there was a parallel increase in the concentration of tubular dolichyl phosphate and dehydrodolichyl diphosphate synthase activity during early stages of spermatogenesis, it is proposed that the level of dolichyl phosphate may be controlled at least in part by the regulation of de novo dehydrodolichyl diphosphate biosynthesis. The synthase was also solubilized from tubular membranes with deoxycholate and partially purified by chromatography.

DNA-polymerase activity detected in situ. Relationship between incorporation of 3H-deoxytriphosphates and 3H-actinomycin D binding during the cell cycle in antheridial filaments of Chara vulgaris L. Effect of auxin and cytokinins.

DNA-polymerase activity during the cell cycle (S + G2 + M + C type) in antheridial filaments cells of Chara vulgaris was studied using the autoradiographic method. Incorporation of 3H-deoxytriphosphates (3H-dTPs) during the whole of interphase indicates, that the cell cycle is not accompanied by distinct changes in enzyme activity. Incorporation of 3H-dTPs was also observed in spermatids and in early stages of spermatogenesis. Intensity of 3H-dTPs incorporation during interphase and spermatogenesis is similar to the intensity of 3H-actinomycin D (3H-AMD) binding. Auxin (IAA) and kinetin stimulate both 3H-AMD binding and 3H-dTPs incorporation; benzyladenine does not affect any of these processes. The in situ autoradiographic method of detecting DNA-polymerase activity reveals availability of DNA template for the enzyme rather than DNA polymerase activity itself.

Developmental activation of phosphoglycerate mutase-2 in the testis of the mouse

The expression of the phosphoglycerate mutase locus Pgam-2 which synthesizes the muscle-specific PGAM-B subunit was analyzed in the testis of the mouse. No PGAM-B activity was detected in testes of newborn mice, in which only the PGAM-AA isozyme was observed. PGAM-B was first observed between Day 14 and Day 16 of postnatal development. In adult males approximately 50% of total PGAM activity is contributed by the PGAM-B subunit and 50% by the PGAM-A subunit. Immunohistochemical studies show that in the testis PGAM-B is localized exclusively in germ cells. PGAM-B is detected in pachytene spermatocytes and in spermatids, but not in earlier stages of spermatogenesis. The muscle-specific PGAM isozyme was also found in testes of bull, cat, and rat, as well as in human sperm. PGAM-B might thus be useful as a marker for germ cell differentiation, along with other germ cell-specific proteins.

Ultrastructural and cytochemical study on interactions between nutrient storage cells and gametogenesis in the mussel Mytilus edulis

The ultrastructural morphology of storage-cell breakdown and replenishment in Mytilus edulis is described for mussels collected at monthly intervals over a period of one year (September 1981 to October 1982) from a site in Cornwall, England. In addition cytochemical staining reactions at light and electron microscope levels are described for the storage cells and the Sertoli cells. The breakdown of adipogranular (ADG) cell components is believed to be a lysosomally-mediated process of controlled autolysis involving fusion of primary lysosomes with the protein granules. Increasing activity for lysosomal enzymes during ADG cell breakdown is demonstrated using quantitative cytochemistry. The presence of glycogen in vesicular connective tissue (VCT) cells is demonstrated using freeze-dried, formaldehyde-vapour-fixed tissue. The breakdown of VCT cells involves sequestration of glycogen from the central reserve into small cytoplasmic vesicles; subsequent release appears to be mediated by eccrine secretion. The presence of arylsulphatase activity in lysosomes within the peripheral cytoplasm of the VCT cells during the winter months indicates that glycogen breakdown may also be lysosomally mediated. The morphology of the Sertoli cells is described; during the early stages of spermatogenesis they are characterized by the presence of numerous lipid droplets which are depleted as development proceeds. Contact between Sertoli cells and developing spermatogonia is maintained by means of long septate junctions. Following spawning, the well developed lysosomal system is evident in the Sertoli cells and appears to be engaged in intracellular digestion of phagocytosed waste sperm and residual cytoplasm. Resorption of the products of gamete degeneration by the gonoduct epithelial cells is described. During regeneration, VCT cells appear to endocytose material; the ADG cells demonstrate extensive arrays of rough endoplasmic reticulum and are occasionally seen undergoing mitosis. A process involving loss of glycogen vesicles from the mantle epithelial cells by means of apocrine sections is described.

The Effects of Gossypol on Spermatogenesis and Development of the Eyefluke, Philophthalmus gralli, and Its Chicken Host

To determine the effects of gossypol, a male antifertility drug, on the eyefluke, P. gralli, this chemical was administered orally to chickens in long-term and short-term regimens. Gossypol acetic acid (GAA), fed to juvenile chickens from 1 to 35 days, caused a decreased weight gain when compared to controls on untreated feed. An FeSO4 supplement to the GAA-fed chickens provided partial protection from the toxic effects of GAA. Worms from GAA-fed chickens were significantly larger than controls, while those from chickens fed GAA + FeSO4 were intermediate in size. Sperm development in these worms was unaffected by GAA. In a second experiment, GAA was administered either in the feed of the hosts from days 35 to 70 or by capsule from days 63 to 77. Worms were exposed to [3H] thymidine, transplanted to the host's eyes, removed on a timed schedule, and processed for autoradiography to determine the rate of spermatogenesis in both GAA-feed and GAA-capsule groups. Early stages of spermatogenesis in both groups were unaffected by GAA and later stages developed at a slightly faster rate than reported for worms from chickens on untreated feed. Higher frequencies of testicular anomalies were observed in both groups including 3 testes, 1 testis, no testes, fused testes, degenerating testes, ovarian tissue in the testes, deformed sperm, and encapsulated sperm. Testes from chickens in both groups showed a significantly lower weight and no signs of spermatogenesis when compared to control chickens.

Isolation and biochemical studies of enriched populations of spermatogonia and early primary spermatocytes from rat testes.

A method to obtain several highly enriched populations of testis cell types from rats of a single age is described. Single cell suspensions from immature rat testes were prepared after enzymatic removal of interstitial cells. Cells were separated on the basis of size into four fractions (bulk preparations) or eight fractions (analytical preparations) by centrifugal elutriation. These elutriator fractions were further separated by equilibrium density centrifugation in Percoll gradients. In this manner, populations of 2 X 10(7) type A spermatogonia (51% purity), 3 X 10(7) type B spermatogonia (76% purity), 5 X 10(7) zygotene/early pachytene spermatocytes (56% purity), 3 X 10(7) midpachytene spermatocytes (70% purity), and 4 X 10(7) Sertoli cells (89% purity) could be obtained from 50 immature rats within 6 h after killing. Purities, determined by examination of cytologic smears, were verified by Coulter volume and flow cytometric DNA determinations. These separation methods were used to obtain cell populations for characterization of levels and synthesis of high mobility group proteins in the early stages of spermatogenesis.

Histone variants in rat spermatogonia and primary spermatocytes

The levels and synthesis of histone variants have been directly measured in spermatogonia and in various stages of primary spermatocytes purified from the rat testis. These measurements were made possible by the development of a procedure, employing centrifugal elutriation and density gradient centrifugation, to separate highly enriched populations of such cells from immature rat testes at the early stages of spermatogenesis. The results show a difference in regulation of the synthesis and accumulation of testis-specific histones H1t, TH2A, TH2B, and TH3. TH3 is present and actively synthesized in A and B spermatogonia. The testis-enriched variants, H2A.X and H1a, are also present at their maximal levels in A spermatogonia. No detectable amounts of H1t, and at most, low levels of TH2A and TH2B could be found in spermatogonia. While TH2A and TH2B are already present and actively synthesized in early primary spermatocytes (around the preleptotene stage), H1t does not accumulate until the pachytene stage.