Spermatogenesis In Rainbow Trout Research Articles

Molecular characterization and expression profiles of cyclin B1, B2 and Cdc2 kinase during oogenesis and spermatogenesis in rainbow trout ( Oncorhynchus mykiss)

The meiotic maturation of oocyte and spermatocyte in animals is controlled by the maturation promotion factor (MPF), a complex of Cdc2 and cyclin B proteins. To better understand the mechanism of oocyte and spermatocyte maturation in fish, the expression of cyclin B1 (CB1), B2 (CB2) and Cdc2 kinase during oogenesis and spermatogenesis in rainbow trout were examined at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that the amount of CB1 and CB2 mRNA was greater at previtellogenesis and late vitellogenesis stages, but less at early vitellogenesis stage and during early embryogenesis. Cdc2 mRNA was continuously present throughout the processes of oogenesis and early embryogenesis except for a decline at early vitellogenesis. In situ hybridization analysis indicated that CB1, CB2 and Cdc2 transcripts were present in oocytes of different developmental stages as well as in all spermatogenic cells except for spermatogonia. Immunohistochemical analysis revealed that CB1 protein was absent in vitellogenic oocytes, but present in young previtellogenic and mature oocytes. In contrast, CB2 and Cdc2 proteins were present at all stages oocyte development. Similarly, CB2 and Cdc2 proteins were present throughout spermatogenesis, whereas CB1 protein was only detected in spermatogonia and spermatocytes, but not in spermatids. Thus, it appears that CB1, CB2 and Cdc2 transcripts have similar expression patterns during oogenesis and spermatogenesis, but CB1 protein varies in amount during these processes. These data suggest that CB1 may have a leading role in the regulation of meiotic maturation of oocytes and spermotocytes.

The Expression of Multiple Connexins Throughout Spermatogenesis in the Rainbow Trout Testis Suggests a Role for Complex Intercellular Communication1

Certain fish, such as rainbow trout (Oncorhynchus mykiss), are seasonal breeders. Spermatogenesis in rainbow trout is synchronous; therefore, at any time point during this process, germ cells are predominantly at the same stage of development. As such, rainbow trout represent an excellent model in which to study spermatogenesis. Gap junctions are composed of connexons, which are themselves formed by six transmembrane proteins termed connexins (Cxs). The objectives of this study were to assess which Cxs are expressed in the rainbow trout testis, and if their expression was stage specific during gonadal maturation. Rainbow trout were killed at various stages of maturation, and total cellular RNA was isolated from the testes. RT-PCR using degenerate primers recognizing all vertebrate Cxs indicates that there are several different Cxs in trout testes. Amplicons were cloned and sequenced. Homology comparisons indicate that these were cx43, cx43.4, cx31, and cx30. Immunolocalization of these Cxs indicate that Cx43 was localized primarily to Sertoli cells, while Cx43.4 was localized along the lateral plasma membranes between adjacent spermatocytes. Cx30 was localized to the interstitial Leydig cells, and Cx31 was localized primarily to the endothelium of interstitial blood vessels. The expression of each Cx varied as a function of the stage of spermatogenesis, suggesting that the expression of these proteins is highly regulated. Together, these results indicate that intercellular communication in the testis is complex, involves several different Cxs, and is a highly regulated process.

Expression of cytochrome P450 11β (11β-hydroxylase) gene during gonadal sex differentiation and spermatogenesis in rainbow trout, Oncorhynchusmykiss

Androgens and especially 11-oxygenated androgens are known to be potent masculinizing steroids in fish. As a first step to study their physiological implication in gonadal sex differentiation in fish, we cloned a testicular cytochrome P450 11β (11β-hydroxylase) cDNA in the rainbow trout, Oncorhynchus mykiss. We isolated a 1882 bp P450 11β cDNA (rt11βH2, AF217273) which contains an open reading frame encoding a 552 putative amino acids protein. This sequence was highly homologous (98% in nucleotides and 96.5% in amino acids) to another rainbow trout P450 11β sequence (AF179894) and also to a Japanese eel P450 11β (68% in amino acids). Northern blot analysis detected a single transcript of 2 kb which was highly expressed in the testis (stage II) and to a lesser degree in the anterior kidney (containing the interrenal tissue). No signal was detected in the posterior kidney, brain, liver, skin, intestine and heart. In the testis this transcript was highly expressed at the beginning of spermatogenesis (stages I and II), followed by a decrease during late spermatogenesis (stages III to V). By semi-quantitative reverse transcription polymerase chain reaction, P450 11β expression during gonadal differentiation was estimated to be at least 100 times higher in male than in female gonads. This difference was first detected at 55 days post-fertilization (dpf), i.e. 3 weeks before the first sign of histological sex differentiation, and was sustained long after differentiation (127 dpf). Specific P450 11β gene expression was also demonstrated before testis differentiation (around 50 dpf) using virtual Northern blot, with no expression detected in female differentiating gonads. From these results, and also based on the already known actions of 11-oxygenated androgens in testicular differentiation in fish, it is now suggested that P450 11β gene expression is a key factor for the testicular differentiation in rainbow trout.

Changes in carbohydrate metabolism in domesticated rainbow trout ( Oncorhynchus mykiss) related to spermatogenesis

1. 1. A study of several carbohydrate metabolism pathways was carried out in three different tissues—liver, testes and kidney—from domesticated rainbow trout males ( Oncorhynchus mykiss) during spermatogenesis. 2. 2. A fall in liver glycogen levels occurred during spermatogenesis and the glucose obtained by glycogen rupture may be mainly used in liver glycolysis. 3. 3. An increase of glucose levels was observed in testes during spermatogenesis and may be mainly due to an increased gluconeogenesis. The higher glucose levels may be used not only in glycogen synthesis but also in generating reducing power. 4. 4. In kidney, only PFK activity was changed (decreased) during spermatogenesis. 5. 5. Spermatogenesis in rainbow trout is mainly associated with a higher use of glucose in the gonad and increased metabolic activity in the liver.

Changes in brain dopamine levels, oocyte growth and spermatogenesis in rainbow trout, Oncorhynchus mykiss, following sublethal cyanide exposure.

Whole brain dopamine (DA) and norepinephrine (NE) levels were measured in sexually maturing (2 years +) male and female rainbow trout, Oncorhynchus mykiss, following exposure to 0.01 mg/L hydrogen cyanide (HCN). Following a 12 day exposure period in July and August 1988, whole brain DA levels of HCN exposed fish were significantly higher (p less than 0.05), relative to control fish, as measured by high performance liquid chromatography (HPLC). Brain NE levels were unaffected by HCN exposure. Whole brain DA and NE levels showed a strong correlation in control fish (r = +0.81), but not in HCN exposed fish (r = +0.28), likely due to altered DA levels in the latter group. No significant differences were found in brain DA and NE levels between males and females. Mean diameters of oocyte from ovaries of the vitellogenic females were significantly (p less than 0.01) reduced from 226 to 183 microns in control and HCN exposed fish respectively. Testes from males revealed significantly (p less than 0.001) higher numbers of spermatogonial cysts in HCN exposed fish. Evidence is given that chronic exposure to sublethal levels of HCN significantly alters brain DA levels in both sexes of rainbow trout, reduces growth in vitellogenic oocytes of the ovary in females and interferes with the passage of spermatogonia to the spermatocyte stage in sexually maturing males. Collectively, these results suggest that sublethal HCN affects the reproductive mechanisms via the hypophyseal-gonad axis in sexually maturing rainbow trout.

Hyperacetylation of histone H4 promotes chromatin decondensation prior to histone replacement by protamines during spermatogenesis in rainbow trout.

During the final stages of spermatogenesis in rainbow trout a dramatic increase in the level of histone H4 hyperacetylation is observed which is closely correlated with the replacement of histones by protamines. In order to understand further how H4 hyperacetylation might assist in protamine replacement of the histones, we have investigated the effect of H4 hyperacetylation on chromatin structure in trout testes actively undergoing the replacement process. Long chromatin fragments enriched in hyperacetylated H4 have been isolated and characterized. Evidence is presented that hyperacetylated H4 is clustered in certain regions (domains) of late stage testis chromatin and within these domains the chromatin exhibits an altered, highly relaxed structure which is believed to be the result of the extensive hyperacetylation. These domains, which are nearly devoid of protamine, are postulated to represent an initial structural transition which is necessary for the proper histone removal and protamine replacement process to take place.

Hyperacetylation of histone H4 correlates with the terminal, transcriptionally inactive stages of spermatogenesis in rainbow trout

Developing trout testis has an unusually high steady-state level of histone H4 hyperacetylation and therefore provides an excellent system in which to investigate the role(s) of this modification. We have studied the pattern of H4 hyperacetylation as a function of trout testis cell differentiation by using (a) whole testes at progressive stages of development, and (b) individual testis cell populations separated in a stage-dependent manner. The transcriptional activity associated with the differentiating cell types has also been investigated. The results provide strong evidence that the proliferative and transcriptionally active spermatogonia and spermatocytes possess nearly undetectable steady-state levels of H4 hyperacetylation. The content of hyperacetylated H4 rises dramatically (approaching 50% of the total H4) during spermiogenesis—the process comprises the transition of spermatids to mature sperm and is characterized by declining transcriptional activity. We conclude that the majority of the hyperacetylation of H4 in this case is not associated with the proliferative, transcriptionally active stages of testis development, but rather is associated with an event occurring late in development, during spermiogenesis. The most obvious process associated with this stage is the complete replacement of the histones with the sperm-specific proteins, the protamines. It is suggested that the unusually high level of histone H4 hyperacetylation observed in trout testis is acting, in some manner, to assist in this replacement process.

Inhibition of spermatogenesis in rainbow trout during chronic cyanide poisoning

This study involves a histological examination of the testicular tissue to assess, both quantitatively and qualitatively, the effects of an 18-day exposure of juvenile male rainbow trout,Salmo gairdneri, to cyanide in laboratory flow-through aquaria at 12.5°C. Test concentrations of 0.01 and 0.03 mg/L HCN reduced the number of dividing spermatogonia by 13 and 50% respectively. The exposure to cyanide led to an increase in the prophase stage spermatogonia which appeared to be due to a blockage of mitotic progress.

Protamine messenger RNA: Evidence for early synthesis and accumulation during spermatogenesis in rainbow trout

Trout testis cells were separated into various developmental classes by velocity sedimentation in bovine serum albumin gradients and were identified morphologically with particular stages of the process of spermatogenesis. The stage of testis cell differentiation at which protamine mRNA appears in the cell cytoplasm for the first time was determined by hybridization of RNA populations extracted from the separated cells to radioactively labeled protamine cDNA. Primary spermatocytes represent the earliest stage of differentiation at which protamine mRNA can be detected in large quantities in the cell cytoplasm, establishing that the synthesis of this class of mRNA occurs at a much earlier stage than the time of its translation at the spermatid stage. Protamine mRNA sequences were found in both the polysomes and postribosomal supernatant of the spermatid cells which are involved in the synthesis of protamine, while primary and secondary spermatocytes contained the mRNA sequences only in their postribosomal supernatant fractions. These findings strongly suggest that protamine mRNA is synthesized, accumulated, and stored in the cell sap of primary and secondary spermatocytes in the form of “inactive” messenger ribonucleoprotein particles, which are “activated” and translated at the spermatid stage.

Sites of in Vivo Acetylation in Trout Testis Histone IV

During the process of spermatogenesis in rainbow trout, there is extensive acetylation of some histone fractions. A large proportion of the acetyl groups is present in histone IV, and the sites of acetylation in this fraction have been determined. Four lysyl residues in the NH2-terminal region at positions 5, 8, 12, and 16 were found to be acetylated in a fraction of the histone IV molecules. Acetylation of each of these 4 lysyl residues can probably occur independently of the others, at least to some extent. This leads to a complex mixture of modified histone IV molecules, which is partially reflected in the starch gel pattern of this protein. There seems to be no acetylation of the other lysyl residues in the molecule.