Differentiation of steroid-producing cells and folliculogenesis in the developing ovary of the Nile tilapia Oreochromis niloticus

Differentiation and development of steroid-producing cells (SPCs) and folliculogenesis during ovarian differentiation in the Nile tilapia Oreochromis niloticus were immunohistochemically and ultrastructurally examined. Clusters of immunopositive cells (IPCs) against antibodies (ABs) of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD), and cytochrome P450aromatase (P450arom) only appeared in the area near blood vessels in the fish ovaries at 50-60 days after hatching (dah). Ultrastructural results showed that differentiation and development of SPCs from undifferentiated to maturation occurred in the area near blood vessels, indicating that it would be the original site of SPCs. At 70-80 dah, IPC clusters invaded the interstices among oocytes at the perinucleolar stage from the area near the blood vessels. IPCs increased in number in the interstices among the previtellogenic oocytes, and some clusters began to enclose the outer thecal layer of the previtellogenic oocytes at 90 dah. The process of folliculogenesis was ultrastructurally observed. SPCs enclosed by fibroblastic cells invaded the interstitial areas among oocytes and some reached the surfaces of oocytes. The upper portions of these elongations opened and began to enclose the outer surfaces of developed oocytes to become thecal layer. Later, newly migrated SPCs reach the thecal layer to become thecal cells. These results indicate that steroid-producing thecal cells originate from the SPCs in the area near blood vessels. After thecal layer formation, an immunopositive reaction against P450arom AB, but not against P450scc or 3β-HSD ABs, appeared first in the granulosa cells enclosing the vitellogenic oocytes at 100 dah. At this time, estrogen production in serum levels rapidly increased. Thus, folliculogenesis could be essential for active production of estrogen in the ovary.     

Steroid Producing Cells during Ovarian Differentiation of the Tilapia, Sarotherodon niloticus

In order to examine the initial appearance and development of the steroid producing cells (SPCs) during the process of ovarian differentiation, histology and ultrastructure of tilapia ( Sarotherodon niloticus ) ovaries were investigated from 10 to 50 days after hatching. In gonads of fry at 23–26 days after hatching, initial ovarian differentiation was confirmed by the differentiation of stromal aggregations in the proximal and distal region of the gonad on the side facing the lateral wall. This represents the initial formation of the ovarian cavity. At the same time as ovarian differentiation, a few large cells appeared initially in the vicinity of blood vessels. They have some of the ultrastructural features characteristic of SPCs such as a moderate number of mitochondria with tubular cristae, a large amount of smooth endoplasmic reticulum and many free ribosomes. Based on these ultrastructural criteria, together with the present finding that these cells further differentiated into the typical SPCs at older stages, these cells were identified as SPCs. Thereafter, by 30–50 days, SPCs increased gradually in number in the area enclosing the blood vessels of ovaries. The increase in SPCs coincided with the development of germ cells, including the multiplication of oogonia and the transformation from oogonia to oocytes.     

Seabream GnRH immunoreactivity in brain and pituitary of XX and XY Nile tilapia, Oreochromis niloticus during early development

Seabream gonadotropin-releasing hormone (sbGnRH)-the chief preoptic area-hypothalamus (POA-H) form of GnRH in tilapia is involved in sexual maturation. In this study, we investigated the qualitative changes in ontogeny of sbGnRH immunoreactivity (ir-), between sexes to understand its impending role during sex differentiation. For this, the differences in immunocytochemical localization of sbGnRH in genetically male (XY) and female (XX) fish were studied from 1 day after hatching (dah), through the critical period of sex differentiation (7-21 dah) to 40 dah and mature Nile tilapia. Specific antisera against sbGnRH were used for immunolocalization. SbGnRH ir- neurons were observed in POA-H as early as 5 and 15 dah in XY fish and XX fish, respectively. Higher ir- was detected in the POA-H of XY tilapia compared with XX population till 10 dah. There was a qualitative drop in sbGnRH ir- neurons/cell bodies in POA-H around 20 dah till 30 dah in XY population compared with other durations. SbGnRH ir- cells were detected in pituitary of XX fish by 15 dah and in XY fish around 10 dah but seemed to drop down by 20 dah in XY whereas it continued to remain steady in XX fish. The sbGnRH ir- in XY fish showed a rise from 35 dah and thence till 40 dah. This study revealed subtle differences in POA-H and pituitary sbGnRH ir- during early development between genetic male and female fish with possible implications in sex differentiation.     

Efficacy of exemestane,a new generation of aromatase inhibitor,on sex differentiation in a gonochoristic fish

We report the first use of exemestane (EM), a steroidal aromatase inhibitor (AI) commercially known as aromasin, in studies of sex differentiation in fish. The effectiveness of EM was examined in two different age groups of the gonochoristic fish, Nile tilapia (Oreochromis niloticus). Untreated control fish (all female) showed normal ovarian differentiation through 120 days after hatching (dah), whereas fish treated with EM at 1000 and 2000 µg/g of feed from 9 dah through 35 dah, the critical period for sex differentiation, exhibited complete testicular differentiation; all stages of spermatogenic germ cells were evident and well developed efferent ducts were present. Fish treated with EM at 1000 µg/g of feed from 70 dah through 100 dah significantly suppressed plasma estradiol-17β level and increased level of 11-ketotestosterone. Furthermore, untreated control fish showed strong gonadal expression of the steroidogenic enzymes P450 cholesterol-side chain-cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and cytochrome P450 aromatase (P450arom). In contrast, EM-treated fish showed immunopositive reactions against P450scc and 3β-HSD but not against P450arom in interstitial Leydig cells. These results indicate that treatment of tilapia juveniles with EM during sex differentiation leads to the development of testes, apparently by a complete suppression of aromatase activity.     

南方鲶性腺分化的组织学观察

用芳香化酶抑制剂(Fadrozole)、雌激素受体拮抗剂(Tamoxifen)对人工孵化的南方鲶(Silurus meridionalis)幼鱼进行雄性化诱导处理(口服),获得雄鱼。对孵化后第5—130d的南方鲶幼鱼性腺进行组织学观察,结果表明,在实验条件下,南方鲶性腺分化发生在孵化后7d左右,雌雄性分化过程差异明显。雌鱼卵巢腔在孵化后12d左右形成,生殖细胞在孵化后35d左右快速增殖,成熟分裂最早发生在孵化后55d左右;雄鱼生殖细胞在孵化后130d左右快速增殖,成熟分裂最早发生在孵化后130d左右。雌性性腺分化早于雄性。     

Induction of XY sex reversal by estrogen involves altered gene expression in a teleost, tilapia

To clarify the importance of endogenous estrogens during sex differentiation in a teleost fish, the Nile tilapia, we examined the target events for endogenous estrogens and their role during gonadal sex differentiation. The expression of CYP19a (P450arom) precedes any morphological gonadal sex differentiation. Further to these findings, the treatment of XX fry with non-steroidal aromatase inhibitor (AI), Fadrozole, from seven to 14 days after hatching caused complete sex reversal to functional males. The XX sex reversal induced by AI was rescued completely with simultaneous estrogen treatment. We also found that XY fry treated with estrogen, before the appearance of morphological sex differences, caused complete sex reversal from males to females. Taken together, these results suggest that endogenous estrogens are required for ovarian differentiation. To identify the down-stream gene products of estrogen during ovarian differentiation, we performed subtractive hybridization using mRNA derived from normal and estrogen treated XY gonads. Two out of ten gene products were expressed in germ cells, whereas the others were expressed in somatic cells.     

Cloning of brain aromatase gene and expression of brain and ovarian aromatase genes during sexual differentiation in genetic male and female Nile tilapia Oreochromis niloticus

A brain aromatase gene was identified from the Nile tilapia Oreochromis niloticus. The cDNA sequence of this gene differed from that of the ovarian aromatase gene previously reported from this species. Tissue specific expression for both brain and ovarian aromatase genes was examined in the tissues of adult tilapia. Brain aromatase mRNA was expressed in the brain, kidney, eye, ovary, and testis, but not in the liver and spleen. Ovarian aromatase mRNA was expressed in the brain, spleen, ovary, and testis but not in the eye, kidney, and liver. Differential aromatase gene expression between the sexes was investigated in all-male (XY) and all-female (XX) groups of tilapia fry from fertilisation throughout the sexual differentiation period. Semi-quantitative RT-PCR analysis revealed that the initiation of expression of both aromatase genes lay between 3 and 4 dpf (days post fertilisation) in both sexes. The level of brain aromatase mRNA gradually increased throughout the period studied with little difference between the sexes. This contrasted with marked sexual dimorphism of ovarian aromatase mRNA expression. In females, the expression level was maintained or increased gradually throughout ontogeny, while the level in males was dramatically down-regulated between 15 and 27 dpf. Subsequently, the level of ovarian aromatase mRNA expression fluctuated slightly in both sexes, with the expression in females always being higher than in males. These findings clearly suggest that ovarian aromatase plays a decisive role in sexual differentiation in this species and that this is achieved by down-regulation of the expression of this gene in males. Mol. Reprod. Dev. 59: 359-370, 2001.     

Differentiation of steroid-producing cells during ovarian differentiation in the protogynous Malabar grouper, Epinephelus malabaricus

To understand the mechanism of sex differentiation in the protogynous Malabar grouper Epinephelus malabaricus, we performed an immunohistochemical investigation of the expression of three steroidogenic enzymes, cholesterol-side-chain-cleavage enzyme (CYP11a), aromatase (CYP19a1a), and cytochrome P45011beta-hydroxylase (CYP11b), in the gonads during ovarian differentiation. Strong positive immunoreactivity against CYP11a, the key enzyme of steroidogenesis, and CYP19a1a which is essential for estrogen (17beta-estradiol) production, appeared first in the somatic cells surrounding gonial germ cells in undifferentiated gonads and throughout ovarian differentiation. However, positive immunoreactivity against CYP11b, which is important for androgen (11-ketotestosterone) production, first appeared in the cluster of somatic cells in the ovary tunica near the dorsal blood vessel after differentiation. CYP19a1a and CYP11b did not co-localize in any cells. These results indicate that there are two types of steroid-producing cells, estrogen-producing cells and androgen-producing cells, in the gonads of this fish, and they are distributed differently, suggesting that these cells are derived from different somatic cells. Estrogen-producing cells appeared prior to ovarian differentiation, while androgen-producing cells were first detected after ovarian differentiation. These results suggest that endogenous estrogen is involved in ovarian differentiation.     

Ultrastructural analysis of the developing follicle during early vitellogenesis in tilapia,Oreochromis niloticus,with special reference to the steroid-producing cells

The development and distribution of steroid producing cells (SPCs) in the ovary of tilapia have been studied by light and electron microscopy. At 40–50 d after hatching, these cells are seen only in the vicinity of blood vessels; there are no SPCs in the interstitial region, nor in the thecal layer enclosing young oocytes at the peri-nucleolus stage. By 70–80 d after hatching, the number of SPCs in the area near blood vessels has increased, and the capillaries have spread among the developing peri-nucleolar stage oocytes, and into the ovarian tunica. Clusters of SPCs have also migrated into the interstitial region and into the tunica along with these capillaries. In the ovary 100 d after hatching, some SPCs can be found in the thecal layer enclosing vitellogenic oocytes. Moreover, masses of SPCs can now be observed infiltrating the thecal layer of the oocyte. Serum testosterone (T) and estradiol-17 (E2) levels at 40–70 d after hatching, are low (T, 0.75–1.10 ng/ml, E2, 0.36–1.08 ng/ml), but at 100 d, plasma E2, but not T, is elevated (T, 1.95 ng/ml, E2, 4.65 ng/ml). These results suggest that SPCs appearing in the vicinity of blood vessels move into the interstitial region between oocytes, and finally enclose the oocytes at an early vitellogenic stage. It is interesting to note that the enclosure of oocytes by SPCs coincides with significant increases in E2 production.     

Expression of aromatase mRNA and effects of aromatase inhibitor during ovarian development in the medaka, Oryzias latipes

In teleost fish, several studies have implicated estrogens in the process of ovarian development, but the exact role of endogenous estrogen is still unclear. We examined the expression of aromatase mRNA with in situ hybridization, and the effects of Fadrozole, a nonsteroidal aromatase inhibitor (AI), during ovarian development in medaka Oryzias latipes. Medaka aromatase was first detected on the ventral side of ovaries from four to 10 days after hatching (dah), after occurrence of oogenesis. AI treatment after hatching suppressed the ovarian cavity formation from 30 dah but did not affect early oogenesis and folliculogenesis during ovarian development. These results suggest that endogenous estrogen is specifically required for formation of the ovarian cavity, but is not essential for early oogenesis and folliculogenesis in medaka.     

Aromatase plays a key role during normal and temperature-induced sex differentiation of tilapia Oreochromis niloticus.

In the tilapia Oreochromis niloticus, sex is determined genetically (GSD), by temperature (TSD) or by temperature/genotype interactions. Functional masculinization can be achieved by applying high rearing temperatures during a critical period of sex differentiation. Estrogens play an important role in female differentiation of non-mammalian vertebrates. The involvement of aromatase, was assessed during the natural (genetic all-females and all-males at 27 degrees C) and temperature-induced sex differentiation of tilapia (genetic all-females at 35 degrees C). Gonads were dissected between 486--702 degree x days. Aromatase gene expression was analyzed by virtual northern and semi-quantitative RT-PCR revealing a strong expression during normal ovarian differentiation concomitant with high levels (465 +/- 137 fg/g) of oestradiol-17 beta (E2-17 beta). This was encountered in gonads after the onset of ovarian differentiation (proliferation of both stromal and germ cells prior to ovarian meiosis). Genetic males exhibited lower levels of aromatase gene expression and E2-17 beta quantities (71 +/- 23 fg/ g). Aromatase enzyme activity in fry heads established a sexual dimorphism in the brain, with high activity in females (377.9 pmol/head/hr) and low activity in males (221.53 pmol/head/hr). Temperature induced the masculinization of genetic females to a different degree in each progeny, but in all cases repression of aromatase expression was encountered. Genetic males at 35 degrees C also exhibited a repression of aromatase expression. Aromatase brain activity decreased by nearly three-fold in the temperature-masculinized females with also a reduction observed in genetic males at 35 degrees C. This suggests that aromatase repression is required in the gonad (and perhaps in the brain) in order to drive differentiation towards testis development. Mol. Reprod. Dev. 59:265-276, 2001.     

Molecular cloning of DAX1 and SHP cDNAs and their expression patterns in the Nile tilapia,Oreochromis niloticus

Piscine DAX1 and SHP cDNAs with an open reading frame encoding 296 and 258 amino acid residues, respectively, as well as SHP partial gene fragment, were cloned from Nile tilapia. Phylogenetic analyses of DAX1s, SHPs, and homologous EST fragments indicate that DAX1 and SHP are conserved in gene structure and are present throughout vertebrates. A single band of approximately 1.4kb for DAX1 and of approximately 1.2kb for SHP was detected in the Northern blot analysis. Tissue distribution analysis by RT-PCR showed that fish DAX1 and SHP mRNAs are widely expressed in adult tissues, with the most abundant expression in gonads and liver, respectively. DAX1 and SHP were also detected in gonads of both sexes at 5-90 days after hatching (dah). However, the expression of DAX1 is weak at 5 and 10dah and then significantly up-regulated between 10 and 15dah, whereas the expression of SHP is moderate and consistent during the ontogeny.     

Mutation of cyp19a1b results in sterile males due to efferent duct obstruction in Nile tilapia

The involvement of estrogen in male fertility has been well established in mammals. However, less is known about the role of estrogen in fish male reproduction. Our recent study revealed that Cyp19a1a deficiency had no effect on fertility in male fish. In this study, expression of Cyp19a1b, but not Cyp19a1a, was detected by immunohistochemistry in Leydig cells of tilapia testes. cyp19a1b mutation resulted in a significant decrease in the concentration of 17β‐estradiol in serum and sterility in XY fish, as no offspring were obtained when crossed with control XX fish at 240 days after hatching (dah). No sperm was obtained from the mature mutants by in vitro extrusion. Further examination of the mutant gonads revealed excessive semen accumulation and testicular hypertrophy. Semen collected from the mutant testes during autopsy contained sperm with a normal morphology that showed no significant differences in motility, VCL, BCF, STR, or fertility compared with control sperm. Efferent ducts from the mutant testes, which had low‐convolution levels, fewer branches, and no blood vessels observed inside the walls, were significantly smaller in size. qRT‐PCR analyses showed downregulated expression of ion exchange genes. There was increased apoptosis in the epithelial cells of the efferent ducts and other somatic cells of the testes as revealed by TUNEL staining, as well as upregulation of apoptosis gene expression in the mutants. At 360 dah, mutant fish showed testicular atrophy and efferent duct fibrosis. These results demonstrated that estrogen deficiency caused by Cyp19a1b mutation resulted in male sterility due to efferent duct obstruction.     

Masculinization of genetic female nile tilapia (Oreochromis niloticus) by dietary administration of an aromatase inhibitor during sexual differentiation

A series of experiments was carried out in which genetically female Nile tilapia (Oreochromis niloticus) fry were treated with Fadrozole, a nonsteroidal aromatase inhibitor (AI), in the diet during the period of sexual differentiation. Batches of tilapia fry treated with AI during the first 30 days following yolk-sac resorption (7-37 days post hatch, dph) showed a dose-dependent increase in the percentage of males from 0 to 200 mg. kg(-1). The percentage of males remained approximately constant (92.5-96.0%) from 200 to 500 mg. kg(-1). Any continuous 2- or 3-week treatment with 500 mg. kg(-1) AI in this 4-week period successfully masculinized the majority of the treated fish (>80%). Treatments of 1 week duration revealed that the most sensitive time to AI lies in the first week (between 7 and 14 dph). Progeny testing of males from AI-treated groups gave results indicating that these were XX males, as expected. These experiments strongly implicate aromatase activity as a key factor in sexual differentiation in the Nile tilapia.