The forkhead gene family in medaka: expression patterns and gene evolution.

Using the genome sequence of the medaka, Oryzias latipes, we examined the genomic complement of Fox genes in this organism to gain insight into the evolutionary relationships and expression patterns of this gene family. We identified 31 Fox genes by searching for Forkhead domains in the medaka genome and by polymerase chain reaction (PCR) using primers designed from protein alignments. All the medaka Fox genes are encoded in 18 subclasses as follows: 5 Fox genes in subclass D; 3 Fox genes in subclass O; 2 Fox genes in each of subclass A, B, E, F, G, I, P, and Q, respectively; 1 Fox gene in each of subclass C, H, J, K, M, N, and R. The gene structures and general features are also discussed. In addition, we examined the expression patterns of some of these genes in different adult tissues and during embryonic development in medaka using quantitative PCR and in situ hybridization. The present study shows that Forkhead transcription factors play an important role during early embryonic development in medaka, and the results will enhance our knowledge in terms of Fox family evolution in different taxa.

Genome-wide analysis of Sox genes in Medaka (Oryzias latipes) and their expression pattern in embryonic development.

Genes of the Sox family encode evolutionarily conserved high-mobility group box containing transcription factors, which play key roles in various events of developmental contexts. In this study, we identified 15 sox genes by searching for the high-mobility group domain in the medaka genome and by polymerase chain reaction using primers designed from the results obtained from homology protein alignment. All medaka sox genes except a novel sox gene, Olsox32, are encoded in 5 groups as follows: 4 sox genes in group B; 3 sox genes in group D and F, respectively; 2 sox genes in group C and E, respectively, while no sox genes were found in groups A, G, H, I, and J. The medaka Olsox32 does not fall within any of the previously defined groups A-J. Here we have assigned it to a new group K. Together with the Sox protein sequences of other species, the phylogenetic relationship was analyzed and compared. Our findings point to recent sox gene loss, duplication and divergence occurring during the evolution of tetrapod and teleost lineages. The expression pattern shows that sox genes play a variety of roles in the early embryonic development of medaka.

Gender differences in tryptophan hydroxylase-2 mRNA, serotonin, and 5-hydroxytryptophan levels in the brain of catfish, Clarias gariepinus, during sex differentiation.

Tryptophan hydroxylase (tph) is the key regulator in serotonin (5-HT) biosynthesis that stimulates the release of GnRH and gonadotropins by acting at the level of hypothalamo-hypophyseal axis. In brain, 5-HT is expressed predominantly in preoptic area-hypothalamus (POA-HYP) region in teleosts. Therefore, in the present study we isolated tph2 from catfish brain to evaluate its expression pattern in male and female brains during early development. Tph2 cloned from catfish brain is 2.768 Kb in length which encodes predicted protein of 488 amino acid residues. The characterization of recombinant tph2 was done by transient transfection in CHO cells. Tissue distribution of tph2 revealed ubiquitous expression except ovary. Real time PCR analysis in discrete regions of adult male brain revealed that tph2 mRNA was abundant in the POA-HYP and optic tectum+cerebellum+thalamus (OCT) regions. Differential expression of tph2 was observed at mRNA and protein levels in the POA-HYP and OCT regions of male and female brains during development that further correlate with the 5-hydroxytryptophan (5-HTP) and 5-HT levels measured using HPLC method in these regions of male and female brains. Tph2 immunoreactive neurons were observed in different regions of brain at 50 days post hatch using catfish specific tph2 antibody. Changes in tph2 mRNA expression, 5-HTP, and 5-HT levels in the POA-HYP+OCT region of brains of methyltestosterone and para-chlorophenylalanine treated fishes during development further endorse our results. Based on our results, we propose that the serotonergic system is involved in brain sex differentiation in teleosts.

Heritable artificial sex chromosomes in the medaka, Oryzias latipes.

Chromosomal sex determination is widely used by vertebrates, however, only two genes have been identified as master sex-determining genes: SRY/Sry in mammals and DMY in the teleost medaka. Transfer of both genes into genetically female (XX) individuals can induce male development. However, transgenic strains have not been established in both cases because of infertility of the transgenic founders in mammals and low germline transmission rates in medaka. In this study, we used a BAC clone containing DMY in a 117 kb genomic region and two types of fluorescent marker to establish two DMY-transgenic medaka strains. In these strains, exogenous DMY is completely linked to a male phenotype and early gonadal development is not different from that of the wild-type strain. Sex-linkage analysis showed that the exogenous DMY was located on linkage group (LG) 23 in one strain and on LG 5 in the other strain, whereas the sex chromosome in medaka is on LG 1. Real-time PCR analysis indicated that these strains have multiple copies of DMY and higher DMY expression levels than the wild-type strain. These results showed that LGs 23 and 5 function as sex chromosomes in the two strains, respectively. This is not only the first example of the artificial generation of heritable sex chromosomes in vertebrates but also the first evidence showing plasticity of homomorphic sex chromosomes. This plasticity appears to be a characteristic of lower vertebrates and the underlying cause of frequent sex chromosome switching, recently reported in several fish and frog species.

Dimorphic expression of tryptophan hydroxylase in the brain of XX and XY Nile tilapia during early development.

Serotonin (5-HT) is well known for modulating the release of GnRH and gonadotropin in teleosts. Reports on increased female:male ratio after the blockade of 5-HT biosynthesis proposed a role for 5-HT in brain sex differentiation. Two types of tryptophan hydroxylase (Tph), rate-limiting enzyme in the biosynthesis of 5-HT were cloned from vertebrates. In the present study, we cloned Tph from brain and evaluated its importance during early development of XX and XY Nile tilapia. Tph cloned from tilapia brain is 1888 bp in length and it encodes predicted protein of 462 amino acid residues. Tph activity of tilapia was confirmed by demonstrating the conversion of L-tryptophan to 5-hydroxy tryptophan by the recombinant protein after transient transfection of this cDNA clone in COS-7 cells. Northern blot identified single transcript around 2kb in male brain. Tissue distribution of Tph revealed high abundance in brain, kidney, liver and testis. Semi-quantitative RT-PCR revealed exclusive expression of Tph in the male brain from 5 to 20 days post hatch (dph) while in the female brain, it was from 25 dph. These results were authenticated by localization of Tph transcripts in olfactory bulb-telencephalon region of 11 dph male brain using in situ hybridization. Tph immunoreactivity (-ir) was also evident in the nucleus preopticus-periventricularis area of male brain as early as 12 dph. However, Tph-ir was observed in several regions of both male and female brain without any distinction from 30 dph. Dimorphic expression pattern of Tph during early brain development around the critical period (7-21 dph) of gonadal sex determination and differentiation may implicate a role for Tph in brain sex differentiation of tilapia.

Novel 3beta-hydroxysteroid dehydrogenases from gonads of the Nile tilapia: phylogenetic significance and expression during reproductive cycle.

Multiple forms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) and their differential tissue expression pattern have not been shown in any lower vertebrates. In the present study, we report cloning of two novel 3beta-HSDs and two variants from gonads of the Nile tilapia. 3beta-HSD cDNAs encode two peptides of 375 (3beta-HSD type-I/variant 1) and 367 (3beta-HSD type-II/variant 1) amino acid residues that share 31.9% homology. 3beta-HSD type-I/variant 1 shared high homology with other piscine counterparts while 3beta-HSD type-II/variant 1 exhibited homology to mammalian DeltaC27-3beta-HSD and multifunctional viral 3beta-HSD. The latter seems to be ancient form among vertebrates. Transiently transfected 3beta-HSDs' open reading frames in COS-7 cells converted exogenous pregnenolone/androsta-5-ene-3beta-17beta-diol to progesterone/testosterone. Tissue distribution pattern of 3beta-HSDs by RT-PCR revealed varied expression pattern. Northern blot analysis of 3beta-HSDs demonstrated steady or gradual rise in transcripts level at different gonadal stages. These data revealed the importance of novel 3beta-HSDs in teleosts and also provided phylogenetic significance.

Initiation of cyclin B degradation by the 26S proteasome upon egg activation.

Immediately before the transition from metaphase to anaphase, the protein kinase activity of maturation or M-phase promoting factor (MPF) is inactivated by a mechanism that involves the degradation of its regulatory subunit, cyclin B. The availability of biologically active goldfish cyclin B produced in Escherichia coli and purified goldfish proteasomes (a nonlysosomal large protease) has allowed the role of proteasomes in the regulation of cyclin degradation to be examined for the first time. The 26S, but not the 20S proteasome, digested recombinant 49-kD cyclin B at lysine 57 (K57), producing a 42-kD truncated form. The 42-kD cyclin was also produced by the digestion of native cyclin B forming a complex with cdc2, a catalytic subunit of MPF, and a fragment transiently appeared during cyclin degradation when eggs were released from metaphase II arrest by egg activation. Mutant cyclin at K57 was resistant to both digestion by the 26S proteasome and degradation at metaphase/anaphase transition in Xenopus egg extracts. The results of this study indicate that the destruction of cyclin B is initiated by the ATP-dependent and ubiquitin-independent proteolytic activity of 26S proteasome through the first cutting in the NH2 terminus of cyclin (at K57 in the case of goldfish cyclin B). We also surmise that this cut allows the cyclin to be ubiquitinated for further destruction by ubiquitin-dependent activity of the 26S proteasome that leads to MPF inactivation.

Molecular characterization and quantification of the gonadotropin receptors FSH-R and LH-R from Atlantic cod (Gadus morhua).

In order to elucidate regulatory mechanisms during puberty final oocyte maturation and spawning, full-length sequences coding for the receptors for follicle-stimulating hormone (FSH-R) and luteinizing hormone (LH-R) were isolated from female Atlantic cod (Gadus morhua) by a RACE-PCR based strategy. The nucleotide and amino acid sequences showed high homologies with the corresponding sequences of other fish species but contained some distinct differences. Conserved features important for functionality, such as a long N-terminal extracellular domain (ECD), seven transmembrane domains and a short C-terminal intracellular domain, were identified in both predicted proteins. Partial genomic sequences for these genes were also determined, allowing the design of mRNA-specific quantitative PCR assays. Due to suspected alternative splicing during expression of these genes, additional real-time PCR assays detecting variants containing the membrane-anchoring domain were established. Besides the expected expression of FSH-R and LH-R mRNA in the gonads similarly strong signals for LH-R were also obtained in male gill, and in female and male brain. When relative expression was analysed at different stages of sexual maturation, levels for FSH-R increased moderately during gonadal growth whereas those of LH-R showed a high peak at spawning.

Expression pattern of prmt5 in adult fish and embryos of medaka, Oryzias latipes.

DNA methylation is an important biochemical epigenetic determinant of gene expression in cells and therefore actively involved in gene regulation, chromosomal conformation, and protein activity. Protein arginine methyltransferases (PRMTs) play a major role in the methylation of proteins that have an arginine residue, catalyzing both the asymmetric dimethylation of arginine (aDMA) and symmetric dimethylation of arginine (sDMA). PRMT5, a type II PRMT which catalyzes sDMA, has been shown to have a pivotal role in pole plasm assembly and germ cell development in Drosophila and also to be an associate factor of Blimp1 for germ cell development in mouse. Here, we report a homolog of prmt5 identified in medaka, Oryzias latipes, which was detected in the brain, gill, muscle, heart, liver, spleen, intestine, testis and ovary of adult fish by reverse transcriptase-PCR. The expression of prmt5 in the gonads is restricted to oocytes of the ovary, spermatogonia, and spermatocytes of testis. The prmt5 transcripts were detected as early as the one-cell stage and in all the tissues of embryos during embryogenesis. In summary, prmt5 is a maternal determinant factor of embryogenesis of medaka, possibly playing an important role in oogenesis and spermatogenesis in adult medaka.

Dmy initiates masculinity by altering Gsdf/Sox9a2/Rspo1 expression in medaka (Oryzias latipes).

Despite identification of several sex-determining genes in non-mammalian vertebrates, their detailed molecular cascades of sex determination/differentiation are not known. Here, we used a novel RNAi to characterise the molecular mechanism of Dmy (the sex-determining gene of medaka)-mediated masculinity in XY fish. Dmy knockdown (Dmy-KD) suppressed male pathway (Gsdf, Sox9a2, etc.) and favoured female cascade (Rspo1, etc.) in embryonic XY gonads, resulting in a fertile male-to-female sex-reversal. Gsdf, Sox9a2, and Rspo1 directly interacted with Dmy, and co-injection of Gsdf and Sox9a2 re-established masculinity in XY-Dmy-KD transgenics, insinuating that Dmy initiates masculinity by stimulating and suppressing Gsdf/Sox9a2 and Rspo1 expression, respectively. Gonadal expression of Wt1a starts prior to Dmy and didn't change upon Dmy-KD. Furthermore, Wt1a stimulated the promoter activity of Dmy, suggesting Wt1a as a regulator of Dmy. These findings provide new insights into the role of vertebrate sex-determining genes associated with the molecular interplay between the male and female pathways.

Molecular cloning of cDNA encoding a ubiquitin-activating enzyme (E1) from goldfish (Carassius auratus) and expression analysis of the cloned gene.

Destruction of cyclin B is required to the mitotic and meiotic cycles. A cyclin-specific ubiquitinating system, including ubiquitin-activating enzyme (E1), is thought to be responsible for cyclin B destruction. Here we present the cloning, sequencing and expression analysis of goldfish, Carassius auratus, E1 from goldfish ovary. The cloned cDNA is 4069 bp long and encodes 1059 amino acids. The deduced amino acid sequence is highly homologous to E1 from other species. Recombinant goldfish E1 could transfer ubiquitin to cyclin-selective ubiquitin-conjugating enzyme. Tissue distribution revealed a single 4.0-kb message ubiquitous among tissues.

1-Methyladenine production from ATP by starfish ovarian follicle cells.

1-Methyladenine (1-MeAde), the oocyte maturation-inducing substance in starfish, is produced by ovarian follicle cells upon stimulation with a gonad-stimulating substance (GSS) released from the radial nerves. We have shown previously that GSS causes a reduction in the intracellular levels of ATP coincident with 1-MeAde production. The present study examined whether the adenine molecule of 1-MeAde is directly derived from ATP. When isolated follicle cells from the starfish Asterina pectinifera were preloaded with [U-14C]adenine or [U-14C]adenosine, there was an increase in the intracellular levels of radiolabeled adenine nucleotides, particularly ATP. Following further incubation with GSS, the intracellular levels of radiolabeled ATP decreased, concomitant with a marked increase in the levels of [14C]1-MeAde in the medium. The amount of ATP consumed under the influence of GSS was similar to the amount of 1-MeAde produced. However, there was no change in the levels of ADP and AMP regardless of the presence or absence of GSS. These findings strongly suggest that 1-MeAde is synthesized from ATP as a substrate in follicle cells under the influence of GSS. Furthermore, using [methyl-3H]methionine, the methyl group of 1-MeAde was found to be derived from methionine. Thus GSS appears to stimulate the synthesis of 1-MeAde from ATP via the methylation process in starfish ovarian follicle cells.

Superoxide dismutase and thioredoxin restore defective p34cdc2 kinase activation in mouse two-cell block.

We recently showed that superoxide dismutase (SOD), a free radical scavenger, and thioredoxin, a potent protein disulfide reductase, release mouse two-cell stage developmental block in vitro. To elucidate the mechanism underlying the two-cell block and the effects of these enzymes, we studied the chronological changes in the kinase activity and the immunoblotting pattern of p34cdc2, a key regulator of the cell cycle, during the first and second cell cycles of the mouse embryonic development. In vivo embryos were freshly collected at fixed times, and in vitro embryos cultured from the pronuclear stage were also sampled with the same time-course. A marked elevation of p34cdc2 kinase activity was observed in vivo at 27-32 and 51 h after an injection of human chorionic gonadotropin. These times coincide with the M-phases of embryo cleavage. In vitro embryos showed high kinase activity during the M-phase of the first cleavage, but this activity was not elevated during the second cell cycle. The addition of recombinant human SOD (200 micrograms/ml) or thioredoxin from Escherichia coli (500 micrograms/ml) to the medium enabled kinase activation with a time course similar to that of in vivo embryos. The immunoblotting patterns suggested the dephosphorylation of p34cdc2 at the M-phase of the first and the second cleavages in vivo. Although p34cdc2 was dephosphorylated at the M-phase of the first cleavage and then rephosphorylated for embryos cultured in vitro, the second dephosphorylation was not observed during the second cell cycle. The addition of SOD or thioredoxin permitted the dephosphorylation at the M-phases of both the first and the second cleavage. These results suggest that one of the chief causes of two-cell block in vitro is the impairment in p34cdc2 dephosphorylation, recently shown to be catalyzed by the cdc25 homologue. This impairment is thought to be due to oxidative stress, because both SOD and thioredoxin are known to play a defensive role against it.

Clinical significance of PQ segment depression in acute Q wave anterior wall myocardial infarction.

OBJECTIVES: This study was designed to evaluate the clinical significance of PQ segment depression and to examine the frequency of PQ segment depression in infarction-associated pericarditis. BACKGROUND: PQ segment deviation is almost as characteristic as the classic ST segment deviation and is detected in most patients with pericarditis. However, the incidence and clinical characteristics of PQ segment depression in acute myocardial infarction are not defined. METHODS: Three hundred four consecutive patients with acute Q wave anterior wall myocardial infarction were examined carefully by auscultation, electrocardiogram, echocardiogram and chest roentgenogram. The diagnosis of pericarditis was made on the basis of pericardial rub detected by more than two observers during the 1st 3 days after hospital admission. At least 0.5 mm of PQ segment depression from the TP segment observed for > 24 h in both limb and precordial leads was considered diagnostic of PQ segment depression. RESULTS: A pericardial rub was present in 65 patients (21%) and absent in 239 patients. PQ segment depression was detected in both limb and precordial leads in 30 patients (10%): 18 patients with pericardial rub and 12 patients without pericardial rub. On the basis of five clinical variables, multivariate analysis was performed to determine the important variables related to the occurrence of PQ segment depression. Pericardial rub was selected with left ventricular segments with advanced asynergy as a significant factor related to PQ segment depression. There were 31 in-hospital deaths, and a significantly higher hospital mortality rate was observed in patients with PQ segment depression (23% vs. 9%). CONCLUSIONS: Although PQ segment depression was observed in a minority of patients with infarction-associated pericarditis, it was one of the clinical signs of larger infarct size and increased hospital deaths.

Cloning, expression and characterization of three types of 17beta-hydroxysteroid dehydrogenases from the Nile tilapia, Oreochromis niloticus.

In order to elucidate the roles of 17beta-HSDs in fish gonadal steroidogenesis, three types of 17beta-HSDs (17beta-HSD1, 17beta-HSD8 and putative 17beta-HSD12) were cloned and characterized from the Nile tilapia, Oreochromis niloticus. The cloned cDNAs of 17beta-HSD type 1, 8 and 12 were 1504, 1006 and 1930 bp long, with open reading frames encoding proteins of 289, 256 and 314 aminoacids, respectively. Tissue distribution pattern analyzed by RT-PCR and Northern blot showed that 17beta-HSD1 was dominantly expressed in the ovary, while the putative 17beta-HSD12, one of the two duplicates found in fish, is a male specific enzyme and expressed exclusively in testis (detected by RT-PCR only). On the other hand, 17beta-HSD8 was expressed in the brain, gill, heart, liver, intestine, gonad, kidney and muscle of both male and female. Enzymatic assays of the three types of 17beta-HSDs were performed using recombinant proteins expressed in E. coli or HEK 293 cells. Tilapia 17beta-HSD1 expressed in E. coli had the preference for NADP(H) as cofactor and could catalyze the inter-conversion between estrone and estradiol efficiently as well as the inter-conversion between androstenedione and testosterone, but less efficiently. Tilapia 17beta-HSD8 recombinant protein expressed in HEK 293 cells could catalyze the conversion of testosterone to androstenedione, as well as the inter-conversion between estrone and estradiol. However, the putative 17beta-HSD12 expressed in E. coli or in HEK 293 cells showed no conversion to any of the four substrates tested in this study. Based on enzyme characterization and tissue distribution, it is plausible to attribute crucial roles to 17beta-HSDs in the gonadal steroidogenesis of teleosts.

Differential expression of vasa homologue gene in the germ cells during oogenesis and spermatogenesis in a teleost fish, tilapia, Oreochromis niloticus.

Vas (a Drosophila vasa homologue) gene expression pattern in germ cells during oogenesis and spermatogenesis was examined using all genetic females and males of a teleost fish, tilapia. Primordial germ cells (PGC) reach the gonadal anlagen 3 days after hatching (7 days after fertilization), the time when the gonadal anlagen was first formed. Prior to meiosis, no differences in vas RNA are observed in male and female germ cells. In the ovary, vas is expressed strongly in oogonia to diplotene oocytes and becomes localized as patches in auxocytes and then strong signals are uniformly distributed in the cytoplasm of previtellogenic oocytes, followed by a decrease from vitellogenic to postvitellogenic oocytes. In the testis, vas signals are strong in spermatogonia and decrease in early primary spermatocytes. No vas RNA expression is evident in either diplotene primary spermatocytes, secondary spermatocytes, spermatids or spermatozoa. The observed differences in vas RNA expression suggest a differential function of vas in the regulation of meiotic progression of female and male germ cells.

Molecular mechanisms of sex determination and gonadal sex differentiation in fish.

We have used various genetic and molecular approaches to investigate the mechanisms of sex determination and gonadal sex differentiation in fish. DMY was identified as the sex-determining gene of medaka. In tilapia, endogenous estrogens act as natural inducers of ovarian differentiation, while DMRT1 may be important for testicular differentiation. The roles of these regulators in sex determination and gonadal sex differentiation were ascertained using a gene or hormonal blockade strategy.

Ontogenic expression patterns of several nuclear receptors and cytochrome P450 aromatases in brain and gonads of the Nile tilapia Oreochromis niloticus suggests their involvement in sex differentiation.

Using semi-quantitative reverse transcriptase polymerase chain reaction we analyzed the ontogenic expression patterns of several nuclear receptors (estrogen receptors [ERalpha and beta], androgen receptors [ARalpha and beta], Ad4BP/SF-1 and Dax-1) and cytochrome P450 aromatases (brain and ovarian types) in whole brain and gonads of the Nile tilapia. ERalpha and beta transcripts were evident in both sexes with a high expression of ERalpha in females at 0 day after hatching (0 dah). ARalpha appeared early (0 dah) in males and while in females at 25 dah. Among the two types of cytochrome P450 aromatases, the expression of the brain type (bP450arom) but not the ovarian type (oP450arom) was evident from 0 to 90 dah in the whole brain of both males and females. Expression of Ad4BP/SF-1 in female brain began from 0 dah but in male brain at 5 dah. Expression of Dax-1 began at 0 dah and it was higher throughout in male brain than that of the female brain. In gonads, ERalpha and beta transcripts were evident in both sexes with slight variation. In females, both oP450arom and Ad4BP/SF-1 amplicons were evident at 15 dah. In males, although faint expressions of Ad4BP/SF-1 amplicons were evident at early duration of development, oP450arom did not appear until 90 dah. Conversely, expression of bP450arom was observed throughout in the developing testis with varied pattern while in developing ovary it was evident till 15 dah and reappeared only after 90 dah. Taken together, present results suggest that brain acts merely as a synchronizer in the sex differentiation process initiated by gonadal cues/factors in the Nile tilapia.

Effect of methyl testosterone- and ethynyl estradiol-induced sex differentiation on catfish, Clarias gariepinus: expression profiles of DMRT1, Cytochrome P450aromatases and 3 beta-hydroxysteroid dehydrogenase.

The objective of the present study is to observe the effect of exogenous steroids, methyl testosterone (MT) and ethynyl estradiol (EEL) on gonadal differentiation and analyze its effect on the expression of several genes during testicular and ovarian differentiation in juvenile catfish. Exogenous hormone treatments (MT and EEL) were given by immersion at different days of hatching. The histological analysis revealed that the EEL- and MT-treatments resulted in the initiation of ovarian and testicular differentiation, respectively. This is further supported by specific expression of two forms of DMRT1 in the MT-treated group but not in the EEL-treated group at 47 days after hatching (dah). The reverse is true for the expression of ovarian aromatase. Results of the semi-quantitative RT-PCR show that brain aromatase transcript levels are high in 47 dah control (histologically female) and 47 dah EEL-treated fish, as compared to 47 dah MT-treated fish. At 60 dah, brain aromatase showed elevation in its expression. Interestingly, the expression pattern of 3 beta-HSD did not show any change in EEL- and MT-treated fish. The present study also provides a strategy to study sex differentiation, for those species where genetic sex population is unavailable.

Immunocytochemical localization of gonadotropins during the development of XX and XY Nile tilapia.

Gonadal development and steroidogenesis in teleosts is regulated by two gonadotropic hormones; luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Earlier studies in tilapia have shown that FSH-beta and LH-beta appear by 14 days after hatching (dah), results from the current study corroborate with these previous reports in tilapia. Here we demonstrate the appearance of LH in pituitary between 14 and 20 dah. In addition to this the present study primarily focuses on any possible differences in appearance of LH-beta and FSH-beta immunoreactivity between XX and XY population of Nile tilapia. LH immunoreactivity was found to be lower in pituitary of XX fish when compared to XY fish. The development of FSH-beta immunoreactivity in pituitary of the Nile tilapia is also presented. Overall, it remains to be established what significance these findings on the appearance of gonadotropins hold for sex differentiation in tilapia.