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.

In order to elucidate the roles of 17β-HSDs in fish gonadal steroidogenesis, three types of 17β-HSDs (17β-HSD1, 17β-HSD8 and putative 17β-HSD12) were cloned and characterized from the Nile tilapia, Oreochromis niloticus. The cloned cDNAs of 17β-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 17β-HSD1 was dominantly expressed in the ovary, while the putative 17β-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, 17β-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 17β-HSDs were performed using recombinant proteins expressed in E. coli or HEK 293 cells. Tilapia 17β-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 17β-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 17β-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 17β-HSDs in the gonadal steroidogenesis of teleosts.

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 experimentally induced thyroxine overdose on the testis and seminal vesicles was studied in the air-breathing catfish, Clarias gariepinus during the preparatory and the pre-spawning phase. The present study revealed a marked reduction in testosterone level in serum, testis and seminal vesicles (SV). Histological examination showed a considerable reduction in the number of spermatozoa/spermatids in the seminiferous tubular lumen as well as depletion of fluid in the loculi of SV. SDS-PAGE analysis of SV fluid proteins demonstrated a significant decrease in the level of a ~27 kDa protein in thyroxine treated fishes. Evidences are presented here to indicate that thyroid hormone plays a role in regulating testis and SV function in catfish.

Estrogens are essential for many reproductive and non-reproductive functions. In teleosts, it is well-known that several subtypes of estrogen receptors are required for the precise action of estrogens. Present study describes the cloning of the third estrogen receptor, ER- beta 2, from the Nile tilapia by EST sequencing coupled microarray. The cloned ER-beta 2 showed 77.7% amino acid identity with the reported Atlantic croaker ER-beta. Three ERs, ER-alpha, ER-beta 1 and ER-beta 2, from the fugu genome were also isolated to analyze their gene structures. Comparison of the intron/exon boundaries and exon numbers of fugu, tilapia, rainbow trout and zebrafish, and phylogenetic analysis of 63 ER sequences revealed that ER-beta probably underwent two successive lineage-specific duplications in teleost. The former took place only in zebrafish lineage, and the latter took place in advanced teleosts without the zebrafish lineage, whereas no duplication of the ER-alpha gene has been detected. Tissue distribution analysis by RT-PCR revealed that tilapia ER-alpha and ER-beta 1 were expressed ubiquitously, whereas ER-beta 2 is expressed only in the pituitary, liver, intestine, kidney and gonads, with the highest expression in the testis and the lowest level in the ovary. Northern blot analysis detected a single transcript of about 3.4 kb in the testis but not in the ovary mRNAs. In transient transfection assays using human embryonic kidney 293 (HEK293) cells, tilapia ER-beta 2 showed estrodiol-17 beta dependent transactivation.

A new tissue kallikrein-like protease, blarinasin, has been purified from the salivary glands of the short-tailed shrew Blarina brevicauda. Blarinasin is a 32-kDa N-glycosylated protease with isoelectric values ranging between 5.3 and 5.7, and an optimum pH of 8.5 for enzyme activity. The cloned blarinasin cDNA coded for a pre-pro-sequence and a mature peptide of 252 amino acids with a catalytic triad typical for serine proteases and 43.7-54.0% identity to other mammalian tissue kallikreins. Blarinasin preferentially hydrolysed Pro-Phe-Arg-4-methylcoumaryl-7-amide (MCA) and N-tert-butyloxycarbonyl-Val-Leu-Lys-MCA, and preferentially converted human high-molecular-weight kininogen (HK) to bradykinin. The activity of blarinasin was prominently inhibited by aprotinin (K(i) =3.4 nM). A similar kallikrein-like protease, the lethal venom blarina toxin, has previously been purified from the salivary glands of the shrew Blarina and shows 67.9% identity to blarinasin. However, blarinasin was not toxic in mice. Blarinasin is a very abundant kallikrein-like protease and represents 70-75% of kallikrein-like enzymes in the salivary gland of B. brevicauda.

Gonadotropins (GTHs; FSH and LH) require two major steroidal mediators, estradiol-17β (E2) and 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP) to act as critical hormones to execute oocyte growth and maturation, respectively. A two-cell type model has been proposed, where the theca cells provide the precursor steroids, and the granulosa cells produce the two steroidal mediators under the direct influence of FSH and LH. A distinct shift in steroidogenesis, i.e. from E2 to 17α,20β-DP as well as the steroidogenic enzyme genes from ovarian cytochrome P450 aromatase (oP450arom) to 20β-hydroxysteroid dehydrogenase (20β-HSD), occurs in the granulosa layers of ovarian follicles prior to oocyte maturation. The triggering of the steroidogenic shift by GTHs in granulosa cells occurs through the subjugation of Ad4BP/SF-1 expression in respect of oP450arom, followed by an over-expression of 20β-HSD probably through the CREB.

The proteasome is involved in the progression of the meiotic cell cycle in fish oocytes. We reported that the alpha4 subunit of the 26S proteasome, which is a component of the outer rings of the 20S proteasome, is phosphorylated in immature oocytes and dephosphorylated in mature oocytes. To investigate the role of the phosphorylation, we purified the protein kinase from immature oocytes using a recombinant alpha4 subunit as substrate. A protein band which well corresponded to the kinase activity was identified as casein kinase Ialpha (CKIalpha). Two-dimensional (2D) PAGE analysis showed that part of the alpha4 subunit was phosphorylated by CKIalpha in vitro. This spot was detected in purified immature 26S proteasome but not in mature 26S proteasome, demonstrate that the alpha4 subunit is phosphorylated by CKIalpha meiotic cell cycle dependently.

In starfish, the peptide hormone gonad-stimulating substance (GSS) secreted from nervous tissue stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) production by ovarian follicle cells. The SALMFamide family is also known to an echinoderm neuropeptide. The present study examined effect of SALMFamide 1 (S1) on oocyte maturation of starfish Asterina pectinifera. Unlike GSS, S1 did not induce spawning in starfish ovary. In contrast, S1 was found to inhibit GSS secretion from radial nerves by treatment with high K+ concentration. Fifty percent inhibition was obtained by 0.1 mM S1. S1 did not have any effect on GSS- and 1-MeAde-induced oocyte maturation. Following incubation with a S1 antibody and subsequently with rhodamine-conjugated second antibody, neural networks were observed in ovaries. The networks were restricted mainly to their surface with little evidence of immunoreactivity inside the basement membranes. This indicates that neural networks are distributed in the ovarian wall. The result further suggests that S1 plays a role in oocyte maturation to regulate GSS secretion from the nervous system.

Venomous mammals are rare, and their venoms have not been characterized. We have purified and characterized the blarina toxin (BLTX), a lethal mammalian venom with a tissue kallikrein-like activity from the submaxillary and sublingual glands of the short-tailed shrew Blarina brevicauda . Mice administered BLTX i.p. developed irregular respiration, paralysis, and convulsions before dying. Based on the amino acid sequence of purified protein, we cloned the BLTX cDNA. It consists of a prosequence and an active form of 253 aa with a typical catalytic triad of serine proteases, with a high identity with tissue kallikreins. BLTX is an N-linked microheterogeneous glycoprotein with a unique insertion of 10 residues, L 106 TFFYKTFLG 115 . BLTX converted kininogens to kinins, which may be one of the toxic pathogens, and had dilatory effects on the blood vessel walls. The acute toxicity and proteolytic activity of BLTX were strongly inhibited by aprotinin, a kallikrein inhibitor, suggesting that its toxicity is due to a kallikrein-like activity of the venom.

Four types of GPHR cDNAs have been cloned from ovary and testis of medaka (Oryzias latipes) and their gene constructions have been determined. Two of them are closely related to known fish receptors for FSH and LH, respectively. Changes in their mRNA levels were examined during the course of oogenesis. FSH receptor mRNA could not be detected from 20 h before ovulation, whereas LH mRNA remained 5 h before ovulation.

Gonadotropin (GTH)α, FSHβ and LHβ cDNAs were cloned from the Nile tilapia. Northern blot analysis detected a single band for each subunit. Preliminary studies indicate that FSHβ is expressed as early as 0 days after hatching (dah) in the fish pituitaries.

A 548 bp partial cDNA fragment of 17β-hydroxysteroid dehydrogenase (17β-HSD1) was obtained by RT-PCR from the ovary of Nile Tilapia. The expression of 17β-HSD1 was high from 0 to 11 days after spawning, but there was a sharp decline at the day of spawning (day 14) indicating its involvement in ovarian cycle.

The steroidogenesis in the granulosa-thecal layers of fish ovarian follicles undergo a distinct shift from the production of estradiol-17β (E2) to 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP, maturation-inducing hormone, MIH) prior to meiotic maturation. This review attempts to explain the underlying mechanisms of steroidogenic shift.

Upon fertilization, two major protein groups, ZI-1,2 and ZI-3, composing the egg envelope are polymerized to insoluble forms via temporary formation of 132 kDa and 61–62 kDa intermediate proteins. We have shown that an astacin-like protease, `alveolin' is released from cortical vesicles into the perivitelline space after egg activation and induces initiation of the polymerization of egg envelope proteins. To clarify initial reactions of the polymerization, the primary structure of the 132 kDa protein was analyzed.

Tilapia riboflavin-binding protein (RfBP) cDNA was isolated by library screening. Even though it shows only 31∼35% similarity to the reported RfBPs, tilapia RfBP is conserved in amino acid residues that are known to be essential for its protein function. The exclusive expression of RfBP in the brain-pituitary-gonadal axis, as revealed by both RT-PCR and Northern blot, suggested a possible role for RfBP in fish reproduction.

To investigate the regulatory mechanism for the proteasome in the meiotic cell cycle, we purified the 26S proteasome from immature (in G2-phase) and mature (in M-phase) oocytes, and compared its subunits by immunoblotting. A monoclonal antibody, GC3β (anti-goldfish 20S proteasome component 3β) cross-reacted with two bands in the 26S proteasome from immature oocytes, however the upper band was absent in the 26S proteasome from mature oocytes. cDNAs which encode the α4 subunit of goldfish 20S proteasome (α4 ca ) were isolated by an immuno-screening method using GC3β. Phosphatase treatment of the 26S proteasome revealed that a part of α4 ca phosphorylated in G2-phase and dephosphorylated in M-phase. By the assay using recombinant α4 ca as a substrate, a kinase was purified by column chromatographs. Amino acid sequence analysis was performed for resulting partial purified fraction. A protein band, which well corresponded to the kinase activity, was identified as Casein kinase-1α (CK-1α). The result suggests that CK-1α phosphorylate α4 subunit of the 26S proteasome in immature oocyte of goldfish.