Induction of Gonadal Development in Protogynous Grouper with Orally Delivered FSH DNA.

The availability of sexually mature fish often dictates the success of its captive breeding. In this study, we induced reproductive development in juvenile protogynous tiger grouper through oral administration of a plasmid (p) containing an engineered follicle-stimulating hormone (FSH). An expression construct (pcDNA3.1) was designed to express a single-chain FSH consisting of giant grouper FSH ß-subunit and glycoprotein subunit-α (CGα), linked by the carboxy-terminal peptide (CTP) sequence from the human chorionic gonadotropin (hCG). Single oral delivery of pFSH encapsulated in liposome and chitosan to tiger grouper yielded a significant increase in plasma FSH protein level after 4 days. Weekly pFSH feeding of juvenile tiger groupers for 8 weeks stimulated ovarian development as indicated by a significant increase in oocyte diameter and progression of oocytes to cortical alveolar stage. As the pFSH treatment progressed from 20 to 38 weeks, female to male sex change was initiated, characterized by oocyte regression, proliferation of spermatogonial cells, and occurrence of spermatogenic cysts. It was also associated with significantly lower mRNA expression of steroidogenic genes (cyp11b, cyp19a1a, and foxl2) and basal plasma levels of sex steroid hormones 17ß-estradiol (E2), testosterone (T), and 11-ketotestosterone (11KT). Results suggest that pFSH stimulates ovarian development up to cortical alveolar stage and then initiates sex change in tiger grouper. These findings significantly contribute to our knowledge on the role of FSH in the development of protogynous hermaphroditic fish. This study is the first to demonstrate induction of reproductive development in fish through oral delivery of plasmid gonadotropin.

Gonadal response of juvenile protogynous grouper (Epinephelus fuscoguttatus) to long-term recombinant follicle-stimulating hormone administration†.

The role of follicle-stimulating hormone (FSH) in the gonadal development of protogynous hermaphroditic grouper (Epinephelus fuscoguttatus) was investigated. Recombinant giant grouper (E. lanceolatus) FSH (rggFSH) was produced in yeast. Its receptor-binding capacity and steroidogenic potency were confirmed in vitro. Weekly injections of rggFSH to juvenile tiger grouper for 8 weeks (100 µg/kg body weight, BW) resulted in significantly larger and more advanced oocytes (cortical alveolar stage vs primary growth stage in control). Sustained treatment with rggFSH (20 to 38 weeks at 200 µg/kg BW) resulted in significant reduction in gonad size, degeneration of oocytes, and proliferation of spermatogonial cells, indicative of female to male sex change. Gene expression analysis showed that, while initiating female to male sex change, the rggFSH significantly suppressed the steroidogenic genes cyp11b, cyp19a1a, and foxl2 which restrained the endogenous production of sex steroid hormones and thus prevented the differentiation of spermatogonial cells. Expression profile of sex markers dmrt1, amh, figla, and bmp15 suggests that the observed sex change was restricted at the initiation stage. Based on these results, we propose that the process of female to male sex change in the protogynous grouper is initiated by FSH, rather than sex steroids, and likely involves steroid-independent pathway. The cortical alveolar stage in oocyte development is the critical point after which FSH-induced sex change is possible in grouper.

mRNA expression patterns for GH, PRL, SL, IGF-I and IGF-II during altered feeding status in rabbitfish, Siganus guttatus.

Feeding time is a major synchronizer of many physiological rhythms in many organisms. Alteration in the nutritional status, specifically fasting, also affects the secretion rhythms of growth hormone (GH) and insulin-like growth factor-I (IGF-I). In this study, we investigated whether the expression patterns for the mRNAs of GH, prolactin (PRL) and somatolactin (SL) in the pituitary gland, and insulin-like growth factor I and II (IGF-I and IGF-II) in the liver of juvenile rabbitfish (Siganus guttatus) follow a rhythm according to feeding time and whether these hormone rhythms changes with starvation. Hormone mRNA levels were determined by real time PCR. The daily expression pattern for the mRNAs of GH, PRL and SL was not altered whether food was given in the morning (10:00 h) or in the afternoon (15:00 h). The daily GH mRNA expression pattern, however, was affected when food was not available for 3 days. In contrast, the daily expression pattern for IGF-I mRNA reaches its peak at roughly 5-6h after feeding. This pattern, however, was not observed with IGF-II mRNA. During 15-day starvation, GH mRNA levels in starved fish were significantly higher than the control fish starting on the 9th day of starvation until day 15. The levels returned to normal after re-feeding. In contrast to GH, PRL mRNA levels in starved fish were significantly lower than the control group starting on the 6th day of starvation until 3 days after re-feeding. SL mRNA levels were not significantly different between the control and starved group at anytime during the experiment. Both IGF-I and IGF-II mRNA levels in starved group were significantly higher than the control fish on the 3rd and 6th day of starvation. mRNA levels of both IGF-I and II in the starved fish decreased starting on the 9th day of starvation. While IGF-I mRNA levels in the starved group continued to decrease as starvation progressed, IGF-II mRNA levels were not significantly different from the control during the rest of the starvation period. The results indicate that aside from GH and IGF-I, PRL and IGF-II are likewise involved in starvation in rabbitfish.

Daily expression patterns for mRNAs of GH, PRL, SL, IGF-I and IGF-II in juvenile rabbitfish, Siganus guttatus, during 24-h light and dark cycles.

Most animals respond to changes in the external environment in a rhythmic fashion. In teleost fishes, daily rhythms are observed in plasma concentrations of some hormones but it is not clear whether these rhythms are exogenous or are entrained by predictable cues. We investigated whether the expression patterns for the mRNAs of growth hormone (GH), prolactin (PRL) and somatolactin (SL) in the pituitary gland, and insulin-like growth factor-I and II (IGF-I and IGF-II) in the liver, follow a daily rhythm when juvenile rabbitfish (Siganus guttatus) are reared under a normal 24-h light and dark cycle (LD), and when they are exposed to either continuous light (LL) or darkness (DD). Hormone mRNA levels were determined by real time PCR. Under LD conditions, GH mRNA expression in the pituitary was significantly lower during the light phase than during the dark phase suggesting a diurnal rhythm of expression. The rhythm disappeared when fish were exposed to LL or DD conditions. PRL mRNA expression pattern was irregular in all 3 conditions. Very low levels of SL mRNA were observed during the mid day under LD conditions. The expression pattern of SL mRNA became irregular under LL and DD conditions. No pattern could be observed in the expression profile of IGF-I and II mRNA in the liver during LD and LL conditions but a single peak in mRNA level was observed under DD conditions in both IGF-I and II. The results indicate that except for GH, the daily expression pattern for the mRNAs of the hormones examined do not seem to follow a rhythm according to light and dark cycles.

Changes in mRNA expression of grouper (Epinephelus coioides) growth hormone and insulin-like growth factor I in response to nutritional status.

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are key links to nutritional condition and growth regulation in teleost. To understand the endocrine mechanism of growth regulation in grouper, we cloned the cDNAs for grouper GH and IGF-I and examined their mRNA expression during different nutritional status. Grouper GH cDNA is 936 base pairs (bp) long excluding the poly-A tail. It contained untranslated regions of 85 and 231bp in the 5'- and 3'-ends, respectively. It has an open reading frame of 612bp coding for a signal peptide of 17 amino acids (aa) and a mature hormone of 187aa residues. Based on the aa sequence of the mature hormone, grouper GH shows higher sequence identity (>76%) to GHs of perciforms than to GHs of cyprinids and salmonids (53-69%). Grouper preproIGF-I cDNA consisted of 558bp, which codes for 186aa. This is composed of 44aa for the signal peptide, 68aa for the mature peptide comprising B, C, A, and D domains, and 74aa for the E domain. Mature grouper IGF-I shows very high sequence identity to IGF-I of teleost fishes (84-97%) compared to advanced groups of vertebrates such as chicken, pig, and human (80%). Using DNA primers specific for grouper GH and IGF-I, the changes in mRNA levels of pituitary GH and hepatic IGF-I in response to starvation and refeeding were examined by a semi-quantitative RT-PCR. Significant elevation of GH mRNA level was observed after 2 weeks of food deprivation, and increased further after 3 and 4 weeks of starvation. GH mRNA level in fed-controls did not change significantly during the same period. Hepatic IGF-I mRNA level decreased significantly starting after 1 week of starvation until the 4th week. There was no significant change in IGF-I mRNA levels in fed-controls. One week of refeeding can restore the GH and IGF-I mRNA back to its normal levels. Deprivation of food for 1-4 weeks also resulted in cessation of growth and decrease in condition factor.

Differential expression of insulin-like growth factor I and II mRNAs during embryogenesis and early larval development in rabbitfish, Siganus guttatus.

In rodents, the expression of insulin-like growth factor II (IGF-II) is higher than that of insulin-like growth factor I (IGF-I) during fetal life while the reverse is true after birth. We wanted to examine whether this is also true in fish and whether IGF-I and IGF-II are differentially regulated during different stages of embryogenesis and early larval development in rabbitfish. We first cloned the cDNAs of rabbitfish IGF-I and IGF-II from the liver. Rabbitfish IGF-I has an open reading frame of 558 bp that codes for a signal peptide of 44 amino acids (aa), a mature protein of 68 aa, and a single form of E domain of 74 aa. Rabbitfish IGF-II, on the other hand, has an open reading frame of 645 bp that codes for a signal peptide of 47 aa, a mature protein of 70 aa, and an E domain of 98 aa. On the amino acid level, rabbitfish IGF-I shares 68% similarity with IGF-II. We then examined the relative expression of the two IGFs in unfertilized eggs, during different stages of embryogenesis, and in early larval stages of rabbitfish by a semiquantitative reverse transcription-polymerase chain reaction. Primers that amplify the mature peptide region of both IGFs were used and PCR for both peptides was done simultaneously, with identical PCR conditions for both. The identity of the PCR products was confirmed by direct sequencing. Contrary to published reports for seabream and rainbow trout, IGF-I mRNA was not detected in rabbitfish unfertilized eggs; it was first expressed in larvae soon after hatching. IGF-II mRNA, however, was expressed in unfertilized eggs, albeit weakly, and was already strongly expressed during the cleavage stage. mRNAs for both peptides were strongly expressed in the larvae, although IGF-II mRNA expression was higher than IGF-I expression.