Citation
Mohamed Zulperi, Zarirah
(2016)
Molecular characterization and recombinant gonadotropin subunit development for improving reproductive performance in female Hemibagrus nemurus valenciennes.
Doctoral thesis, Universiti Putra Malaysia.
Abstract
Hemibagrus nemurus, locally known as ‘ikan baung’, is a valuable cultured
catfish in Malaysia, but the production is limited due to problems associated
with its reproduction. Most hatcheries encountered problems in induced
spawning due to the fact that the fish do not expose to their natural spawning
habitat, especially the failure of female brood stock to undergo final oocyte
maturation (FOM), preventing its ability to ovulate and spawn. Gonadotropin
(GtH), comprised of follicle-stimulating hormone (FSH) and luteinizing
hormone (LH), play an important role in gametogenesis and sexual
maturation in mammals as well as in fish. This research was conducted to
study the molecular characterization and to monitor changes in the mRNA
expression of three GtH subunits, , FSH, and LH, during the reproductive
cycle of the female H. nemurus. Next, a single-chain recombinant LH (rLH) of
H. nemurus was expressed using E. coli expression system. The bioactivity
of the recombinant hormone was assessed for their potency in stimulating
the production of 17-estradiol (E2), ovarian development and mRNA
expression level of GtH subunits in experimental fish. Molecular
characterization using PCR amplifications revealed that the cDNA of ,
FSH and LH in H. nemurus were 656, 728 and 602 bp, and encoded for
116, 134 and 140 amino acids, respectively. Multiple sequence alignment
showed all the three subunits shared the highest homology with Siluriformes
and Anguilliformes, and least similar to higher vertebrates. Phylogenetic
analysis of and FSH grouped fishes and higher vertebrates together,
while in LH, the tree was divided in two distinct groups, the first group
contained all fishes together, and the second group only contained higher
vertebrates. The abundance of transcripts encoding the GtH subunits, ,
FSH and LH subunits in pituitaries of female H. nemurus was tracked
throughout the reproductive cycle using quantitative real-time PCR. Data
were collected from August 2013 until October 2015, with fish aged between
1 to 27 month-old. The mRNA expression of GtH subunits increased together with ovarian development throughout the reproductive cycle. The FSH
mRNA level increased during ovarian development and reached its peak in
mid-vitellogenesis and reduced during ovulation and spawning, whereas the
LH mRNA level significantly increased (P<0.05) in late vitellogenesis and
reached its peak during ovulation and spawning period. In subunit, the
mRNA level corresponded to FSH and LH, which showed its highest
expression during ovulation. This suggest that FSH could be involved in
vitellogenesis, while LH subunit raised at the end of reproductive cycle,
determined the involvement of LH in ovulation and final oocyte maturation. A
single chain recombinant LH (rLH) from H. nemurus consisted of LHβα
fusion proteins was constructed and derived through gene-synthesis. The
rLH construct was successfully cloned into pET-32 Ek/LIC and expressed in
E. coli BL21 (DE3) cells. A specific band at 45.4 kDa was detected which
corresponded to the molecular size of fusion rLH resolved by SDS PAGE.
Two monoclonal antibodies, His-Tag and S-tag, were used to confirm the
same expressed bands in Western immunoblotting. In order to investigate
the bioactivity of the rLH hormone, fish trial was performed to assess the
effect of exogenous rLH on the plasma level of E2, their mRNA level of GtH
subunits and oocyte development in the gonad of female H. nemurus.
Immature female H. nemurus were divided into four different treatments of
ten fish/group, and each group received a single injection of: Group 1= 1X
PBS (sham control), Group 2= 0.5 ml/kg Ovaprim (positive control), Group
3= rLH 50 g/kg, and Group 4= rLH 150 g/kg, respectively. Study
parameters including the oocyte development through observation on gonad
histology, the E2 plasma levels using enzyme-linked immunosorbent assay
(ELISA) and the mRNA transcript level of , FSH and LH subunits using
real-time PCR. Based on the histological observation of the gonad, fish
received treatment with rLH 50 g/kg, rLH 150 g/kg, as well as Ovaprim
were able to induce the maturation of oocyte after 48 hr p.i. The E2 plasma
level in fish treated with rLH 50 g/kg raised after 6 hr p.i., significantly
increased (p<0.05) after 12 hr p.i., and the level was sustained up to 24 hr
p.i. The mRNA expression levels of , FSH and LH also showed
significant increment (p<0.05) in fish treated with rLH 50 g/kg after 48 hr p.i.
Fish treated with Ovaprim showed significant increased (p<0.05) of E2
plasma level after 6 hr p.i., but the level was reduced after 12 hr p.i. There
was no significant increased (p>0.05) of mRNA subunits in fish treated with
Ovaprim after 48 hr p.i., as compared to sham control fish. Meanwhile,
treatment with rLH 150 g/kg showed a weak stimulatory effect on the E2
production and mRNA transcript level of GtH subunits. The results revealed
that treatment with rLH at 50 g/kg is the best dosage to induce spawning
and oocyte maturation in maturing female H. nemurus. These findings
enhanced the understanding of GtH subunit in H. nemurus, and the temporal
profile helped to estimate the correct time for hormonal therapies to induce
ovulation and spawning in female H. nemurus reared in captivity. The
production of recombinant GtH from H. nemurus would offer the
establishment of a potentially efficient method to induce the gonadal
development in this species and possibly in other catfish species.
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