Karen Jackson

Growth differences and growth hormone expression in male and female European eels [Anguilla anguilla (L.)].

In this study, we examined the growth differences of males and females following a sex reversion, and the growth hormone (GH) expression variation between sexes of European eels [Anguilla anguilla (L.)]. A high percentage of females (88%) was found in the group fed with estradiol 17beta compared to the control group (comprised of only 6% female eels), which was defined as the male population. Significant differences between growth rate and size were found following 480 days of growth, whereby the males reached 60+/-4.3 g (means+/-SE) in size and the females 73.4+/-5.9 (g+/-SE); after 600 days, the males reached 114.1+/-4.3 and the females 171+/-11.7 (g+/-SE). A cDNA coding for the complete growth hormone of the European eel A. anguilla (eeGH) was cloned by RACE PCR using several sets of degenerate oligonucleotides. The eeGH cDNA coding region is 627 bp long. A sequence comparison of eeGH with Anguilla japonica GH (jeGH) cDNA showed a 98% identical base. Comparison of the deduced amino acid sequence revealed 99% identical residues, meaning that a difference exists in only two of the 209 residues. In both cases, the differing residues in the eeGH amino acid sequence are lysine. We measured the mRNA levels of growth hormone in the pituitaries of male and female eels growing at different rates. A significantly higher expression of eeGH was found in the female eels in comparison to the males. These results show that different levels of GH transcription eeGH can explain the growth rate differences between male and female European eels.

βFSH, βLH and growth hormone gene expression in blue gourami (Trichogaster trichopterus, Pallas 1770) during spermatogenesis and male sexual behavior

Abstract The relationship between gonadal development (histological evidence for spermiogenesis and/or spermatogenesis), sexual behavior (nest-building) and mRNA levels of gonadotropins (βFSH and βLH) and growth hormone (GH) in the male pituitary was investigated. Amplification of βFSH cDNA showed a significantly higher mRNA level in mature males (whether sexually active or not) than in juveniles. However, following PCR amplification of βLH cDNA, a significantly higher mRNA level was found in the sexually active group compared to the sexually inactive group. These results suggest that FSH may participate in spermatogenesis, whereas LH is more involved in spermiogenesis. The GH mRNA level increased slightly during the maturation process but no significant differences were found between the groups studied.

Growth hormone, sexual maturity and steroids in male carp (Cyprinus carpio)

Samples of pituitary, blood plasma and gonad were taken from male carp. The growth hormones (GH) in the pituitary and plasma were measured in fish of various body weights (BW) and degrees of gonad development, and compared with the levels of 17 beta-estradiol (E2), testosterone (T), 17 alpha-hydroprogesterone (17-P), 11-ketotesterone (11-KT) and progesterone (P) in the testes and plasma. The gonadosomatic index increased rapidly with BW from 100 to 600 g, and then decreased at 900 g. The pituitary GH did not change with BW, but plasma GH was higher in fish weighing 300 +/- 50 and 600 +/- 50 g, than in fish weighing 900 +/- 50 g. In fish weighing 150 +/- 50 to 300 +/- 50 g, the level of T rose significantly in the testes (2.27 ng g-1) and plasma (1.3 ng g-1); E2 was very low in both testes (0-30 pg g-1) and plasma (11-28 pg ml-1), increasing as BW rose from 150 to 600 g. The level of P rose mainly at BW of 300 +/- 50 and 600 +/- 50 g: from 0 to 25 ng g-1 in the testes and from 0 to 17 ng ml-1 in the plasma. The level 17-P rose from 2.5 to 20 ng g-1 in the testes at 600 +/- 50 g BW, but no significant change was recorded in the plasma. The level of 11-KT rose significantly in the tests of fish at 300, 600 and 900 g (0.5-6 ng g-1). The application of different steroids (E2, T and 17-P) on a primary culture of pituitary cells led to the release of GH. Release was significantly higher (P < 0.05) after 4 h at steroid concentrations of 10(-6) M.

cDNA cloning of blue gourami (Trichogaster trichopterus) prolactin and its expression during the gonadal cycles of males and females

Background: The blue gourami fish (Trichogaster trichopterus) provides a unique model for the study of reproduction endocrinology in teleost fish. Its oocyte development may be controlled easily, and the vitellogenic and final maturation phases may be separated artificially in the laboratory. Moreover, this gourami exhibits exclusive parental behavior. Aim: The aim of the present study was to clone and sequence the blue gourami PRL (bgPRL) cDNA in order to enable the determination of its mRNA levels in the male and female blue gourami during the gonadal cycles. Materials and methods: bgPRL was cloned by extracting total RNA from freshly excised pituitaries of gourami fish, followed by cDNA synthesis, rapid amplification of cDNA ends (RACE)-PCR and finally, sequencing. bgPRL mRNA expression was determined by realtime PCR, and results were normalized with 18S RNA. Results: When bgPRL was compared to PRLs of other fish, it had the most homology with PRL of Perciformes and the least with those of Anguilliformes. bgPRL was expressed during the entire gonadal cycle in males and females. The average levels of PRL mRNA in juvenile and low vitellogenetic females were lower than in mature females (at high vitellogenesis and maturation), but the differences were not significant. On the other hand, the PRL mRNA levels in mature reproductive males (nest-builders) and non-reproductive (non-nest-builders) were significantly higher in comparison to young males. Conclusions: The results of this study imply that PRL has a possible role in the endocrine control of gonadal development in fish, in addition to its role in reproductive behavior.

Cloning of European eel (Anguilla anguilla) FSH-β subunit, and expression of FSH-β and LH-β in males and females after sex determination

The European eel (Anguilla anguilla) is a catadromic teleost species with a complex life cycle, both in sea and freshwater environments. The sex determination phase of gonadal development occurs in a freshwater environment. Polymorphism occurs in increasing rates with respect to gender. While males stop growing at approximately 150 g, females continue to grow to being much larger. In this study, we cloned the cDNA FSH-β subunit of the European eel (A. anguilla), and measured the mRNA levels of FSH-β and LH-β in males and females after sex determination. The FSH-β subunit cDNA consisted of 1068 bp, encoding a 127 amino acid peptide. A comparison between European and Japanese eels of the FSH-β amino acid sequence showed 98% similarity.

Association between pituitary adenylate cyclase-activating polypeptide and reproduction in the blue gourami

In order to gain a better understanding of the roles of pituitary adenylate cyclase-activating polypeptide (PACAP) in reproduction and growth, the expression of the PACAP gene during the reproduction cycle and its potential role in regulating gonadotropin and growth hormone (GH) gene transcription in blue gourami were investigated. The cDNA sequences of the full-length blue gourami brain PACAP and that of its related peptide (PRP) were acquired. PACAP cDNA had two variants, obtainable by alternative splicing: a long form encoding for both PRP and PACAP and a short form encoding only for PACAP. In females, mRNA levels of PACAP were very high only in individuals with oocytes in the maturation stage, as compared to levels in unpaired vitellogenic and non-vitellogenic fish. The PACAP mRNA levels in males were high only in nest builders, as opposed to in non-nest building males and juveniles. In pituitary culture cells from high vitellogenic females, PACAP38 (the 38 amino acid form) only brought about an increase in betaFSH levels, without altering GH and betaLH mRNA levels. On the other hand, in adult non-reproductive male pituitary cells, PACAP38 decreased the GH mRNA level. Based on these results, we propose that in the blue gourami, PACAP is involved in the final oocyte maturation stage in females, whereas in males, it is associated with sexual behavior. In addition, the effect of PACAP38 on pituitary hormone gene expression is different in females and males, indicating that PACAP38 is potentially a hypophysiotropic regulator of reproduction, which mediates pituitary hormone expression.

GROWTH HORMONE, GONAD DEVELOPMENT, AND STEROID LEVELS IN FEMALE CARP

The pituitary and plasma growth hormone (GH) levels of female carp (Cyprinus carpio L.) were measured in fish of various sizes and degrees of maturity, and were matched against the levels of 17 beta-estradiol (E2), testosterone (T), 17 alpha-hydroxyprogesterone (17-P), and progesterone (P) in the ovary and plasma. The short-term action of the above hormones and 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20-P) on the release of GH was examined in vitro in primary culture pituitary cells. The gonadosomatic index (%GSI) increased rapidly in specimens when they had attained 900 +/- 50 g body weight (BW). The pituitary and plasma GH levels increased between 150 and 600 g BW (when oocytes reached the stage at which lipoprotein appeared in the cytoplasm), but at 900 g BW (with oocytes in vitellogenesis) the plasma GH dropped, while pituitary GH remained high. E2 increased with BW, reaching its maximum at 600 and 900 g BW in the ovary and plasma, respectively. Similar patterns were found in the levels of T and P, both hormones reaching their maximum levels at 900 g BW. The level of 17-P was very low and did not increase in proportion to BW. The application of various concentrations of different steroids on a primary culture of pituitary cells led to release of GH. The highest degrees of release were obtained from 10(-6) and 10(-7) M E2, 10(-6) M T, 10(-7) M 17-P and 10(-8) M 17,20-P. In all these cases, hormone treatment effected higher release of GH than was found in the control. A model of the relationship between GH and the steroids associated with maturation is proposed.

Cloning of Russian sturgeon ( Acipenser gueldenstaedtii ) growth hormone and insulin-like growth factor I and their expression in male and female fish during the first period of growth

In this study, the GH and IGF-I of the Russian sturgeon (rs), Acipenser gueldenstaedtii, were cloned and sequenced, and their mRNA gene expression determined. In addition, to improve our understanding of the GH function, the expression of this hormone was assessed in young males and females. Moreover, IGF-I expression was quantified in young males and compared to that in older ones. The nucleotide sequence of the rsGH cDNA was 980 bp long and had an open reading frame of 642 bp, beginning with the first ATG codon at position 39 and ending with the stop codon at position 683. A putative polyadenylation signal, AATAAA, was recognized 42 bp upstream of the poly (A) tail. The position of the signal-peptide cleavage site was predicted to be at position 111, yielding a signal peptide of 24 amino-acids (aa) and a mature peptide of 190 aa. When the rsGH aa sequence was compared with other species, the highest degree of identity was found to be with mammalians (66–70% identity), followed by anguilliformes and amphibia (61%) and other fish (39–47%). The level of rsGH mRNA was discovered to be similar in pituitaries of females and males of 5 age groups (1, 2, 3,4, and 5-yr-old). In females and males, the levels did not change dramatically during the first 5 yr of growth. The partial nucleotide sequence of the rsIGF-I was 445 bp long and had an open reading frame of 396 bp, beginning with the ATG codon at position 50. The position of the signal-peptide cleavage site was predicted to be at position 187, yielding a signal peptide of 44 aa. The highest level of IGF-I mRNA expression was recorded in the kidney of adult sturgeons. The IGF-I mRNA expression levels in the intestine, pituitary gland, and liver were not significantly different. Low levels of expression were found in the brain, heart, and muscle. In most tissues, there was no significant difference between mRNA levels of one and 5-yr-old fish. In conclusion, based on the GH-sequence analysis, A. gueldenstaedtii is genetically distant from other teleosts. The expression of the GH mRNA was similar in males and females, and its level remained constant during the first 5 yr of growth. While the IGF-I mRNA expression differed amongst various tissues, the level in each tissue was similar in 1 and 5-yr-old fish.

Expression of the two cytochrome P450 aromatase genes in the male and female blue gourami (Trichogaster trichopterus) during the reproductive cycle.

In this study, the involvement of the cytochrome P450 aromatase gene (CYP19) in the gametogenesis of the teleost blue gourami (Trichogaster trichopterus) is described. The blue gourami brain CYP19 (bgCYP19b) and gonadal CYP19 (bgCYP19a) aromatase genes were cloned and their expression analyzed during the different reproductive stages. The cloned cDNAs of the bgCYP19b and bgCYP19a were found to contain segments of 1518 bp (an open reading frame encoding a deduced protein of 506 residues) and 489 bp (encoding a peptide of 163 residues), respectively. Although the mRNA levels of bgCYP19b were very low in females until the vitellogenic phase, they were significantly higher in the final oocyte maturation stage. The aromatase gene mRNA levels in the gonads were significantly lower in females in the high vitellogenic stage, as compared to females during early vitellogenesis or maturation. In males, the mRNA levels of bgCYP19b were significantly lower in juveniles than in mature individuals. However, no significant differences were observed between mature non-reproductive and reproductive males. In addition, there was no significant difference between the expression of bgCYP19a in juvenile and non-nest building mature males, although a significant increase was detected in mature reproductive males. Although CYP19b expression was similar in both sexes, the expression of CYP19a was significantly different between males and females.

Antimicrobial susceptibility of Clostridium difficile isolates in Israel

OBJECTIVES An increase of Clostridium difficile isolates with reduced susceptibility to various antimicrobial agents has been observed, including isolates that are non-susceptible to antibiotics that are routinely used for treatment of C. difficile, such as vancomycin and metronidazole. We determined the susceptibility rates of C. difficile isolates from hospitals in northern Israel to various antibiotics including tigecycline, which was not previously reported from Israel. METHODS A total of 81 stool samples were collected from three hospitals in northern Israel from patients with C. difficile infection. Specimens were screened for BI/NAP1/027 ribotype, cultured, and sensitivity tests were performed for vancomycin, metronidazole, moxifloxacin, and tigecycline. Statistical tests were applied for analysing the differences in distribution of resistance between the different antibiotics and between BI/NAP1/027 and resistance of antibiotics. RESULTS Reduced susceptibility was found among 6/81 isolates for vancomycin, 4/81 for metronidazole, and 17/81 for moxifloxacin. Only 1 isolate had reduced susceptibility to tigecycline, with a mean MIC of 0.05μg/mL. Reduced susceptibility to moxifloxacin was significantly associated with reduced susceptibility to vancomycin (p=0.016) and to metronidazole (p=0.0276), and reduced susceptibility to metronidazole was associated with reduced susceptibility to vancomycin (p=0.0259). Eight of 81 isolates (9.9%) were positive for BI/NAP1/027 ribotype and had significantly higher non-susceptibility rates to moxifloxacin and vancomycin compared with BI/NAP1/027 negative isolates (p<0.0001 and p=0.0113, respectively). CONCLUSIONS We found higher non-susceptibility rates to vancomycin and metronidazole than most previous studies, while tigecycline resistance rates are very low in northern Israel, rendering it a potential agent for treating CDI.