Radha Chaube

Intracellular levels of hydrogen peroxide and nitric oxide in oocytes at various stages of meiotic cell cycle and apoptosis

The objective was to find out the functional roles of hydrogen peroxide (H2O2) and nitric oxide (NO) during various stages of meiotic cell cycle and apoptosis in rat oocytes. For this purpose, 30 oocytes from each stage such as diplotene, metaphase-I (M-I), metaphase-II (M-II) and apoptosis were collected and intracellular H2O2, total nitrite level and inducible nitric oxide synthase (iNOS) expression were analysed. This study demonstrated that generation of a tonic level of H2O2 induces meiotic resumption in diplotene-arrested oocytes and further increase may lead to apoptosis. Conversely, reduction in iNOS expression and total nitrite level are associated with meiotic resumption in diplotene-arrested oocytes, but induce apoptosis in aged oocytes. These results suggest that generation of a tonic level of H2O2, reduced iNOS expression and total nitrite level are associated with meiotic resumption, while more generation of H2O2 and sustained reduced total nitrite level are linked with oocyte apoptosis in rat.

Sex differences in aromatase gene expression in the medaka brain.

The brain of teleost fish exhibits a significant degree of sexual plasticity, even in adulthood. This unique feature is almost certainly attributable to a teleost‐specific sexual differentiation process of the brain, which remains largely unknown. To dissect the molecular basis of sexual differentiation of the teleost brain, we searched for genes differentially expressed between both sexes in the medaka brain. One gene identified in the screen, cyp19a1b, which encodes the steroidogenic enzyme aromatase, was selected for further analysis. As opposed to the situation in most vertebrates, medaka cyp19a1b is expressed at higher levels in the adult female brain than the male brain. The female‐biased expression in the brain is consistent regardless of reproductive or diurnal cycle. Medaka cyp19a1b is expressed throughout the ventricular zones in wide areas of the brain, where, in most regions, females have a greater degree of expression compared to males, with the optic tectum exhibiting the most conspicuous predominance in females. Contrary to what is known in mammals, cyp19a1b expression exhibits neither a transient elevation nor a sex difference in medaka embryos. It is not until just before the onset of puberty that cyp19a1b expression in the medaka brain is sexually differentiated. Finally, cyp19a1b expression in the medaka brain is not under the direct control of sex chromosome genes but relies mostly, if not solely, on oestrogen derived from the gonad. These unique properties of aromatase expression in the brain probably contribute substantially to the less rigid sexual differentiation process, thus ensuring remarkable sexual plasticity in the teleost brain.

Brain tyrosine hydroxylase in the catfish Heteropneustes fossilis: annual and circadian variations, and sex and regional differences in enzyme activity and some kinetic properties.

Dynamics of tyrosine hydroxylase (TH) was studied in various brain regions and pituitary in relation to annual/seasonal and circadian variations in either sex of the catfish Heteropneustes fossilis. alpha-Methylparatyrosine inhibited TH activity significantly in a time-(in vivo study) and concentration-(in vitro study) dependent manner. The inhibition was higher in resting phase than preparatory phase and in females than males (time-course study). The apparent kinetic constant of inhibition (K(i)) varied both seasonally and regionally. The values were higher in resting phase and in the forebrain regions (telencephalon and hypothalamus) than medulla oblongata. No significant sex difference was noticed in the K(i) values in the same season. TH activity showed significant annual/seasonal variations with telencephalon and hypothalamus showing higher activity than medulla oblongata. The females showed significantly higher enzyme activity than males with a distinct activity peak in June (prespawning phase). Such an activity peak was apparently absent in males. TH activity showed significant circadian/diurnal variations, the highest activity was noticed at 12 h and the lowest at 24 h. The apparent Michaelis-Menten constant (K(m)) values (hypothalamic TH) for substrate (L-tyrosine) and cofactor (DMPH(4)) showed seasonal variations with the highest values in the resting phase, decreasing through preparatory and prespawning phases, to the lowest values in spawning phase. V(max) was the lowest in the resting phase and highest in the spawning phase. TH activity was low in the pituitary and could be detected in pooled samples from March (preparatory phase) to July (spawning phase). Activity showed significant variations, which could be correlated with the gonadosomatic index. The results show that TH activity could be positively correlated with the annual reproductive cycle. The enzyme activity could be also correlated with seasonal, sex, and regional variations in the apparent K(m) and V(max) values suggesting apparent differences in the affinity of the enzyme towards substrate and cofactor.

In vitro effects of lead nitrate on steroid profiles in the post-vitellogenic ovary of the catfish Heteropneustes fossilis.

Heavy metals are endocrine disruptors with the ability to cause hormonal imbalances, affecting various physiological processes such as reproduction. In this study, in vitro effects of exposure (12 or 24h) of lead nitrate [Pb(NO(3))(2)] (0, 0.01, 0.1, 1, 3 and 10 μg/ml) on steroid levels in post-vitellogenic catfish (Heteropneustes fossilis) ovary was investigated. Steroids were assayed by HPLC/ELISA. Lead (Pb) elicited biphasic effects on estradiol-17β, testosterone and cortisol: stimulatory at lower concentrations and inhibitory at higher concentrations. In contrast, progesterone, 17-hydroxyprogesterone, 17,20β-dihydroxyprogesterone, corticosterone, 21-deoxycortisol and deoxycorticosterone were inhibited in a dose-dependent manner. Thus, the present results suggest that short-term Pb response can be a potent endocrine disruptor of normal follicular steroidogenesis. The stimulatory effect on E(2) suggests that Pb in trace amounts may be beneficial. The cortisol elevation may be indicative of the metal/stress insult. Nevertheless, further studies are required to understand the mechanism of action of lead toxicity.

Vasotocin--A new player in the control of oocyte maturation and ovulation in fish.

In this article, the physiological role of ovarian vasotocin (VT) on fish final oocyte maturation (FOM) and ovulation is reviewed based on the studies mainly available in the catfish Heteropneustes fossilis. The VT system is characterized in the follicular layer of the oocytes by both immunocytochemical and in situ hybridization techniques. The distribution was confirmed in isolated follicular layer preparations by HPLC characterization and quantification. Three VT receptor subtype genes are identified: V1a1 and V1a2 subtypes are distributed in the follicular layer and V2 subtype is present along the granulosa-oocyte membrane junction. The expression of peptide, VT precursor gene and VT receptor genes shows seasonal and periovulatory changes in the ovary. VT secretion is modulated by E2 differentially in a season-specific manner, and by progestin steroids positively. VT modulates E2 in a biphasic manner in early recrudescent phase and induces a steroidogenic shift inhibiting E2 and stimulating progestin steroid (P4, 17P4 and 17,20β-DP) pathways in the late recrudescent phase. VT stimulates prostaglandin secretion, germinal vesicle breakdown (GVBD), oocyte hydration and ovulation. VT acts through different receptors to stimulate these processes. It uses the V1 type receptor to stimulate GVBD and ovulation, and the V2 type to stimulate oocyte hydration. VT acts as an important link in the cascade of gonadotropin control of FOM and ovulation. More research is required in other species.

An insight into the molecular basis for convergent evolution in fish antifreeze Proteins

Antifreeze proteins (AFPs) prevent the growth of ice-crystals in order to enable certain organisms to survive under sub-zero temperature surroundings. These AFPs have evolved from different types of proteins without having any significant structural and sequence similarities among them. However, all the AFPs perform the same function of anti-freeze activity and are a classical example of convergent evolution. We have analyzed fish AFPs at the sequence level, the residue level and the physicochemical property group composition to discover molecular basis for this convergent evolution. Our study on amino acid distribution does not reveal any distinctive feature among AFPs, but comparative study of the AFPs with their close non-AFP homologs based on the physicochemical property group residues revealed some useful information. In particular (a) there is a similar pattern of avoidance and preference of amino acids in Fish AFP subtypes II, III and IV-Aromatic residues are avoided whereas small residues are preferred, (b) like other psychrophilic proteins, AFPs have a similar pattern of preference/avoidance for most of the residues except for Ile, Leu and Arg, and (c) most of the computed amino acids in preferred list are the key functional residues as obtained in previous predicted model of Doxey et al. For the first time this study revealed common patterns of avoidance/preference in fish AFP subtypes II, III and IV. These avoidance/preference lists can further facilitate the identification of key functional residues and can shed more light into the mechanism of antifreeze function.

Temporal and periovulatory changes in ovarian catecholamines in the catfish Heteropneustes fossilis

A high performance liquid chromatography-fluorescence detection method was employed to demonstrate temporal and periovulatory changes in ovarian catecholamines in the catfish Heteropneustes fossilis. Tyrosine, L-DOPA, dopamine (DA), norepinephrine (NE) and epinephrine showed significant seasonal and diurnal changes during the reproductive cycle. A high concentration of tyrosine was detected, the values increased in day and decreased in night during recrudescence from preparatory to spawning phases. Similarly, L-DOPA increased during the breeding phase giving the day value concentration peak in the spawning phase and the night value peak in the postspawning phase. DA activity or turnover index (calculated as a ratio of DA to 3,4-dihydroxyphenylacetic acid, DOPAC) showed a bimodal pattern with the major activity peak in the postspawning phase and the minor one in the prespawning phase, the spawning phase registered the lowest activity. NE activity or turnover index (ratio of NE to normetanephrine, NME) increased during the recrudescent phase to give the peak in the spawning phase and decreased in the quiescent phase. Epinephrine elicited an inverse relationship in the day-night pattern, the day values increased to the peak in the spawning phase. All the study correlates showed significant periovulatory changes after hCG treatment. DA activity dropped to the nadir at 8h but recovered at 16 and 24h. NE activity showed only a marginal decrease up to 16h but decreased drastically at 24h. Epinephrine levels remained unchanged but only to increase at 24h. The seasonal patterns and periovulatory changes strongly point to important functions for catecholamines in the ovary. The occurrence of tyramine (an invertebrate neurotransmitter) suggests the presence of alternate catecholamine pathway in fish ovary.

Molecular cloning and characterization of brain and ovarian cytochrome P450 aromatase genes in the catfish Heteropneustes fossilis: Sex, tissue and seasonal variation in, and effects of gonadotropin on gene expression.

Cytochrome P450 aromatase (Cyp19arom) is the rate-limiting enzyme controlling estrogen biosynthesis, coded by Cyp19a1 in most gnathostomes. Most teleosts have two forms expressed differentially in ovary (cyp19a1a) and neural tissue (cyp19a1b). In this study, full length cDNAs of 2006 bp and 1913 bp with ORFs of 1575 bp and 1488 bp were isolated from the brain and ovary, respectively, of the catfish Heteropneustes fossilis, an air-breathing species with high aquaculture potential. The ORFs encode predicted proteins of 495 and 524 amino acid residues, respectively. The proteins show 62% identity with each other and cluster in two distinct clades (the brain type and ovary type) in the teleost taxon, separated from the tetrapod type. In the in situ localization study, both cyp19a1a and cyp19a1b transcripts were localized in the brain but the signal intensity was higher for the brain type paralog. The transcript signals were observed in the radial glial cells and in neuronal populations of the dorso-lateral region of the telencephalon, pre-tectum, hypothalamus and medulla oblongata. In the ovary, both paralogs were expressed in the follicular layer with a high signal intensity of the ovarian type (cyp19a1a). The differential expression of the gene paralogs was evident from qPCR analysis. Cyp19a1b has relatively a high abundance in the female brain, followed by other peripheral tissues (gonads, liver, gill, kidney and muscle). On the other hand, cyp19a1a has relatively a high transcript abundance in the ovary and female brain, followed by the testis and male brain, and female liver and muscle. The expression was low in male liver and muscle, and the lowest in the gill and kidney. The expression of the two paralogs exhibit brain regional differences; both types have relatively a high transcript abundance in telencephalon-preoptic area with the cyp19a1b expression higher in females than males. In hypothalamus, the expression of both types is higher in males than females. In medulla, the expression of the cyp19a1b is higher than cyp19a1a, and the transcript abundance of the ovarian type is higher in females than males. The expression of the gene paralogs elicits significant seasonal variations in the ovary and brain. In both tissues, the expression increases from the resting to preparatory phases, and decreases through the prespawning phase to low levels in spawning phase. In vivo and/or in vitro treatments with human chorionic gonadotropin (hCG) stimulated the expression of the gene paralogs in the brain and ovary, time-dependently. In conclusion, both paralogs have an overlapping distribution at different levels of the brain-pituitary-gonad axis and may function as a single functional unit as far as the estrogen synthesis is concerned.

Effects of ovaprim, a commercial spawning inducer, on vasotocin and steroid hormone profiles in the catfish Heteropneustes fossilis: in vivo and in vitro studies.

Ovaprim (OVP) is used as an effective spawning inducer for artificial breeding of fishes and contains a salmon gonadotropin-releasing hormone analogue and a dopamine receptor-2 antagonist, domperidone. Previously, we have shown that vasotocin (VT) stimulates ovarian final oocyte maturation, hydration, and ovulation through a mechanism involving induction of a steroidogenic shift, favouring the production of a maturation-inducing hormone (MIH). In the present study, we demonstrated that OVP stimulated brain, plasma and ovarian VT levels, suggesting multiple sites of action, apart from its well established role in the induction of a preovulatory LH surge. An intraperitoneal injection of 0.5μL/g body weight of OVP for different time intervals (0, 4, 8, 12, 16 and 24h) induced ovulation as well as increased significantly brain and plasma VT levels in a time-dependent manner. Plasma steroids were differentially altered; the levels of estradiol-17β (E2) and testosterone (T) decreased, and the MIH (17, 20β-dihydroxy-4-pregnen-3-one; 17, 20β-DP) level increased time-dependently. In order to demonstrate whether OVP acts at the level of the ovary directly, in vitro experiments were conducted. The incubation of ovarian slices/follicles with OVP (1, 5 and 10μL/mL) for different time points (0, 4, 8, 12, 16 and 24h) induced germinal vesicle breakdown (GVBD) in a concentration- and time-dependent manner. Ovarian VT increased significantly in a concentration- and time-dependent manner with a maximal increment at 16h. Ovarian T and E2 levels decreased concurrently with the rise in the MIH level, dose- and duration-dependently. The results show that OVP stimulates VT at the brain and ovarian level. The direct OVP-VT cascade has the potential to stimulate FOM and ovulation, sidelining the pituitary glycoprotein hormone (LH) surge.

Structural and functional diversity of nonapeptide hormones from an evolutionary perspective: A review

The article presents an overview of the comparative distribution, structure and functions of the nonapeptide hormones in chordates and non chordates. The review begins with a historical preview of the advent of the concept of neurosecretion and birth of neuroendocrine science, pioneered by the works of E. Scharrer and W. Bargmann. The sections which follow discuss different vertebrate nonapeptides, their distribution, comparison, precursor gene structures and processing, highlighting the major differences in these aspects amidst the conserved features across vertebrates. The vast literature on the anatomical characteristics of the nonapeptide secreting nuclei in the brain and their projections was briefly reviewed in a comparative framework. Recent knowledge on the nonapeptide hormone receptors and their intracellular signaling pathways is discussed and few grey areas which require deeper studies are identified. The sections on the functions and regulation of nonapeptides summarize the huge and ever increasing literature that is available in these areas. The nonapeptides emerge as key homeostatic molecules with complex regulation and several synergistic partners. Lastly, an update of the nonapeptides in non chordates with respect to distribution, site of synthesis, functions and receptors, dealt separately for each phylum, is presented. The non chordate nonapeptides share many similarities with their counterparts in vertebrates, pointing the system to have an ancient origin and to be an important substrate for changes during adaptive evolution. The article concludes projecting the nonapeptides as one of the very first common molecules of the primitive nervous and endocrine systems, which have been retained to maintain homeostatic functions in metazoans; some of which are conserved across the animal kingdom and some are specialized in a group/lineage-specific manner.