Centre for Nanosciences
Amrita Institute of Medical Sciences,
Amrita Vishwa Vidyapeetham
AIMS Ponekkara P. O., Kochi, Kerala - 682 041, India.

0484 285 8750


Dr. Bindhu Paul joined Amrita Centre for Nanosciences and Molecular Medicine as Assistant Professor in January, 2011 and currently heads the laboratory of reproductive and developmental biology. She took M.Sc. in Zoology with specialization in Molecular Genetics and Cytogenetics from Banaras Hindu University, India, and Ph.D. in Population Genetics from Cochin University of Science and Technology, India. She pursued post-doctoral research in Japan at the National Institute for Basic Biology (National Institute of Natural Sciences). She spent 8 years in Japan doing research in the area of reproductive physiology/endocrinology/developmental biology. She has over 18 international publications to her credit. Her research focused on vertebrate sex determination and differentiation and how exposure to endocrine disruptors/synthetic hormones during early development influenced gonadal morphogenesis in Japanese medaka, which is widely used as a model organism for biomedical research. At Amrita, Dr. Bindhu Paul is investigating the interactions between engineered nanoparticles and germ cells, which are responsible for the transmission of genetic material between generations. Other major thrust areas of her research includes transgenerational toxicity potential of engineered nanoparticles, developmental origin of adulthood diseases and germline stem cell biology. A well-established fish facility holding natural as well as transgenic and mutant strains of Japanese Medaka is being maintained at Amrita by Dr. Bindhu’s team.

Role of Engineered Nanoparticles on Reproductive and Developmental Toxicity: A Multi-generational Approach

The project intends to critically analyze the potential of engineered nanoparticles, both organic (Graphene oxide) and inorganic (Zinc oxide), to induce anomalies in the reproduction and development of Japanese medaka as well as their ability to exert damaging effects on the germ line through mitotically stable epigenetic modifications that could result in transgenerational toxicities.

Development of Pluripotent Stem Cells from Human Ovarian Germ Line Stem Cells and Their Differentiation into Adipocytes

The centre of interest of this project is to isolate ovarian germ line stem cells (OGSCs) and examine whether these cells could be induced to generate pluripotent stem cells, which in turn could be differentiated into other lineages using biomimetic scaffolds such as hydrogels (3D environment) for regenerative engineering applications.

Development of New Assay for the Evaluation of Pesticides used in India for Endocrine Disruption using Japanese Medaka (Orzyias Latipes)

The project focuses on developing an assay for the detection of different endocrine disruptors which are constantly getting introduced into our premises. Specifically, we aim to develop an assay that can confirm the endocrine disrupting potential of a substance within a short span of time using Japanese medaka embryos and adults as the model organisms.

Vinod Paul

Vinod, an INSPIRE fellow is doing research on reproductive toxicity of zinc oxide nanoparticles. His work focuses on biodistribution, biological consequences and mechanisms of zinc oxide exposure to males and females.

Sreelakshmi Krishnakumar

Sreelakshmi, a Women Scientist Scheme-A fellow is working on transgenerational toxicity of organic and inorganic nanoparticles in Japanese medaka. Her focus is on the germline-mediated transmission of toxic effects from one generation to the other.

Ajish A.

Ajish is working as Junior Research Fellow on the project dealing with the isolation and characterization of human ovarian germline stem cells and generation of induced pluripotent stem cells from these cells.

Anoop P.D.

Anoop is working as Junior Research Fellow on the project dealing with development of an assay to detect the potency of various chemicals that can cause endocrine disruption using Japanese medaka as the animal model.

Lucienne Mathai

Lucienne is an M.Tech student and her project entails the toxicity of endosulfan as an endocrine disrupter on the sexual behaviour of male Japanese medaka and the pathological changes that occur in the brain and gonads.

Elizabeth C. Varghese

Elizabeth is an M.tech student doing her project which investigates the role of germline stem cells in dehydroepiandrosterone-mediated enhancement of ovarian reserve.


Publication Type: Journal Article

Year of Publication Title


S.K. Kanthlal, Jipnomon Joseph, Dr. Bindhu Paul, M, V., and Dr. Umadevi P., “Antioxidant and vasorelaxant effects of aqueous extract of large cardamom in L-NAME induced hypertensive rats”, Clinical and Experimental Hypertension, vol. 42, no. 7, pp. 581-589, 2020.[Abstract]

ABSTRACT Purpose The present work aimed to study the effect of aqueous extract of large cardamom (AELC) to prevent vascular remodeling and oxidative stress in Nω-Nitro-L-arginine methyl ester (L-NAME)-induced hypertension. Method Male Wistar rats were administered with L-NAME 40 mg/kg/day for 28 days by oral gavage. The treatments included captopril (20 mg/kg/day) or AELC (100, 200 and 400 mg/kg/day) along with L-NAME administration. Results L-NAME treated rats showed high systolic, diastolic and mean arterial pressure, decreased nitric oxide level, increased level of malondialdehyde in plasma, heart, aorta and kidney, hypertrophy of the vascular wall and reduced vascular response to acetylcholine in phenylephrine-precontracted aorta. Treatment with AELC markedly reduced the blood pressure, restored the nitric oxide level, reduced the malondialdehyde level, alleviated the hypertrophy in L-NAME-induced hypertensive rats. Additionally, it also improved the vascular response to acetylcholine in phenylephrine pre-contracted aorta. Conclusion In conclusion, our results demonstrate the preventive effect of AELC in L-NAME-induced hypertensive model, which is possibly related to antioxidant activities and restoration of nitric oxide level.

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K. S, Dr. Bindhu Paul, A, A., PD, A., S, S., and D, M., “Graphene Quantum Dots Alter Proliferation and Meiosis of Germ Cells Only in Genetic Females of Japanese Medaka during Early Embryonic Development”, ACS Applied Bio Materials , vol. 2, no. 2, pp. 737-746, 2019.[Abstract]

Graphene quantum dots (GQDs) have emerged as promising biolabeling agents owing to their stable innate fluorescence, photostability, and biocompatibility as opposed to semiconductor quantum dots. While several studies reported GQDs to be cytocompatible, their potential for reproductive toxicity, particularly to germ cells that can cause transgenerational toxicity, remains unexplored. Here we report the intrinsic toxicity of 2–3 nm sized GQDs synthesized from glucose by a novel bottom-up green chemistry technique on germ cell proliferation and meiosis during early embryonic development. In vitro cell viability studies with a normal ovarian cell line, Chinese Hamster Ovarian cells (CHO), and a primary cell type, Human Umbilical Vein Endothelial Cells (HUVEC), portrayed good cytocompatibility even up to a high concentration of 800 μg/mL. When embryos of Japanese medaka were exposed to GQDs, no developmental toxicity was observed up to 250 μg/mL, beyond which hatchability and survival were affected adversely. In contrast, toxicity to germ cells in developing gonads was apparent in genetically female (XX) embryos exposed to much lower doses (50, 75, and 100 μg/mL), at which in vitro cytotoxicity and embryo hatchability and survival were unaffected. A drastic decline in germ cell number and meiosis was observed at these doses in XX embryos implying anomalies in sexual differentiation of the gametes. Conversely, germ cells of genetically male embryos exposed to GQDs were unaltered. Significantly high levels of reactive oxygen species (ROS) were detected in the XX larvae exposed to GQDs; however, there was no DNA damage, suggesting ROS to be responsible for the adverse effects observed.

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S. Padmakumar, Dr. Bindhu Paul, Pavithran, K., Vijaykumar, D. Kottarathi, Rajanbabu, A., Sivanarayanan, T. Balakrishn, Kadakia, E., Amiji, M. M., Shantikumar V Nair, and Dr. Deepthy Menon, “Long-term Drug Delivery using Implantable Electrospun Woven Polymeric Nanotextiles”, Nanomedicine: Nanotechnology, Biology, and Medicine, vol. 15, no. 1, pp. 274-284, 2019.[Abstract]

A woven nanotextile implant was developed and optimized for long-term continuous drug delivery for potential oncological applications. Electrospun polydioxanone (PDS) nanoyarns, which are twisted bundles of PDS nanofibres, were loaded with paclitaxel (PTX) and woven into nanotextiles of different packing densities. A mechanistic modeling of in vitro drug release proved that a combination of diffusion and matrix degradation controlled the slow PTX-release from a nanoyarn, emphasizing the role of nanostructure in modulating release kinetics. Woven nanotextiles, through variations in its packing density and thereby architecture, demonstrated tuneable PTX-release. In vivo PTX-release, pharmacokinetics and biodistribution were evaluated in healthy BALB/c mice by suturing the nanotextile to peritoneal wall. The slow and metronomic PTX-release for 60 days from the loosely woven implant was extremely effective in enhancing its residence in peritoneum, in contrast to intraperitoneal injections. Such an implantable matrix offers a novel platform for therapy of solid tumors over prolonged durations

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Dr. Bindhu Paul, Nair, R., Victor, A. C., and Paul-Prasanth, B., “Effects of N-Nitrosodiethylamine, a Potent Carcinogen, on Sexual Development, Gametogenesis, and Oocyte Maturation”, Sexual Development, vol. 11, no. 3, pp. 161-167, 2017.[Abstract]

N-Nitrosodiethylamine (DEN), a well-known hepatocarcinogen, is found in certain food products as such or as a metabolic byproduct. This study investigated the effects of DEN on sexual development, gametogenesis, and oocyte maturation in Japanese medaka (Oryzias latipes). DEN reduced the germ cell number dose-dependently during early stages of sexual differentiation in XX larvae, resulting in underdeveloped ovaries in adulthood at low doses. This effect was sex-specific as no such changes were seen in XY larvae. Furthermore, XX and XY larvae that were exposed at a low dose during early life showed a significant reduction in body weight in adulthood. Gonads in sexually immature adult medaka males and females exposed to DEN were in advanced stages in comparison to that of the controls. Gonado-somatic indices were significantly high in treated males and females. DEN induced oocyte maturation in vitro, which was inhibited by cordycepin, demonstrating that it stimulated oocyte maturation through polyadenylation of cyclin B mRNA as in the case of the endogenous maturation-inducing hormone. Altogether, our results have proven that DEN could disrupt or mimic the signaling pathways involved in germ cell development, proliferation, and maturation.

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M. Chatterjee, D'Morris, S., Paul, V., Warrier, S., Vasudevan, A. Kumar, Muralidharan Vanuopadath, Nair, S. Sadasivan, Dr. Bindhu Paul, Dr. Gopi Mohan C., and Dr. Raja Biswas, “Mechanistic Understanding of Phenyllactic Acid Mediated Inhibition of Quorum Sensing and Biofilm Development in Pseudomonas Aeruginosa”, Appl Microbiol Biotechnol, vol. 101, no. 22, pp. 8223-8236, 2017.[Abstract]

Pseudomonas aeruginosa depends on its quorum sensing (QS) system for its virulence factors' production and biofilm formation. Biofilms of P. aeruginosa on the surface of indwelling catheters are often resistant to antibiotic therapy. Alternative approaches that employ QS inhibitors alone or in combination with antibiotics are being developed to tackle P. aeruginosa infections. Here, we have studied the mechanism of action of 3-Phenyllactic acid (PLA), a QS inhibitory compound produced by Lactobacillus species, against P. aeruginosa PAO1. Our study revealed that PLA inhibited the expression of virulence factors such as pyocyanin, protease, and rhamnolipids that are involved in the biofilm formation of P. aeruginosa PAO1. Swarming motility, another important criterion for biofilm formation of P. aeruginosa PAO1, was also inhibited by PLA. Gene expression, mass spectrometric, functional complementation assays, and in silico data indicated that the quorum quenching and biofilm inhibitory activities of PLA are attributed to its ability to interact with P. aeruginosa QS receptors. PLA antagonistically binds to QS receptors RhlR and PqsR with a higher affinity than its cognate ligands N-butyryl-L-homoserine lactone (C-HSL) and 2-heptyl-3,4-dihydroxyquinoline (PQS; Pseudomonas quinolone signal). Using an in vivo intraperitoneal catheter-associated medaka fish infection model, we proved that PLA inhibited the initial attachment of P. aeruginosa PAO1 on implanted catheter tubes. Our in vitro and in vivo results revealed the potential of PLA as anti-biofilm compound against P. aeruginosa.

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S. Fazal, Dr. Bindhu Paul, Shantikumar V Nair, and Dr. Deepthy Menon, “Theranostic Iron Oxide/Gold Ion Nanoprobes for MR Imaging and Noninvasive RF Hyperthermia.”, ACS Applied Materials and Interfaces, vol. 9, no. 34, pp. 28260-28272, 2017.[Abstract]

<p>This work focuses on the development of a nanoparticulate system that can be used for magnetic resonance (MR) imaging and E-field noninvasive radiofrequency (RF) hyperthermia. For this purpose, an amine-functional gold ion complex (GIC), [Au(III)(diethylenetriamine)Cl]Cl, which generates heat upon RF exposure, was conjugated to carboxyl-functional poly(acrylic acid)-capped iron-oxide nanoparticles (IO-PAA NPs) to form IO-GIC NPs of size ∼100 nm. The multimodal superparamagnetic IO-GIC NPs produced T2-contrast on MR imaging and unlike IO-PAA NPs generated heat on RF exposure. The RF heating response of IO-GIC NPs was found to be dependent on the RF power, exposure period, and particle concentration. IO-GIC NPs at a concentration of 2.5 mg/mL showed a high heating response (δT) of ∼40 °C when exposed to 100 W RF power for 1 min. In vitro cytotoxicity measurements on NIH-3T3 fibroblast cells and 4T1 cancer cells showed that IO-GIC NPs are cytocompatible at high NP concentrations for up to 72 h. Upon in vitro RF exposure (100 W, 1 min), a high thermal response leads to cell death of 4T1 cancer cells incubated with IO-GIC NPs (1 mg/mL). Hematoxylin and eosin imaging of rat liver tissues injected with 100 μL of 2.5 mg/mL IO-GIC NPs and exposed to low RF power of 20 W for 10 min showed significant loss of tissue morphology at the site of injection, as against RF-exposed or nanoparticle-injected controls. In vivo MR imaging and noninvasive RF exposure of 4T1-tumor-bearing mice after IO-GIC NP administration showed T2 contrast enhancement and a localized generation of high temperatures in tumors, leading to tumor tissue damage. Furthermore, the administration of IO-GIC NPs followed by RF exposure showed no adverse acute toxicity effects in vivo. Thus, IO-GIC NPs show good promise as a theranostic agent for magnetic resonance imaging and noninvasive RF hyperthermia for cancer.</p>

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S. Narayanan, Dr. Ullas Mony, Vijaykumar, D. K., Dr. Manzoor K., Dr. Bindhu Paul, and Dr. Deepthy Menon, “Sequential release of epigallocatechin gallate and paclitaxel from PLGA-casein core/shell nanoparticles sensitizes drug-resistant breast cancer cells”, Nanomedicine: Nanotechnology, Biology, and Medicine, vol. 11, pp. 1399-1406, 2015.[Abstract]

Nanomedicines consisting of combinations of cytotoxic drugs and molecular targeted therapeutics which inhibit specific downstream signals are evolving as a novel paradigm for breast cancer therapy. This research addresses one such combination of Paclitaxel (Ptx), having several adversities related to the activation of NF-κB pathway, with Epigallocatechin gallate (EGCG), a multiple signaling inhibitor, encapsulated within a targeted core/shell PLGA-Casein nanoparticle. The sequential release of EGCG followed by Ptx from this core/shell nanocarrier sensitized Ptx resistant MDA-MB-231 cells to Ptx, induced their apoptosis, inhibited NF-κB activation and downregulated the key genes associated with angiogenesis, tumor metastasis and survival. More importantly, Ptx-induced expression of P-glycoprotein was repressed by the nanocombination both at the protein and gene levels. This combination also offered significant cytotoxic response on breast cancer primary cells, indicating its translational value. From the Clinical Editor: Breast cancer is the most common cancer in women worldwide. As well as surgery, chemotherapy plays a major role in the treatment of breast cancer. The authors investigated in this article the combination use of a chemotherapeutic agent, Paclitaxel (Ptx), and an inhibitor of NF-?B pathway, packaged in a targeted nano-based delivery platform. The positive results provided a new pathway for future clinical use of combination chemotherapy in breast cancer. © 2015 Elsevier Inc.

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Dr. Bindhu Paul, Bhandari, R. K., Kobayashi, T., Horiguchi, R., Kobayashi, Y., Nakamoto, M., Shibata, Y., Sakai, F., Nakamura, M., and Nagahama, Y., “ERRATUM: Estrogen oversees the maintenance of the female genetic program in terminally differentiated gonochorists”, Scientific reports, vol. 4, 2014.[Abstract]

The sexual plasticity of the gonads is not retained after the completion of sex differentiation in vertebrates, except in some hermaphroditic species. Here, we report that the depletion of estradiol-17β (E2) by aromatase inhibitors (AI) for up to six months resulted in a functional female-to-male sex reversal in sexually-mature adults of two gonochoristic fish species, Nile tilapia and medaka. The sex-reversed fish showed a typical male pattern of E2 and androgen levels, secondary sexual characteristics, and male-like sex behavior, producing fertile sperm. Conversely, co-treatment of E2 inhibited AI-induced sex reversal. In situ hybridization of medaka gonads during AI-induced sex reversal indicated that cysts on the dorsal side of the adult ovaries are the origin of germ cells and Sertoli cells in the newly formed testicular tissue. Gonochoristic fish maintain their sexual plasticity until adulthood and E2 plays a critical role in maintaining the female phenotype. More »»


S. Maehiro, Takeuchi, A., Yamashita, J., Hiraki, T., Kawabata, Y., Nakasone, K., Hosono, K., Usami, T., Dr. Bindhu Paul, Nagahama, Y., Oka, Y., and Okubo, K., “Sexually dimorphic expression of the sex chromosome-linked genes cntfa and pdlim3a in the medaka brain”, Biochemical and Biophysical Research Communications, vol. 445, pp. 113 - 119, 2014.[Abstract]

Abstract In vertebrates, sex differences in the brain have been attributed to differences in gonadal hormone secretion; however, recent evidence in mammals and birds shows that sex chromosome-linked genes, independent of gonadal hormones, also mediate sex differences in the brain. In this study, we searched for genes that were differentially expressed between the sexes in the brain of a teleost fish, medaka (Oryzias latipes), and identified two sex chromosome genes with male-biased expression, cntfa (encoding ciliary neurotrophic factor a) and pdlim3a (encoding \{PDZ\} and \{LIM\} domain 3 a). These genes were found to be located 3–4 Mb from and on opposite sides of the Y chromosome-specific region containing the sex-determining gene (the medaka X and Y chromosomes are genetically identical, differing only in this region). The male-biased expression of both genes was evident prior to the onset of sexual maturity. Sex-reversed \{XY\} females, as well as wild-type \{XY\} males, had more pronounced expression of these genes than \{XX\} males and \{XX\} females, indicating that the Y allele confers higher expression than the X allele for both genes. In addition, their expression was affected to some extent by sex steroid hormones, thereby possibly serving as focal points of the crosstalk between the genetic and hormonal pathways underlying brain sex differences. Given that sex chromosomes of lower vertebrates, including teleost fish, have evolved independently in different genera or species, sex chromosome genes with sexually dimorphic expression in the brain may contribute to genus- or species-specific sex differences in a variety of traits.

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H. Zhao, Guan, G., Duan, J., Cheng, N., Wang, J., Matsuda, M., Dr. Bindhu Paul, and Nagahama, Y., “Ol4E-T, a Eukaryotic Translation Initiation Factor 4E-Binding Protein of Medaka Fish (Oryzias latipes), Can Interact With Nanos3 and Vasa In Vitro”, Journal of Experimental Zoology Part B: Molecular and Developmental Evolution, vol. 320, pp. 10–21, 2013.[Abstract]

Maternal factors have essential roles in the specification and development of germ cells in metazoans. In Drosophila, a number of genes such as oskar, vasa, nanos, and tudor are required for specific steps in pole cell formation and further germline development. Drosophila cup, another maternal factor, is confirmed as a main factor in normal oogenesis, maintenance, and survival of female germ-line stem cells by interaction with Nanos. Through searching for the homolog of Drosophila cup in the medaka, the homolog of eukaryotic translation initiation factor 4E (eIF4E)-transporter, named Ol4E-T, was identified. Reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization revealed that Ol4E-T is maternally deposited in the embryo and Ol4E-T expression is maintained throughout embryogenesis. Ol4E-T is predominantly expressed in the adult gonads. In the testes, Ol4E-T is expressed in the same regions where medaka vasa, named olvas is expressed. In the ovary, expression of Ol4E-T conforms to that of nanos3 and olvas. Ol4E-T harbors a well-conserved eIF4E-binding motif, YTKEELL, by which Ol4E-T interacts with eIF4E in medaka. Additionally, Ol4E-T can interact with medaka Nanos3 and Olvas, as shown by yeast two hybridization. The spatial expression and interactions between Ol4E-T with germ cell markers Olvas and Nanos3 suggest a role for Ol4E-T in germ-line development in medaka. J. Exp. Zool. (Mol. Dev. Evol.) 320B:10–21, 2013. © 2012 Wiley Periodicals, Inc. More »»


B. Swarnalatha, Nair, S. L., Shalumon, K. T., Milbauer, L. C., Dr. Jayakumar Rangasamy, Dr. Bindhu Paul, Menon, K. K., Hebbel, R. P., Somani, A., and Shantikumar V. Nair, “Poly (lactic acid)–chitosan–collagen composite nanofibers as substrates for blood outgrowth endothelial cells”, International Journal of Biological Macromolecules, vol. 58, pp. 220 - 224, 2013.[Abstract]

Abstract In this work, the attachment, viability and functionality of rat Blood Outgrowth Endothelial Cells (rBOEC) and genetically modified rBOEC (rBOEC/eNOS-GFP), which over express endothelial nitric oxide synthase (eNOS), were investigated on Poly(lactic acid) (PLA)–chitosan and PLA–chitosan–collagen nanofibrous scaffolds. Both the cell types displayed good attachment, remained viable and functional on both scaffolds. Moreover, incorporation of collagen in the scaffold helped in sustaining the rBOEC for upto one week, although collagen was not found necessary for rBOEC/eNOS-GFP. We conclude that PLA–chitosan based nanofibrous scaffolds can be a potential candidate for \{BOEC\} based wound healing applications. More »»


Dr. Bindhu Paul, Bhandari, R. K., Kobayashi, T., Horiguchi, R., Kobayashi, Y., Nakamoto, M., Shibata, Y., Sakai, F., Nakamura, M., and Nagahama, Y., “Estrogen oversees the maintenance of the female genetic program in terminally differentiated gonochorists”, Scientific reports, vol. 3, p. 2862, 2013.[Abstract]

The sexual plasticity of the gonads is not retained after the completion of sex differentiation in vertebrates, except in some hermaphroditic species. Here, we report that the depletion of estradiol-17β (E2) by aromatase inhibitors (AI) for up to six months resulted in a functional female-to-male sex reversal in sexually-mature adults of two gonochoristic fish species, Nile tilapia and medaka. The sex-reversed fish showed a typical male pattern of E2 and androgen levels, secondary sexual characteristics, and male-like sex behavior, producing fertile sperm. Conversely, co-treatment of E2 inhibited AI-induced sex reversal. In situ hybridization of medaka gonads during AI-induced sex reversal indicated that cysts on the dorsal side of the adult ovaries are the origin of germ cells and Sertoli cells in the newly formed testicular tissue. Gonochoristic fish maintain their sexual plasticity until adulthood and E2 plays a critical role in maintaining the female phenotype. More »»


Dr. Bindhu Paul and Nagahama, Y., “Sexual Behavior in Teleosts: Role of Gonadal Hormones”, Indian Journal of Science and Technology, vol. 4, pp. 14–14, 2011.


Dr. Bindhu Paul, Nakamura, M., and Nagahama, Y., “Sex determination in fishes”, Hormones and reproduction of vertebrates: fishes, vol. 1, pp. 1–14, 2011.


J. I. Fernandino, Popesku, J. T., Dr. Bindhu Paul, Xiong, H., Hattori, R. S., Oura, M., Strüssmann, C. A., Somoza, G. M., Matsuda, M., Nagahama, Y., and , “Analysis of sexually dimorphic expression of genes at early gonadogenesis of pejerrey Odontesthes bonariensis using a heterologous microarray”, Sexual development, vol. 5, pp. 89–101, 2011.[Abstract]

The process of morphological development of a differentiated gonad from an undifferentiated primordium is a very important step of gonadogenesis. Studies on sexually dimorphic gene expression are important to increase our understanding of this process and to investigate how environmental factors such as temperature can regulate gonadal development. The aim of this study was to identify putative genes involved in sex differentiation in pejerrey (Odontesthes bonariensis) reared at male- and female-producing temperatures (MPT and FPT, respectively) using a microarray heterologous from the medaka (Oryzias latipes), a closely phylogenetic species. Genes related to numerous processes presented higher expression at MPT, including those involved in muscular contraction, metabolic pathways, developmental processes, and reproduction. Genes induced by FPT were classified under the gene ontology terms of response to stimulus, transport and proteolysis. From genes selected for validation, at MPT ndrg3 expression was observed in the somatic cells, whereas pen-2 was detected in germ cells in the caudal portion of the gonads, where no apoptotic signals were observed. Finally, hsp90 was highly expressed in somatic cells of the gonads at the FPT. The results suggest that the interplay of pro-apoptotic and anti-apoptotic genes is important during the masculinization process and for the prevention of sterility following exposure to warm temperatures. More »»


Dr. Bindhu Paul, Shibata, Y., Horiguchi, R., and Nagahama, Y., “Exposure to diethylstilbestrol during embryonic and larval stages of medaka fish (Oryzias latipes) leads to sex reversal in genetic males and reduced gonad weight in genetic females”, Endocrinology, vol. 152, pp. 707–717, 2011.[Abstract]

Complete feminization of Dmy/Gsdf-expressing somatic cells by diethylstilbestrol is essential for induction of stable ovarian development in XY gonads of medaka. Molecular and cellular mechanisms involved in artificially induced ovarian differentiation were analyzed by exposing embryos of medaka (Oryzias latipes) to a potent nonsteroidal estrogen, diethylstilbestrol (DES). Embryos were exposed for short-exposure (SE) [from 0 to 8 d postfertilization (dpf)] and long-exposure (LE) periods (from 0 to 18/28 dpf) to 1 ng/ml of DES, and status of sexual differentiation in somatic and germ cells of these gonads was analyzed at 8, 18, and 28 dpf by histology, cell proliferation assays, TUNEL assay, and in situ hybridization using sex-specific somatic and germ cell markers. Additionally, gonads of exposed fry were examined after withdrawal of DES to see whether effects of DES in exposed fish were reversible or not. DES induced germ cell proliferation and meiosis in XY fry of SE and LE groups. However, SE induced only a partial reduction in expression of gonadal soma-derived factor, the male-dominant somatic cell marker, and was not sufficient to induce ovarian development after withdrawal of DES. On the contrary, LE resulted in complete loss of such male-specific gene expression in somatic cells of XY gonads, and these gonads underwent sustained ovarian development even after withdrawal of DES. Importantly, LE to DES affected germ cell proliferation in XX gonads adversely during early stages of sexual differentiation, leading to reduced gonad weight in adulthood. Interestingly, apoptosis was not the cause for reduction in germ cell number. Taken together, these results indicated that DES exposure has long-lasting effects on the gonadal development in genetic males (sex reversal) and females (reduced gonad weight) of medaka. More »»


K. Okubo, Takeuchi, A., Chaube, R., Dr. Bindhu Paul, Kanda, S., Oka, Y., and Nagahama, Y., “Sex differences in aromatase gene expression in the medaka brain”, Journal of neuroendocrinology, vol. 23, pp. 412–423, 2011.[Abstract]

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. More »»


Y. Shibata, Dr. Bindhu Paul, Suzuki, A., Usami, T., Nakamoto, M., Matsuda, M., and Nagahama, Y., “Expression of gonadal soma derived factor (GSDF) is spatially and temporally correlated with early testicular differentiation in medaka”, Gene Expression Patterns, vol. 10, pp. 283–289, 2010.[Abstract]

In the teleost fish, medaka (Oryzias latipes), the sex is genetically determined at the time of fertilization. The males are heterogametic with XY chromosome composition, while females are of XX chromosome composition. The male sexual differentiation is initiated in XY embryos of medaka by the sex-determining gene Dmy. In this study, we have cloned the gonadal soma derived factor (Gsdf) from medaka and characterized its expression pattern during the initiation of morphological testicular differentiation. By real-time PCR, an XY-specific up-regulation was detected in the expression levels of Gsdf in the whole embryos of medaka at 6 days post fertilization (dpf), coincident with the initiation of testicular differentiation in the XY gonads. Whole mount and section in situ hybridizations reaffirmed that Gsdf was expressed exclusively in primordial gonads of only the genetic males at 6 dpf. Conversely, the expression of Gsdf was found to be very weak in the XX gonads during embryogenesis. Importantly, Gsdf and Dmy were found to be co-localized in the samesomatic cells in the XY gonads. When the XY embryos were treated with estradiol-17β, in order to reverse their phenotypic sex, a decline was observed in the expression of Gsdf in these embryos by real-time PCR. More »»


Y. Kobayashi, Nakamura, M., Sunobe, T., Usami, T., Kobayashi, T., Manabe, H., Dr. Bindhu Paul, Suzuki, N., and Nagahama, Y., “Sex change in the Gobiid fish is mediated through rapid switching of gonadotropin receptors from ovarian to testicular portion or vice versa”, Endocrinology, vol. 150, pp. 1503–1511, 2009.[Abstract]

Sex-changing fish Trimma okinawae can change its sex back and forth from male to female and then to male serially, depending on the social status in the harem. T. okinawae is well equipped to respond to its social status by possessing both ovarian and testicular tissues even though only one gonad remains active at one time. Here we investigated the involvement of gonadotropins in sex change by determining the changes in gonadotropin receptor (GtHR) gene expression during the onset of sex change from female to male and male to female. The expression of the GtHR was found to be confined to the active gonad of the corresponding sexual phase. During the sex-change from female to male, initially the ovary had high levels of FSHR and LHR, which eventually went up in the testicular tissue if the fish was bigger. Changing of the gonads started with switching of GtHR expression discernible within 8–12 h of the visual cue. Further in vitro culture of the transitional gonads with a supply of exogenous gonadotropin (human chorionic gonadotropin) revealed that the to-be-active gonad acquired the ability to produce the corresponding sex hormone within 1 d of the activation of GtHR. Conversely, the to-be-regressed gonad did not respond to the exogenous gonadotropin. Our findings show that the gonads of successive sex-changing fish possess the intrinsic mechanism to respond to the social cue differentially. Additionally, this location switching of GtHR expression also could substantiate the importance of the hypothalamo-pituitary-gonadotropic axis. Gonadal sex-change in goby, in response to social cue, is mediated through a rapid switching in the expression of GtH receptors in the gonads. More »»


M. Mita, Yoshikuni, M., Ohno, K., Shibata, Y., Dr. Bindhu Paul, Pitchayawasin, S., Isobe, M., and Nagahama, Y., “A relaxin-like peptide purified from radial nerves induces oocyte maturation and ovulation in the starfish, Asterina pectinifera”, Proceedings of the National Academy of Sciences, vol. 106, pp. 9507–9512, 2009.[Abstract]

Gonad-stimulating substance (GSS) of starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to the vertebrate luteinizing hormone (LH). Here, we purified GSS of starfish, Asterina pectinifera, from radial nerves and determined its amino acid sequence. The purified GSS was a heterodimer composed of 2 different peptides, A and B chains, with disulfide cross-linkages. Based on its cysteine motif, starfish GSS was classified as a member of the insulin/insulin-like growth factor (IGF)/relaxin superfamily. The cDNA of GSS encodes a preprohormone sequence with a C peptide between the A and B chains. Phylogenetic analyses revealed that starfish GSS was a relaxin-like peptide. Chemically synthesized GSS induced not only oocyte maturation and ovulation in isolated ovarian fragments, but also unique spawning behavior, followed by release of gametes shortly after the injection. Importantly, the action of the synthetic GSS on oocyte maturation and ovulation was mediated through the production of cAMP by isolated ovarian follicle cells, thereby producing the maturation-inducing hormone of this species, 1-methyladenine. In situ hybridization showed the transcription of GSS to occur in the periphery of radial nerves at the side of tube feet. Together, the structure, sequence, and mode of signal transduction strongly suggest that GSS is closely related to the vertebrate relaxin. More »»


Dr. Bindhu Paul, Matsuda, M., Kobayashi, T., Suzuki, A., and Nagahama, Y., “Functional analysis of the medaka sex determining gene, DMY-A minireview”, CYBIUM, vol. 32, pp. 72–73, 2008.[Abstract]

DMY is the second vertebrate sex-determining gene identified from the fish, Oryzias latipes. Overexpres- sion of genomic DNA carrying DMY in the XX fish induced development of functional testis in these fish. Further, DMY knock-down and estradiol-17b (E2) treatment, to determine the possible function of DMY during early gonadal sex differ- entiation in XY medaka, revealed that the mitotic and meiotic activities of the germ cells in the 0 day after hatching (dah) DMY knock-down XY larvae were identical to that of the normal XX larvae. Conversely, E2 treatment failed to initiate mitosis in the XY gonad, possibly due to an active DMY, even though it could initiate meiosis. More »»


S. Ijiri, Kaneko, H., Kobayashi, T., Wang, D. - S., Sakai, F., Dr. Bindhu Paul, Nakamura, M., and Nagahama, Y., “Sexual dimorphic expression of genes in gonads during early differentiation of a teleost fish, the Nile tilapia Oreochromis niloticus”, Biology of reproduction, vol. 78, pp. 333–341, 2008.[Abstract]

The Nile tilapia, a gonochoristic teleost fish with an XX/XY sex-determining system, provides an excellent model for studying gonadal sex differentiation because genetic all-females and all-males are available. In this study, we used quantitative real-time RT-PCR to determine the precise timing of the gonadal expression of 17 genes thought to be associated with gonadal sex differentiation in vertebrates. Gonads were isolated from all-female and all-male tilapia before (5–15 days after hatching [dah]) and after (25–70 dah) morphological sex differentiation. The transcript of aromatase (cyp19a1a), an enzyme responsible for producing estradiol-17beta, was expressed only in XX gonads at 5 dah, with a marked elevation in expression thereafter. In contrast, mRNA expression of steroid 11beta-hydroxylase (cyp11b2), an enzyme responsible for the synthesis of 11-ketotestosterone (11-KT, a potent androgen in fish), was found in XY gonads from 35 dah only. These results, combined with the presence of transcripts for other steroidogenic enzymes and estrogen receptors in XX gonads at 5–7 dah, are consistent with our earlier suggestion that estradiol-17beta plays a critical role in ovarian differentiation in tilapia, whereas a role for 11-KT in testicular differentiation is questionable. A close relationship between the expression offoxl2, but not nr5a1 (Ad4BP/SF-1), and that of cyp19a1a in XX gonads suggests an important role for Foxl2 in the transcriptional regulation ofcyp19a1a. Dmrt1 exhibited a male-specific expression in XY gonads from 6 dah onward, suggesting an important role for Dmrt1 in testicular differentiation. Sox9 and amh (anti-Mullerian hormone) showed a testis-specific expression, being evident only in the later stages of testicular differentiation. It is concluded that the sex-specific expression of foxl2 andcyp19a1a in XX gonads and dmrt1 in XY gonads during early gonadal differentiation (5–6 dah) is critical for undifferentiated gonads to differentiate into either the ovary or testis in the Nile tilapia. More »»


L. - Y. Zhou, Wang, D. - S., Shibata, Y., Dr. Bindhu Paul, Suzuki, A., and Nagahama, Y., “Characterization, expression and transcriptional regulation of P450c17-I and-II in the medaka, Oryzias latipes”, Biochemical and biophysical research communications, vol. 362, pp. 619–625, 2007.[Abstract]

Recently we reported the isolation of two types of cytochrome P450c17s (steroid 17α-hydroxylase/C17, 20 lyase) encoded by two different genes, from genomes of teleost fish. In this study, we characterized the expression profile, enzymatic activity and transcriptional regulation of P450c17-I and -II in the medaka ovary. Similar to tilapia, medaka P450c17-I possessed both hydroxylase and lyase activities, while P450c17-II possessed only the hydroxylase activity. In situ hybridization and gene expression profiles during 48 h prior to spawning indicated that P450c17-I is responsible for the production of estradiol-17β during oocyte growth, while P450c17-II for the production of 17α, 20β-dihydroxy-4-pregnen-3-one during oocyte maturation and cortisol production in the head kidney. Luciferase assays and expression profiles of transcription factors as revealed by real time PCR suggested that P450c17-I and -II expression are tightly controlled by Ad4BP/SF-1, Lrh-1, Foxl2, and Dax1 during the 48 h prior to spawning. More »»


L. - Y. Zhou, Wang, D. - S., Kobayashi, T., Yano, A., Dr. Bindhu Paul, Suzuki, A., Sakai, F., and Nagahama, Y., “A novel type of P450c17 lacking the lyase activity is responsible for C21-steroid biosynthesis in the fish ovary and head kidney”, Endocrinology, vol. 148, pp. 4282–4291, 2007.[Abstract]

Cytochrome P450c17 is the single enzyme that mediates the 17α-hydroxylase and 17, 20 lyase activities during the biosynthesis of steroid hormones in the gonads and adrenal gland. However, the mechanism underlying its dual action continues to be a controversy in the field of steroidogenesis in fish. In an attempt to resolve this issue, we identified a novel type of P450c17 (P450c17-II) by an in silico analysis from the genomes of six fish species. We cloned P450c17-II from tilapia and medaka, and comparison with the conventional P450c17-I revealed that they differ in gene structure and enzymatic activity. Enzymatic assays by thin-layer chromatography revealed that P450c17-II possesses only the 17α-hydroxylase activity without any 17, 20 lyase activity, unlike P450c17-I, which has both these activities. In testis, both P450c17-I and -II express in the interstitial cells. Remarkable differences, revealed by in situ hybridization, in the expression patterns of the P450c17-I and -II in the ovary and head kidney of tilapia during various stages of development strongly suggest that P450c17-I is responsible for the synthesis of estradiol-17β in the ovary, whereas P450c17-II is required for the production of C21 steroids such as cortisol in the head kidney. More interestingly, a temporally controlled switching is observable in the expression of these two genes during the steroidogenic shift from estradiol-17β to the C21 steroid, 17α, 20β-dihydroxy-4-pregnen-3-one (maturation-inducing hormone of fish oocytes) in the fish ovary, revealing a role for P450c17-II in the production of hormones that induce oocyte maturation in fish. More »»


M. Matsuda, Shinomiya, A., Kinoshita, M., Suzuki, A., Kobayashi, T., Dr. Bindhu Paul, Lau, E. -lieng, Hamaguchi, S., Sakaizumi, M., and Nagahama, Y., “DMY gene induces male development in genetically female (XX) medaka fish”, Proceedings of the National Academy of Sciences, vol. 104, pp. 3865–3870, 2007.[Abstract]

Although the sex-determining gene SRY/Sry has been identified in mammals, homologues and genes that have a similar function have yet to be identified in nonmammalian vertebrates. Recently, DMY (the DM-domain gene on the Y chromosome) was cloned from the sex-determining region on the Y chromosome of the teleost fish medaka (Oryzias latipes). DMY has been shown to be required for the normal development of male individuals. In this study, we show that a 117-kb genomic DNA fragment that carries DMY is able to induce testis differentiation and subsequent male development in XX (genetically female) medaka. In addition, overexpression of DMY cDNA under the control of the CMV promoter also caused XX sex reversal. These results demonstrate that DMY is sufficient for male development in medaka and suggest that the functional difference between the X and Y chromosomes in medaka is a single gene. Our data indicate that DMY is an additional sex-determining gene in vertebrates. More »»


D. - S. Wang, Kobayashi, T., Zhou, L. - Y., Dr. Bindhu Paul, Ijiri, S., Sakai, F., Okubo, K., Morohashi, K. -ichirou, and Nagahama, Y., “Foxl2 up-regulates aromatase gene transcription in a female-specific manner by binding to the promoter as well as interacting with ad4 binding protein/steroidogenic factor 1”, Molecular Endocrinology, vol. 21, pp. 712–725, 2007.[Abstract]

Increasing evidence suggests the crucial role of estrogen in ovarian differentiation of nonmammalian vertebrates including fish. The present study has investigated the plausible role of Foxl2 in ovarian differentiation through transcriptional regulation of aromatase gene, using monosex fry of tilapia. Foxl2 expression is sexually dimorphic, like Cyp19a1, colocalizing with Cyp19a1 and Ad4BP/SF-1 in the stromal cells and interstitial cells in gonads of normal XX and sex-reversed XY fish, before the occurrence of morphological sex differentiation. Under in vitro conditions, Foxl2 binds to the sequence ACAAATA in the promoter region of the Cyp19a1 gene directly through its forkhead domain and activates the transcription of Cyp19a1 with its C terminus. Foxl2 can also interact through the forkhead domain with the ligand-binding domain of Ad4BP/SF-1 to form a heterodimer and enhance the Ad4BP/SF-1 mediated Cyp19a1 transcription. Disruption of endogenous Foxl2 in XX tilapia by overexpression of its dominant negative mutant (M3) induces varying degrees of testicular development with occasional sex reversal from ovary to testis. Such fish display reduced expression of Cyp19a1 as well as a drop in the serum levels of 17β-estradiol and 11-ketotestosterone. Although the XY fish with wild-type tilapia Foxl2 (tFoxl2) overexpression never exhibited a complete sex reversal, there were significant structural changes, such as tissue degeneration, somatic cell proliferation, and induction of aromatase, with increased serum levels of 17β-estradiol and 11-ketotestosterone. Altogether, these results suggest that Foxl2 plays a decisive role in the ovarian differentiation of the Nile tilapia by regulating aromatase expression and possibly the entire steroidogenic pathway. More »»


Dr. Bindhu Paul, Matsuda, M., Lau, E. -lieng, Suzuki, A., Sakai, F., Kobayashi, T., and Nagahama, Y., “Knock-down of DMY initiates female pathway in the genetic male medaka, Oryzias latipes”, Biochemical and biophysical research communications, vol. 351, pp. 815–819, 2006.[Abstract]

DMY is the second vertebrate sex-determining gene identified from the fish, Oryzias latipes. In this study, we used two different ways of sex reversal, DMY knock-down and estradiol-17β (E2) treatment, to determine the possible function of DMY during early gonadal sex differentiation in XY medaka. Our findings revealed that the mitotic and meiotic activities of the germ cells in the 0 day after hatching (dah) DMY knock-down XY larvae were identical to those of the normal XX larvae, suggesting the microenvironment of these XY gonads to be similar to that of the normal XX gonad, where DMY is naturally absent. Conversely, E2 treatment failed to initiate mitosis in the XY gonad, possibly due to an active DMY, even though it could initiate meiosis. Present study is the first to prove that the germ cells in the XY gonad can resume the mitotic activity, if DMY was knocked down. More »»

Dr. Bindhu Paul
Asst. Professor, Nanosciences,
Center for Nanosciences, Kochi