Ramji Kumar Bhandari

Environmentally induced transgenerational epigenetic reprogramming of primordial germ cells and the subsequent germ line.

A number of environmental factors (e.g. toxicants) have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. Transgenerational inheritance requires the germline transmission of altered epigenetic information between generations in the absence of direct environmental exposures. The primary periods for epigenetic programming of the germ line are those associated with primordial germ cell development and subsequent fetal germline development. The current study examined the actions of an agricultural fungicide vinclozolin on gestating female (F0 generation) progeny in regards to the primordial germ cell (PGC) epigenetic reprogramming of the F3 generation (i.e. great-grandchildren). The F3 generation germline transcriptome and epigenome (DNA methylation) were altered transgenerationally. Interestingly, disruptions in DNA methylation patterns and altered transcriptomes were distinct between germ cells at the onset of gonadal sex determination at embryonic day 13 (E13) and after cord formation in the testis at embryonic day 16 (E16). A larger number of DNA methylation abnormalities (epimutations) and transcriptional alterations were observed in the E13 germ cells than in the E16 germ cells. These observations indicate that altered transgenerational epigenetic reprogramming and function of the male germline is a component of vinclozolin induced epigenetic transgenerational inheritance of disease. Insights into the molecular control of germline transmitted epigenetic inheritance are provided.

Gonadal Restructuring and Correlative Steroid Hormone Profiles during Natural Sex Change in Protogynous Honeycomb Grouper (Epinephelus merra)

Abstract The honeycomb grouper shows protogynous hermaphroditism. The endocrine mechanisms involved in gonadal restructuring throughout protogynous sex change are largely unknown. In the present study, we investigated changes in the gonadal structures and levels of serum sex steroid hormones during female to male sex change in the honeycomb grouper. On the basis of histological changes, entire process of sex change was assigned into four developmental phases: female, early transition (ET), late transition (LT), and male phase. At the female phase, the oocytes of several developmental stages were observed including gonial germ cells in the periphery of ovigerous lamellae. At the beginning of ET phase, perinucleolar and previtellogenic oocytes began degenerating, followed by proliferation of spermatogonia toward the center of lamella. The LT phase was characterized by further degeneration of oocytes and rapid proliferation of spermatogenic germ cells throughout the gonad. At the male phase, no ovarian cells were observed and testis had germ cells undergoing active spermatogenesis. Serum levels of estradiol-17β (E2) were high in females in the breeding season, but low in the non-breeding female, transitional and male phase, and those of 11-ketotestosterone (11-KT) and testosterone (T) were low in females and gradually increased in the transitional and male phase. The present results suggest that low serum E2 levels and degeneration of oocytes accompanied by concomitant increase in the 11-KT levels and proliferation of spermatogenic germ cells are probably the events mediating protogynous sex change in the honeycomb grouper.

The role estrogens play in sex differentiation and sex changes of fish

Using genetically controlled all-female and all-male tilapia (Oreochromis niloticus), the role steroid hormones play in the sex differentiation was analyzed histologically, ultrastructurally, immunohistichemically and experimenntally. The results strongly suggest that endogenous estrogen acts as an ovarian inducer, and that the lack of steroid hormone including androgen is important for testicular differentiation. Moreover, the roles of steroid hormones in protogynous sex change of three-spotted wrasse (Halichoeres trimaculatus) and saddleback wrasse (Tharassoma duperrey) were examined. The results strongly support the hypothesis that the endogenous estrogen plays an important role in protogynous sex change.

Seasonal changes of responses to gonadotropin-releasing hormone analog in expression of growth hormone/prolactin/somatolactin genes in the pituitary of masu salmon

Gonadotropin-releasing hormone (GnRH) is considered to stimulate secretion of growth hormone (GH), prolactin (PRL), and somatolactin (SL) at particular stages of growth and sexual maturation in teleost fishes. We therefore examined seasonal variation in the pituitary levels of GH/PRL/SL mRNAs, and tried to clarify seasonal changes of responses to GnRH in expression of GH/PRL/SL genes, in the pituitaries of growing and maturing masu salmon (Oncorhynchus masou). Pituitary samples were monthly collected one week after implantation with GnRH analog (GnRHa). The levels of mRNAs encoding GH, PRL, and SL precursors in single pituitaries were determined by a real-time polymerase chain reaction method. The fork lengths and body weights of control and GnRHa-implanted fish of both sexes gradually increased and peaked out in September of 2-year-old (2+) when fish spawned. GnRHa implantation did not stimulate somatic growth, nor elevate gonadosomatic index (GSI) of 1+ and 2+ males, whereas it significantly increased GSI of 2+ females in late August to early September. The GnRHa-implanted 1+ males had higher levels of GH and PRL mRNAs in July, and SL mRNA from June to August than the control males. The levels of GH, PRL, and SL mRNAs in the control and GnRHa-implanted 1+ females, however, did not show any significant changes. Afterward, the PRL mRNA levels elevated in the control 2+ fish of both sexes in spring. GnRHa elevated the GH mRNA levels in both males and females in 2+ winter, and the PRL mRNA levels in females in early spring. Regardless of sex and GnRHa-implantation, the SL mRNA levels increased during sexual maturation. In growing and maturing masu salmon, expression of genes encoding GH, PRL, and SL in the pituitary is thus sensitive to GnRH in particular seasons probably in relation to physiological roles of the hormones.

Transgenerational effects from early developmental exposures to bisphenol A or 17α-ethinylestradiol in medaka, Oryzias latipes

The transgenerational consequences of environmental contaminant exposures of aquatic vertebrates have the potential for broad ecological impacts, yet are largely uninvestigated. Bisphenol A (BPA) and 17α-ethinylestradiol (EE2) are two ubiquitous estrogenic chemicals present in aquatic environments throughout the United States and many other countries. Aquatic organisms, including fish, are exposed to varying concentrations of these chemicals at various stages of their life history. Here, we tested the ability of embryonic exposure to BPA or EE2 to cause adverse health outcomes at later life stages and transgenerational abnormalities in medaka fish. Exposures of F0 medaka to either BPA (100 μg/L) or EE2 (0.05 μg/L) during the first 7 days of embryonic development, when germ cells are differentiating, did not cause any apparent phenotypic abnormalities in F0 or F1 generations, but led to a significant reduction in the fertilization rate in offspring two generations later (F2) as well as a reduction of embryo survival in offspring three generations later (F3). Our present observations suggest that BPA or EE2 exposure during development induces transgenerational phenotypes of reproductive impairment and compromised embryonic survival in fish of subsequent generations. These adverse outcomes may have negative impacts on populations of fish inhabiting contaminated aquatic environments.

Induction of Female-to–male Sex Change in the Honeycomb Grouper (Epinephelus merra) by 11-ketotestosterone Treatments

Abstract The honeycomb grouper, Epinephelus merra, is a protogynous hermaphrodite fish. Sex steroid hormones play key roles in sex change of this species. A significant drop in endogenous estradiol- 17β (E2) levels alone triggers female-to–male sex change, and the subsequent elevation of 11-ketotestosterone (11KT) levels correlates with the progression of spermatogenesis. To elucidate the role of an androgen in sex change, we attempted to induce female-to–male sex change by exogenous 11KT treatments. The 75-day 11KT treatment caused 100% masculinization of pre-spawning females. Ovaries of the control (vehicle-treated) fish had oocytes at various stages of oogenesis, while the gonads of the 11KT-treated fish had transformed into testes; these contained spermatogenic germ cells at various stages, including an accumulation of spermatozoa in the sperm duct. In the sex-changed fish, plasma levels of E2 were significantly low, while both testosterone (T) and 11KT were significantly increased. Our results suggest that 11KT plays an important role in sex change in the honeycomb grouper. Whether the mechanism of 11KT-induced female-to–male sex change acts through direct stimulation of spermatogenesis in the ovary or via the inhibition of estrogen synthesis remains to be clarified.

Global Genome Analysis of the Downstream Binding Targets of Testis Determining Factor SRY and SOX9

A major event in mammalian male sex determination is the induction of the testis determining factor Sry and its downstream gene Sox9. The current study provides one of the first genome wide analyses of the downstream gene binding targets for SRY and SOX9 to help elucidate the molecular control of Sertoli cell differentiation and testis development. A modified ChIP-Chip analysis using a comparative hybridization was used to identify 71 direct downstream binding targets for SRY and 109 binding targets for SOX9. Interestingly, only 5 gene targets overlapped between SRY and SOX9. In addition to the direct response element binding gene targets, a large number of atypical binding gene targets were identified for both SRY and SOX9. Bioinformatic analysis of the downstream binding targets identified gene networks and cellular pathways potentially involved in the induction of Sertoli cell differentiation and testis development. The specific DNA sequence binding site motifs for both SRY and SOX9 were identified. Observations provide insights into the molecular control of male gonadal sex determination.

Sex Inversion of Sexually Immature Honeycomb Grouper (Epinephelus merra) by Aromatase Inhibitor

Abstract Previous studies have shown that estrogen plays an important role in sex change of protogynous honeycomb grouper, and that the treatments with aromatase inhibitor (AI) cause estrogen depletion and complete sex inversion of pre-spawning females into functional males. In the present study, we examined whether AI causes sex inversion of sexually immature females. Female honeycomb groupers were implanted with various doses of Fadrozole (0, 100, 500 and 1000 μg/fish) in the non-breeding season, and resultant changes in the gonadal structures and the plasma levels of sex steroid hormones (estradiol-17β, E2; testosterone, T; 11-ketotestosterone, 11-KT) were examined three months after implantation. Vehicle-implanted groups did not change sex, while 100 and 500 μg AI-implanted groups had turned into transitionals with intersex gonad. In contrast, the highest dose receiving group exhibited both transitional and male phases. Transitional phase gonad had atretic oocytes and spermatogenic germ cells at the late stages of spermatogenesis, while male phase testis contained spermatozoa accumulated in the seminiferous tubules. All males released sperm upon slight pressure on the abdomen. In the AI-implanted fish, plasma levels of E2 decreased in a dose-dependent manner, while the levels of 11-KT were high in the highest dose receiving group. Present results suggest that estrogen plays an important role in sex change of protogynous honeycomb grouper, and that treatments with AI potentially inhibits endogenous E2 production in vivo, causing oocyte degeneration and subsequently the sex inversion from female to male. The Fadrozole could be an important tool for manipulating the sex of hermaphrodite fishes.

Sex change in coral reef fish.

Gonadal differentiation can take many forms in fish, ranging from gonochorism, where individuals directly develop as male or female and finally possess only testis or ovaries at sexual maturation, to hermaphroditism where the same individuals can produce mature male and female gametes at some time in their lives. Hermaphrodite fish are, thus, an excellent model for studying the plasticity of sex determination and differentiation in vertebrates. We have shown that sex steroids play a principal role in sex differentiation and sex change in fish. Our laboratory implements several fish models that undergo sex change from female to male or male to female or in both directions. In this review, we will briefly discuss recent advances in our understanding of the mechanism of sex change in coral reef fish.

Immunohistochemical evidence identifying the site of androgen production in the ovary of the protogynous grouper Epinephelus merra.

Androgen plays an important role in the developing ovaries of female fish. However, little is known regarding either the sites of production of androgen or its functional roles. In the present study, we investigated immunohistochemically the localization of cholesterol-side-chain-cleavage (P450scc) and cytochrome P45011β-hydroxylase (P45011β) with antibodies P450scc and P45011β in the ovary of the female honeycomb grouper Epinephelus merra during its reproductive cycle. Clusters of strongly immunopositive cells, with 100–1000 cells in each cluster, against both P450scc and P45011β, were observed throughout the annual reproductive cycle in tissue near blood vessels in the tunica ovary surrounding the outer periphery of the ovary. The ultrastructural characteristics of these cells showed that they were steroid-producing cells. In contrast, immunopositive cells against P450scc but not against P45011β were localized in the theca layer surrounding the outer periphery of oocytes. These results suggest that two distinct steroid biosynthesis sites exist in the ovary and that cells at the two sites differ functionally. The only cells that biosynthesize 11-ketotestosterone are found in clusters in the vicinity of blood vessels; they possibly play a physiological role in oocyte growth and gonadal restructuring during the sex change of individuals of this species.