Developmental dynamics of primordial germ cells, their genetic determinants and role in sex differentiation in Gambusia holbrooki

Primordial germ cells (PGC) are the progenitors of life; they develop into gametes and safeguard gonad function in sexually reproducing animals. By virtue of diverse reproductive strategies, fishes provide an invaluable field to study germ cell emergence and fate. The current knowledge of PGC development in live-bearing fish (poeciliids) is limited and instead it is predominantly restricted to well-studied oviparous teleost models such as zebrafish, Danio rerio and medaka, Oryzias latipes. To address this basic knowledge gap and to assist future development of species-specific reproductive biotechnologies to control invasive populations, this thesis focused on Gambusia holbrooki, a notorious pest fish. Beginning with a review of current knowledge, the developmental dynamics of germ cells in this species was evaluated employing the spatio-temporal expression of key germline markers and screening the morphological changes of gonads during the early stages of development. Also, this study discussed the potency of G. holbrooki as a model organism for fertility research, based on its developmental similarities with mammalian models. First, the study systematically reviewed the available literatures on PGC development, with a particular emphasis on teleosts, to synthesise evolutionary origin and mechanism of germ plasm (Gp) acquisition in fish taxa (Chapter 1). Specifically, the review identified two modes of germ cell development as distinguished by PGC specification of being either maternal (e.g., in teleost) or zygotic (e.g., in mammals) origin based on the role of epigenetic machinery in teleostean PGC emergence and fate. Further, prior knowledge of the interplay between molecular pathways and effector genes involved in germ cell signalling, mobilization and homing, and the regulatory RNA interference (RNAi) mechanisms leading to PGC specificity, their en routemigration and homing were distilled. These along with the role of non-coding RNAs (ncRNA), e.g., mi-RNA and pi-RNA, in germ line development was graphically illustrated to identify crucial roles and pathways that occur in fish PGC development. The contextual role of the nested germ cells in determining sexual fate of gonad and gonado-soma sexual identity via cell-cycle decisions and the timing of sex-biased germline stem cell (GSC) differentiation was also reviewed. This review formed the basis to address critical questions, including PGC specification mode, the pattern of germline migration, the temporal and spatial expression of key PGC markers during germline formation, and early gonadogenesis in G holbrooki. Spatio-temporal expression of germline markers is a powerful tool to investigate the processes of gonad formation and gametogenesis. Correspondingly, cloning, and characterisation of the target genes is a fundamental need when investigating non-model organisms whose genetic information is scarce and patchy. To address this, the full-length cDNA of the key germline markers including cxcr4, dazl, dnd, nanos1, piwi II and vasa in G. holbrooki were cloned and their identity established (Chapter 2). Standard rapid amplification of cDNA ends (RACE) techniques were used to establish testis and ovary specific libraries, followed by amplification of G holbrooki homologues using degenerate primers. Subsequently, the full-length cDNAs were cloned, characterised, the functional domains annotated and their evolutionary relationship to other poeciliid homologues established. The Dazl exhibited RNA recognition motif (RRM) and poly(A) binding protein (PABP) domains, while Deadend (Dnd) was encoded by two alternatively spliced variants, with conserved RRM and DSRM-DND domains in the predicted polypeptide. As expected, Piwi II had PAZ domain contained nucleic acid-binding interface in N-terminus and 5‚Äövт⧠RNA guide anchoring site at C-terminus. DEAD-box helicase motif was also conserved in predicted Vasa containing RNA- and ATP-binding sites. Similarly, chemokine binding site and zinc-finger motif in predicted Cxcr4 and Nanos1, respectively were present. Overall, the predicted amino acids of the studied genes showed a strong similarity with respective homologues of other poeciliids. In teleosts with preformation mode of PGC specification, maternal inheritance of Gp provides sufficient factors to form the germline fate of nascent PGCs protecting their identity, in contrast to the surrounding soma, right from post zygotic activation. Later, during embryonic patterning and cell determination, the Gp components are involved in proliferation, migration, and colonisation of germ cell precursors. To understand how and when PGC are formed, migrate, and colonise putative gonad in G holbrooki and how well these events are conserved across teleosts, in situ hybridisation and quantitative expression profiles of five Gp markers, namely dazl, dnd1, nanos, piwi II and vasa were studied (Chapter 3). Based on expression of PGC markers, a group of Gp components was detected in the ovum suggesting maternal inheritance mode of PGC specification in G. holbrooki. Subsequent to fertilisation, the results showed that vasa positive cells were detectable from late cleavage, later forming as two PGC clusters at the early gastrula stage, migrating anteriorly during somitogenesis and eventually colonised at the genital ridge before the pharyngula stage. Of all the markers only vasa and a dnd variant signals were restricted to PGCs. The embryonic expression of piwi II, dazl dnd-˜í‚⧠were detected in PGC clusters as well as the central nervous system. Interestingly, the embryonic expression of nanos1an indispensable PGC marker in zebrafish, was only found in neural tube. The quantitative gene expression profiles showed a transient female-biased surge of the PGC markers at the gastrula stage, suggesting an early onset of PGC proliferation in females. Strictly zygotic expression pattern of dnd-˜í‚⧠and nanos1 also showed maternal to zygotic transition (MZT) occurs before the mid-cleavage stage, in G. holbrooki. Collectively, the results suggest that despite sharing preformation mode of PGC specification, their migration pattern in G. holbrooki is unlike any other fish species studied so far. Instead, somatic expression of teleostean PGC markers (e.g., piwi II, nanos1 and dazl), splicing variation (e.g., dnd) and early MZT appear more similar to those of mammalian models. Gp is an electron dense body in ooplasm involved in germline determination of organisms with preformation mode of PGC specification including teleost. However, the expression patterns of Gp factors during gonadogenesis and their storage in gametes of poeciliids are unknown. To address this, quantitative gene expression of six markers of Gp components namely cxcr4, dazl, dnd-˜í¬±, piwi II, tdrd6 and vasa, with essential roles in gonad function among vertebrate models were evaluated for their expression profile in pre- (i.e., the pharyngula stage and right before parturition) and post-natal (i.e.,juvenile, and adult phase) stages of genetic male and female G. holbrooki (chapter 4). The overall results showed the relative accumulation of Gp components were comparable in prenatal stages of gonadogenesis between genetic males and females. However, their post-natal expression in gonads was female-biased (p<0.05), particularly as the animal reached puberty. Interestingly, the existing Gp transcripts and their abundance showed dissimilar patterns between ovum and spermatozoa of G. holbrooki. Collectively, this study, for the first time, revealed the Gp components involved in early germline determination are detectable not only in ovum, but also in mature spermatozoa indicating a potency for the contribution of paternal factors in PGC specification and function. Gonadogenesis is triggered with successful germ cell colonisation and gonadosoma emergence at the genital ridge followed by sexual identity acquisition through sex determination mechanisms or environmental cues. Among vertebrates, teleosts are known to exhibit diversified plans of gonadogenesis and sex differentiation, however, little is known in poeciliids. To ascertain when and what cellular and molecular determinants trigger sex differentiation in G. holbrooki, this study (Chapter 5) also documented critical events during early gonadogenesis using gonad histology, in situ hybridisation of germ cell markers and quantitative expression of gonadosoma markers. The histological observations showed that germ cells were first colonised at the genital ridge prior to complete somitogenesis to form presumptive gonads. Thereafter, germ cells underwent mitotic proliferation before acquisition of their sexual identity and this pattern was sex-dimorphic in G. holbrooki. Moreover, it was shown that sex differentiation of early gonads in G. holbrooki occurs right before parturition evinced from emerging meiotic primary oocytes stage I in ovary and formation of spermatogonial stem cell cysts in testis. Gonad histology and germ cell labelling showed that presumptive gonad in G. holbrooki undergoes major morphological transformation during its early development. Specifically, presumptive gonads first developed as two distinctive lobes, however, the lobes grew convergently and fused around the parturition stage to form single lobed testis or ovary, as occurs in adults. The quantitative expression pattern of a group of gonadosoma markers, foxl2, cyp19˜í¬±, amh, dmrt1, in pre- and post-natal developmental stages was consistent with morphological changes in early gonad; they were activated at the onset of gonad formation followed by sex-dimorphic expression pattern concurrent with sex differentiation of gonads. In conclusion, this study enlightened aspects of PGC development, their developmental dynamics and events involved in gonadogenesis of an invasive poeciliid fish, G. holbrooki. In concurrence with its ovo-viviparity, it also revealed resemblance between some aspects of its PGC d...