Zebrafish are an important model species in developmental biology. However, their potential in reproductive biology research has yet to be realized. In this study, we established See-Thru-Gonad zebrafish, a transparent line with fluorescently labeled germ cells visible throughout the life cycle, validated its gonadal development features, and demonstrated its applicability by performing a targeted gene knockdown experiment using vivo-morpholinos (VMOs). To establish the line, we crossed the zf45Tg and mitfa(w2/w2); mpv17(b18/b18) zebrafish lines. We documented the in vivo visibility of the germline-specific fluorescent signal throughout development, from gametes through embryonic and juvenile stages up to sexual maturity, and validated gonadal development with histology. We performed targeted gene knockdown of the microRNA (miRNA) miR-92a-3p through injection of VMOs directly to maturing ovaries. After the treatment, zebrafish were bred naturally. Embryos from miR-92a-3p knockdown ovaries had a significant reduction in relative miR-92a-3p expression and a higher percentage of developmental arrest at the 1-cell stage as compared with 5-base mismatch-treated controls. The experiment demonstrates that See-Thru-Gonad line can be successfully used for vertical transmission of the effects of targeted gene knockdown in ovaries into their offspring.
Metazoans exhibit two modes of primordial germ cell (PGC) specification that are interspersed across taxa. However, the evolutionary link between the two modes and the reproductive strategies of lecithotrophy and matrotrophy is poorly understood. As a first step to understand this, the spatio-temporal expression of teleostean germ plasm markers was investigated in Gambusia holbrooki, a poecilid with shared lecitho- and matrotrophy. A group of germ plasm components was detected in the ovum suggesting maternal inheritance mode of PGC specification. However, the strictly zygotic activation of dnd-β and nanos1 occurred relatively early, reminiscent of models with induction mode (e.g., mice). The PGC clustering, migration and colonisation patterns of G. holbrooki resembled those of zebrafish, medaka and mice at blastula, gastrula and somitogenesis, respectively—recapitulating features of advancing evolutionary nodes with progressive developmental stages. Moreover, the expression domains of PGC markers in G. holbrooki were either specific to teleost (vasa expression in developing PGCs), murine models (dnd spliced variants) or shared between the two taxa (germline and somatic expression of piwi and nanos1). Collectively, the results suggest that the reproductive developmental adaptations may reflect a transition from lecithotrophy to matrotrophy.
Built on our recent work that heart rates and function in G holbrooki are sexually dimorphic, this study assessed whether the species is an appropriate model to study sex-hormone effects on heart physiology. With a hypothesis that 17β-estradiol (E2) and 17α-methyltestosterone (MT) regulate the heart rate of juvenile G. holbrooki in a sex-specific manner, genetic males and females were treated with E2 and MT, respectively and the HR (bpm) was measured an hour following treatment using light-cardiogram. Results showed the HR (bpm) of both sexes were significantly (p < 0.05) altered compared to controls. Specifically, the E2 accelerated HR in the males and conversely MT decelerated the HR in the females. The normal expression levels of estrogen (erα and erβ) and G protein-coupled estrogen (gper) receptor genes were significantly higher (p < 0.05) in female than male hearts. Interestingly, the activity of the erβ in the heart of the MT-treated females reversed and was significantly lower (p < 0.05) than those of males while erα and gper were non-responsive. In contrast, significant down- and up-regulation of erα and gper, respectively occurred in the liver of MT-treated females. Morphological observations suggest that MT caused hepatomegaly, somewhat resembling an inflating balloon, perhaps induced by the accumulation of unexpelled gases. While E2 induced ventricular angiogenesis in males was likely due to an influx of blood supply caused by the increased heart rates. Collectively, the results demonstrate that the juvenile G. holbrooki heart readily responds to E2/MT in a sex-specific manner.
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