Apoptosis and autophagy-related gene transcription during ovarian follicular atresia in European hake (Merluccius merluccius).

Follicular atresia is an energy-saving oocyte resorption process that can allow the survival of female fish when environmental conditions are unfavourable and at the expense of fecundity. This study investigated the transcription levels of apoptosis and autophagy-related genes during atresia in the European hake that can show episodes of increased follicular atresia throughout the reproductive cycle. 169 female individuals were collected from the Bay of Biscay, and the ovaries were analysed using histological and molecular methods. Different levels of atresia were histologically detected in 73.7% of the ovaries analysed and the TUNEL assay identified apoptotic nuclei in follicles from both previtellogenic and vitellogenic stages. Transcripts of beclin-1 and ptenb were up-regulated in the ovaries containing atretic follicles, whereas p53, caspase-3, cathepsin D and dapk1 were up-regulated only in ovaries presenting vitellogenic atretic follicles. Our results indicate different implications of apoptotic vs autophagic processes leading to atresia during oocyte development, vitellogenesis being the moment of maximal apoptotic and autophagic activity in atretic hakes. The analysed genes could provide early warning biomarkers to identify follicular atresia in fish and evaluate fecundity in fish stocks.

Transcription pattern of reproduction relevant genes along the brain-pituitary-gonad axis of female, male and intersex thicklip grey mullets, Chelon labrosus, from a polluted harbor.

The reproductive cycle of teleost fishes is regulated by the brain-pituitary-gonad (BPG) axis. The transcription profile of genes involved in the reproduction signalling in the BPG-axis differs in females and males during the gametogenic cycle. Impacts of endocrine disrupting chemicals on these signalling pathways in fish are known, but the participation of the BPG-axis in the development of the intersex condition is not well understood. Intersex thicklip grey mullets (Chelon labrosus) have been identified in several estuaries from the SE Bay of Biscay, revealing the presence of feminizing contaminants in the area. In previous studies, transcription patterns of genes related with steroidogenesis and gamete growth have been shown to differ among female, male and intersex mullets. However, many components of the reproduction control have not been studied yet. The aim of this study was to assess the transcription levels of target BPG-axis genes in female, male and intersex mullets captured in the polluted harbour of Pasaia, during their gametogenic cycle. After histologically examining the gonads, the transcription levels of previously sequenced target genes were measured by qPCR: kiss2, gpr54 and gnrh1 in brain, fshß and lhß in pituitary and fshr and lhr in gonads. In both females and males, brain genes were most transcribed in early gametogenesis, proving their relevance in the onset of both oogenesis and spermatogenesis. Pituitary gonadotropins in females showed upregulation as oogenesis progressed, reaching the highest transcription levels at vitellogenic stage, while in males transcript levels were constant during spermatogenesis. Transcription levels of gonadotropin receptors showed different patterns in ovaries and testes, suggesting differing function in relation to gametogenesis and maturation. Intersex mullets showed transcription levels of brain target genes similar to those observed in females at cortical alveoli stage and to those in mid spermatogenic males. In intersex testes the transcription pattern of gonadotropin receptor fshr was downregulated in comparison to non-intersex testes.

Transcription of ribogenesis genes in fish gonads: Applications in the identification of stages of oogenesis and in environmental monitoring of intersex condition.

One of the best described effects of environmental xenoestrogens in fish is the generation of intersex gonads in males. Considering 5S rRNA a marker of the presence of oocytes, a 5S/18S rRNA index was calculated in 296 thicklip grey mullets (Chelon labrosus) from polluted environments. In addition, qPCR analysis of transcription factors gtf3a and ubtf1, related respectively to 5S and 18S rRNA synthesis, was conducted along female-oogenesis. 5S/18S rRNA index identified sex with a threshold value of 0.4521 separating males from females. Histological analysis identified 38 intersex individuals. Intersex severity and 5S/18S rRNA indexes were correlated. 5S/18S rRNA index identified ovarian developmental stage with high 5S rRNA levels during early oogenesis and 18S rRNA relative values increasing towards maturation. gtf3a and ubtf1 transcription levels followed the pattern of 5S rRNA accumulation. Thus, ribogenesis genes provide easy/quantitative methods to molecularly identify the sex, female gametogenic stage and intersex severity in mullets.

Alteration in molecular markers of oocyte development and intersex condition in mullets impacted by wastewater treatment plant effluents.

Wastewater Treatment Plant (WWTP) discharges are an important source of endocrine disrupting chemicals (EDCs) into the aquatic environment. Fish populations inhabiting downstream of WWTP effluents show alterations in gonad and gamete development such as intersex condition, together with xenoestrogenic effects such as vitellogenin up-regulation. However, the molecular mechanisms participating in the development of intersex condition in fish are not elucidated. The aim of this study was to assess the impact of two WWTPs effluents (Gernika and Bilbao-Galindo situated in the South East Bay of Biscay) with different contaminant loads, in thicklip grey mullet (Chelon labrosus) populations inhabiting downstream, examining the presence and severity of intersex condition, during two seasons. Molecular markers of xenoestrogenicity and oocyte differentiation and development (vtgAa, cyp19a1a, cyp19a1b, cyp11b, foxl2, dmrt1 and gtf3a) were also studied. Intersex mullets were identified downstream of both WWTPs and vtgAa was upregulated in intersex and non intersex males. Sex dependent differential transcription levels of target genes were detected in mullets from Galindo. However, no such pattern was observed in mullets from Gernika, suggesting an attenuating effect over studied genes caused by a higher presence of EDCs in this site, as indicated by the elevated prevalence of intersex mullets in this population. In conclusion, no direct association between xenoestrogenic responses and intersex condition was established. Mullets from Gernika showed signs of severe EDC exposure compared to those from Galindo, as demonstrated by the higher prevalence of intersex males and the reduction in transcription profile differences between sexes of gametogenic gene markers.