RESUMEN
The central roles of luteinizing hormone (LH), progestin and their receptors for initiating ovulation have been well established. However, signaling pathways and downstream targets such as proteases that are essential for the rupture of follicular cells are still unclear. Recently, we found anovulation in nuclear progestin receptor (Pgr) knockout (Pgr-KO) zebrafish, which offers a new model for examining genes and pathways that are important for ovulation and fertility. In this study, we examined expression of all transcripts using RNA-Seq in preovulatory follicular cells collected following the final oocyte maturation, but prior to ovulation, from wild-type (WT) or Pgr-KO fish. Differential expression analysis revealed 3567 genes significantly differentially expressed between WT and Pgr-KO fish (fold change⩾2, p<0.05). Among those, 1543 gene transcripts were significantly more expressed, while 2024 genes were significantly less expressed, in WT than those in Pgr-KO. We then retrieved and compared transcriptional data from online databases and further identified 661 conserved genes in fish, mice, and humans that showed similar levels of high (283 genes) or low (387) expression in animals that were ovulating compared to those with no ovulation. For the first time, ovulatory genes and their involved biological processes and pathways were also visualized using Enrichment Map and Cytoscape. Intriguingly, enrichment analysis indicated that the genes with higher expression were involved in multiple ovulatory pathways and processes such as inflammatory response, angiogenesis, cytokine production, cell migration, chemotaxis, MAPK, focal adhesion, and cytoskeleton reorganization. In contrast, the genes with lower expression were mainly involved in DNA replication, DNA repair, DNA methylation, RNA processing, telomere maintenance, spindle assembling, nuclear acid transport, catabolic processes, and nuclear and cell division. Our results indicate that a large set of genes (>3000) is differentially regulated in the follicular cells in zebrafish prior to ovulation, terminating programs such as growth and proliferation, and beginning processes including the inflammatory response and apoptosis. Further studies are required to establish relationships among these genes and an ovulatory circuit in the zebrafish model.
Asunto(s)
Perfilación de la Expresión Génica , Ovulación/genética , Transcriptoma/genética , Pez Cebra/genética , Pez Cebra/fisiología , Animales , Regulación hacia Abajo/genética , Femenino , Técnicas de Inactivación de Genes , Humanos , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Progesterona/metabolismo , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN , Regulación hacia Arriba/genéticaRESUMEN
Our previous studies suggested that 17α,20ß-dihydroxy-4-pregnen-3-one (DHP), an oocyte maturation inducing progestin, also acts as a sex pheromone in Chinese black sleeper Bostrichthys sinensis, a fish species that inhabits intertidal zones and mates and spawns inside a muddy burrow. The electro-olfactogram response to DHP increased during the breeding season. In the present study, we cloned the cDNAs of the nine progestin receptors (pgr, paqr5, 6, 7(a, b), 8, 9, pgrmc1, 2) from B. sinensis, analyzed their tissue distribution, and determined the expression in the olfactory rosette during the reproductive cycle in female and male fish. The deduced amino acid sequences of the nine progestin receptors share high sequence identities with those of other fish species and relatively lower homology with their mammalian counterparts, and phylogenetic analyses classified the nine B. sinensis progestin receptors into their respective progestin receptor groups. Tissue distribution of B. sinensis progestin receptors showed differential expression patterns, but all these nine genes were expressed in the olfactory rosette. Interestingly, paqr5 mRNA was found in the intermediate and basal parts of the olfactory epithelium but not in the central core using in situ hybridization, and its expression level was the highest in the olfactory rosette among the tissues examined. These results suggested Paqr5 may have an important role for transmitting progestin signaling in the olfactory system. The expression levels of paqr7a and paqr7b, pgr and pgrmc2 mRNA peaked around the mid meiotic stage, and that of paqr8 peaked at late meiotic stage in the olfactory rosette in males, while the olfactory expression of paqr5 decreased gradually as spermatogenesis progressed. In contrast, the expression of the progestin receptors did not change significantly during the development of the ovary in the olfactory rosette in females, except that of pgr. Interestingly, the changes of paqr8 expression in the olfactory rosette in males mirrored the changes of plasma DHP levels in females during the reproductive cycle, suggesting the Paqr8 may also be important for deciphering progestin signaling released by female. To our knowledge, this is the first time to demonstrate the presence of all known progestin receptors in a teleost olfactory rosette, and to show different expressions between the males and females during the reproductive cycle. This study provides the first evidence on changes of all purported progestin receptors during a reproductive cycle in teleost olfactory rosette, and suggests that distinct olfactory sensitivities to DHP may be due to the changes and compositions of each progestin receptor in B. sinensis.
Asunto(s)
Peces/metabolismo , Filogenia , Receptores de Progesterona/metabolismo , Animales , Clonación Molecular , Femenino , Peces/genética , Masculino , Ovario/metabolismo , Progestinas/metabolismo , ARN Mensajero/genética , Receptores de Progesterona/genética , Reproducción/fisiología , EspermatogénesisRESUMEN
ABSTRACT: Meiosis is the process of producing haploid gametes through a series of complex chromosomal events and the coordinated action of various proteins. The mitochondrial protease complex (ClpXP), which consists of caseinolytic mitochondrial matrix peptidase X (ClpX) and caseinolytic protease P (ClpP) and mediates the degradation of misfolded, damaged, and oxidized proteins, is essential for maintaining mitochondrial homeostasis. ClpXP has been implicated in meiosis regulation, but its precise role is currently unknown. In this study, we engineered an inducible male germ cell-specific knockout caseinolytic mitochondrial matrix peptidase X (ClpxcKO) mouse model to investigate the function of ClpX in meiosis. We found that disrupting Clpx in male mice induced germ cell apoptosis and led to an absence of sperm in the epididymis. Specifically, it caused asynapsis of homologous chromosomes and impaired meiotic recombination, resulting in meiotic arrest in the zygotene-to-pachytene transition phase. The loss of ClpX compromised the double-strand break (DSB) repair machinery by markedly reducing the recruitment of DNA repair protein RAD51 homolog 1 (RAD51) to DSB sites. This dysfunction may be due to an insufficient supply of energy from the aberrant mitochondria in ClpxcKO spermatocytes, as discerned by electron microscopy. Furthermore, ubiquitination signals on chromosomes and the expression of oxidative phosphorylation subunits were both significantly attenuated in ClpxcKO spermatocytes. Taken together, we propose that ClpX is essential for maintaining mitochondrial protein homeostasis and ensuring homologous chromosome pairing, synapsis, and recombination in spermatocytes during meiotic prophase I.
RESUMEN
Accumulating evidence suggest that membrane progestin receptor α (mPRα) is the membrane receptor mediating nongenomic progestin signaling that induces oocyte maturation in teleost. However, the involvement of other members of mPR family in oocyte maturation is still unclear. In this study, we found impaired oocyte maturation in zebrafish lacking mPRα1, mPRα2, mPRß, or mPRγ2. In contrast, no difference was observed in oocyte maturation in the single knockout of mPRγ1, mPRδ, or mPRε. To study possible redundant functions of different mPRs in oocyte maturation, we generated a zebrafish line lacking all seven kinds of mPRs (mprs-/-). We found oocyte maturation was further impaired in mprs-/-. In addition, oocyte ovulation delay was observed in mprs-/- females, which was associated with low levels of nuclear progestin receptor (Pgr), a key regulator for ovulation. We also found reduced fertility in mprs-/- female zebrafish. Furthermore, eggs spawned by mprs-/- females were of poor quality.
Asunto(s)
Diferenciación Celular , Técnicas de Inactivación de Genes , Oocitos/metabolismo , Oocitos/patología , Ovulación , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Femenino , Fertilidad/efectos de los fármacos , Mutación/genética , Oocitos/efectos de los fármacos , Oogénesis/efectos de los fármacos , Ovulación/efectos de los fármacos , Progestinas/farmacología , Proteínas de Pez Cebra/genética , Cigoto/efectos de los fármacos , Cigoto/metabolismoRESUMEN
Previously we had identified adamts9 as a downstream target of Pgr, which is essential for ovulation in zebrafish. The primary goal of this study is to determine whether human chorionic gonadotropin (hCG, LH analog) also regulate adamts9 expression prior to ovulation. The expression of adamts9 was induced by hCG in a dose and time dependent manner in zebrafish preovulatory follicles in vitro. Interestingly, the stimulatory effect of hCG on adamts9 expression was not blocked in pgr-/- follicles but blocked in lhcgr-/-. This effect of hCG was via Lhcgr and its associated cAMP and PKC signaling pathways. Reduced fecundity and reduced expression of adamts9 were also found in lhcgr-/- females in vivo. Therefore, we have provided the first evidence of gonadotropin (hCG) regulated adamts9 in zebrafish, which could be important for ovulation.
Asunto(s)
Proteína ADAMTS9/genética , Gonadotropina Coriónica/farmacología , Folículo Ovárico/metabolismo , Receptores de HL/genética , Receptores de Progesterona/genética , Proteínas de Pez Cebra/genética , Pez Cebra/fisiología , Proteína ADAMTS9/metabolismo , Animales , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Fertilidad/efectos de los fármacos , Técnicas de Inactivación de Genes , Humanos , Folículo Ovárico/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Regulación hacia Arriba , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Toll-like receptors (TLRs) are major molecular pattern recognition receptors, which are essential for triggering a series of innate immune responses against invading pathogens by recognizing their evolutionary conserved molecular patterns. The mudskipper, Boleophthalmus pectinirostris is exceptional among fishes due to its amphibious lifestyle and adaptation to living on mudflats. The whole-genome sequencing of B. pectinirostris has revealed that this species possesses an expansion of Tlr11 family [12 Tlr11 family genes (one tlr21, 4 tlr22, and 7 tlr23)] that we focused on in the present study. The full-length cDNA sequences of the 12 tlrs in B. pectinirostris were cloned and their deduced amino acid sequences possessed a typical TLR domain arrangement. Likelihood tests of selection revealed that these 12 Tlr11 family genes are under diversifying selection. A total of 13 sites were found to be positively selected by more than one evolution model, of which 11 were located in the ligand-binding ectodomain. The observed non-synonymous substitutions may have functional implications in antigen and pathogen recognition specificity. These 12 tlrs were highly expressed in immune-related tissues, i.e. spleen and kidney. Tlr21 and tlr22b transcripts were significantly up-regulated by LPS, whereas tlr22a, tlr22d, tlr23b, tlr23e, tlr23g were significantly up-regulated by poly(I:C) in the spleen or/and kidney, which implies that the expanded Tlr11 family genes may play roles in protecting the fish from the invasion of gram-negative bacteria and double-stranded RNA viruses. The results from the present study suggested that the expansion of Tlr11 family genes in B. pectinirostris may recognize ligands from various pathogens found in the intertidal zone.
Asunto(s)
Evolución Molecular , Peces/genética , Peces/inmunología , Lipopolisacáridos/inmunología , Familia de Multigenes , Poli I-C/inmunología , Selección Genética , Receptores Toll-Like/genética , Animales , Mapeo Cromosómico , Peces/clasificación , Sitios Genéticos , Inmunidad Innata , Modelos Moleculares , Filogenia , Conformación Proteica , Análisis de Secuencia de ADN , Receptores Toll-Like/químicaRESUMEN
Ovulation requires proteinases to promote the rupture of ovarian follicles. However, the identity of these proteinases remains unclear. In our previous studies using RNA-seq analysis of differential expressed genes, we found significant down-regulation of five metalloproteinases: adam8b (a disintegrin and metalloproteinase domain 8b), adamts8a (a disintegrin and metalloproteinase with thrombospondin motif 8a), adamts9, mmp2 (matrix metalloproteinase 2), and mmp9 in the nuclear progestin receptor knockout (pgr -/-) zebrafish that have failed to ovulate. We hypothesize that these metalloproteinases are responsible for ovulation and are regulated by progestin and Pgr. In this study, we first determined the expression of these five metalloproteinases and adamts1 in preovulatory follicles at different times within the spawning cycle in pgr -/- and wildtype (wt) zebrafish and under varying hormonal treatments. We found that transcripts of adam8b, adamts1, adamts9, and mmp9 increased drastically in the preovulatory follicular cells of wt female zebrafish, while changes of adamts8a and mmp2 were not significant. This increase of adam8b, adamts9, and mmp9 was significantly reduced in pgr -/-, whereas expression of adamts1 was not affected in pgr -/- zebrafish. Among upregulated metalloproteinases, adamts9 mRNA was found to be expressed specifically in follicular cells. Strong immunostaining of Adamts9 protein was observed in the follicular cells of wt fish, and this expression was reduced drastically in pgr -/-. Interestingly, about an hour prior to the increase of metalloproteinases in wt fish, both Pgr transcript and protein increased transiently in preovulatory follicular cells. The results from in vitro experiments showed that adamts9 expression markedly increased in a dose, time and Pgr-dependent manner when preovulatory follicles were exposed to a progestin, 17α,20ß-dihydroxy-4-pregnen-3-one (DHP). Taken together, our results provide the first evidence that upregulation of adamts9 occurs specifically in preovulatory follicular cells of zebrafish prior to ovulation. Progestin and its receptor (Pgr) are essential for the upregulation of metalloproteinases.
RESUMEN
Our previous studies showed that 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP) acted as a sex pheromone to induce reproductive success in Chinese black sleeper (Bostrychus sinensis), but its functional mechanism remains unclear. In the present study, we cloned the cDNAs of the gonadotropin subunits (cgα, fshß, and lhß), and found that, in exposure to 5 nM DHP, transcript levels of lhß significantly increased in the pituitary at 6 h post exposure; plasma 11-KT levels increased at 24 h post exposure in mature male fish. In contrast, DHP exposure failed to increase the transcript levels of lhß in the pituitary of immature male fish, suggesting that the responsiveness to DHP depends on reproductive status. Interestingly, expression of progestin and adipoQ receptor 8 (paqr8, also known as mPRß) and progesterone receptor membrane component 2 significantly increased in the olfactory rosette of male fish at late meiosis stage following a co-injection of human chorionic gonadotropin (HCG) and luteinizing hormone releasing hormone-A3 (LHRH-A3), while no increases of other progestin receptors were observed. Moreover, Paqr8 protein was localized in the dendritic knobs of the olfactory sensory neurons, which were activated following the in vivo exposure to DHP. The DHP-induced expression of lhß in pituitary was not inhibited by RU486, an antagonist of nuclear progesterone receptor. Taken together, our results suggested that sex pheromone DHP increased the expression of lhß transcript in the pituitary and plasma 11-KT levels of mature male, important for reproduction; and Paqr8 might be involved in responding to sex pheromone DHP in the olfactory rosette of male B. sinensis.