17ß-Estradiol Induced Sex Reversal and Gonadal Transcriptome Analysis in the Oriental River Prawn (Macrobrachium nipponense): Mechanisms, Pathways, and Potential Harm.

Sex reversal induced by 17ß-estradiol (E2) has shown the potential possibility for monoculture technology development. The present study aimed to determine whether dietary supplementation with different concentrations of E2 could induce sex reversal in M. nipponense, and select the sex-related genes by performing the gonadal transcriptome analysis of normal male (M), normal female (FM), sex-reversed male prawns (RM), and unreversed male prawns (NRM). Histology, transcriptome analysis, and qPCR were performed to compare differences in gonad development, key metabolic pathways, and genes. Compared with the control, after 40 days, feeding E2 with 200 mg/kg at PL25 (PL: post-larvae developmental stage) resulted in the highest sex ratio (female: male) of 2.22:1. Histological observations demonstrated the co-existence of testis and ovaries in the same prawn. Male prawns from the NRM group exhibited slower testis development without mature sperm. RNA sequencing revealed 3702 differentially expressed genes (DEGs) between M vs. FM, 3111 between M vs. RM, and 4978 between FM vs. NRM. Retinol metabolism and nucleotide excision repair pathways were identified as the key pathways for sex reversal and sperm maturation, respectively. Sperm gelatinase (SG) was not screened in M vs. NRM, corroborating the results of the slice D. In M vs. RM, reproduction-related genes such as cathepsin C (CatC), heat shock protein cognate (HSP), double-sex (Dsx), and gonadotropin-releasing hormone receptor (GnRH) were expressed differently from the other two groups, indicating that these are involved in the process of sex reversal. Exogenous E2 can induce sex reversal, providing valuable evidence for the establishment of monoculture in this species.

Sex Reversal Induced by Dietary Supplementation with 17α-Methyltestosterone during the Critical Period of Sex Differentiation in Oriental River Prawn (Macrobrachium nipponense).

The steroid 17α-methyltestosterone (MT) inhibits ovarian function and is often used to induce sex reversal artificially in vertebrates. In the present study, different concentrations of MT were added as dietary supplementation, and the effects on sex ratio, growth, and gonadal development were examined. After 40 days, the sex ratio (male:female) in each group increased at different degrees with 50 (1.36:1), 100 (1.57:1), and 200 (2.61:1) mg/kg MT, and neo-males with testis-ovary coexistence were observed in the 200 mg/kg MT group. Furthermore, 50 and 100 mg/kg MT could induce female reversion in neo-males. Histologically, the development of the testes in experimental groups was slower, but the ovaries of the experimental and control groups had similar developmental rates. The expression levels of DMRT11E, Foxl2, and SoxE1 in males at 200 mg/kg MT were 8.65-, 3.75-, and 3.45-fold greater than those of the control group. In crustaceans, sex reversal through vertebrate sex hormones can be observed. Neo-males (sex-reversed female prawns) were maintained by exogenous androgen, and over-reliance led to slow testis growth, small body size, and low growth rate, but sperm was still produced. In female prawns, MT inhibited ovary development and promoted growth.

Self-Prepared Hyaluronic Acid/Alkaline Gelatin Composite with Nano-Hydroxyapatite and Bone Morphogenetic Protein for Cranial Bone Formation.

New bone-forming substitute materials are highly useful in dental implantology. The purpose of this study was to prepare cross-linked hyaluronic acid (cHLA)/cross-linked alkaline gelatin (cAG)/nano-hydroxyapatite (nHAp)/bone morphogenic protein (BMP) constructs; and evaluate their bone-forming capabilities in rat cranial bone defects. The cHLA and cAG liquids processed with an epoxy cross-linker were blended with a 3:1 volume ratio, followed by freeze-drying. The dry composites were further infiltrated with water containing nHAp only (BMP (−)) or with water containing nHAp and BMP (BMP (+)). Prepared wet constructs (BMP (−) and BMP (+)) were implanted in rat cranial bone defects, while defects only were also made, and animals were fed for 8 weeks, followed by subsequent soft X-ray measurements and histological observations. The X-ray results showed that BMP (+) constructs disappeared, though caused inward extension of peripherical bone from defect edges with an increase in length of approximately 24%, larger than those of BMP (−) constructs and defect only with approximately 17% and 8% increments, respectively (p < 0.05). Histological observations of BMP (+) construct samples clearly indicated active bone extension consisting of an array of island-like bones. It was concluded that cHLA/cAG/nHAp/BMP could be used as novel bone-substitute materials.

Identification of potential functions of polo-like kinase 1 in male reproductive development of the oriental river prawn (Macrobrachium nipponense) by RNA interference analysis.

Polo-like kinase 1 (Plk1) has multiple functions in the cell cycle, including in the maturation of centrosomes during the G2/M transition, the separation of centrosomes, and the activation of cyclin-dependent kinase 1 expression and spindle assembly. In this study, we investigated the potential regulatory roles of Plk1 in the reproductive development of the male oriental river prawn (Machrobrachium nipponense). The full cDNA sequence of Mn-Plk1 was 2360 base pairs long, with an open reading frame of 1836 base pairs encoding 611 amino acids. Protein sequence alignment identified a conserved serine/threonine kinase domain and two Polo-boxes. Phylogenetic tree analysis revealed that Mn-Plk1 had the closest evolutionary distance with Plk1s of freshwater prawns and then with those of crustacean species, whereas the evolutionary distance with mollusks was much more distant. Quantitative PCR analysis predicted that Mn-Plk1 plays essential roles in the regulation of gonad development. RNA interference analysis and histological observations showed that expression of insulin-like androgenic gland hormone decreased as the expression of Mn-Plk1 decreased, and fewer than 5% of cells were sperm cells at day 14 in the dsPlk1 injected prawns. This result indicated that Plk1 positively regulated testis development in M. nipponense by affecting the expression of this hormone. Our results highlight the functions of Plk1 in M. nipponense and provide valuable information that can be applied to establish artificial techniques to regulate testis development in this species.

Morphological and biochemical changes during somatic embryogenesis in mahogany, Swietenia macrophylla (Meliaceae)

Intensive exploitation of mahogany wood (Swietenia macrophylla, Meliaceae) has resulted in the loss of natural populations. Somatic embryogenesis offers an alternative to clonal propagation and conservation of mahogany. This study describes biochemical (carbohydrates, total phenols, total flavonoids, protein, and plant growth regulators content) and histological characteristics of the somatic embryogenesis process in mahogany. Calli were obtained by culturing cotyledons of seeds from immature fruits for six weeks on semi-solid MS medium supplemented with 1.0 mgL-1 of kinetin and 4.0 mgL-1 of 2, 4-D. Primary callus was cultured on half strength semi-solid MS medium supplemented with 1.0 mgl-1 6-BA (6-benzylaminopurine) and embryogenic structures were obtained. Embryo development from globular-shaped somatic embryos to the cotyledonary stage was confirmed by histology and scanning electron microscopy. Shoot initiation was observed after somatic embryos were transferred to germination and maturation medium. Endogenous concentrations of carbohydrates, total phenols, total flavonoids, protein, and plant growth regulators were determined in embryogenic (EC) and non-embryogenic (NEC) calli of mahogany. Embryogenic cultures contained significantly higher concentrations of IAA (indoleacetic acid), ABA (abscisic acid), and GAs (Gibberellins 1+3+20), whereas non-embryogenic calli contained more total phenols, flavonoids and resistant starch. Fructose and glucose were not present at detectable levels in EC or NEC, whereas soluble starch and sucrose were only detectable in EC. Concentrations of total proteins, Z/ZR (Zeatin/zeatin riboside) and iP/iPA (N6-(Δ2-isopentenyl) adenine and N6-(Δ2-isopentenyl) adenosine) were similar in EC and NEC.

La explotación intensiva de la madera de caoba (Swietenia macrophylla) ha provocado la pérdida de poblaciones naturales. La embriogénesis somática ofrece una alternativa a la propagación clonal y la conservación de esta especie. Este estudio describe las características bioquímicas (contenido de carbohidratos, fenoles totales, flavonoides totales, proteínas y reguladores del crecimiento) e histológicas del proceso de embriogénesis somática en caoba. Los callos se obtuvieron cultivando cotiledones de semillas de frutos inmaduros durante seis semanas en medio MS semisólido suplementado con 1.0 mgL-1 de kinetina y 4.0 mgL-1 de 2, 4-D. Luego se cultivó el callo primario en medio MS semisólido y un suplemento de 1.0 mgl-1 BA y se obtuvieron estructuras embriogénicas. El desarrollo de embriones somáticos de forma globular a la etapa cotiledonar se confirmó por histología y microscopía electrónica de barrido. La iniciación del brote se observó después de que los embriones somáticos se transfirieron a un medio de germinación y maduración. Se determinaron las concentraciones endógenas de carbohidratos, fenoles totales, flavonoides totales, proteínas y reguladores del crecimiento en callos embriogénicos (EC) y no embriogénicos (NEC) de caoba. Los cultivos embriogénicos contenían concentraciones significativamente más altas de IAA, ABA y GA, mientras que los callos no embriogénicos contenían más fenoles totales, flavonoides y almidón resistente. La fructosa y la glucosa no estaban presentes en niveles detectables en EC o NEC, mientras que el almidón soluble y la sacarosa solo se detectaron en el EC. Las concentraciones de proteínas totales, Z / ZR e iP / iPA fueron similares en EC y NEC.

The Influence of Light on Olive (Olea europaea L.) Fruit Development Is Cultivar Dependent.

In olive, the response to environmental conditions, such as light availability, is under genetic control and requires a combination of biochemical and physiological events. We investigated the effect of irradiance in fruit development in two Italian cultivars, Leccino and Frantoio. Morphological and cyto-histological analyses, as well as water and oil content determination, were carried out in fruits exposed to a different light regime (named as light and shade fruits). Results demonstrated that the influence of light availability on fruit development depends on the cultivar. In Leccino, the fresh and the dry weight, the percentage of dry matter, the kernel and fruit diameter, the mesocarp thickness and the mesocarp cell size were higher in the light exposed fruits than in the ones grown in the shade. In Frantoio, differences between light and shade fruits were observed only at 140 DAF (Days After Flowering) and only in the kernel and fruit diameter and in the dry and fresh weight, which were higher in the light exposed fruits. Leccino, therefore, showed a greater sensitivity to the light availability. This may be related to the observed delay in the endocarp lignification as compared to the Frantoio cultivar. In each cultivar, moreover, shade and light fruits did not show differences in the timing of cell differentiation. Finally, the investigation of oil storage carried out in cyto-histological studies demonstrated that differences in oil content between fruit subjected to different light regimes correlated with the number of oil containing cells, rather than the oil content per cell. A different behaviour was observed in the two cultivars: in Leccino, the mesocarp cell size was almost twice of Frantoio, while oil drops were only 30% larger; therefore, the percentage of cell volume occupied by the oil drops was lower in Leccino than in Frantoio. The chemical analysis confirmed this observation.

The VviMYB80 Gene is Abnormally Expressed in Vitis vinifera L. cv. 'Zhong Shan Hong' and its Expression in Tobacco Driven by the 35S Promoter Causes Male Sterility.

Anther development is a very precise and complicated process. In Arabidopsis, the AtMYB80 transcription factor regulates genes involved in pollen development and controls the timing of tapetal programmed cell death (PCD). In this study, we isolated and characterized cDNA for VviMYB80 expressed in flower buds of male-sterile Vitis vinifera L. cv. 'Zhong Shan Hong', a late-maturing cultivar derived from self-progeny of cv. 'Wink'. VviMYB80 belongs to the MYB80 subfamily and clusters with AtMYB35/TDF1 in a distinct clade. We found that in flower buds, expression of the VviMYB80 gene in cv. 'Zhong Shan Hong' sharply increased at the tetrad stage, resulting in a higher and earlier transcript level than that found in cv. 'Wink'. Expression of the VviMYB80 gene, driven by the 35S promoter, caused pleiotropic effects on the stamens, including smaller and shriveled anthers, delayed dehiscence, fewer seeds, shorter anther filaments, distorted pollen shape and a lack of cytoplasm, with the tapetum exhibiting hypertrophy in transformed tobacco. These results suggest that VviMYB80 may play an important role in stamen development and that expression of VviMYB80 driven by the 35S promoter in tobacco induces male sterility.

Cloning, expression and methylation analysis of piwil2 in half-smooth tongue sole (Cynoglossus semilaevis).

piwi is an important regulator gene in germ cell division during spermatogenesis. piwi homologous genes are involved in gametogenesis and germline specification, and knocking down these genes could affect germ cell meiotic progression. To understand the function of piwi-related genes in spermatogenesis, we cloned a Piwi-subfamily member (piwil2 gene) from the gonad of Cynoglossus semilaevis. The full-length of piwil2 cDNA was 3314bp, including a 3162bp open reading frame (ORF), a 60bp 5'-UTR, along with a 92bp 3'-UTR, and encoded a predicted protein of 1053 amino acid residues. Phylogenetic analysis showed that the PIWIL2 putative protein belonged to the Argonaute protein family, and Piwi-subfamily, with typical PAZ and Piwi domains. Ultrathin sections of different gonadal stages, and real-time quantitative PCR showed that the relative expression of the piwil2 gene couldn't be detected until day 95 (95days) larvae, when germ cell divided rapidly in C. semilaevis. The piwil2 transcripts were more abundant in gonads of males and neo-males than in females, and weakly expressed in other tissues and organs. Compared with the relative expression of piwil2 in gonads, the CpG methylation levels were significantly higher in females. Chromosomal fluorescence in situ hybridization (FISH) showed that the piwil2 gene was located on the Z sex chromosome of C. semilaevis. These results suggest that piwil2 plays an important role in spermatogenesis of C. semilaevis.