Hepatoprotective mechanism of freshwater clam extract alleviates non-alcoholic fatty liver disease: elucidated in vitro and in vivo models.

Freshwater clams (Corbicula fluminea) have long been used as a folk remedy in Chinese tradition. Their hot-water extract has been commercialized as a functional drink for liver protection. The objective of this study was to develop a product of the residual clam meat (FCR) and assess its functional compounds. The ethanol extract of FCR, designated FCRE, was identified to comprise phytosterols, polyunsaturated fatty acids (PUFAs) and carotenoids. FCRE significantly reduced lipid accumulation and cell death in HepG2 cells via decreased fatty acid synthase (FAS) activity and increased activities of carnitine palmitoyltransferase (CPT) and acyl-CoA oxidase (ACO), indicative of suppressed lipogenesis and increased ß-oxidation of fatty acids. In tilapia fed with high-fat diet (HFD), FCRE mitigated nonalcoholic steatohepatitis (NASH), which was evidenced by decreased levels of plasma aspartate transaminase (AST) and alanine transaminase (ALT), in addition to reduced total cholesterol and accumulation of triacylglycerols, particularly those of saturated and monounsaturated fatty acids. FCRE also suppressed stearoyl-CoA desaturase-1 (SCD-1) index, increased the PUFAs' n3/n6 ratio, and reduced prostaglandin E2 (PGE2) and inflammatory infiltrates in tilapia liver. Tilapia fed with HFD for 2 weeks displayed NASH symptoms, while mice took 10 weeks to display NASH symptoms. No previous study has been reported on the potential use of tilapia as an NASH model for pre-screening hepatoprotective-functional foods.

Morphology of reproductive accessory glands in female Sepia pharaonis (Cephalopoda: Sepiidae) sheds light on egg encapsulation.

The pharaoh cuttlefish, Sepia pharaonis, is an important cephalopod fishery species in southeastern Asia, with understudied reproductive physiology. The present study aimed to investigate the cellular characteristics of epithelial cells found in the nidamental glands (NGs) and accessory NGs (ANGs), as well as the structural connections between these two glands in mature female S. pharaonis. A histological analysis revealed two types of epithelial cells in NGs: Alcian blue-positive, PAS-negative mucosubstance-secreting cells and eosinophilic, PAS-positive granule-secreting cells. Using transmission electron microscopy, three types of epithelial cells were identified: cells with electron-dense granules, cells with electron-lucent granules, and cells with both cilia and microvilli in the apex. Mature ANGs contain an abundance of tubular units composed of epithelial cells resting on a thin layer of basal lamina. Innervated muscle cells are tightly adhered to the basal lamina. In addition, we observed epithelial canalization of ANG tubules penetrating through the connective tissue linking NGs and the walls of the tubules in ANGs, which allows the contents of the ANG tubules to be transported to the NGs. Our results suggest that ANGs participate in the encapsulation of the ova via the same pathway as NGs, which provides an important basis for future studies on the mechanism of protection provided by NGs and ANGs during embryonic development in S. pharaonis.

Reproductive Behavior and Embryonic Development of the Pharaoh Cuttlefish, Sepia pharaonis (Cephalopoda: Sepiidae).

Mong-Fong Lee, Chun-Yen Lin, Chuan-Chin Chiao, and Chung-Cheng Lu (2016) The pharaoh cuttlefish, Sepia pharaonis, is one of the most important cephalopod fishery species in southeastern Asia. In the present study, we described their reproductive behavior and characterized their embryonic development. Sperm competition during mating was high in S. pharaonis; therefore, consort males always escorted their mates after pairing, although sneaker males were frequently observed. Their egg-laying behavior can be divided into three phases. Females first retracted and bent their arms into a fist-like posture to spawn eggs. They then extended their arms forward and used funnels to blow the spawning ground. Finally, they extended their arms again to deposit eggs onto appropriate substrata. Based on the characteristics of the embryos, a set of easily distinguished criteria was developed to define 30 stages of embryonic development. This classification scheme was consistent with that of S. officinalis. The present study provided an important basis for future investigations of the reproductive biology and aquaculture in the pharaoh cuttlefish, S. pharaonis.

Development in a naturally acidified environment: Na+/H+-exchanger 3-based proton secretion leads to CO2 tolerance in cephalopod embryos.

BACKGROUND: Regulation of pH homeostasis is a central feature of all animals to cope with acid-base disturbances caused by respiratory CO2. Although a large body of knowledge is available for vertebrate and mammalian pH regulatory systems, the mechanisms of pH regulation in marine invertebrates remain largely unexplored. RESULTS: We used squid (Sepioteuthis lessoniana), which are known as powerful acid-base regulators to investigate the pH regulatory machinery with a special focus on proton secretion pathways during environmental hypercapnia. We cloned a Rhesus protein (slRhP), V-type H+-ATPase (slVHA) and the Na+/H+ exchanger 3 (slNHE3) from S. lessoniana, which are hypothesized to represent key players in proton secretion pathways among different animal taxa. Specifically designed antibodies for S. lessoniana demonstrated the sub-cellular localization of NKA, VHA (basolateral) and NHE3 (apical) in epidermal ionocytes of early life stages. Gene expression analyses demonstrated that slNHE3, slVHA and slRhP are up regulated in response to environmental hypercapnia (pH 7.31; 0.46 kPa pCO2) in body and yolk tissues compared to control conditions (pH 8.1; 0.045 kPa pCO2). This observation is supported by H+ selective electrode measurements, which detected increased proton gradients in CO2 treated embryos. This compensatory proton secretion is EIPA sensitive and thus confirms the central role of NHE based proton secretion in cephalopods. CONCLUSION: The present work shows that in convergence to teleosts and mammalian pH regulatory systems, cephalopod early life stages have evolved a unique acid-base regulatory machinery located in epidermal ionocytes. Using cephalopod molluscs as an invertebrate model this work provides important insights regarding the unifying evolutionary principles of pH regulation in different animal taxa that enables them to cope with CO2 induced acid-base disturbances.

Conserved sex-specific timing of meiotic initiation during sex differentiation in the protandrous black porgy Acanthopagrus schlegelii.

Meiosis is an essential mechanism of gametogenesis for all sexually reproducing species. In vertebrates, one conserved aspect of sex differentiation is that female embryonic germ cells enter meiosis earlier than male germ cells. In some lower vertebrates, female germ cells proliferate prior to entering meiosis, whereas male cells remain in mitotic arrest. Protandrous black porgy fish, Acanthopagrus schlegelii, have a dramatic life cycle involving a characteristic sex change. Black porgy are functional males for their first and second spawning seasons, but approximately half of the fish transform into females during their third year. We cloned the black porgy homologs of dosage suppressor of mck1 homolog (dmc1) and synaptonemal complex protein 3 (sycp3), and examined their expression profiles as well as those of cytochrome P450 family 26 genes (cyp26: cyp26a and cyp26b), retinaldehyde dehydrogenases (raldh: raldh2 and raldh3), retinoic acid receptors (rars: raralpha, rarbeta, rargamma, and rargammab), retinoid X receptors (rxrs: rxralpha, rxrbeta, and rxrgamma) and deleted azoospermia-like (dazl) during gonadal sex differentiation by RT-PCR, quantitative RT-PCR, and immunohistochemistry. Our results show that during gonadal development, germ cells located in ovarian tissue proceed into meiosis earlier than germ cells in testicular tissue. Furthermore, treatment with estradiol-17beta, which induced cyp26 expression, blocked dazl and raldh expression and reduced the expression of rars, rxrs, dmc1, and sycp3. This unique model therefore suggests that the temporal differences in meiosis initiation between females and males are conserved during gonadal sex differentiation in hermaphroditic vertebrates.

Development of the genital duct system in the protandrous black porgy, Acanthopagrus schlegeli.

Protandrous black porgies, Acanthopagrus schlegeli, have a striking life cycle, which includes early sex differentiation, bisexual gonads, and a male-to-female sex change at three years of age. We found novel features of and insights into the development of the genital duct system in relation to the gonadal stage during early gonadal development and natural sex change. We found that the genital ducts developed at 16-20 weeks of age during sex differentiation. The gonad developed "ovarian cavity cracks" and became "four-stranded" during the first prespawning period and then proceeded to the development of genital ducts before 1 year of age. Two ovarian cavities of the paired gonads combined, developed, and extended caudally to form the oviduct, making up the inner duct of the genital ducts. The testicular main cavities also extended and fused together to form the outer duct of the genital duct system, that is, the sperm duct. The coexistence of an outer sperm duct and an inner oviduct constituted a unique "double cannula genital duct" structure. Gradually the inferior walls of the oviduct intermingled with those of the sperm duct, and the circular lumen of the sperm duct changed into an "M-shaped canal." Finally, the sperm duct and oviduct separated completely at the distal part of genital duct system. During natural sex change, the male reproductive passage regressed and degenerated and was replaced by connective tissue. The oviduct arrested as a blunt end during male phase and, finally, extended and connected to the genital pore during the female phase.

Regulation of two forms of gonadotropin-releasing hormone receptor gene expression in the protandrous black porgy fish, Acanthopagrus schlegeli.

Two GnRH receptors (GnRH-R I and GnRH-R II) were obtained in protandrous black porgy (Acanthopagrus schlegeli). We investigated their tissue distribution, developmental/seasonal changes and regulation of expression using in vivo and in vitro (primary cultures of dispersed pituitary cells) approaches. The relative expressions of GnRH-Rs in the pituitary and gonad were as follows: pituitary: GnRH-R I > GnRH-R II; testicular tissue: GnRH-R I > GnRH-R II; ovarian tissue: GnRH-R I = GnRH-R II. GnRH-R I but not GnRH-R II expression was higher in the pituitary during the spawning period as compared to the prespawning. The expression profiles of both forms of GnRH-R were variable in the gonads according to the gonadal stage and season. In vivo, hCG stimulated GnRH-R I and GnRH-R II expression in testis and ovary. The LHRH analog also up-regulated both receptors in testis and but increased only GnRH-R II in the ovary. Sex steroids (estradiol, E2 and testosterone, T) increased the expression of both receptors in the testis and ovary. In the pituitary, sex steroids (E2 and T) increased the expression of GnRH-R I, but not GnRH-II, both in vivo and in vitro. The LHRH analog also specifically up-regulated the expression of GnRH-R I, but not GnRH-R II, by pituitary cells in vitro. All these data suggest that GnRH-R I rather than GnRH-R II may play a major physiological role in the pituitary. In contrast, both GnRH-R I and GnRH-R II may participate in the regulation of gonadal functions, including a possible role during sex change.

Sex differentiation and sex change in the protandrous black porgy, Acanthopagrus schlegeli.

Protandrous black porgy fish, Acanthopagrus schlegeli, have a striking life cycle with a male sex differentiation at the juvenile stage and male-to-female sex change at 3 years of age. We had characterized the sex differentiation and sex change in this species by the integrative approaches of histology, endocrine and molecular genetics. The fish differentiated in gonad at the age around 4-months and the gonad further developed with a bisexual gonad for almost for 3 years and sex change at 3 year of age. An antagonistic relationship in the testicular and ovarian tissues was found during the development of the gonadal tissue. Male- (such as sf-1, dmrt1, dax-1 and amh) and female- (such as wnt4, foxl2 and cyp19a1a) promoting genes were associated with testicular and ovarian development, respectively. During gonadal sex differentiation, steroidogenic pathway and estrogen signaling were also highly expressed in the brain. The increased expression of sf-1 and wnt4, cyp19a1a in ovarian tissue and decreased expression of dax-1 in the ovarian tissue may play important roles in sex change from a male-to-female. Endocrine factors such as estradiol and luteinizing hormone may also involve in the natural sex change. Estradiol induced the expression of female-promoting genes and resulted in the precocious sex change in black porgy. Our series of studies shed light on the sex differentiation and sex change in protandrous black porgy and other animals.

Current status of genetic and endocrine factors in the sex change of protandrous black porgy, Acanthopagrus schlegeli (Teleostean).

Black porgy, Acanthopagrus schlegeli Bleeker, a marine protandrous hermaphrodite fish, is functionally male for the first 2 years of life, but begins to sexually change to female after the third year. Testicular tissue and ovarian tissue are separated by connective tissue in the bisexual gonad. This sex pattern provides a unique model to study the mechanism of sex change in fish. The annual profiles of plasma estradiol, vitellogenin, and 11-ketotestosterone concentrations in males were significantly different from those in the 3-year-old females. Oral administration of estradiol stimulated high levels of gonadal aromatase activity, plasma luteinizing hormone (LH) levels, and sex change in the 2-year-old fish. Oral administration with aromatase inhibitors for 1 year further blocked the natural sex change in 3-year-old black porgy and all fish became functional males. Transcripts of estrogen receptor (ER), androgen receptor, and gonadotropin receptors in the ovarian tissue of bisexual gonad were significantly less expressed than those in the bisexual testicular tissue. ER and aromatase transcripts were much higher in the vitellogenic ovary than those in the bisexual ovarian tissue. Plasma LH levels were higher in male fish than sex-changing fish during postspawning and nonspawning season in 2(+)-year-old black porgy. We are also conducting investigations on the role of the genetic factors (Dmrt 1, Sox 9, Sf-1, and Dax-1) in sex development and sex change. An endocrine mechanism of sex change in black porgy is proposed.