Metallated phthalocyanines and their hydrophilic derivatives for multi-targeted oncological photodynamic therapy.

BACKGROUND AND AIM: A photosensitizer (PS) delivery and comprehensive tumor targeting platform was developed that is centered on the photosensitization of key pharmacological targets in solid tumors (cancer cells, tumor vascular endothelium, and cellular and non-cellular components of the tumor microenvironment) before photodynamic therapy (PDT). Interstitially targeted liposomes (ITLs) encapsulating zinc phthalocyanine (ZnPC) and aluminum phthalocyanine (AlPC) were formulated for passive targeting of the tumor microenvironment. In previous work it was established that the PEGylated ITLs were taken up by cultured cholangiocarcinoma cells. The aim of this study was to verify previous results in cancer cells and to determine whether the ITLs can also be used to photosensitize cells in the tumor microenvironment and vasculature. Following positive results, rudimentary in vitro and in vivo experiments were performed with ZnPC-ITLs and AlPC-ITLs as well as their water-soluble tetrasulfonated derivatives (ZnPCS4 and AlPCS4) to assemble a research dossier and bring this platform closer to clinical transition. METHODS: Flow cytometry and confocal microscopy were employed to determine ITL uptake and PS distribution in cholangiocarcinoma (SK-ChA-1) cells, endothelial cells (HUVECs), fibroblasts (NIH-3T3), and macrophages (RAW 264.7). Uptake of ITLs by endothelial cells was verified under flow conditions in a flow chamber. Dark toxicity and PDT efficacy were determined by cell viability assays, while the mode of cell death and cell cycle arrest were assayed by flow cytometry. In vivo systemic toxicity was assessed in zebrafish and chicken embryos, whereas skin phototoxicity was determined in BALB/c nude mice. A PDT efficacy pilot was conducted in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts. RESULTS: The key findings were that (1) photodynamically active PSs (i.e., all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells; (2) following PDT, photodynamically active PSs were highly toxic-to-potent as per anti-cancer compound classification; (3) the photodynamically active PSs did not elicit notable systemic toxicity in zebrafish and chicken embryos; (4) ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum-containing PSs did not exert detectable skin phototoxicity; and (5) ITL-delivered ZnPC and AlPC were equally effective in their tumor-killing capacity in human tumor breast cancer xenografts and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis. CONCLUSIONS: AlPC(S4) are the safest and most effective PSs to integrate into the comprehensive tumor targeting and PS delivery platform. Pending further in vivo validation, these third-generation PSs may be used for multi-compartmental tumor photosensitization.

Pituitary Gonadotropin Gene Expression During Induced Onset of Postsmolt Maturation in Male Atlantic Salmon: In Vivo and Tissue Culture Studies.

Precocious male maturation causes reduced welfare and increased production costs in Atlantic salmon (Salmo salar) aquaculture. The pituitary produces and releases follicle-stimulating hormone (Fsh), the gonadotropin triggering puberty in male salmonids. However, little is known about how Fsh production is regulated in Atlantic salmon. We examined, in vivo and ex vivo, transcriptional changes of gonadotropin-related genes accompanying the initial steps of testis maturation, in pituitaries of males exposed to photoperiod and temperature conditions promoting maturation (constant light and 16°C). Pituitary fshb, lhb and gnrhr2bba transcripts increased in vivo in maturing males (gonado-somatic index > 0.1%). RNA sequencing (RNAseq) analysis using pituitaries from genetically similar males carrying the same genetic predisposition to mature, but differing by responding or not responding to stimulatory environmental conditions, revealed 144 differentially expressed genes, ~2/3rds being up-regulated in responders, including fshb and other pituitary hormones, steroid-related and other puberty-associated transcripts. Functional enrichment analyses confirmed gene involvement in hormone/steroid production and gonad development. In ex vivo studies, whole pituitaries were exposed to a selection of hormones and growth factors. Gonadotropin-releasing hormone (Gnrh), 17ß-estradiol (E2) and 11-ketotestosterone (11-KT) up-regulated gnrhr2bba and lhb, while fshb was up-regulated by Gnrh but down-regulated by 11-KT in pituitaries from immature males. Also pituitaries from maturing males responded to Gnrh and sex steroids by increased gnrhr2bba and lhb transcript levels, but fshb expression remained unchanged. Growth factors (inhibin A, activin A and insulin-like growth factor 1) did not change gnrhr2bba, lhb or fshb transcript levels in pituitaries either from immature or maturing males. Additional pituitary ex vivo studies on candidates identified by RNAseq showed that these transcripts were preferentially regulated by Gnrh and sex steroids, but not by growth factors, and that Gnrh/sex steroids were less effective when incubating pituitaries from maturing males. Our results suggest that a yet to be characterized mechanism up-regulating fshb expression in the salmon pituitary is activated in response to stimulatory environmental conditions prior to morphological signs of testis maturation, and that the transcriptional program associated with this mechanism becomes unresponsive or less responsive to most stimulators ex vivo once males had entered pubertal developmental in vivo.

Insulin-like 3 affects zebrafish spermatogenic cells directly and via Sertoli cells.

Pituitary hormones can use local signaling molecules to regulate target tissue functions. In adult zebrafish testes, follicle-stimulating hormone (Fsh) strongly increases the production of insulin-like 3 (Insl3), a Leydig cell-derived growth factor found in all vertebrates. Little information is available regarding Insl3 function in adult spermatogenesis. The Insl3 receptors Rxfp2a and 2b were expressed by type A spermatogonia and Sertoli and myoid cells, respectively, in zebrafish testis tissue. Loss of insl3 increased germ cell apoptosis in males starting at 9 months of age, but spermatogenesis appeared normal in fully fertile, younger adults. Insl3 changed the expression of 409 testicular genes. Among others, retinoic acid (RA) signaling was up- and peroxisome proliferator-activated receptor gamma (Pparg) signaling was down-regulated. Follow-up studies showed that RA and Pparg signaling mediated Insl3 effects, resulting in the increased production of differentiating spermatogonia. This suggests that Insl3 recruits two locally active nuclear receptor pathways to implement pituitary (Fsh) stimulation of spermatogenesis.

PGE2 inhibits spermatogonia differentiation in zebrafish: interaction with Fsh and an androgen.

Changes in zebrafish testicular gene expression induced by follicle-stimulating hormone (Fsh) or anti-Mullerian hormone (Amh) suggested that Amh inhibition and Fsh stimulation of spermatogenesis involved up and downregulation, respectively, of prostaglandin (PG) signaling. We found that Sertoli cells contacting type A undifferentiated (Aund) and differentiating (Adiff) spermatogonia expressed a key enzyme of PG production (Ptgs2); previous work showed that Sertoli cells contacting Adiff and B spermatogonia and spermatocytes showed ptges3b expression, an enzyme catalyzing PGE2 production. In primary testis tissue cultures, PGE2, but not PGD2 or PGF2α, reduced the mitotic activity of Adiff and their development into B spermatogonia. Vice versa, inhibiting PG production increased the mitotic activity of Adiff and B spermatogonia. Studies with pharmacological PG receptor antagonists suggest that an Ep4 receptor mediates the inhibitory effects on the development of spermatogonia, and cell-sorting experiments indicated this receptor is expressed mainly by testicular somatic cells. Combined inhibition of PG and steroid production moreover reduced the mitotic activity of Aund spermatogonia and led to their partial depletion, suggesting that androgens (and/or other testicular steroids), supported by PGE2, otherwise prevent depletion of Aund. Androgens also decreased testicular PGE2 production, increased the transcript levels of the enzyme-catabolizing PGs and decreased PGE2 receptor ptger4b transcript levels. Also Fsh potentially reduced, independent of androgens, PGE2 production by decreasing ptges3b transcript levels. Taken together, our results indicate that PGE2, via Ep4 receptors, favors self-renewal in conjunction with androgens and, independent of Fsh and androgens, inhibits differentiating divisions of spermatogonia.

Gnrh receptor gnrhr2bbα is expressed exclusively in lhb-expressing cells in Atlantic salmon male parr.

Gonadotropin-releasing hormone (Gnrh) plays a major role in the regulation of physiological and behavioural processes related to reproduction. In the pituitary, it stimulates gonadotropin synthesis and release via activation of Gnrh receptors (Gnrhr), belonging to the G protein-coupled receptor superfamily. Evidence suggests that differential regulation of the two gonadotropins (Fsh and Lh) is achieved through activation of distinct intracellular pathways and, probably, through the action of distinct receptors. However, the roles of the different Gnrhr isoforms in teleosts are still not well understood. This study investigates the gene expression of Gnrhr in the pituitary gland of precociously maturing Atlantic salmon (Salmo salar) male parr. A total of six Gnrhr paralogs were identified in the Atlantic salmon genome and named according to phylogenetic relationship; gnrhr1caα, gnrhr1caß, gnrhr1cbα, gnrhr1cbß, gnrhr2bbα, gnrhr2bbß. All paralogs, except gnrhr1caα, were expressed in male parr pituitary during gonadal maturation as evidenced by qPCR analysis. Only one gene, gnrhr2bbα, was differentially expressed depending on maturational stage (yearly cycle), with high expression levels in maturing fish, increasing in parallel with gonadotropin subunit gene expression. Additionally, a correlation in daily expression levels was detected between gnrhr2bbα and lhb (daily cycle) in immature fish in mid-April. Double fluorescence in situ hybridization showed that gnrhr2bbα was expressed exclusively in lhb gonadotropes in the pituitary, with no expression detected in fshb cells. These results suggest the involvement of receptor paralog gnrhr2bbα in the regulation of lhb cells, and not fshb cells, in sexually maturing Atlantic salmon male parr.

Endocrine and local signaling interact to regulate spermatogenesis in zebrafish: follicle-stimulating hormone, retinoic acid and androgens.

Retinoic acid (RA) is crucial for mammalian spermatogonia differentiation, and stimulates Stra8 expression, a gene required for meiosis. Certain fish species, including zebrafish, have lost the stra8 gene. While RA still seems important for spermatogenesis in fish, it is not known which stage(s) respond to RA or whether its effects are integrated into the endocrine regulation of spermatogenesis. In zebrafish, RA promoted spermatogonia differentiation, supported androgen-stimulated meiosis, and reduced spermatocyte and spermatid apoptosis. Follicle-stimulating hormone (Fsh) stimulated RA production. Expressing a dominant-negative RA receptor variant in germ cells clearly disturbed spermatogenesis but meiosis and spermiogenesis still took place, although sperm quality was low in 6-month-old adults. This condition also activated Leydig cells. Three months later, spermatogenesis apparently had recovered, but doubling of testis weight demonstrated hypertrophy, apoptosis/DNA damage among spermatids was high and sperm quality remained low. We conclude that RA signaling is important for zebrafish spermatogenesis but is not of crucial relevance. As Fsh stimulates androgen and RA production, germ cell-mediated, RA-dependent reduction of Leydig cell activity may form a hitherto unknown intratesticular negative-feedback loop.

Regulation of spermatogonial development by Fsh: The complementary roles of locally produced Igf and Wnt signaling molecules in adult zebrafish testis.

Spermatogenesis is a cellular developmental process characterized by the coordinated proliferation and differentiation activities of somatic and germ cells in order to produce a large number of spermatozoa, the cellular basis of male fertility. Somatic cells in the testis, such as Leydig, peritubular myoid and Sertoli cells, provide structural and metabolic support and contribute to the regulatory microenvironment required for proper germ cell survival and development. The pituitary follicle-stimulating hormone (Fsh) is a major endocrine regulator of vertebrate spermatogenesis, targeting somatic cell functions in the testes. In fish, Fsh regulates Leydig and Sertoli cell functions, such as sex steroid and growth factor production, processes that also control the development of spermatogonia, the germ cell stages at the basis of the spermatogenic process. Here, we summarize recent advances in our understanding of mechanisms used by Fsh to regulate the development of spermatogonia. This involves discussing the roles of insulin-like growth factor (Igf) 3 and canonical and non-canonical Wnt signaling pathways. We will also discuss how these locally active regulatory systems interact to maintain testis tissue homeostasis.

Correction to: Entry into puberty is reflected in changes in hormone production but not in testicular receptor expression in Atlantic salmon (Salmo salar).

Following publication of the original article [1], the authors would like to apologize for an error in Fig. 5e, the correct graph is presented below and shows the significant increase in pituitary mRNA levels of fshb in recruited males in the SGA stage.

The initiation of puberty in Atlantic salmon brings about large changes in testicular gene expression that are modulated by the energy status.

BACKGROUND: When puberty starts before males reach harvest size, animal welfare and sustainability issues occur in Atlantic salmon (Salmo salar) aquaculture. Hallmarks of male puberty are an increased proliferation activity in the testis and elevated androgen production. Examining transcriptional changes in salmon testis during the transition from immature to maturing testes may help understanding the regulation of puberty, potentially leading to procedures to modulate its start. Since differences in body weight influence, via unknown mechanisms, the chances for entering puberty, we used two feed rations to create body weight differences. RESULTS: Maturing testes were characterized by an elevated proliferation activity of Sertoli cells and of single undifferentiated spermatogonia. Pituitary gene expression data suggest increased Gnrh receptor and gonadotropin gene expression, potentially responsible for the elevated circulating androgen levels in maturing fish. Transcriptional changes in maturing testes included a broad variety of signaling systems (e.g. Tgfß, Wnt, insulin/Igf, nuclear receptors), but also, activation of metabolic pathways such as anaerobic metabolism and protection against ROS. Feed restriction lowered the incidence of puberty. In males maturing despite feed restriction, plasma androgen levels were higher than in maturing fish receiving the full ration. A group of 449 genes that were up-regulated in maturing fully fed fish, was up-regulated more prominently in testis from fish maturing under caloric restriction. Moreover, 421 genes were specifically up-regulated in testes from fish maturing under caloric restriction, including carbon metabolism genes, a pathway relevant for nucleotide biosynthesis and for placing epigenetic marks. CONCLUSIONS: Undifferentiated spermatogonia and Sertoli cell populations increased at the beginning of puberty, which was associated with the up-regulation of metabolic pathways (e.g. anaerobic and ROS pathways) known from other stem cell systems. The higher androgen levels in males maturing under caloric restriction may be responsible for the stronger up-regulation of a common set of (449) maturation-associated genes, and the specific up-regulation of another set of (421) genes. The latter opened regulatory and/or metabolic options for initiating puberty despite feed restriction. As a means to reduce the incidence of male puberty in salmon, however, caloric restriction seems unsuitable.

Entry into puberty is reflected in changes in hormone production but not in testicular receptor expression in Atlantic salmon (Salmo salar).

BACKGROUND: Puberty in male Atlantic salmon in aquaculture can start as early as after the first winter in seawater, stunts growth and entails welfare problems due to the maturation-associated loss of osmoregulation capacity in seawater. A better understanding of the regulation of puberty is the basis for developing improved cultivation approaches that avoid these problems. Our aim here was to identify morphological and molecular markers signaling the initiation of, and potential involvement in, testis maturation. METHODS: In the first experiment, we monitored for the first time in large Atlantic salmon males several reproductive parameters during 17 months including the first reproductive cycle. Since testicular growth accelerated after the Winter solstice, we focused in the second experiment on the 5 months following the winter solstice, exposing fish from February 1 onwards to the natural photoperiod (NL) or to continuous additional light (LL). RESULTS: In the first experiment, testis weight, plasma androgens and pituitary gonadotropin transcript levels increased with the appearance of type B spermatogonia in the testis, but testicular transcript levels for gonadotropin or androgen receptors did not change while being clearly detectable. In the second experiment, all males kept under NL had been recruited into puberty until June. However, recruitment into puberty was blocked in ~ 40% of the males exposed to LL. The first morphological sign of recruitment was an increased proliferation activity of single spermatogonia and Sertoli cells. Irrespective of the photoperiod, this early sign of testis maturation was accompanied by elevated pituitary gnrhr4 and fshb and testicular igf3 transcript levels as well as increased plasma androgen levels. The transition into puberty occurred again with stable testicular gonadotropin and androgen receptor transcript levels. CONCLUSIONS: The sensitivity to reproductive hormones is already established before puberty starts and up-regulation of testicular hormone receptor expression is not required to facilitate entry into puberty. The increased availability of receptor ligands, on the other hand, may result from an up-regulation of pituitary Gnrh receptor expression, eventually activating testicular growth factor and sex steroid release and driving germ and Sertoli cell proliferation and differentiation.

Japanese medaka as a model for studying the relaxin family genes involved in neuroendocrine regulation: Insights from the expression of fish-specific rln3 and insl5 and rxfp3/4-type receptor paralogues.

The goal of this paper is to establish Japanese medaka (Oryzias latipes) as a model for relaxin family peptide research, particularly for studying the functions of RLN3 and INSL5, hormones playing roles in neuroendocrine regulation. Medaka, like other teleosts, retained duplicate copies of rln3, insl5 and their rxfp3/4-type receptors following fish-specific whole genome duplication (WGD) and paralogous copies of these genes may have sub-functionalised providing an intuitive model for teasing apart the pleiotropic roles of the corresponding genes in mammals. To this end, we provide experimental evidence for the expression of the relaxin family genes in medaka that had previously only been identified in-silico, confirm the gene structure of five of the ligand genes, characterise gene expression across multiple tissues and during embryonic development, perform in situ hybridization with anti-sense insl5a on embryos and in adult brain and intestinal samples, and compare these results to the data available in zebrafish. We find broad similarities but also some differences in the expression of relaxin family genes in zebrafish versus medaka, and find support for the hypothesis that the rln3a/rln3b and insl5a/insl5b paralogues have been subfunctionalized. Given that medaka has a suite of relaxin family genes more similar to other teleosts, and has retained the gene for rxfp4 (which is lost in zebrafish), our results suggest that O. latipes may be a good model for delineating the ancestral function of the relaxin family genes involved in neuroendocrine regulation.

Fsh stimulates Leydig cell Wnt5a production, enriching zebrafish type A spermatogonia.

Follicle-stimulating hormone (Fsh) modulates vertebrate spermatogenesis by regulating somatic cell functions in the testis. We have found previously that zebrafish Fsh stimulated the differentiating proliferation of type A undifferentiated spermatogonia (Aund) in an androgen-independent manner by regulating the production of growth factors and other signaling molecules in both Sertoli (SCs) and Leydig cells (LCs). For example, Fsh triggered the release of Igf3 that subsequently activated ß-catenin signaling to promote the differentiating proliferation of Aund. In the present study, we report that Fsh moreover uses the non-canonical Wnt pathway to promote the proliferation and accumulation of Aund. Initially, we found that the stimulatory effect of Fsh on the proliferation activity of Aund was further strengthened when ß-catenin signaling was inhibited, resulting in an accumulation of Aund. We then showed that this Fsh-induced accumulation of Aund was associated with increased transcript levels of the non-canonical Wnt ligand, wnt5a. In situ hybridization of insl3 mRNA, a gene expressed in LCs, combined with Wnt5a immunocytochemistry identified LCs as the cellular source of Wnt5a in the adult zebrafish testis. Addition of an antagonist of Wnt5a to incubations with Fsh decreased both the proliferation activity and the relative section area occupied by Aund, while an agonist of Wnt5a increased these same parameters for Aund. Taken together, our data suggest that Fsh triggered LCs to release Wnt5a, which then promoted the proliferation and accumulation of Aund. Hence, Fsh uses non-canonical Wnt signaling to ensure the production of Aund, while also triggering ß-catenin signaling via Igf3 to ensure spermatogonial differentiation.

Igf3 activates ß-catenin signaling to stimulate spermatogonial differentiation in zebrafish.

Follicle-stimulating hormone (Fsh) is a major regulator of spermatogenesis, targeting somatic cell functions in the testes. We reported previously that zebrafish Fsh promoted the differentiation of type A undifferentiated spermatogonia (Aund) by stimulating the production of factors that advance germ cell differentiation, such as androgens, insulin-like peptide 3 (Insl3) and insulin-like growth factor 3 (Igf3). In addition, Fsh also modulated the transcript levels of several other genes, including some belonging to the Wnt signaling pathway. Here, we evaluated if and how Fsh utilizes part of the canonical Wnt pathway to regulate the development of spermatogonia. We quantified the proliferation activity and relative section areas occupied by Aund and type A differentiating (Adiff) spermatogonia and we analyzed the expression of selected genes in response to recombinant proteins and pharmacological inhibitors. We found that from the three downstream mediators of Fsh activity we examined, Igf3, but not 11-ketotestosterone or Insl3, modulated the transcript levels of two ß-catenin sensitive genes (cyclinD1 and axin2). Using a zebrafish ß-catenin signaling reporter line, we showed that Igf3 activated ß-catenin signaling in type A spermatogonia and that this activation did not depend on the release of Wnt ligands. Pharmacological inhibition of the ß-catenin or of the phosphoinositide 3-kinase (PI3K) pathways revealed that Igf3 activated ß-catenin signaling in a manner involving PI3K to promote the differentiation of Aund to Adiff spermatogonia. This mechanism represents an intriguing example for a pituitary hormone like Fsh using Igf signaling to recruit the evolutionary conserved, local ß-catenin signaling pathway to regulate spermatogenesis.

Estrogen-induced inhibition of spermatogenesis in zebrafish is largely reversed by androgen.

The hormonal regulation of spermatogenesis involves both gonadotropins and steroid hormones. Long-term in vivo exposure of adult zebrafish to estrogen impaired spermatogenesis associated with an androgen insufficiency, possibly induced by inhibiting gonadotropin release. Using this experimental model, we investigated if androgen treatment could enhance spermatogenesis, while maintaining the inhibition of gonadotropin release through continued estrogen exposure. Moreover, we also exposed animals to androgen alone, in order to examine androgen effects in the absence of estrogen-induced gonadotropin inhibition. Estrogen exposure depleted type B spermatogonia, meiotic and postmeiotic germ cells from the adult testis, but promoted the proliferation of type A undifferentiated spermatogonia, which accumulated in the testis. This change in germ cell composition was accompanied by reduced mRNA levels of those growth factors (e.g. insl3 and igf3) expressed by testicular somatic cells and known to stimulate spermatogonial differentiation in zebrafish. Additional androgen (11-ketoandrostenedione, which is converted to 11-ketotestosterone) treatment in vivo reversed most of the effects of estrogen exposure on spermatogenesis while insl3 and igf3 transcript levels remained suppressed. When androgen treatment was given alone, it promoted the production of haploid cells at the expense of spermatogonia, and increased transcript levels of some growth factor and hormone receptor genes, but not those of insl3 or igf3 We conclude that estrogen exposure efficiently inhibits spermatogenesis because it induces androgen insufficiency and suppresses gonadotropin-regulated growth factors known to stimulate germ cell differentiation. Moreover, our results suggest that androgens and the growth factors Insl3 and Igf3 stimulate spermatogenesis via independent pathways.

The relaxin receptor as a therapeutic target - perspectives from evolution and drug targeting.

The peptide relaxin was first identified as an important circulating hormone during pregnancy over 90 years ago. Research over many years defined the numerous biological roles that relaxin plays throughout pregnancy in many mammalian species. These important biological actions have led to the testing of relaxin as a therapeutic agent for a number of indications. The discovery of the relaxin receptor, RXFP1, in 2002 facilitated the better understanding of the cellular targets of relaxin, its mechanism of action and enabled the development of relaxin mimetics and screening for small molecule agonists. Additionally, the rapid expansion of the genome databases and bioinformatics tools has significantly advanced our understanding of the evolution of the relaxin/RXFP1 signaling system. It is now clear that the relaxin-RXFP1 signaling axis is far more ancient than previously appreciated with important roles for invertebrate relaxin-like peptides in reproductive and non-reproductive functions. This review summarizes these advances as well as developments in drug targeting of RXFP1. Hence the complex mode of activation of RXFP1 is discussed as is the discovery and development of a peptide mimetic and small molecule agonist. Detailed signaling studies are summarized which highlight the cell specific signaling of a peptide mimetic and biased signaling of a small molecule agonist. These studies highlight the complexities of targeting peptide GPCRs such as RXFP1.

Characterization of vasa homolog in a neotropical catfish, Jundiá (Rhamdia quelen): Molecular cloning and expression analysis during embryonic and larval development.

We have characterized the full-length vasa cDNA from Jundiá, Rhamdia quelen (Heptapteridae, Siluriformes). vasa encodes a member of the DEAD-box protein family of ATP-dependent RNA helicases. This protein is highly conserved among different organisms and its role is associated with RNA metabolism. In the majority of the investigated species, vasa is restricted to the germ cell lineage and its expression has been used to study germline development in many organisms, including fish. The deduced R. quelen vasa amino acid sequence displayed high similarity with Vasa protein sequences from other organisms, and did not cluster with PL10 or P68 DEAD-box protein subfamilies. We also reported that there is no other isoform for vasa mRNA in R. quelen gonads. Expression analysis by RT-PCR and qPCR showed vasa transcripts exclusively expressed in the germ cells of R. quelen gonads. R. quelen vasa mRNA was maternally inherited, and was detected in the migrating primordial germ cells (PGCs) until 264 h post-fertilization during embryonic and larval development. This work has characterized for the first time the full-length R. quelen vasa cDNA, and describes its expression patterns during R. quelen embryonic and larval development. Our results will contribute to the basic reproductive biology of this native species, and will support studies using vasa as a germ cell marker in different biotechnological studies, such as germ cell transplantation.

Follicle-Stimulating Hormone Regulates igfbp Gene Expression Directly or via Downstream Effectors to Modulate Igf3 Effects on Zebrafish Spermatogenesis.

Previous work showed that pharmacological inactivation of Igf-binding proteins (Igfbps), modulators of Igf activity, resulted in an excessive differentiation of type A undifferentiated (Aund) spermatogonia in zebrafish testis in tissue culture when Fsh was present in the incubation medium. Using this testis tissue culture system, we studied here the regulation of igfbp transcript levels by Fsh and two of its downstream effectors, Igf3 and 11-ketotestosterone (11-KT). We also explored how Fsh-modulated igfbp expression affected spermatogonial proliferation by adding or removing the Igfbp inhibitor NBI-31772 at different times. Fsh (100 ng/mL) decreased the transcript levels of igfbp1a, -3, and -6a after 1 or 3 days, while increasing igfbp2a and -5b expression, but only after 5 days of incubation. Igf3 down-regulated the same igfbp transcripts as Fsh but with a delay of at least 4 days. 11-KT increased the transcripts (igfbp2a and 5b) that were elevated by Fsh and decreased those of igfbp6a, as did Fsh, while 11-KT did not change igfbp1a or -3 transcript levels. To evaluate Igfbps effects on spermatogenesis, we quantified under different conditions the mitotic indices and relative section areas occupied by the different spermatogonial generations (type Aund, type A differentiating (Adiff), or type B (B) spermatogonia). Igf3 (100 ng/mL) increased the area occupied by Adiff and B while decreasing the one for Aund. Interestingly, a concentration of Igf3 that was inactive by itself (25 ng/mL) became active in the presence of the Igfbp inhibitor NBI-31772 and mimicked the effect of 100 ng/mL Igf3 on spermatogonia. Studies exploiting the different dynamics of igfbp expression in response to Fsh and adding or removing NBI-31772 at different times showed that the quick downregulation of three igfbp as well as the delayed upregulated of two igfbps all support Igf3 bioactivity, namely the stimulation of spermatogonial differentiation. We conclude that Fsh modulates, directly or via androgens and Igf3, igfbp gene expression, supporting Igf3 bioactivity either by decreasing igfbp1a, -3, -6a or by increasing igfbp2a and -5b gene expression.

Sex steroid production associated with puberty is absent in germ cell-free salmon.

In all vertebrates studied so far, germ cells are not required for pubertal maturation of the gonadal steroidogenic system, subsequent development of secondary sex characteristics and reproductive behavior. To explore if the absence of germ cells affects puberty or growth in Atlantic salmon, germ cell-free (GCF), dnd knockout and wild type (WT) postsmolts were stimulated to enter puberty. No GCF fish entered puberty, whereas 66.7% (males) and 30% (females) WT fish completed or entered puberty, respectively. Expression of genes related to steroidogenesis (star, cyp17a1, cyp11ß, cyp19a1a), gonadal somatic cells (insl3, amh, igf3), oocytes (bmp15), gonadotropin receptors (fshr, lhcgr), and pituitary gonadotropic cells (fshb, lhb, gnrhr4) showed an immature status and failure to up-regulate gonadal sex steroid production in male and female GCF fish was also reflected in low or undetectable plasma sex steroids (11-ketotestosterone, estradiol-17ß and testosterone). A gender difference (high in females, low in males) was found in the expression of star and cyp17a1 in GCF fish. No clear difference in growth was detected between GCF and immature WT fish, while growth was compromised in maturing WT males. We demonstrate for the first time in a vertebrate that germ cells are required for pubertal activation of the somatic steroidogenic cells.

Gonadotropin subunits of the characiform Astyanax altiparanae: Molecular characterization, spatiotemporal expression and their possible role on female reproductive dysfunction in captivity.

To better understand the endocrine control of reproduction in Characiformes and the reproductive dysfunctions that commonly occur in migratory fish of this order when kept in captivity, we chose Astyanax altiparanae, which has asynchronous ovarian development and multiple spawning events, as model species. From A. altiparanae pituitary total RNA, we cloned the full-length cDNAs coding for the follicle-stimulating hormone ß subunit (fshb), the luteinizing hormone ß subunit (lhb), and the common gonadotropin α subunit (gpha). All three sequences showed the highest degree of amino acid identity with other homologous sequences from Siluriformes and Cypriniformes. Real-time, quantitative PCR analysis showed that gpha, fshb and lhb mRNAs were restricted to the pituitary gland. In situ hybridization and immunofluorescence, using specific-developed and characterized polyclonal antibodies, revealed that both gonadotropin ß subunits mRNAs/proteins are expressed by distinct populations of gonadotropic cells in the proximal pars distalis. No marked variations for lhb transcripts levels were detected during the reproductive cycle, and 17α,20ß-dihydroxy-4-pregnen-3-one plasma levels were also constant, suggesting that the reproductive dysfunction seen in A. altiparanae females in captivity are probably due to a lack of increase of Lh synthesis during spawning season. In contrast, fshb transcripts changed significantly during the reproductive cycle, although estradiol-17ß (E2) levels remained constant during the experiment, possibly due to a differential regulation of E2 synthesis. Taken together, these data demonstrate the putative involvement of gonadotropin signaling on the impairment of the reproductive function in a migratory species when kept in captivity. Future experimental studies must be carried to clarify this hypothesis. All these data open the possibility for further basic and applied studies related to reproduction in this fish model.

Igf Binding Proteins Protect Undifferentiated Spermatogonia in the Zebrafish Testis Against Excessive Differentiation.

IGF binding proteins (IGFBPs) modulate the availability of IGFs for their cognate receptors. In zebrafish testes, IGF3 promotes the proliferation and differentiation of type A undifferentiated (Aund) spermatogonia, and igf3 expression is strongly elevated by FSH but also responds to T3. Here we report the effects of FSH and T3 on igfbp transcript levels in adult zebrafish testis. We then examined T3 and FSH effects on zebrafish spermatogenesis and explored the relevance of IGFBPs in modulating these T3 or FSH effects, using a primary tissue culture system for adult zebrafish testis. T3 up-regulated igfbp1a and igfbp3 expression, whereas FSH reduced igfbp1a transcript levels. To quantify effects on spermatogenesis, we determined the mitotic index and relative section areas occupied by Aund, type A differentiating, or type B spermatogonia. In general, T3 and FSH stimulated spermatogonial proliferation and increased the areas occupied by spermatogonia, suggesting that both self-renewal and differentiating divisions were stimulated. Preventing IGF/IGFBP interaction by NBI-31772 further increased T3- or FSH-induced spermatogonial proliferation. However, under these conditions the more differentiated type A differentiating and B spermatogonia occupied larger surface areas at the expense of the area held by Aund spermatogonia. Clearly decreased nanos2 transcript levels are in agreement with this finding, and reduced amh expression may have facilitated spermatogonial differentiation. We conclude that elevating IGF3 bioactivity by blocking IGFBPs shifted T3- or FSH-induced signaling from stimulating spermatogonial self-renewal as well as differentiation toward predominantly stimulating spermatogonial differentiation, which leads to a depletion of type Aund spermatogonia.