Targeted mutation of secretogranin-2 disrupts sexual behavior and reproduction in zebrafish.

The luteinizing hormone surge is essential for fertility as it triggers ovulation in females and sperm release in males. We previously reported that secretoneurin-a, a neuropeptide derived from the processing of secretogranin-2a (Scg2a), stimulates luteinizing hormone release, suggesting a role in reproduction. Here we provide evidence that mutation of the scg2a and scg2b genes using TALENs in zebrafish reduces sexual behavior, ovulation, oviposition, and fertility. Large-scale spawning within-line crossings (n = 82 to 101) were conducted. Wild-type (WT) males paired with WT females successfully spawned in 62% of the breeding trials. Spawning success was reduced to 37% (P = 0.006), 44% (P = 0.0169), and 6% (P < 0.0001) for scg2a-/- , scg2b-/- , and scg2a-/-;scg2b-/- mutants, respectively. Comprehensive video analysis indicates that scg2a-/-;scg2b-/- mutation reduces all male courtship behaviors. Spawning success was 47% in saline-injected WT controls compared to 11% in saline-injected scg2a-/-;scg2b-/- double mutants. For these mutants, spawning success increased 3-fold following a single intraperitoneal (i.p.) injection of synthetic secretoneurin-a (P = 0.0403) and increased 3.5-fold with injection of human chorionic gonadotropin (hCG). Embryonic survival at 24 h remained on average lower in scg2a-/-;scg2b-/- fish compared to WT injected with secretoneurin-a (P < 0.001). Significant reductions in the expression of gonadotropin-releasing hormone 3 in the hypothalamus, and luteinizing hormone beta and glycoprotein alpha subunits in the pituitary provide evidence for disrupted hypothalamo-pituitary function in scg2a and scg2b mutant fish. Our results indicate that secretogranin-2 is required for optimal reproductive function and support the hypothesis that secretoneurin is a reproductive hormone.

Secretoneurin is a secretogranin-2 derived hormonal peptide in vertebrate neuroendocrine systems.

Secretogranin-2 (SCG2) is a large precursor protein that is processed into several potentially bioactive peptides, with the 30-43 amino acid central domain called secretoneurin (SN) being clearly evolutionary conserved in vertebrates. Secretoneurin exerts a diverse array of biological functions including regulating nervous, endocrine, and immune systems in part due to its wide tissue distribution. Expressed in some neuroendocrine neurons and pituitary cells, SN is a stimulator of the synthesis and release of luteinizing hormone from both goldfish pituitary cells and the mouse LßT2 cell line. Neuroendocrine, paracrine and autocrine signaling pathways for the stimulation of luteinizing hormone release indicate hormone-like activities to regulate reproduction. Mutation of the scg2a and scg2b genes using TALENs in zebrafish reduces sexual behavior, ovulation, oviposition, and fertility. A single injection of the SNa peptide enhanced reproductive outcomes in scg2a/scg2b double mutant zebrafish. Evidence in goldfish suggests a new role for SN to stimulate food intake by actions on other feeding-related neuropeptides. Expression and regulation of the Scg2a precursor mRNA in goldfish gut also supports a role in feeding. In rodent models, SN has trophic-like properties promoting both neuroprotection and neuronal plasticity and has chemoattractant properties that regulate neuroinflammation. Data obtained from several cellular models suggest that SN binds to and activates a G-protein coupled receptor (GPCR), but a bona fide SN receptor protein needs to be identified. Other signaling pathways for SN have been reported which provides alternatives to the GPCR hypothesis. These include AMP-activated protein kinase (AMPK), extracellular signal-regulated kinases (ERK), mitogen-activated protein kinase (MAPK)and calcium/calmodulin-dependent protein kinase II in cardiomyocytes, phosphatidylinositol 3-kinase (PI3K) and Akt/Protein Kinase B (AKT, and MAPK in endothelial cells and Janus kinase 2/signal transducer and activator of transcription protein (JAK2-STAT) signaling in neurons. Some studies in cardiac cells provide evidence for cellular internalization of SN by an unknown mechanism. Many of the biological functions of SN remain to be fully characterized, which could lead to new and exciting applications.

Estrogen-dependent expression and function of secretogranin 2a in female-specific peptidergic neurons.

Secretogranin 2 (Scg2) is a member of the secretogranin/chromogranin family of proteins that is involved in neuropeptide and hormone packaging to secretory granules and serves as a precursor for several secreted pleiotropic peptides. A recent study in zebrafish showed that the teleost Scg2 orthologs, scg2a and scg2b, play an important role in mating behavior, but its modes of action and regulatory mechanisms remain unclear. In this study, we identify scg2a in another teleost species, medaka, by transcriptomic analysis as a gene that is expressed in an ovarian secretion-dependent manner in a group of neurons relevant to female sexual receptivity, termed FeSP neurons. Investigation of scg2a expression in the FeSP neurons of estrogen receptor (Esr)-deficient medaka revealed that it is dependent on estrogen signaling through Esr2b, the major determinant of female-typical mating behavior. Generation and characterization of scg2a-deficient medaka showed no overt changes in secretory granule packaging in FeSP neurons. This, along with the observation that Scg2a and neuropeptide B, a major neuropeptide produced by FeSP neurons, colocalize in a majority of secretory granules, suggests that Scg2a mainly serves as a precursor for secreted peptides that act in conjunction with neuropeptide B. Further, scg2a showed sexually biased expression in several brain nuclei implicated in mating behavior. However, we found no significant impact of scg2a deficiency on the performance of mating behavior in either sex. Collectively, our results indicate that, although perhaps not essential for mating behavior, scg2a acts in an estrogen/Esr2b signaling-dependent manner in neurons that are relevant to female sexual receptivity.

Characterization of blood-derived exosomal proteins after exercise.

OBJECTIVE: To assess changes in plasma exosome levels and protein content in mice after long-term exercise. METHODS: We subjected 9-month-old adult C57BL/6J mice to daily treadmill running exercise for 4 weeks prior to the isolation of blood-derived exosomes. Exosomal proteins were identified using mass spectrometry. RESULTS: Extracellular bodies were successfully isolated from mouse blood. Protein levels were altered in blood-derived exosomes after chronic treadmill exercise. Levels of the secretagogue secretogranin 2 were markedly elevated in exercise-induced exosomes. CONCLUSION: Our data suggest that levels of secretogranin 2 were increased in mouse exosomes following chronic treadmill exercise. We conclude that exercise increases exocrine secretion of secretogranin 2.

Striatal Neurotransmitter Release-related Presynaptic Proteins in L-dopa Induced Dyskinesia in a Model of Parkinsonism.

INTRODUCTION: In Parkinson's disease, L-dopa-induced dyskinesia (LID) and motor fluctuations incapacitate patients as much as the disease itself. Many studies demonstrated that postsynaptic alterations and striatal synaptic plasticity changes play a role in LID development. Here, we aimed to study the role of striatal presynaptic proteins in LID pathogenesis. METHODS: For this purpose, 6-hydroxydopamine model of parkinsonism was used. To induce LID, these rats were treated with intraperitoneal injections of L-dopa 25 mg/kg with benserazide 6.25 mg/kg b.i.d for 21 days. Rats with parkinsonism treated with saline and control rats treated with saline or L-dopa/ benserazide were also included. Behaviors of rats were videotaped and scored according to dyskinesia scale. Striatal tissue was analysed with immunofluorescence staining and immunoblotting to confirm loss of tyrosine hydroxylase (TH) expression due to dopaminergic denervation and to explore the alterations in the expression of presynaptic proteins, secretogranin 2 (SG2), synaptophysin (Syp) and synaptotagmin 7 (Syt7). RESULT: LID developed only in rats with parkinsonism treated with chronic L-dopa. Immunofluorescence and immunoblotting studies for TH confirmed depletion of dopaminergic neurons, which was also strongly and negatively correlated with severity of LID. Striatal SG2 and Syp levels were found increased in parkinsonian rats. Chronic L-dopa treatment further increased SG2 levels in denervated striatum. Striatal SG 2 level showed a significant moderate, positive correlation with LID severity. Immunofluorescence studies also demonstrated increased expression of these presynaptic proteins in the denervated striatum. CONCLUSION: As, severity of LID was clearly correlated with striatal SG2 expression; there is supposedly a functional relationship between striatal SG2 and LID. Further studies are needed to find out molecular mechanisms linking increased SG2 expression and LID.