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.publishedVersio

Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, and expression analysis

<p>Abstract</p> <p>Background</p> <p>In recent years, the relaxin family of signaling molecules has been shown to play diverse roles in mammalian physiology, but little is known about its diversity or physiology in teleosts, an infraclass of the bony fishes comprising ~ 50% of all extant vertebrates. In this paper, 32 relaxin family sequences were obtained by searching genomic and cDNA databases from eight teleost species; phylogenetic, molecular evolutionary, and syntenic data analyses were conducted to understand the relationship and differential patterns of evolution of relaxin family genes in teleosts compared with mammals. Additionally, real-time quantitative PCR was used to confirm and assess the tissues of expression of five relaxin family genes in <it>Danio rerio </it>and <it>in situ </it>hybridization used to assess the site-specific expression of the insulin 3-like gene in <it>D. rerio </it>testis.</p> <p>Results</p> <p>Up to six relaxin family genes were identified in each teleost species. Comparative syntenic mapping revealed that fish possess two paralogous copies of human <it>RLN3</it>, which we call <it>rln3a </it>and <it>rln3b</it>, an orthologue of human <it>RLN2</it>, <it>rln</it>, two paralogous copies of human <it>INSL5</it>, <it>insl5a and insl5b</it>, and an orthologue of human <it>INSL3</it>, <it>insl3</it>. Molecular evolutionary analyses indicated that: <it>rln3a, rln3b </it>and <it>rln </it>are under strong evolutionary constraint, that <it>insl3 </it>has been subject to moderate rates of sequence evolution with two amino acids in <it>insl3/INSL3 </it>showing evidence of positively selection, and that <it>insl5b </it>exhibits a higher rate of sequence evolution than its paralogue <it>insl5a </it>suggesting that it may have been neo-functionalized after the teleost whole genome duplication. Quantitative PCR analyses in <it>D. rerio </it>indicated that <it>rln3a </it>and r<it>ln3b </it>are expressed in brain, <it>insl3 </it>is highly expressed in gonads, and that there was low expression of both <it>insl5 </it>genes in adult zebrafish. Finally, <it>in situ </it>hybridization of <it>insl3 </it>in <it>D. rerio </it>testes showed highly specific hybridization to interstitial Leydig cells.</p> <p>Conclusions</p> <p>Contrary to previous studies, we find convincing evidence that teleosts contain orthologues of four relaxin family peptides. Overall our analyses suggest that in teleosts: 1) <it>rln3 </it>exhibits a similar evolution and expression pattern to mammalian <it>RLN3</it>, 2) <it>insl3 </it>has been subject to positive selection like its mammalian counterpart and shows similar tissue-specific expression in Leydig cells, 3) <it>insl5 </it>genes are highly represented and have a relatively high rate of sequence evolution in teleost genomes, but they exhibited only low levels of expression in adult zebrafish, 4) <it>rln </it>is evolving under very different selective constraints from mammalian <it>RLN</it>. The results presented here should facilitate the development of hypothesis-driven experimental work on the specific roles of relaxin family genes in teleosts.</p

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

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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, gnrhr1caβ, gnrhr1cbα, gnrhr1cbβ, gnrhr2bbα, gnrhr2bbβ. 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.publishedVersio

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

Rehabilitation in patients with radically treated respiratory cancer: A randomised controlled trial comparing two training modalities.

INTRODUCTION: The evidence on the effectiveness of rehabilitation in lung cancer patients is limited. Whole body vibration (WBV) has been proposed as an alternative to conventional resistance training (CRT). METHODS: We investigated the effect of radical treatment (RT) and of two rehabilitation programmes in lung cancer patients. The primary endpoint was a change in 6-min walking distance (6MWD) after rehabilitation. Patients were randomised after RT to either CRT, WBVT or standard follow-up (CON). Patients were evaluated before, after RT and after 12 weeks of intervention. RESULTS: Of 121 included patients, 70 were randomised to either CON (24), CRT (24) or WBVT (22). After RT, 6MWD decreased with a mean of 38m (95% CI 22-54) and increased with a mean of 95m (95% CI 58-132) in CRT (p<0.0001), 37m (95% CI -1-76) in WBVT (p=0.06) and 1m (95% CI -34-36) in CON (p=0.95), respectively. Surgical treatment, magnitude of decrease in 6MWD by RT and allocation to either CRT or WBVT were prognostic for reaching the minimally clinically important difference of 54m increase in 6MWD after intervention. CONCLUSIONS: RT of lung cancer significantly impairs patients' exercise capacity. CRT significantly improves and restores functional exercise capacity, whereas WBVT does not fully substitute for CRT

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), 17β-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.publishedVersio

Effects of re-stripping on the seminal characteristics of pacu (Piaractus mesopotamicus) during the breeding season

Seminal characteristics in teleost fish with an annual reproductive period, such as pacu (Piaractus mesopotamicus), may vary during the breeding season. The sperm formed before the beginning of the spawning period may be stored for a long time, causing damage to the cells. Therefore, re-stripping may be an important way to eliminate the "old" and allow for the collection of "new" spermatozoids. In this study, we analyzed the seminal characteristics of hormonally induced pacu at the beginning, middle and end of the breeding season, and we analyzed samples from re-stripped males (stripped first at the beginning, re-stripped in the middle, and re-stripped again at the end of the season) during two breeding seasons. The sperm density, ionic composition, pH, and osmolality were similar among the groups. The semen volume, seminal plasma protein concentration and incidence of morphologically anomalous sperm increased over time. In addition, some parameters that are associated with good-quality semen decreased, such as sperm motility, viability and DNA integrity. Moreover, we observed a positive association among motility, viability and DNA integrity for sperm with elevated 11-ketotestosterone, but there was no such association for fshb or lhb mRNA levels in the pituitary. The semen that was obtained earlier (at the beginning) or from re-stripped males exhibited better characteristics than the other samples collected. In conclusion, collecting semen from pacu at the end of breeding season should be avoided; it is preferable to strip early and then re-strip later in the season, and this approach may be used for diverse aquaculture purposes

Characteristics, Treatment Strategies and Outcome in Cardiogenic Shock Complicating Acute Myocardial Infarction:A Contemporary Dutch Cohort

Cardiogenic shock (CS) complicating acute myocardial infarction (AMI) is associated with high morbidity and mortality. Our study aimed to gain insights into patient characteristics, outcomes and treatment strategies in CS patients. Patients with CS who underwent percutaneous coronary intervention (PCI) between 2017 and 2021 were identified in a nationwide registry. Data on medical history, laboratory values, angiographic features and outcomes were retrospectively assessed. A total of 2328 patients with a mean age of 66 years and of whom 73% were male, were included. Mortality at 30 days was 39% for the entire cohort. Non-survivors presented with a lower mean blood pressure and increased heart rate, blood lactate and blood glucose levels (p-value for all &lt;0.001). Also, an increased prevalence of diabetes, multivessel coronary artery disease and a prior coronary event were found. Of all patients, 24% received mechanical circulatory support, of which the majority was via intra-aortic balloon pumps (IABPs). Furthermore, 79% of patients were treated with at least one vasoactive agent, and multivessel PCI was performed in 28%. In conclusion, a large set of hemodynamic, biochemical and patient-related characteristics was identified to be associated with mortality. Interestingly, multivessel PCI and IABPs were frequently applied despite a lack of evidence.</p

Definition of the interacting interfaces of Apobec3G and HIV-1 Vif using MAPPIT mutagenesis analysis

The host restriction factor Apobec3G is a cytidine deaminase that incorporates into HIV-1 virions and interferes with viral replication. The HIV-1 accessory protein Vif subverts Apobec3G by targeting it for proteasomal degradation. We propose a model in which Apobec3G N-terminal domains symmetrically interact via a head-to-head interface containing residues 122 RLYYFW 127. To validate this model and to characterize the Apobec3G–Apobec3G and the Apobec3G–Vif interactions, the mammalian protein–protein interaction trap two-hybrid technique was used. Mutations in the head-to-head interface abrogate the Apobec3G–Apobec3G interaction. All mutations that inhibit Apobec3G–Apobec3G binding also inhibit the Apobec3G–Vif interaction, indicating that the head-to head interface plays an important role in the interaction with Vif. Only the D128K, P129A and T32Q mutations specifically affect the Apobec3G–Vif association. In our model, D128, P129 and T32 cluster at the edge of the head-to-head interface, possibly forming a Vif binding site composed of two Apobec3G molecules. We propose that Vif either binds at the Apobec3G head-to-head interface or associates with an RNA-stabilized Apobec3G oligomer