Hormonal changes over the spawning cycle in the female three-spined stickleback, Gasterosteus aculeatus.

Female three-spined sticklebacks are batch spawners laying eggs in a nest built by the male. We sampled female sticklebacks at different time points, when they were ready to spawn and 6, 24, 48 and 72h post-spawning (hps) with a male. Following spawning, almost all females (15 out of 19) had ovulated eggs again at Day 3 post-spawning (72hps). At sampling, plasma, brain and pituitaries were collected, and the ovary and liver were weighed. Testosterone (T) and estradiol (E2) were measured by radioimmunoassay. Moreover, the mRNA levels of follicle-stimulating hormone (fsh-ß) and luteinizing hormone (lh-ß) in the pituitary, and of the gonadotropin-releasing hormones (GnRHs: gnrh2, gnrh3) and kisspeptin (kiss2) and its G protein-coupled receptor (gpr54) in the brain were measured by real-time qPCR. Ovarian weights peaked in "ready to spawn" females, dropped after spawning, before again progressively increasing from 6 to 72hps. Plasma T levels showed peaks at 24 and 48hps and decreased at 72hps, while E2 levels increased already at 6hps and remained at high levels up to 48hps. There was a strong positive correlation between T and E2 levels over the spawning cycle. Pituitary lh-ß mRNA levels showed a peak at 48hps, while fsh-ß did not change. The neuropeptides and gpr54 did not show any changes. The changes in T and E2 over the stickleback spawning cycle were largely consistent with those found in other multiple-spawning fishes whereas the marked correlation between T and E2 does not support T having other major roles over the cycle than being a precursor for E2.

Developmental exposure to progestins causes male bias and precocious puberty in zebrafish (Danio rerio).

Progestins are aquatic contaminants that in low concentrations can impair fish reproduction. The mechanisms are likely multiple since different progestins interact with other steroid receptors in addition to progesterone receptors. Puberty is the process when animals first acquire the capability to reproduce and it comprises maturation of sperm and eggs. In zebrafish, puberty is initiated around 45days post fertilization (dpf) in females and around 53-55 dpf in males, and is marked by increased production of pituitary gonadotropins. We exposed juvenile zebrafish from 20 to 80 dpf to the androgenic progestin levonorgestrel at concentrations of 5.5, 79 and 834ngL(-1) and to the non-androgenic progestin progesterone at concentrations of 3.7, 77 and 1122ngL(-1), during sexual differentiation and puberty. Levonorgestrel exposure caused 100% males even at the lowest concentration tested whereas progesterone did not affect the sex ratio. Transcript levels of the gonadal genes amh, CYP11B and CYP19a1a indicated that the masculinizing effect of levonorgestrel occurred very rapidly. Transcript concentrations of gonadotropins in pituitaries were low in control fish at 44 dpf, but high at 55 dpf and onward. In fish exposed to levonorgestrel or progesterone gonadotropin transcript concentrations were high already at 44 dpf, indicating that both progestins caused precocious puberty. Gonad histology at 50 dpf confirmed a well advanced sexual maturation, but only in males. Our results show that progestins can affect sexual development in fish and that the androgenic progestin levonorgestrel induces a male phenotype at concentrations similar to those detected in aquatic environments.

Schistocephalus solidus infections increase gonadotropins and gonadotropin releasing hormone (GnRH3) mRNA levels in the three-spined stickleback, Gasterosteus aculeatus.

Parasites often impair the reproduction of their hosts, one well known case being the cestode Schistocephalus solidus which is a common parasite in three-spined sticklebacks, Gasterosteus aculeatus. One of the possible ways that this could be exerted is by suppression on the brain-pituitary-gonadal (BPG) axis. In this study, mRNA levels of FSH-ß and LH-ß and of GnRH2 (cGnRH II) and GnRH3 (sGnRH) were measured via Q-PCR in infected and uninfected fish sampled from the field a few weeks before the onset of breeding. The pituitary mRNA levels of both FSH-ß and LH-ß were higher in infected males than in uninfected males. Also in females, FSH-ß mRNA levels were higher in infected individuals than in others, whereas there was no significant difference found in LH-ß expression. Brain mRNA levels of GnRH3 were higher in infected fish than in uninfected fish in both sexes, but no difference was found in GnRH2 mRNA levels. Thus, infection by S. solidus was able to alter the expressions not only of gonadotropins (GtHs), but also of GnRH which has not been observed previously. However, the effects are opposite to what should be expected if the parasite suppressed reproduction via actions on the brain-pituitary level. The gonads are perhaps more likely to be impaired by the parasites in other ways, and changed feedbacks on the BPG axis could then lead to the increases in GtHs and GnRH.

Effects of castration and androgen-treatment on the expression of FSH-beta and LH-beta in the three-spine stickleback, gasterosteus aculeatus--feedback differences mediating the photoperiodic maturation response?

In many animals, including the three-spine stickleback (Gasterosteus aculeatus), photoperiod strongly influences reproduction. The aim of this study was to investigate if feedback mechanisms on the brain-pituitary-gonadal axis play a role in mediating the photoperiodic response in the stickleback. To that end, stickleback males, exposed to either non-stimulatory short photoperiod (light/dark 8:16) or under stimulatory long photoperiod (LD 16:8), were subjected to either sham-operation, castration, castration combined with treatment with the androgens 11-ketoandrostenedione (11KA) and testosterone (T), and the effects on levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH)-beta mRNA were analyzed. During breeding season the kidney of the stickleback male hypertrophies and produces a glue used for building nests. Kidney weight and expression of both LH-beta and FSH-beta were higher in sham-operated fish kept under long than under short photoperiod. Under both photoperiods, LH-beta mRNA levels were lower in castrated males compared to sham-operated males and treatment with 11KA and T increased expression, indicating a positive feedback. A positive feedback was also found on FSH-beta expression under long photoperiod, where castration decreased, and androgen replacement restored FSH-beta mRNA expression. On the contrary, castration under short photoperiod instead increased FSH-beta levels whereas treatment with 11KA and T decreased FSH-beta expression, indicating a negative feedback on FSH-beta under these conditions. The positive feedback on FSH-beta expression under stimulatory photoperiod may accelerate maturation, whereas the negative feedback under inhibitory photoperiod may suppress maturation. This could be part of the mechanisms by which photoperiod controls maturation.

Comparable long-term survival after bone marrow versus peripheral blood progenitor cell transplantation from matched unrelated donors in children with hematologic malignancies.

Despite the increasing use of peripheral blood progenitor cells (PBPC) instead of bone marrow (BM) for allogeneic hematopoietic stem cell transplantation (allo HSCT) from human leukocyte antigen (HLA)-matched unrelated donors in children, the relative benefits and risks of both stem cell sources in the pediatric setting remain largely unknown. Recently, the only larger study comparing the value of the 2 stem cell sources in a young patient group was confined to transplantation from HLA-identical sibling donors in older children and adolescents with acute leukemia. Based on the paucity of data in pediatric HLA-matched unrelated donor transplantation, we analyzed the outcome of 23 BM and 38 PBPC transplantations performed at our center. Neutrophil and platelet engraftment were achieved significantly faster in PBPC compared to BM recipients (18 versus 22 days and 26 versus 33 days; P < .001 and P = .03) whereas the risk for grade II-IV acute graft-versus-host disease (aGVHD) (62% versus 55%; P = .53) and chronic GVHD (cGVHD 65% versus 59%; P = .54) was comparable. As overall survival (OS; PBPC versus BM: 47.5% +/- 8.6% versus 51.8% +/- 10.5%; P = .88) and relapse-free survival (43.3% +/- 8.3% versus 51.8% +/- 10.5%; P = .60) are without detectable difference, PBPC and BM appear both as a valid stem cell source for HLA-matched unrelated donor transplantation in children with hematologic malignancies.

Seasonal changes in expression of LH-beta and FSH-beta in male and female three-spined stickleback, Gasterosteus aculeatus.

In teleost fishes, like in other vertebrates, the gonadal development is stimulated by two gonadotropic hormones; luteinizing hormone (LH) and follicle-stimulating hormone (FSH). To achieve a better understanding of the role of gonadotropins in teleost reproduction; expression of LH-beta and FSH-beta mRNA and the status of gonads and secondary sexual characters were analyzed over the annual cycle in male and female three-spined sticklebacks, a species in which the development of male secondary sexual characters and spermatogenesis are separated in time. The kidney in the male stickleback hypertrophies during the breeding season and produces a glue used when building nests. Kidney weights, as well as levels of 11-ketotestosterone (11KT), reached a peak in May. Both testosterone (T) levels and the gonadosomatic index (GSI, gonad weight/body weight x 100) in females started to increase in April, and peaked in May as well. Later in summer, after the breeding season, these features declined. In females, LH-beta expression followed the GSI and T levels closely, levels were low during winter and early spring, increased to a peak in late May and declined to low levels again in July. FSH-beta expression peaked earlier, in January and declined slowly over spring. In males, LH-beta expression peaked in May. During June-September, when spermatogenesis was active, LH-beta levels were very low. FSH-beta expression peaked in January, earlier than LH-beta expression did, and reached the lowest levels in July. Thus, when spermatogenesis started at the end of summer, the expression of both GTH-beta mRNAs, and circulating 11KT, displayed their lowest levels.

Differential regulation of luteinizing hormone and follicle-stimulating hormone expression during ovarian development and under sexual steroid feedback in the European eel.

Pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are, in teleosts as in mammals, under the control of hypothalamic factors and steroid feedbacks. In teleosts, feedback regulations largely vary depending on species and physiological stage. In the present study the regulation of FSH and LH expression was investigated in the European eel, a fish of biological and phylogenetical interest as a representative of an early group of teleosts. The eel FSHbeta subunit was cloned, sequenced and together with earlier isolated eel LHbeta and glycoprotein hormone alpha (GPalpha) subunits used to study the differential regulation of LH and FSH. In situ hybridization indicated that FSHbeta and LHbeta are expressed by separate cells of the proximal pars distalis of the adenohypophysis, differently from the situation in mammals. The profiles of LHbeta and FSHbeta subunit expression were compared during experimental ovarian maturation, using dot-blot assays. Expression levels for LHbeta and GPalpha increased throughout ovarian development with a positive correlation between these two subunits. Conversely, FSHbeta mRNA levels decreased. To understand the role of sex steroids in these opposite variations, immature eels were treated with estradiol (E2)and testosterone (T), both steroids being produced in eel ovaries during gonadal development. E2 treatment induced increases in both LHbeta and GPalpha mRNA levels, without any significant effect on FSHbeta. In contrast, T treatment induced a decrease in FSHbeta mRNA levels, without any significant effect on the other subunits. These data demonstrate that steroids exert a differential feedback on eel gonadotropin expression, with an E2-specific positive feedback on LH and a T-specific negative feedback on FSH, leading to an opposite regulation of LH and FSH during ovarian development.

Endocrine evidence that silvering, a secondary metamorphosis in the eel, is a pubertal rather than a metamorphic event.

Silvering (transition from yellow to silver eel) has been traditionally considered as a metamorphosis in view of the numerous morphological, physiological and behavioral changes preparing the eel for the oceanic migration. However, some changes, such as increases in gonad weight and steroidogenesis, suggest that silvering could also be considered as a pubertal event. In order to assess which endocrine axis may be involved in the induction of silvering, we compared the profiles of pituitary and peripheral hormones during the transition from yellow to silver female eels. A strong activation of the gonadotropic axis was shown during silvering. Follicle-stimulating hormone (FSH) mRNA levels increased during the early stages of silvering, followed by a later increase in luteinizing hormone (protein and mRNA) levels. In addition, plasma levels of sexual steroids (estradiol, E2; testosterone, T, and 11-ketotestosterone) and of vitellogenin significantly increased. In contrast, thyrotropin mRNA levels did not change and no or weak variations in plasma thyroid hormones were observed, indicating no or moderate change of the thyrotropic axis during silvering. Similarly, the somatotropic axis was not activated, as shown by pituitary growth hormone expression (protein and mRNA) and plasma levels. In addition, we studied the effects of chronic treatments of female yellow eels with thyroid hormone (thyroxine, T4) and sex steroids (T and E2) on biometrical parameters characteristics of silvering. T induced an increase in eye size and a reduction of digestive tract, whereas T4 and E2 had no effect. These hormonal profiles and experimental data lead to the conclusion that eel silvering should be considered as an onset of puberty rather than a 'genuine' metamorphosis.

Cloning and sequencing of the FSH-beta and LH beta-subunit in the three-spined stickleback, Gasterosteus aculeatus, and effects of photoperiod and temperature on LH-beta and FSH-beta mRNA expression.

The aim of this study was to characterize the FSH-beta and LH-beta subunit in the three-spined stickleback, a fish used extensively in experimental studies, and to use the cloned cDNAs as probes for measuring FSH-beta and LH-beta mRNA expression in sticklebacks treated with different photoperiods and temperatures. A first strand cDNA was prepared from 10 pituitaries from male sticklebacks, and cDNA fragments were amplified by PCR using degenerated primers based on highly conserved regions of known teleost FSH-beta and LH-beta cDNA sequences. To obtain full-length cDNAs of FSH-beta and LH-beta, RACE amplifications were performed. The cDNA of FSH-beta was 540 bp long, encoding a protein of 122 amino acids and LH-beta cDNA was 568 bp long, encoding a protein of 148 amino acids. Of gonadotropin (GTH) beta-subunits published so far, those most similar to stickleback GTHs are found among other percomorph fishes, with amino acid similarities of 46-55% for FSH-beta and 75-77% for LH-beta subunits.The cloned cDNAs were used as probes to analyze LH and FSH mRNA expression in pituitaries from sticklebacks exposed to different photoperiods and temperatures. Two hundred males were divided into four groups and kept under different photoperiods and temperatures; light:dark (LD) 16:8, 20 degrees C; LD 16:8, 7 degrees C; LD 8:16, 20 degrees C, and LD 8:16, 7 degrees C. Red breeding colors and a marked kidney hypertrophy, androgen-dependent male secondary sexual characters in the stickleback, appeared in the group kept under LD 16:8 at 20 degrees C, but not in the other groups. The kidney epithelium height (KEH) was significantly lower in the LD 8:16, 20 degrees C group than in all other groups, and this was also the only group with active spermatogenesis. The LD 8:16 20 degrees C had significantly lower expression of both FSH-beta and LH-beta than all other groups. Thus, both GTHs displayed their lowest expression when spermatogenesis was active and when low KEH indicated that androgens levels were at their lowest.